KR20120065654A - A composition comprising sialic acid-containing whey protein for preventing animal feeding addition and inclused animal feeding of influenza virus infectious disease - Google Patents

Abstract

PURPOSE: An additive for animal feed for preventing the influenza virus infection, and the animal feed containing thereof are provided to use sialic acid-containing whey protein as an active ingredient. CONSTITUTION: An additive for animal feed for preventing the influenza virus infection contains sialic acid-containing whey protein as an active ingredient. The additive contains approximately more than 4wt% of sialic acid for the total amount of the sialic acid-containing whey protein. The additive additionally contains 0.1-50wt% sialic acid-containing whey protein.

Description

Additives for the prevention of influenza virus infection containing sialic acid-containing whey protein as an active ingredient and composition for animal feed comprising the same (A composition comprising sialic acid-containing whey protein for preventing animal feeding addition and inclused animal feeding of influenza virus infectious disease}

An object of the present invention is to provide a feed and additive composition of influenza virus infection having an inhibitory effect against influenza virus containing sialic acid-containing whey protein as an active ingredient.

Selmons et al., Avian Dis., 18 (1), pp 119-124, 1974.

Document 2 Webster RG et al., Microbiol Rev., 56 (1), pp152-179, 1992

Document 3 Alexander DJ, Vet. Microbiol., 74 (1-2), pp3-13, 2000

Document 4 Grmek MD, Les Maladies a L'aube de la Civilization Accidentale, Payot, Paris, 1893

Document 5 JH, Bull. NY Acad. Med., 54, pp 855-864, 1978

Reference 6 Taubenberger JK et al., Science, 275, pp1793-1796, 1997

7 Potter CW, J. appl. Microbiol., 91, pp 572-579, 2001

[Reference 8] Oxford JS, Rev. Med. Virol., 10 (2), pp119-133, 2000

9 Suarez DL et al., J. Virol., 72 (8), pp6678-6688, 1998

Ward P et al., J. antimicrob. Chemother., 55 (supp1), ppi5-i21, 2005

11 Rhicha Sinha et al., 2007, Food Chemistry, 101, pp 1484-1491

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Influenza virus is an RNA virus belonging to the family Orthomyxoviridae, and serotypes are classified into three types, type A, type B and type C. Among them, hepatitis B and C have been identified only in humans, while hepatitis A has been identified in humans, horses, pigs, other mammals and various types of poultry and wild birds (Selmons et al., Avian Dis. , 18 (1), pp 119-124, 1974; Webster RG et al., Microbiol Rev., 56 (1), pp 152-179, 1992).

The serotypes of influenza A virus are classified according to the two types of proteins on the surface of the virus: hemagglutinin (HA) and neuraminidase (NA), and 144 types (HA protein) 16 species and 9 NA proteins). HA acts as a virus to attach to somatic cells, and NA allows the virus to penetrate into cells (Alexander DJ, Vet. Microbiol., 74 (1-2), pp3-13, 2000). The normal natural host of influenza A virus is known as wild waterfowl such as ducks and seagulls.As a result of epidemiological investigations of influenza infection in wild birds around the world, there are 16 existing HA and 9 NA influenza species. The virus was confirmed to be infected in wild birds (Selmons et al., Avian Dis., 18 (1), pp119-124, 1974). Avian influenza virus, classified as type A, is a common infectious disease virus, a non-pathogenic avian influenza that causes mild respiratory symptoms when infected with chickens, and low pathogenicity that causes mortality and spawning degradation of 1-30%. Low pathogenic avian influenza (LPAI) and high pathogenic avian influenza (HPAI) with over 95% mortality and high pathogenic avian influenza (HPAI), also known as "bird flu." (Alexander DJ, Vet. Microbiol., 74 (1-2), pp3-13, 2000). Among them, highly pathogenic avian influenza is classified as Class A by the International Water Office (OIE) and domestic type 1 infectious disease.

The first record of human influenza dates back to 412 B.C.E., but the first pandemic influenza record of mankind is estimated to have occurred throughout Europe from 1173 to 1174 (Grmek MD, Les Maladies a L '). aube de la Civilization Accidentale, Payot, Paris, 1893). Since the 20th century, records of influenza have been more scientifically treated, based on the data that have been left behind, since the 20th century there have been three pandemic influenza. Since the twentieth century, the world-wide pandemic between 1918 and 1920, the pandemic of the first influenza pandemic (aka Spanish flu) has been recorded as the greatest damage to humanity, and between 20 and 50 million people died during that period. Walter JH, Bull.NY Acad.Med., 54, pp 855-864, 1978). The causative agent of the Spanish flu virus has been identified as serotype A / H1N1, which is very similar to the swine influenza virus and is still a major epidemic of pandemic flu, which is still prevalent worldwide every year (Taubenberger JK et al., Science, 275, pp1793-1796, 1997). Since the 20th century, which occurred between 1957 and 1958, the second influenza pandemic began in China and rapidly spread along the coast to nearby Hong Kong, Singapore, Japan and Taiwan in six to seven months. About 40-50% of the world's population was infected during this period, of which 25% had clinical symptoms, mainly infants and middle-aged people who died of infection, and about 1 million people died. (Potter CW, J. appl. Microbiol., 91, pp572-579, 2001). The causative virus was identified as serotype A / H2N2 influenza virus. In addition, the third pandemic of the influenza pandemic between 1968 and 1969 was confirmed to originate in Hong Kong and spread rapidly to Taiwan, the Philippines, Singapore and Vietnam. The causative virus is serotype A / H3N2, which differs from the previous serotype H2N2 virus and HA, which has been analyzed to be transmitted from birds, and is still the main epidemic of pandemic flu, which is still prevalent worldwide every year. (Oxford JS, Rev. Med. Virol., 10 (2), pp 119-133, 2000).

Some serotypes of AIV, a major concern in birds, are infected by humans, causing flu and causing death. Several strains of avian-induced avian influenza viruses, including serotype A / H5N1, which has been called "Hong Kong bird flu" to date, and serotype A / H7N7, and serotype A / H9N2, can infect humans. It is estimated that there is a possibility. Therefore, the present study on the mutant viruses derived from these three serotypes is being conducted worldwide (Suarez DL et al., J. Virol., 72 (8), pp6678-6688, 1998).

The case of human infection that overcomes the barriers between avian influenza virus (serum-type A / H5N1 avian influenza virus) recently reported in Vietnam is the prelude to the pandemic of the fourth human influenza pandemic since the 20th century. Attention has been paid to the spread of the mutant virus, including direct transmission from person to person. The mutant virus is an avian influenza virus belonging to serotype A / H5N1, which is also avian influenza virus that has been prevalent only in birds for the first time among humans.

Influenza viruses can infect the respiratory tract, cause systemic symptoms, change their appearance periodically, and move to other hosts without killing them, leading scientists to assume that influenza viruses will survive until the end of humanity. Although it is the virus that causes the greatest economic loss to human beings and preventive vaccines have been developed, they have not caught up with the mutation of the virus, and there is no fundamental virus treatment yet.

Amantadine and rimantadine are substances that inhibit the function of M2 ion channel proteins of influenza viruses and are representative antiviral agents that inhibit the growth of influenza viruses in vivo. However, these two antiviral agents were found to be effective only against serotype A influenza virus and not against serotype B influenza virus without M2 protein. In addition, amantadine and rimantadine have been found to have the disadvantage that the emergence of a mutant virus that does not affect the ion channel function of influenza virus M2 protein when used. Zanamivir and oseltamivir, which are developed to compensate for these drawbacks, inhibit the function of the neuraminidase protein of influenza viruses and are representative of the suppression of influenza virus proliferation in vivo. Antiviral agents. These two antiviral agents are known to be effective against all 16 serotype A influenza viruses and serotype B influenza viruses. However, zanamivir has the disadvantage of inhalation and intravenous administration, while oseltamivir can be administered orally, but it has been pointed out as a disadvantage due to side effects such as recent reports of resistant virus and vomiting and dizziness upon oral administration (Ward P et. al., J. antimicrob.Chemother., 55 (supp1), ppi5-i21, 2005).

Whey contains proteins of high nutritional value and various biological activities. Whey protein refers to a protein contained in milk serum (whey) from which casein is removed from milk, and includes β-lactoglobulin, α-lactoglobulin and trace amounts of BSA, It consists of various bioactive protein factors such as immunoglobulins, phospholipoproteins, lactoferrins, and enzymes (Rhicha Sinha et al., 2007, Food Chemistry, 101, pp1484-). 1491). Whey protein is known to have endurance enhancement, fatigue recovery, and immunity enhancement, and is also used as a protein dispensing material for exercise nutrition foods and diet foods.

Sialic acid is a generic term for several types of neuramic acid, and sialic acid commonly found in glycoproteins is N-acetyl neuraminic acid (Neu5Ac) or N-glyco. N-glycosyl neuraminic acid (Neu5Gc), usually α2-3 or α2-6 bonds, and non-sugar substances such as sulfates, phosphates, 2-aminoethyl phosphates, alkyls, acyl groups Sometimes. Sialic acid is an acidic sugar of gut sugar and does not exist freely in nature except for some metabolic processes.It is composed of oligosaccharides, polysaccharides, glycoproteins, and glycolipids of organs, tissues, blood, mucus, and milk of animals. It is widely present as a component and has not been found in plants, but is relatively widely distributed in microbial systems.

Sialic acid is a component of glycoproteins or glycolipids on the cell surface of the cell membrane and is located at the terminal to exert a variety of biological functions, due to the recent active research, various physiological functions have been elucidated. For example, sialic acid is synthesized from fructose-6-phospate via N-acetylamannoseamine in mammals in vivo. Since sialic acid is a component of gangliosides or glycoproteins in the brain, it is particularly high in the brain and central nervous system, and its amount increases rapidly in infancy, so sialic acid plays an important role in the expression and development of function of these tissues. (SE Carlson, Am. J. Clin. Neut., 41, 720-729, 1985). In addition, the infection of viruses and bacteria begins by binding the virus or bacteria to the sugar group in the mucosal epithelial cells. Toxins produced by Escherichia coli or cholera bacteria bind to the sugar group containing sialic acid in the gut epithelial cells. Causes diarrhea (S. Fukuta et al., Infect. Immun., 56, 1748-1753, 1988). In addition, as an indispensable component of mucoprotein (mucoprotein) has been found to play a variety of roles, such as inhibiting the hemolytic action of influenza virus, in addition to the development of anticancer drugs, neutralization of the toxin components, anti-inflammatory action has been reported. It is also found that there is a non-specific hemagglutination inhibition factor in milk (Saito et al., Agri. Biol. Chem. 36, 1437-1439, 1972), among which sialic acid And oligosaccharides and gangliosides.

Accordingly, the present inventors completed the present invention by confirming an excellent inhibitory effect on influenza virus by confirming an excellent hemagglutination inhibitory ability while studying the effect of sialic acid-containing whey protein on influenza virus.

In order to achieve the above object, the present invention provides an animal feed additive for preventing influenza virus infection containing sialic acid-containing whey protein as an active ingredient and a composition for animal feed comprising the same.

Sialic acid as defined herein is represented by the following structural formula (A).

     Influenza viruses as defined herein are H1N1, H1N2, H2N2, Human B, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H9N1, or preferably H10N7, or H10N7, or H10N7. It is characterized by having a mold.

     Sialic acid-containing whey proteins as defined herein are characterized by comprising products from Japan, China, Europe, and the like, preferably from Japan, more preferably from JFLA, Japan.

     As defined herein, sialic acid is about 4% by weight or more, preferably about 4 to 50% by weight, more preferably about 4 to 20% by weight, and even more preferably about 4% by weight of sialic acid relative to the total weight of the protein. To 10 wt%.

     The preventive animal feed additive for influenza virus infection of the present invention and the composition for animal feed comprising the same include 0.1 to 50% by weight of the sialic acid-containing whey protein based on the total weight of the composition.

     Such animals as defined herein include mammals such as pigs, cows, goats: fish such as carps, goldfish and the like, and poultry such as pheasants, chickens, ducks, turkeys and the like.

     The present invention provides an animal feed additive for preventing influenza virus infection containing sialic acid-containing whey protein as an active ingredient and a composition for animal feed comprising the same.

     The sialic acid-containing whey protein may include organic acids such as citric acid, fumaric acid, adipic acid, lactic acid and malic acid, phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate and polyphosphate (polyphosphate), polyphenols and catechins (catechin). It may further include any one or more of natural antioxidants, such as alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid.

Animal feed additives for the prevention of influenza virus infections containing the sialic acid-containing whey protein of the present invention as an active ingredient and the composition for animal feed comprising the same are amino acids, inorganic salts, vitamins, antibiotics, antibacterial substances, antioxidants, Various adjuvants such as antifungal enzymes, live microbial agents and the like may be used in cereals such as wheat, oats, barley, corn and rice; Vegetable protein feed, such as rape, soybeans and sunflower; Animal protein feeds such as blood meal, meat meal, bone meal and fish meal; Sugars and dairy products, for example, a dry ingredient consisting of various powdered milk and whey powder, and a drying additive, all mixed together with a liquid component, and a component which becomes a liquid after heating, that is, a lipid, for example, an animal liquefied by heating. In addition to the main components such as fats and vegetable fats can be used with substances such as nutritional supplements, digestion and absorption enhancers, growth promoters, disease prevention agents and the like.

The sialic acid-containing whey protein may be administered to an animal alone in combination with other feed additives in an edible carrier.

In addition, sialic acid-containing whey proteins can be easily administered as top dressings or directly mixed into animal feed or separately from the feed, in separate oral formulations, by injection or transdermal or in combination with other ingredients. Typically, a single daily dose or divided daily doses can be used, as is well known in the art.

When the sialic acid-containing whey protein is administered separately from the animal feed, the dosage form of the dry powder or extract, as is well known in the art, is an immediate release or sustained release formulation in combination with a non-toxic pharmaceutically acceptable edible carrier. It can be prepared by. Such edible carriers may be solid or liquid, for example corn starch, lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil and propylene glycol. When a solid carrier is used, the dosage form of the dry powder or extract may be tablets, capsules, powders, torokies or sugar-containing tablets or top dressings in microdisperse form. If a liquid carrier is used, it may be in the form of soft gelatin capsules or syrups or liquid suspensions, emulsions or solutions. In addition, the dosage form may contain auxiliaries such as preservatives, stabilizers, wetting or emulsifying agents, solution promoters and the like.

In addition, the composition for animal feed, wherein the sialic acid-containing whey protein is included as an animal feed additive, may be any protein-containing organic cereal meal commonly used to meet the dietary needs of animals. Such protein-containing flours typically consist mainly of corn, soy flour, or corn / bean flour mixtures.

The animal feed additive may be used by adding to the animal feed composition by dipping, spraying or mixing.

The invention is applicable to a number of animal diets, including mammals, poultry and fish. More specifically, the diet is commercially important mammals such as pigs, cows, sheep, goats, laboratory rodents (rats, mice, hamsters and gerbils), furry animals (eg mink and fox), and Zoo animals (eg monkeys and tailless monkeys), as well as livestock (eg cats and dogs). Commercially important poultry typically include chickens, turkeys, ducks, geese, pheasants and quails. Commercially raised fish, such as trout, may also be included.

Animal feed formulation method comprising sialic acid-containing whey protein according to the present invention, the sialic acid-containing whey protein is incorporated into the animal feed in an amount of about 1 g to 100 g per kg of feed on a dry weight basis.

In the animal feed according to the present invention, it is preferable to vary the dosage according to the weight or growth stage of the target animal. When the animal's weight is 1 kg or less, 0.001 to 10% by weight relative to the weight, and when the body weight is 1 to 5 kg 0.001 to 5% by weight relative to the body weight, if the body weight is 5kg or more, 0.001 to 3% by weight relative to the body weight is administered several times a day or divided.

In addition, the feed mixture is thoroughly mixed and then a viscous granulated or granular material is obtained depending on the degree of grinding of the components. It is fed as a mash or formed into the desired discrete shape for further processing and packaging. At this time, in order to prevent separation during storage, it is preferable to add water to the animal feed, followed by a conventional pelleting, expansion, or extrusion process. Excess water may be removed to dryness.

An object of the present invention is to provide an animal feed additive for preventing influenza virus infection and an animal feed composition comprising the same by showing an inhibitory effect on influenza virus infection using sialic acid-containing whey protein.

1 is a diagram showing the results of hemagglutination and hemagglutination inhibition test,
Figure 2 is a diagram showing the results of influenza virus inhibitory effect using the MDCK cell line,
3 is a diagram showing the effect on hemagglutination inhibition,
Figure 4 is a diagram showing the results of sialic acid HI test,
5 is a diagram showing the results of the cytotoxicity test (A: 72 hours culture of L929 cells treated with 5% HOE; B: 72 hours culture of L929 cells treated with 2.5% HOE; C: 72 hours of L929 cells treated with 1.25% HOE; Incubation; D: 72 h incubation of 0.625% HOE treated L929 cells; E: control).

Hereinafter, the present invention will be described in detail with reference to the following examples and experimental examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following Experimental Examples.

Example 1 Preparation of Sialic Acid-Containing Whey Protein

More than 4% of the sialic acid-containing whey protein based on the total weight was purchased from JFLA (Lot. No. MT050227, www.j-fla.com/foodbio , Japan Food & Liquor Alliance, Japan). Named 'SY-1A'.

Reference Example 1. Preparation of hemagglutination and hemagglutination inhibition test

The virus strain used A / Beijing / 262/95 (H1N1) virus, distributed by the National Institutes of Health, and at 0.03% SWP (30mg) mix with DW and natural antibiotics, 0.3% SWP (0.3g) mix with DW and natural antibiotics at room temperature. 0.1% SWP (0.1g) mix with DW and natural antibiotics, 0.6% SWP (0.6g) mix with DW and natural antibiotics were stored and used in the following experiment.

Experimental Example 1. Hemagglutination test using blood cells

In order to confirm the hemagglutination test using blood cells, the test was carried out according to the method of the Infectious Disease Laboratory Diagnosis Guideline (National Institutes of Health, p28-30, 1996).

A / Beijing / 262/95 (H1N1) virus (prepared from the National Institutes of Health) prepared in Reference Example 1 was diluted with PBS in two dilutions. RBC control was added with the same amount (100 μl) of PBS. Add 0.5% GP RBC suspension to each well equally (100µl). After mixing well with a shaker (Fine Mold, SH30), the plate was left at room temperature (23 ℃) and left for about 30 minutes until the RBC control becomes a negative pattern. After 30 minutes, the dilution factor of the virus to be used for the hemagglutination inhibition test was determined to be 4 HAU. In this experiment, the titer of the virus was confirmed to be HA 1: 4 as shown in FIG. 1, and when the hemagglutination inhibition test was performed, the virus was used without dilution (see FIG. 1).

Experimental Example 2. Hemagglutination inhibition test

In order to confirm the hemagglutination inhibition test using blood cells, the test was carried out according to the method of infectious disease laboratory diagnosis guideline (National Institutes of Health, p28-30, 1996).

Each sample obtained in Reference Example 1 was repeatedly added to the well three times. Viruses diluted to 4HAU in Experimental Example 1 were added in equal amounts (50 μl), mixed well, and sensitized at room temperature for 30 minutes. Virus control group and blood cell control group were placed for comparison. 0.5% GP RBC suspension was added to each well in the same amount (100 μl), and then mixed well with a shaker (Fine Mold, SH30). The plate was left at room temperature (23 ° C.) and left for about 30 minutes until the RBC control became a negative pattern. After 30 minutes, read and record (see FIG. 1).

As a result, the titer of the virus in the hemagglutination test was HAU 1: 4. In the hemagglutination inhibition test with 4 HAU, the sample showed that the whole sample inhibited the virus in all three replicates. Samples 0.6 and 0.3 showed a clear inhibitory effect. Samples 0.1 and 0.03 also inhibited the virus clearly but not as clearly as 0.6 and 0.3.

Reference Example 2 Preparation of Influenza Virus Inhibitory Effect Experiment

The virus line was used A / Beijing / 262/95 (H1N1) virus distributed by the National Institutes of Health, and the cell line was used MDCK (Marbin & Darby Canine Kidney) Cell distributed from the National Institutes of Health. Samples were stored at room temperature, and the samples were white powdery white SWP when preparing 2% aqueous solution.

Experimental Example 3. Influenza virus inhibition effect experiment using MDCK cell line

R Rott, M Orlich, HD Klenk and DC Wiley (1984) Studies on the adaptation of influenza virus to MDCK cells.EMBO J. 1984 December 20: 3 (13) 3329 According to the method of -3332) and the like was tested as follows.

Cell culture media A 2% Whey aqueous solution was prepared using EMEM (eagle's minimal essential medium), completely dissolved, and then passed through a 0.22μm filter (Millipore, SLGV033RB) as a sample solution. The sample solution was diluted stepwise by four-fold using EMEM without serum. 50 μl of the sample solution diluted in multiples of 4 in a 96well culture plate was dispensed. An equal amount (50 μl) of 100TCID50 virus was dispensed into the well containing the sample solution. After standing at 37 ° C. for 30 minutes, MDCK cells prepared on 96well culture plates were aliquoted to 2 × 100,000 cells / ml using 1% FBS (fetal bovine serum) EMEM medium. 72 hours of incubation at 37 ° C. in a 5% CO ₂ incubator observed abnormal cell lesions in an inverted microscope. When cell lesions appeared in virus control wells, 96 well plates were removed and subjected to MTT assay. MTT assay results were read by ELISA reader at 450nm.

As a result of the experiment, it was observed that cell lesions of influenza virus did not appear in the wells containing 2%, 0.5%, 0.125%, and 0.03% of SWP (visual observation through microscope) and Elisa reader. Cell lesions were observed from C5 and D5 wells. Therefore, up to 0.03% SWP concentration, Whey protein was found to inhibit MDCK cell adsorption of A / HINI / Beijing virus (Table 1; see FIG. 2).

Experimental Example  3. Hemagglutination inhibition test method ( HI Assay )

In Example 1 to determine the virus titer hemagglutination change by a SY-1A obtained was subjected to hemagglutination test (Donald HB, Isaacs A, J. Gen. Microbiol. 10 (3) pp457-64, 1954) .

In this experiment, H1N1 (A / Puerto Rico8 / 34 (PR8)), H3N2 (A / Aichi / 2/68 (Aic68) and A / Memphis / 1/71 (M71)), which are influenza A viruses including avian influenza, H5N3 (A / duck / 313/4/78 (D313)) was used. Phosphate buffered saline (PBS) in a 96-well polyvinylchloride microtiter plate (Falcon # 3591196) with a U-shaped bottom; pH 7.2, 131 mM NaCl, 14 mM Na ₂ HPO ₄ , 1.5 mM KH ₂ PO _To each well of ₄ and 2.7 mM KCl) add 25 μl of diluted SY-1A (final concentrations are 10, 5, 2.5, 1.25, 0.625, 0.313 mg / ml). Then add 25 μL of 4 hemagglutination unit (HAU) influenza virus-PBS suspension. The mixture is mixed for 30 seconds by a microplate mixer and then reacted on ice for 1 hour. 50 µl of 0.5% guinea pig erythrocyte-PBS suspension was added, mixed for 30 seconds by a microplate mixer, incubated on ice overnight, and then hemagglutination titers were measured. Hemagglutination titer was the dilution factor of the final well in which aggregation occurred.

As a result of the experiment, as shown in Figure 3 below, the circle of blood cells in the middle of the well gathered means that the influenza virus first reacts with SY-1A and can not fuse with sialic acid on the surface of the guinea pig, so the blood cells sink intact. The blood cells are gathered in the middle of the U-shaped well, and the clouded well means no SY-1A, so the virus fuses directly with the sialic acid of the guinea pig blood cells and is electrically positive. Attached to the wall, it looks cloudy when viewed from above. In the middle of the well, the shape of the ring at the center of the well means that some of the virus reacted with the surplus SY-1A first, but the amount is not high enough to form a ring shape. After all of the SY-1A is exhausted in response, the virus is fused with sialic acid on the surface of the guinea pig blood without interruption of SY-1A, and thus some of the cloudiness is clouded (see FIG. 3).

Therefore, it was confirmed that all four types of A-type viruses were suppressed up to a dilute fold of 0.625 mg / ml of SY-1A, which was faintly ring-shaped in the wells.

Experimental Example  4. Sialic acid HI Test  ( Hemmaglutinin Inhibition test )

HI test of sialic acid was carried out according to the method described in Clarke DH, Casal J (1958) Techniques for hemagglutination and hemagglutination-inhibition with arthropod borne viruses.Am J Trop Med Hyg 7: 561. .

It was experimented with Experimental Example 3 with sialic acid 99% chemically synthesized (JFLA Bioresearch Center).

As a result of the experiment, at 1% and 0.5%, all of the blood cells were destroyed because of toxicity. 0.25% showed complete virus inhibition as the toxicity was diluted, and 0.125% showed slight inhibition (see FIG. 4).

Reference Example  3. Preparation of Cytotoxicity Test

Cell line L929 from the National Institutes of Health; Human diploid cells were used, and samples were used with sialic acid contained whey protein. The sample was stored at room temperature and used as a white turbid liquid sample when preparing a 5% aqueous solution as a white powder.

Experimental Example  5. Cytotoxicity Test

In order to confirm the cytotoxicity test by HOE (skim milk powder), it tested as follows according to the method of Keisari Y. J. Immunol Methods. 1992, 146 (2): 155-61.

Cell culture media 5% HOE aqueous solution was made with EMEM (eagle's minimal essential medium) completely dissolved, and then passed through a 0.45μm filter (Acrodisc, # 4614) was used as a sample solution. The sample solution was diluted stepwise in two-fold using EMEM without serum.

In a 24-well plate, L929 cells (Human diploid cell, National Institutes of Health) were aliquoted to 2 X 100,000 cells / ml using EMEM medium (eagle's minimal essential medium) with 10% FBS, followed by 37 ° C, 5% CO ₂ Monolayer incubation for 24 hours. After the culture solution was removed and washed with serum-free EMEM medium, 500 μl / well of 2% FBS EMEM medium was dispensed, and then 5% aqueous solution of SY-1A obtained in Example 1 was dissolved in EMEM to completely dissolve 0.45. Samples made by passing through the filter are step-diluted in a double-fold using a medium without serum, and then 500 μl are added to each well (final treatment ratio: 0, 0.625%, 1.25%, 2.5%, 5%). After 24 hours, 48 hours, and 72 hours of incubation in a 37 ° C. and 5% CO ₂ incubator, the presence of abnormalities (cell lesions, shape deformation, growth slowdown, etc.) was observed using an inverted microscope. Recorded.

As a result of the experiment, HOE was dissolved in EMEM to the maximum amount that could be dissolved, and then diluted in two stages, treated with L929 cells, cultured for 72 hours, and cytotoxicity was observed. 5).

Hereinafter, an example of the formulation of a feed composition comprising the sialic acid-containing whey protein of the present invention will be described, but the present invention is not intended to be limited thereto but merely to be described in detail.

Feed composition example 1.

Example 1 (SY-1A) 100 mg

0.7 mg of extract containing β-glucan

Yeast culture 24 mg

Vitamin E 0.7 mg

L-carnitine 0.7 mg

Claims (6)

Animal feed additive for the prevention of influenza virus infection containing sialic acid-containing whey protein as an active ingredient. The animal feed additive of claim 1 wherein the sialic acid-containing whey protein comprises a Japanese, Chinese or European product. The animal feed additive of claim 1, comprising at least about 4% by weight of sialic acid relative to the total weight of the protein as defined herein. The method of claim 1, wherein the influenza virus is H1N1, H1N2, H2N2, Human B, H3N2, H3N8, H5N1, Animal feed additives having a serotype of H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H9N2 or H10N7. According to claim 1, Animal feed additive, characterized in that containing 0.1 to 50% by weight based on the total weight of the composition. Claim 1 animal feed composition comprising the animal feed additive of claim 1.

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