KR20200057147A - Compositions for immuno-enhancing food, pharmaceutical or feed comprising a stabilized amino acid mineral complex and a lactic acid bacteria - Google Patents

Abstract

The present invention relates to an amino acid mineral complex having immunity enhancing activity and a food, pharmaceutical or feed composition for containing the same. More specifically, the present invention relates to a composition for food (or food additives), medicines, feed (or feed additives) which can enhance immunity of humans or non-human animals, or can increase the antibody production rate of an antiviral vaccine by containing the amino acid mineral complex as an effective component.

Description

Compositions for immuno-enhancing food, pharmaceutical or feed comprising a stabilized amino acid mineral complex and a lactic acid bacteria}

The present invention relates to a composition for food, pharmaceutical or feed for immunity enhancement, comprising an amino acid mineral complex compound and lactic acid bacteria, and more specifically, to be actively absorbed at a high absorption rate in the small intestine, and has no stability in the long-term use due to no accumulation in vivo. , It relates to a food (or food additive), drug, feed (or feed additive) composition that can effectively enhance the immunity of humans or animals other than humans.

The advantage of the amino acid mineral complex in mineral nutrition is the fact that it is easily absorbed by mucosal and plant cells by active transport or other mechanisms. That is, when the amino acid is absorbed by using an amino acid as a transport molecule, there is an advantage of avoiding the problem related to competition for the active site for absorption and the function of inhibiting absorption of special trace elements between minerals.

The mineral amino acid complex is generally produced by a reaction between an alpha-amino acid and a metal ion, and a metal ion having a valence of 2 or more is required for the complex to have a ring structure. In this reaction, the positive charge of the metal ion is neutralized by reacting with the negative charge of the amino group or carboxyl group of the alpha-amino acid.

Lactic acid bacteria, which are abundant in fermented food, are responsible for decomposing fibrous and complex proteins into important nutrients while symbiotic with the human digestive system, and thus improving the microbial environment of the host's gut in the gastrointestinal tract of animals, including humans. Live microorganisms that have a beneficial effect on the health of the host are collectively called probiotics. Lactic acid bacteria are bacteria that break down carbohydrates and make lactic acid by using them, which are common anaerobic bacteria or organized anaerobic bacteria that proliferate well in low oxygen. Among them, the microorganism of the genus Lactobacillus is a lactic acid bacterium that is homozygous or heterozygous, and is commonly found in the fermentation process of intestinal tracts of animals, including humans, and dairy products or vegetables. So far, there is an example in which the physiological activity of the lactic acid bacteria or the mineral amino acid complex alone is verified, but a method for enhancing their effect has not been considered.

The literature related to the structure and chemical formula of a mineral amino acid complex and bioavailability are very diverse, and representatively, Ashmead et al., Chelated Mineral Nutrition, (1982), Chas. C. Thomas Publishers, Springfield, Ill. ), Ashmead et al., Intestinal Absorption of Metal Ions (1985), Ashmead et al., Foliar Feeding of Plants with Amino Acid Chelates, (1986) and U.S. Patent No. 4,020,158 , 4,167,564, 4,216,143, 4,721,644, 4,599,152, 4,774,089, 4,830,716, 4,863,898, 4,725,427, and the like.

U.S. Patent No. 4,020,158 U.S. Patent No. 4,167,564

Ashmead et al., Chelated Mineral Nutrition, (1982), Chas. C. Thomas Publishers, Springfield, Ill. Chas. C. Thomas Publishers, Springfield, Ill. Ashmead et al., Intestinal Absorption of Metal Ions, (1985) Ashmead et al., Foliar Feeding of Plants with Amino Acid Chelates, (1986)

The present invention applies a water-soluble amino acid mineral complex compound and a lactic acid bacterium as a composition for enhancing immune activity, enhances the active absorption of minerals in the small intestine, improves immunity of animals other than humans or humans, and is stable by taking a long period of time without accumulation in vivo It is intended to provide a food, pharmaceutical or feed composition that can exhibit an effect.

The present invention relates to a composition for enhancing immune activity using water-soluble amino acid mineral complexes and lactic acid bacteria in which zinc and aspartic acid are combined at a molar ratio of 1: 1.5 to 2.5 as active ingredients.

The lactic acid bacteria include Lactobacillus plantarum, L. casei, L. acidophilus, Lactobacillus L. bulgaricus, Bifidobacterium It can be any one or more selected from the group consisting of B. longum, B. bifidum, Actiregularis and L. rhamnosus.

The composition for enhancing immune activity may be a food composition, a food additive composition, a pharmaceutical composition, a feed composition, or a feed additive composition.

The composition for enhancing immune activity may be administered in parallel or sequentially with an antiviral vaccine.

The anti-virus vaccine, Newcastle disease, infectious bronchitis, coccidium diarrhea, avian mama, avian cholera, rheovirus-induced tendonitis (viral arthritis), avian laryngitis, avian encephalomyelitis, infectious F-cystitis (IBD), Marek disease , Salmonella infections, Mycoplasma gallicepticum infections, avian rhinitis, avian herpes and Mycoplasma hyopneumoniae, spawning syndrome, infectious nasal congestion (Hemophilis fasagalinarum), Mycoplasma sinobies or avian reoviruses A vaccine for birds for prevention;

Pleural pneumococcus, atrophic rhinitis, pseudorabies, swine alone, porcine parvovirus, E. coli enterotoxin, Mycoplasma hyopneumoniae, influenza, leptospira, E. coli infection, porcine genital and respiratory syndrome (PRRS), Bordetella and type A And mammalian livestock vaccines for the prevention or treatment of D-type Multoshita infection, Haemophilus parasuis infection, Welch infection, Rotavirus infection, Streptococcal infection, Glaser disease, pneumonia, Bordetella bronchiseptica infection; Or influenza, hepatitis A, hepatitis B, hepatitis C, herpes simplex virus (type 2), polio, diphtheria, whooping cough, haemophilus type B influenza (Hib), measles, mumps, rubella, typhoid fever, chickenpox (chicken) Fox), Dengue fever, Epstein-Barr virus infection, human papilloma virus infection, pneumococcal infection, meningococcal infection, pneumococcal infection, viral meningitis, rotavirus infection, tick-mediated encephalitis, traveling diarrhea, cholera, yellow fever or tuberculosis It may be a human vaccine for the prevention of.

In addition, the present invention comprises the steps of 1) adding a zinc precursor and aspartic acid to water at a molar ratio of 1: 1.5 to 2.5;

2) heating and reacting the mixed aqueous solution of zinc precursor and aspartic acid in step 1) at 50 to 100 ° C. for 10 to 24 hours; And

3) preparing a composition for enhancing immune activity by mixing lactic acid bacteria in the amino acid mineral complex;

Including the amino acid mineral complex and lactic acid bacteria of the present invention, even when formulated into tablets such as powders, granules, effervescent tablets as water-soluble, it can be quickly dissolved in water and does not precipitate even when prepared as a liquid and has a mineral absorption rate. outstanding.

In addition, the composition for enhancing the immune activity using the amino acid mineral complex and lactic acid bacteria of the present invention as an active ingredient may enhance immunity of humans or animals other than humans, or increase the antibody production rate of antiviral vaccines.

The composition for enhancing the immune activity of the present invention has an effect of enhancing the proliferation activity of beneficial bacteria in the host and inhibiting harmful bacteria, and improves the intestinal environment of the host, thereby improving intestinal immunity.

In addition, the composition for enhancing the immune activity of the present invention exhibits an effect of significantly reducing the amount of ammonia and hydrogen sulfide generated in the manure, so in the case of humans, it can be utilized as a deodorizing composition that reduces the smell of fart and the smell of the elderly. Excluded animals, especially cattle and pigs, can be used as a composition for reducing the smell of livestock manure, which can contribute to improving the environment of livestock farmers.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a composition for enhancing immune activity using water-soluble amino acid mineral complexes and lactic acid bacteria in which zinc and aspartic acid are combined at a molar ratio of 1: 1.5 to 2.5 as active ingredients.

The composition for enhancing immune activity is a single agent formed by mixing a prebiotic composition of a water-soluble amino acid mineral complex in which zinc and aspartic acid are combined in a molar ratio of 1: 1.5 to 2.5 and a probiotic composition called lactic acid bacteria, and the minerals are better in the small intestine. It is absorbed and has the effect of enhancing the immune activity in the intestine.

The immunity enhancement means to increase the immune response or activity of the immune system in vivo, and is effective in alleviating or reducing the immune function caused by aging, pregnancy, and immunosuppressive treatment, or preventing diseases caused by the reduced immune function. In addition, the disease caused by the reduced immune function may be any one or more selected from viral-sensitive diseases such as colds and inflammatory diseases, chronic fatigue and cancer.

The lactic acid bacteria means a beneficial bacteria that inhabit the intestine of the host and a strain that has a beneficial effect on the intestinal environment when it is ingested and reaches the intestine. They survive and survive in gastric and bile acids, multiply and settle in the intestine, exhibit useful effects in the intestinal tract, and mean bacteria that satisfy non-pathogenic and non-pathogenic conditions, and can be understood as probiotics.

The lactic acid bacteria include Lactobacillus plantarum, L. casei, L. acidophilus, Lactobacillus L. bulgaricus, Bifidobacterium It can be any one or more selected from the group consisting of B. longum, B. bifidum, Actiregularis and L. rhamnosus.

The water-soluble amino acid mineral complex may include adding a zinc precursor and aspartic acid to water in a molar ratio of 1: 1.5 to 2.5; And heating and reacting the mixed mineral precursor and aspartic acid aqueous solution at 50 to 100 ° C. for 10 to 24 hours.

Finally, a composition for enhancing immune activity is prepared by mixing lactic acid bacteria in the amino acid mineral complex.

The zinc precursor is a zinc salt or zinc oxide that can be used as a food, drug, or feed, preferably a water-soluble zinc salt such as zinc gluconate or zinc sulfate, or zinc oxide insoluble in water, which has been confirmed to be safe as a food additive. It can be used, and more preferably, zinc oxide is more excellent in activity to increase the antibody production rate of the antiviral vaccine. In particular, in the conventional preparation of zinc aspartic acid complex, it was natural to use a water-soluble zinc salt soluble in water, but rather zinc zinc aspartic acid complex, which is more effective when heated over a certain temperature and time while using zinc oxide insoluble in water, is more effective. It was confirmed that it was formed.

The molar ratio of the zinc precursor and aspartic acid means a molar ratio of zinc and aspartic acid, and 1: 1.5 to 2.5 mol, preferably 1: 1.8 to 2.2 mol, and more preferably 1: 2 mol remains after forming the complex It is advantageous in that it can minimize reactants or insoluble materials.

The step of reacting is 10 minutes to 24 hours at 50 to 100 ° C, especially in the case of a water-soluble zinc salt, only relatively low temperature and short time at the above range, for example, heating at 50 to 80 ° C for 10 minutes to 1 hour The reaction may be terminated, but in the case of zinc oxide, heating is performed at 80 to 100 ° C. for 1 hour or more, preferably 1.5 hours. On the other hand, since both as water-soluble zinc salt and zinc oxide react aspartic acid in about 95%, preferably 99% or more, within 3 hours, even if heated for more than 3 hours, the reduction of insoluble or unreacted substances may not be significant. In the reaction step, ultrasonic treatment may be used to shorten the reaction time.

The step of removing the insoluble material from the reaction solution of the step may further include. The step of removing the insoluble material may be added even when using a water-soluble zinc salt as a zinc precursor, but is particularly necessary when using zinc oxide as a zinc precursor.

The step of removing the insoluble material may be filtered with a number of µm or less, for example, 0.2 μm filter paper or filter cloth, and the insoluble material may be removed before or after the filtration through centrifugation.

Unlike the conventional method of manufacturing a zinc aspartic acid complex, the manufacturing method has the advantage of not requiring a separate additive in addition to the zinc precursor, aspartic acid and water, and the manufacturing process is simple, thereby reducing process cost.

The lactic acid bacteria are mixed in the amino acid mineral complex, and the lactic acid bacteria may be cultured in MRS, a culture medium of lactic acid bacteria, at 30-40 ° C. for 12 hours. Specifically, it is preferable that the lactic acid bacteria of the present invention are inoculated into the medium in an amount of 10 ⁴ to 10 ⁵ CFU / g.

The composition for enhancing the immune activity of the present invention has remarkably superior immunity and antiviral activity, and the proliferative activity of beneficial bacteria in the intestine is higher than that of using the lactic acid bacteria or the amino acid mineral complex alone, respectively. I could confirm that it is better.

The lactic acid bacteria based on 10 parts by weight of the amino acid mineral complex is preferably mixed by 1 part by weight.

On the other hand, in this specification, the term 'containing an active ingredient' means that it contains an amount sufficient to achieve the efficacy or activity of the mineral amino acid complex and lactic acid bacteria of the present invention. In one embodiment of the present invention, the food composition, pharmaceutical composition or feed composition in the composition of the present invention is a mixture of mineral amino acid complex and lactic acid bacteria, for example, 0.001 mg / kg or more, preferably 0.1 mg / kg or more, more It is preferably 10 mg / kg or more, even more preferably 100 mg / kg or more, even more preferably 250 mg / kg or more, and most preferably 0.1 g / kg or more. However, the mineral amino acid complex in the food additive composition or the feed additive composition is 10 g / kg or more, preferably 50 g / kg or more, more preferably 100 g / kg or more, and may be used purely as a mineral amino acid complex.

The pharmaceutical composition of the present invention includes, as well as pharmaceutical compositions for humans, pharmaceutical compositions for veterinary medicine such as birds or mammals other than humans. Pharmaceutical compositions may be prepared using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the active ingredients, and as adjuvants, excipients, disintegrants, sweeteners, binders, coating agents, expanders, lubricants, lubricants or flavors Can be used.

The pharmaceutical composition may be administered in parallel or sequentially with an antiviral vaccine. To this end, the pharmaceutical composition of the present invention may be formulated together with an antiviral vaccine, or may be formulated separately.

The antiviral vaccines include Newcastle disease, infectious bronchitis, coccidium diarrhea, avian mama, avian cholera, rheovirus-induced tendonitis (viral arthritis), avian laryngitis, avian encephalomyelitis, infectious F-cystitis (IBD), Marek disease, Prevention of Salmonella infection, Mycoplasma gallicepticum infection, avian rhinitis, avian herpes and Mycoplasma hyopneumoniae, spawning syndrome, infectious nasal congestion (Hemophilis fasagalinarum), Mycoplasma sinobiae or avian reovirus Vaccine for birds; Pleural pneumococcus, atrophic rhinitis, pseudorabies, swine alone, porcine parvovirus, E. coli enterotoxin, Mycoplasma hyopneumoniae, influenza, leptospira, E. coli infection, porcine genital and respiratory syndrome (PRRS), Bordetella and type A And mammalian livestock vaccines for the prevention or treatment of D-type Multoshita infection, Haemophilus parasuis infection, Welch infection, Rotavirus infection, Streptococcal infection, Glaser disease, pneumonia, Bordetella bronchiseptica infection; Or influenza, hepatitis A, hepatitis B, hepatitis C, herpes simplex virus (type 2), polio, diphtheria, whooping cough, haemophilus type B influenza (Hib), measles, mumps, rubella, typhoid fever, chickenpox (chicken) Fox), Dengue fever, Epstein-Barr virus infection, human papilloma virus infection, pneumococcal infection, meningococcal infection, pneumococcal infection, viral meningitis, rotavirus infection, tick-mediated encephalitis, traveling diarrhea, cholera, yellow fever or tuberculosis It may be a human vaccine for the prevention of.

The pharmaceutical composition may be preferably formulated into a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration.

Formulations of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops or injectables. For example, for formulation in the form of tablets or capsules, the active ingredient can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and colorants can also be included in the mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, trachocanth or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

Acceptable pharmaceutical carriers in compositions formulated as liquid solutions are sterile and biocompatible, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and these One or more of the components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets.

Furthermore, it can be preferably formulated according to each disease or component using methods disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA by appropriate methods in the art.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc., preferably oral administration.

Suitable dosages of the pharmaceutical compositions of the invention vary by factors such as formulation method, mode of administration, age or weight of the patient or animal, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion, and response responsiveness. In general, a skilled doctor or veterinarian can easily determine and prescribe a dose effective for the desired treatment or prevention. According to a preferred embodiment of the invention, the daily dosage of the pharmaceutical composition of the invention is 0.001-10 g / kg.

The pharmaceutical composition of the present invention is prepared in a unit dose form by formulating using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily carried out by those skilled in the art to which the present invention pertains, or It can be manufactured by incorporating into a multi-dose container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of ex, powder, granule, tablet, or capsule, and may further include a dispersant or stabilizer.

As used herein, the term “effective amount” refers to the amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as considered by a researcher, veterinarian, physician or other clinician, as applicable It includes an amount that induces relief of symptoms of a disease or disorder. It will be apparent to those skilled in the art that the effective amount and the number of administrations to the active ingredient of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be easily determined by those skilled in the art, the type of disease, the severity of the disease, the content of active ingredients and other ingredients in the composition, the type of formulation, and the age or weight of the patient or animal, It can be adjusted according to a variety of factors, including general health status, sex and diet, time of administration, route of administration and secretion rate of the composition, duration of treatment, and drugs used simultaneously. In the method of prevention, treatment or improvement of the present invention, in the case of adults or adults, it is preferable to administer the mixture of the mineral amino acid complex and lactic acid bacteria once or several times a day at a dose of 0.001 mg / kg to 10 g / kg. Do.

The food composition or food additive composition according to the present invention may be formulated in the same manner as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the food additive composition can be added include, for example, beverages, alcoholic beverages, confectionery, diet bars, dairy products, meat, chocolate, pizza, instant noodles, other noodles, gums, ice creams, vitamin complexes, and health supplements And so on.

The food composition of the present invention may include mineral amino acid complexes and lactic acid bacteria as active ingredients, as well as ingredients commonly added in food production, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavoring agents. Includes. Examples of the aforementioned carbohydrates include monosaccharides, such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides, etc .; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a flavoring agent, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizine, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is made of a drink and a beverage, in addition to the extract of rice, rice bran or rice husk of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, and various plant extracts are additionally included. I can do it.

The present invention provides a health functional food comprising a food composition for enhancing immune activity comprising the mineral amino acid complex and lactic acid bacteria as an active ingredient. Health functional foods are foods made by adding rice bran extract or rice bran powder to food materials such as beverages, teas, spices, chewing gum, confectionery, or by encapsulation, powdering, suspension, etc. It means to bring, but it has the advantage that there is no side effect that can occur when taking the drug for a long time by using food as a raw material, unlike ordinary drugs. The health functional food of the present invention thus obtained is very useful because it can be consumed on a daily basis. The amount of rice bran extract or rice bran powder added in such a health functional food cannot be uniformly defined depending on the type of the health functional food, but may be added in a range that does not impair the original taste of the food. It is usually in the range of 0.01 to 50% by weight, preferably 0.1 to 20% by weight. In addition, in the case of a health functional food in the form of pills, granules, tablets or capsules, it is usually added in the range of 0.1 to 100% by weight, preferably 0.5 to 80% by weight. In one embodiment, the dietary supplement of the present invention may be in the form of pills, tablets, capsules or beverages.

In the feed additive composition according to the present invention, the feed additive composition is added to 0.01-1% by weight based on the solid content of the feed composition of livestock such as pig, chicken, duck, cow, sheep, goat, and dog, and mixed with various animals. It can be applied by administration, and more preferably, it is used as an additive in pig feed.

The feed additive composition of the present invention may be prepared as a feed composition in the form of fermented feed, blended feed, pellet form, and silage, but is not limited thereto, and may be prepared by modification as necessary.

The present invention can provide a composition for inhibiting growth of beneficial bacteria in the intestine and growth of harmful bacteria in the intestine containing the composition, as described above.

The intestinal beneficial bacteria means a beneficial bacteria that inhabit the intestine of the host and a strain that has a beneficial effect on the intestinal environment when it is ingested and reaches the intestine.

Harmful bacteria in the intestine refers to a strain that has a harmful effect on the intestinal environment when it reaches the intestine and ingested harmful bacteria existing in the host's intestine.

The intestinal beneficial bacteria may be at least one strain selected from the genus Bifidobacterium, genus Leukonostock, genus Pediococcus, genus Weisella, streptococcus, Lactobacillus, and combinations thereof.

More preferably, the strain of the genus Bifidobacterium is at least one selected from Bifidobacterium adolescentis, Bifidobacterium brebe, Bifidobacterium plantarum, and combinations thereof, and the genus Leuconostock The strain is at least one selected from Leukonostock mesenteroides, Leukonostock pseudomesenteroides, and combinations thereof, and the genus Pediococcus is Pediococcus pentosaceus, Pediococcus halopilus and their At least one selected from the combination, wherein the strain of Wessela is at least one selected from Weisella cybaria, Wessella choreensis, Wessella paramethentroid, and combinations thereof, and the strain of the genus S. streptococcus is Streptococcus thermophilus, At least one selected from Streptococcus thermophilus, Streptococcus faecalis, and combinations thereof, and the strain of the genus Lactobacillus is Lactobacillus luteri, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus rhamnosus, and the like. It may be at least one selected from combinations. Most preferably, Leuconostock mesenteroides KSD-2152, Pediococcus pentosaceus KSD-PP2, Weisella civaria KSD-914, Lactobacillus plantarum KCCM 12116, Lactobacillus acidophilus KCTC 3164, Lactobacillus Rhamnosus GG.

The intestinal harmful bacteria may be at least one strain selected from Clostridium genus, Eubacterium genus, Bacteroid genus, E. coli genus, Bacillus genus, Salmonella genus, and combinations thereof. More preferably, the strain of the genus Clostridium is at least one selected from Clostridium difficile, Clostridium perfringens, and combinations thereof,

The strain of Eubacterium genus is Eubacterium limosum, and the bacterium genus strain is at least one selected from bacteroid furagilis, bacteroid prazilis, and combinations thereof, and the strain of Escherichia genus is Escherichia Collie, the Bacillus strain is at least one selected from Bacillus celeus, Bacillus anthracis, and combinations thereof, and the Salmonella strain is Salmonella enterica, Salmonella bongori, Salmonella typhimurium, Salmonella gallinalium, Salmonella pullorium And combinations thereof. Most preferably, it can be E. coli O157: H7, Bacillus seleus KACC 11240, Salmonella enterica DSM 14846, Salmonella ring DSM 13772.

The present invention will be described in more detail through the following examples, but the scope and content of the present invention may be reduced or limited by the following examples, etc., and cannot be interpreted. In addition, if it is based on the disclosure of the present invention including the following examples, it is obvious that a person skilled in the art can easily carry out the present invention in which experimental results are not specifically presented, and patents to which such modifications and corrections are attached Naturally, it is within the scope of the claims.

Example 1-1: Preparation of a composition for enhancing immune activity of zinc aspartic acid complex and Lactobacillus plantarum

60 g of aspartic acid was added to 500 ml of water while stirring, zinc zinc sulfate (35 wt% zinc) was added thereto, and the mixture was sufficiently reacted at a temperature of 60 ° C. for 30 minutes or more, followed by centrifugation to lyophilize the supernatant. 70 g of a water-soluble zinc aspartic acid complex was obtained. However, the zinc sulfate and the aspartic acid were added in a content ratio of 1 to 2 moles.

The strain used in the experiment was Lactobacillus plantarum (Lactobacillus plantarum). The Lactobacillus plantarum strain was cultured for 12 hours at 37 ° C in MRS, a culture medium of lactic acid bacteria, and then mixed with 4 g of 40 g of the zinc aspartic acid complex to prepare a composition for enhancing the immune activity of the present invention. This was referred to as the composition of Example 1-1 and was used in the following experiment.

Example 1-2: Preparation of composition for enhancing immune activity of zinc aspartic acid complex and mixed bacteria

60 g of aspartic acid was added to 500 ml of water while stirring, zinc zinc sulfate (35 wt% zinc) was added thereto, and the mixture was sufficiently reacted at a temperature of 60 ° C. for 30 minutes or more, followed by centrifugation to lyophilize the supernatant. 70 g of a water-soluble zinc aspartic acid complex was obtained. However, the zinc sulfate and the aspartic acid were added in a content ratio of 1 to 2 moles.

The strain used in the experiment was a mixture of Lactobacillus plantarum and B. bifidum (B. bifidum). The strains were cultured for 12 hours at 37 ° C. in MRS, which is a culture medium of each lactic acid bacteria, and then mixed with 2 g of 40 g of the zinc aspartic acid complex to prepare a composition for enhancing the immune activity of the present invention. This was referred to as the composition of Example 1-2, and was used in the following experiment.

Example 1-3: Preparation of composition for enhancing immune activity of zinc aspartic acid complex and Lactobacillus plantarum

The composition was prepared in the same manner as in Example 1-1, except that the zinc aspartic acid complex prepared by zinc oxide was mixed instead of the zinc aspartic acid complex.

The zinc aspartic acid complex made of zinc oxide was prepared as follows. 57.5 g of aspartic acid was added to 500 ml of water and stirred, while 17.5 g of zinc oxide (81% by weight of zinc) was added thereto while stirring, and sufficiently reacted at a temperature of 90 ° C. for at least 120 minutes, and then the reaction solution was filtered with 0.2 μm filter paper. Then, the filtrate was concentrated to 80% for 3 hours at 65 ° C., followed by vacuum drying to obtain a water-soluble zinc aspartic acid complex. This was referred to as the composition of Examples 1-3 and was used in the following experiments.

Example 1-4: Preparation of composition for enhancing immune activity of zinc aspartic acid complex and mixed bacteria

The composition was prepared in the same manner as in Example 1-2, except that the zinc aspartic acid complex prepared by zinc oxide was mixed instead of the zinc aspartic acid complex.

The zinc aspartic acid complex made of zinc oxide was prepared as follows. 57.5 g of aspartic acid was added to 500 ml of water and stirred, while 17.5 g of zinc oxide (81% by weight of zinc) was added thereto while stirring, and sufficiently reacted at a temperature of 90 ° C. for at least 120 minutes, and then the reaction solution was filtered with 0.2 μm filter paper. Then, the filtrate was concentrated to 80% for 3 hours at 65 ° C., followed by vacuum drying to obtain a water-soluble zinc aspartic acid complex. This was referred to as the composition of Examples 1-4 and was used in the following experiments.

Comparative Example 1-1: Preparation of composition for enhancing immune activity of zinc glutamic acid complex and Lactobacillus plantarum

The composition was prepared in the same manner as in Example 1-1, except that the zinc glutamic acid complex was mixed instead of the zinc aspartic acid complex.

At this time, the zinc glutamic acid complex was prepared as follows. 60 g of glutamic acid was added to 500 ml of water and stirred, while zinc sulfate (35% zinc) was added thereto, and sufficiently stirred at a temperature of 50 to 100 ° C for 30 minutes or more, followed by centrifugation to lyophilize the supernatant, and 70 g of a water-soluble zinc glutamic acid complex was obtained. However, the zinc sulfate and the glutamic acid were added in a content ratio of 1 to 2 moles.

Comparative Example 1-2: Preparation of composition for enhancing immune activity of iron aspartic acid complex and Lactobacillus plantarum

The composition was prepared in the same manner as in Example 1-1, except that the iron aspartic acid complex was mixed in place of the zinc aspartic acid complex.

At this time, the iron aspartic acid complex was prepared as follows. 60 g of aspartic acid was added to 500 ml of water while stirring, iron iron acetate (35% iron) was added thereto, and the mixture was sufficiently reacted at a temperature of 50 to 100 ° C for 30 minutes or more, followed by centrifugation to lyophilize the supernatant. About 70 g of iron lactate aspartic acid complex was obtained. However, the iron lactate and the aspartic acid were added in a content ratio of 1 to 2 moles.

Experimental Example 1: Evaluation of reaction yield

In Examples 1-1 and 1-2 and Comparative Examples 1-1 and 1-2, the reaction yields of the mineral amino acid complexes prepared before mixing lactic acid bacteria were first compared and are shown in Table 1 below. The reaction yield was calculated by dividing the content of the mineral contained in the generated mineral amino acid complex by ICP analysis and dividing it by the theoretical mineral content (zinc or iron) of the injected mineral precursor.

division Example 1-1 Example 1-2 Comparative Example 1-1 Comparative Example 1-2 Reaction yield (%) 100% 100% 48% 60%

As a result, in the case of Examples 1-1 and 1-2, the water-soluble zinc aspartic acid complex was prepared by participating in the reaction with a reaction yield of 100%. On the other hand, in the case of Comparative Example 1-1 using glutamic acid, the reaction yield was significantly reduced to less than about 50% because the glutamic acid was not completely dissolved in water, and the reaction yield was also increased in Comparative Example 1-2 using iron lactate. Significantly reduced to about 60%.

Experimental Example 2: Confirming the effect on the growth activity of beneficial bacteria in the intestine

To determine the effect of the composition of the present invention on the growth activity of Bifidobacterium longum, Lactobacillus sp, and Lactobacillus acidophilus strains among the beneficial bacteria in the intestine, the following experiment was performed. Experiments on the effect of intestinal beneficial bacteria on the growth activity were repeated 3 times, and the results of the experiments were treated with Examples 1-1, 1-2, 1-3, and 1-4 with respect to the control group treated with water. OD It was expressed as a ratio to the value.

1) Preparation of intestinal bacteria

As an index of beneficial bacteria in the intestine, Bifidobacterium longum ATCC 15707, Lactobacillus sp.KCTC 3930, and Lactobacillus acidophilus ATCC 4356 were used. The strains were cultured in anaerobic conditions in BBL GasPak (Becton Dickinson and Company, USA) at 37 ° C in Reinforced Clostridial Media (RCM, Difco, USA) medium.

2) Growth activity test by Agar diffusion method

After sterilizing the EG agar medium, it was cooled to 50 ° C. and three plates inoculated with monarchs were included so that 3 strains were included respectively, and the medium was hardened at room temperature. After 0.1 mg of the compositions of Examples 1-1, 1-2, 1-3, and 1-4 were added dropwise to a 6 mm diameter disc paper (Whatman paper No. 41), they were placed at regular intervals on the hardened medium. . The medium was incubated at 37 ° C for 48 hours under anaerobic conditions, and then the size of the growth active ring was observed.

Strain Example 1-1 Example 1-2 Example 1-3 Example 1-4 Bifidobacterium long sword + ++ + +++ Lactobacillus sp + + ++ ++ Lactobacillus acidophilus - + + ++

As a result, as shown in Table 2, in the solid medium in which the compositions of Examples 1-1, 1-2, 1-3, and 1-4 were disposed, the Bifidobacterium longum strain exhibited a growth activity of + The Lactobacillus sp. Strain showed a positive growth active ring of + and the Lactobacillus acidophilus strain showed a wide growth active ring of +++, so that the composition for enhancing the immune activity of the present invention has high growth activity against beneficial bacteria in the intestine. It was confirmed to have efficacy.

Example 2-1: Preparation of feed additive composition

After adding and mixing 20 parts by weight of the composition of Example 1-1 to 100 parts by weight of rice bran, dried in a dryer at 80 ° C, dried to a solid having a moisture content of less than 10%, crushed in a roll mill grinder, processed into a fine powder, and added as a feed additive. The composition was prepared.

Example 2-2: Preparation of feed additive composition

The same procedure as in Example 2-1 was carried out, but instead of Example 1-1, the composition of Example 1-2 was used to prepare a feed additive composition.

Comparative Example 2-1: Preparation of feed additive composition

The same as in Example 2-1, but using the composition of Comparative Example 1-1 instead of Example 1-1 to prepare a feed additive composition.

Comparative Example 2-2: Preparation of feed additive composition

The same as in Example 2-1, but using the composition of Comparative Example 1-2 instead of Example 1-1 to prepare a feed additive composition.

Experimental Example 2: Evaluation of pig feed efficiency and malodorous substances in minutes

In order to determine the effect on feed efficiency and development by feeding the feed additive composition to pigs, the feed additive composition prepared by Example 2-1, Example 2-2 and Comparative Examples 2-1 and 2-2 was used. Table 3 shows the results of feeding the pig feed with 0.2% by weight.

division Example 2-1 Example 2-2 Comparative Example 2-1 Comparative Example 2-2 Breeding head 30 horses 30 horses 30 horses 30 horses Feed intake 223 kg 220 kg 285 kg 281 kg 110 kg reach 160.7 days 159.7 days 189.8 days 181.1 days

As shown in Table 3, when the feed additive compositions of Example 2-1 and Example 2-2 were added to the pig feed, the result of a 110 kg reduction period was reduced by about 20 days, and thus the feed intake was also reduced. It can be seen that the decrease was about 20.7%.

In addition, in order to analyze the amount of odor-related ammonia and hydrogen sulfide generated from pig powder, fresh feces are collected at the end of the experiment and contained in 100 g of specially manufactured plastic containers, and the concentration of ammonia and hydrogen sulfide in the gas generated at room temperature is Measurement was performed using a digital gas meter (Multi Gas Monitor PGM-7800, RAE Systems Inc., USA) as it progressed (after 3 hours and after 24 hours), and the results are shown in Table 4 below.

Category (ppm) ammonia Hydrogen sulfide 3 hours 24 hours 3 hours 24 hours Example 2-1 4.3 20.5 28.1 3.8 Example 2-2 4.2 18.1 24.4 2.3 Comparative Example 2-1 4.5 45.5 38.8 4.2 Comparative Example 2-2 4.4 35.8 45.5 6.3

As shown in Table 4, the concentrations of ammonia and hydrogen sulfide in the odor gas generated from manure show significantly lower results in Examples 2-1 and 2-2 than Comparative Examples 2-1 and 2-2. You can see that

Experimental Example 3: Porcine foot and mouth disease antibody forming ability

In order to investigate the effect of the feed additive composition on the blood characteristics and foot-and-mouth antibody production capacity of pigs, lyophilized powder of zinc oxide powder, zinc sulfate powder, Lactobacillus plantarum bacteria, the composition of Example 1-1, Example 1 The composition of -2, the composition of Examples 1-3 and the composition of Examples 1-4 were added to the feed, respectively, and the animal experiment was carried out for 6 weeks. The body weight at the start of the experiment was 25.56 W 2.22 kg.

For the feed, corn-soybean meal-based feed blended according to NRC (2012) requirements was used, and 0.3 wt% (CON 1) of zinc oxide powder, 0.3 wt% of zinc sulfate powder (CON 2), Example 1- 0.3% by weight of the composition of Example 1 (Example 1-1-0.3), 0.3% by weight of the composition of Example 1-2 (Example 1-2-0.3), 0.3% by weight of the composition of Example 1-3 (Example 1) -3-0.3), and 0.3% by weight of the composition of Example 1-4 (Example 1-4-0.3) was added to the treatment of ternary hybrid (Duroc x Yorkshire x Landrace) breeding pigs 4 treatments, 8 repetitions per treatment, repeated Completely randomized placement of 5 heads per sugar. The feed was made to be free-to-vegetarian, and water was freely eaten using an automatic water dispenser.

1) Weight change

Table 5 shows the weight change by measuring the weight at the start and end of feeding.

division CON 1 CON 2 Example 1-1-0.3 Examples 1-2-0.3 Examples 1-3-0.3 Example
1-4-0.3 Starting point 22.56 22.56 22.56 22.56 22.56 22.56 End point 53.62b 53.74b 54.43ab 55.40a 54.76ab 54.54ab

No significant change in body weight was observed in the specification experiment for 6 weeks, but pigs to which the compositions of Examples 1-1, 1-2, 1-3, and 1-4 were administered orally were administered to the control (CON 1, CON 2). Compared, it was confirmed that the weight gained somewhat.

2) Content of zinc and immunoglobulins in the blood

To measure the content of zinc and immunoglobulin in the blood, 5 ml of blood was collected using E3 EDTA vacuum tube (Becton Dickinson Vacutainer systems, Granklin Lakes, NJ) in the jugular vein of the same individual at the end of the experiment (6 weeks). After collection, zinc content and immunoglobulin content in serum obtained after centrifugation for 15 minutes at 3000 rpm at 4 ° C. are shown in Table 6.

division CON 1 CON 2 Example 1-1-0.3 Examples 1-2-0.3 Examples 1-3-0.3 Example
1-4-0.3 Zinc (µg / dL) 100c 98c 161b 180ab 205a 228a IgG (mg / dL) 472b 466b 485b 524ab 557a 562a

In Examples 1-1, 1-2, 1-3, and 1-4, zinc content in the blood was significantly increased compared to CON 1 and CON 2, and through this, high bioavailability of the composition for enhancing immune activity of the present invention was obtained. Show.

The immunoglobulin content in the blood was not significantly different in Example 1-1 compared to CON 1 and CON 2, but increased significantly in Example 1-2. In particular, in Examples 1-3 and 1-4, a tendency to increase clearly was observed.

3) Foot and mouth antibody formation rate

Foot and mouth disease antibody (FMDV-O Type) analysis was performed on the serum obtained in 2) of Experimental Example 3. Foot-and-mouth disease antibody titer assay was performed by SP ELISA each time SP antibody test was performed to analyze the individual antibody titer (percentage inhibition titer, PI value) to investigate the persistence of the antibody and the degree of antibody formation by vaccination time. The results were verified using simple linear regression analysis. SP antibody test was carried out according to the method provided by the manufacturer using the PrioCHECK FMDV Type O ELISA kit (Prionics Lelystad B. V., Netherlands). ELISA results were determined by the manufacturer's calculation formula {100- (corrected OD450 test sample / correcte OD 450 max) x 100} dp.

division CON 1 CON 2 Example 1-1-0.3 Examples 1-2-0.3 Examples 1-3-0.3 Example
1-4-0.3 Antibody (PI) 47.38b 47.52b 55.8b 68.6b 72.1a 85.51a Positive rate 50 50 89 100 100 100

Foot-and-mouth disease antibody formation rate was slightly increased in Examples 1-1 and 1-2 compared to CON 1 and CON 2, and significant in Examples 1-3 and 1-4 compared to CON 1 and CON 2 Increased.

The foot and mouth antibody positive rate was 50% in CON 1 and CON 2, but 89% in Examples 1-1-0.3 and 100% in Examples 1-2 and Examples 1-3 and 1-4.

All of the data in Experimental Example 3 were processed using Duncan's multiple range test through linear, quadratic, and cubic analysis to determine the efficacy according to the addition level using the general linear model procedure of SAS (2013). Was tested for significance.

Experimental Example 4: Influenza H1N1 inhibitory effect

29 ㅁ 1 g of 12 week old female (ICR) mice (Orient Bio Inc., Seoul, Korea) were used for the experiment. Before the experiment, the mice were used for the experiment after 7 days of purification, and during the purification, they were bred freely in a breeding room with a temperature of 22 ㅁ 1 ° C and a humidity of 55 ㅁ 10%, and the contrast cycle was adjusted to a 12-hour cycle.

The mice were divided into 10 mice, and each sample was diluted in physiological saline for 5 days after administration of the H1N1 virus to the virus control group, CON 1, Example 1-1, and Example 1-2 administration groups to 20 μg / kg / d. It was administered orally. The negative control used in this experiment is physiological saline, and the virus control is a group infected with a virus but not administered with a reagent.

1) Weight change

Table 8 shows changes in body weight by measuring body weight at the start and end of feeding.

division Eumseong Control Virus control CON 2 Example 1-1 Example 1-2 Start time (0 days) 100.0 100.0 100.0 100.0 100.0 End time (5 days) 106.0b 99.1a 99.9a 106.1ab 107.3b

After administration of the virus, the weight was reduced in the virus control group and CON2 compared to the negative control group, but in Examples 1-1 and 1-2, there was no significant difference in weight gain from the negative control group.

2) H1N1 virus content in lung tissue

After the sacrifice of the mouse, the content of the virus remaining in the lung tissue of the mouse was measured by titer and is shown in Table 9.

division Eumseong Control Virus control CON 2 Example 1-1 Example 1-2 Tissues virus titers
(Log ₁₀ TCID ₅₀ ) 0.0a 5.2b 4.4b 3.9b 1.9a

It was confirmed that the virus content in the lung tissue was significantly reduced in Example 1-2.

Claims (6)

A composition for enhancing immune activity, wherein the water-soluble amino acid mineral complex and lactic acid bacteria in which zinc and aspartic acid are combined at a molar ratio of 1: 1.5 to 2.5 are used as active ingredients. According to claim 1,
The lactic acid bacteria include Lactobacillus plantarum, L. casei, L. acidophilus, Lactobacillus L. bulgaricus, Bifidobacterium Immune activity characterized by at least one selected from the group consisting of B. longum, B. bifidum, Actiregularis and L. rhamnosus Composition for reinforcement. According to claim 1,
The composition is a composition for enhancing immune activity, characterized in that the food composition, food additive composition, pharmaceutical composition, feed composition or feed additive composition. The composition for enhancing immune activity according to claim 1, wherein the composition for enhancing immune activity is administered in parallel or sequentially with an antiviral vaccine. According to claim 4, The anti-virus vaccine,
Newcastle disease, infectious bronchitis, coccidium diarrhea, avian mama, avian cholera, rheovirus-induced tendonitis (viral arthritis), avian laryngotracheitis, avian encephalomyelitis, infectious F-cystitis (IBD), Marek's disease, Salmonella infection, Mycoplasma gallicep A vaccine for birds for the prevention of ticcum infections, avian rhinitis, avian herpes and mycoplasma hyopneumoniae, spawning depressive syndrome, infectious nasal congestion (hemophilis pasagalinarum), mycoplasma sinobies or avian reoviruses;
Pleural pneumococcus, atrophic rhinitis, pseudorabies, swine alone, porcine parvovirus, E. coli enterotoxin, Mycoplasma hyopneumoniae, influenza, leptospira, E. coli infection, porcine genital and respiratory syndrome (PRRS), Bordetella and type A And mammalian livestock vaccines for the prevention or treatment of D-type Multoshita infection, Haemophilus parasuis infection, Welch infection, Rotavirus infection, Streptococcal infection, Glaser disease, pneumonia, Bordetella bronchiseptica infection; or
Influenza, hepatitis A, hepatitis B, hepatitis C, herpes simplex virus (type 2), polio, diphtheria, whooping cough, haemophilus type B influenza (Hib), measles, mumps, rubella, typhoid fever, chickenpox (chicken fox) ), Dengue fever, Epstein-Barr virus infection, human papilloma virus infection, pneumococcal infection, meningococcal infection, pneumococcal infection, viral meningitis, rotavirus infection, tick-mediated encephalitis, traveling diarrhea, cholera, yellow fever or tuberculosis A composition for enhancing immune activity, characterized in that it is a human vaccine for prevention. 1) Injecting zinc precursor and aspartic acid into water at a molar ratio of 1: 1.5 to 2.5;
2) heating and reacting the mixed aqueous solution of zinc precursor and aspartic acid of step 1) at 50 to 100 ° C. for 10 minutes to 24 hours; And
3) preparing a composition for enhancing immune activity by mixing lactic acid bacteria in the amino acid mineral complex;