KR101894241B1 - A bioconversion product comprising Sichuan pepper - Google Patents

Abstract

The present invention relates to a biotransformation product obtained by biotransformation of a microorganism (microbial mycelium) fermentation and an enzyme-treated biotransformation process. Since the biotransformation product of the present invention has a great effect on reducing salmonella infection and sepsis, It is expected to contribute greatly to production.

Description

A bioconversion product comprising Sichuan pepper is obtained by fermentation of microorganisms (mycelium mycelium)

The present invention relates to a biotransformed biosynthesized biotransformation product through fermentation of a microorganism (mycelium mycelium) and an enzymatic treatment biotransformation process.

Livestock products are important as a major source of food for humans, but they are also a major mediator of food poisoning bacteria. Salmonella infections account for the highest percentage of water-borne foodborne illnesses in the United States and Europe, and are the second highest percentage in domestic surveys in 2008, causing many public health problems. According to the FAO / WHO (2002) report, about 26% of food poisoning in Europe is from poultry, including eggs and meat, and 77.1% of food poisoning cases are due to Salmonella. S. enteritidis was the major cause of Salmonella, accounting for more than one - third of the cases. In the United States, the cause of death is higher than other foodborne pathogens. In Korea, S. enteritidis was the highest among 17 isolates (70.8%) among 24 isolates from chicken carcasses. Salmonella is causing problems not only by poultry but also by meat. According to a European report in 2008, salmonella cases accounted for about 50 ~ 70% of the cases of salmonella contamination by poultry, followed by pork and beef by 10 ~ 20%, respectively.

Infection of Salmonella spp. In pig farms is a species-specific Salmonella infection in pigs, S. choleraesuis causing acute septicemia resulting in high mortality and S. typhimurium and S. enteritidis causing diarrhea due to colonization with acquired salmonella . In particular, infections caused by common causative microorganisms can cause food poisoning due to pork contamination in humans, which is an important disease in terms of food hygiene. Although S. choleraesuis is the most isolated salmonella in pigs with clinical symptoms worldwide, the incidence of this disease is very low in Korea.

S. Typhimurium, which causes predominantly chronic gastrointestinal disease in pigs, has a wide variety of contaminants, including polluted feed, water, or wild animals around the farm (birds, rodents, etc.) It can be an important infectious source. In particular, it is not only difficult to differentiate the health insurance funds, but also because there are infinite number of hosts and a strong resistance.

Bacterial food poisoning has increased worldwide, and statistics show that there were 17,468 bacterial food poisoning cases in the United States in 2009, and Salmonella food poisoning is the highest percentage of all food poisoning cases. Enteritidis 1,226 (19.2%), Typhimurium 1,024 (16.1%), Newport 772 (12.1%), Javiana 544 (8.5%) and Heidelberg 230 (3.6%) were among the samples of Salmonella serotype, Montevideo 206 (3.2%), Muenchen 170 (2.7%), Saintpaul 157 (2.5%) and Oranienburg 132 (2.1%) while S. Enteritidis and S. Typhimurium accounted for 35.2% of food poisoning cases caused by Salmonella (CDC, 2010).

Salmonella enters live slaughterhouses and processing plants through live chickens, ducks, pigs, and conductors, which pollute the facility along the line and endanger the microbiological quality of the finished livestock products. Considering the fact that Salmonella from various sources including Salmonella from S. enteritidis has been isolated in the domestic environment and Salmonella derived from the slaughterhouse has been transferred to slaughterhouses through hatcheries and broiler farms, Systematic control techniques at all stages from breeding to processing are needed.

Bacteriocins, bacteriophages, antioxidants, antioxidants, antioxidants, antioxidants, antioxidants, antioxidants, antioxidants, antioxidants, antioxidants, antioxidants, And antibiotics have been used (US registered patent US 8062645, Korean registered patent KR 542484, Korean registered patent KR 941892, Korean registered patent KR 1178173), and food poisoning by salmonella continues to occur. to be.

Accordingly, the present invention relates to biotransformed biosynthesized biosynthesized products through fermentation of microorganisms (mycelium mycelia) and biosynthesis processes with enzymes, and the biosynthesized products of the present invention have a great effect on reducing salmonella infection and sepsis, It is expected to contribute greatly to the production of safe livestock products.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above-mentioned problems occurring in the prior art, and an object of the present invention is to provide a biosynthetic biotransformation product obtained by fermentation of microorganisms However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

Hereinafter, various embodiments described herein will be described with reference to the drawings. In the following description, for purposes of complete understanding of the present invention, various specific details are set forth, such as specific forms, compositions and processes, and the like. However, certain embodiments may be practiced without one or more of these specific details, or with other known methods and forms. In other instances, well-known processes and manufacturing techniques are not described in any detail in order not to unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "embodiment" means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Accordingly, the appearances of the phrase " in one embodiment "or" an embodiment "in various places throughout this specification are not necessarily indicative of the same embodiment of the present invention. In addition, particular features, forms, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

Unless defined otherwise in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In one embodiment of the present invention, " Salmonella " is a gram-negative mixed anaerobic bacterium belonging to Enterobacteriaceae and is divided into S. enterica and S. bongori. Among these two species, S. enterica is a pathogenic organism in humans and animals. It is divided into six subspecies (subspecies): enterica, salamae, arizonae, diarizoanae, houtenae and indica do. Among the most subspecies, S. enterica subsp. enterica, which are further subdivided by serotype, biotype and phage type. The surface antigens of Salmonella strains are O-antigen, H-antigen and Vi-antigen, and they are detected by the Kauffman-White serological scheme Type. There are about 2,500 serotypes known to date, of which about 200 species are relatively high and widely distributed in nature.

Salmonella is highly adaptive to the host and is distinguishable from species-specific and non-species-specific strains. S. Typhi and S. Paratyphi among species-specific serotypes were found in humans, S. pullorum and S. gallinarum in chickens, S. choleraesuis in pigs, S. Dublin in cows, and S. abortusovis She is infected with sheep. S. Enteritidis and S. Typhimurium are representative serotypes with low species specificity. These strains are common pathogens that infect almost all animals including humans and cause enteritis and sepsis in humans.

The source of salmonella food poisoning varies, but poultry products, including eggs and meat, are considered to be representative sources of contamination, as well as food products contaminated with processed products or by-products of animals such as less cooked meat. Poultry is easy to breed because it is more susceptible to pathogenic microorganisms than other species, and it is difficult to control when disease occurs. Ducks, unlike chickens, are basically resistant to Salmonella and appear to be predominantly infectious, but can act as mediators of Salmonella infection.

In one embodiment of the present invention, " abatement " means " lowering ". In the present specification, the term " reducing agent " means " a preparation capable of being lowered ", and the reducing agent in the present invention is preferably a salmonella reducing agent or an agent for reducing sepsis. Is used to mean an agent capable of inhibiting the growth of a microorganism at the onset of infection or sepsis, or inhibiting the disease at the onset of salmonella infection or sepsis.

In one embodiment of the present invention, " turmeric " is a perennial plant belonging to the genus Ginger, also called < RTI ID = 0.0 > Ulgum, < / RTI > Yellow flowers bloom on the leaf axils in April-June. The upper bract is slightly narrower of the butterfly, the tip is fuchsia, and the flower does not run on the leaf axil. Root stalk is light yellow on the outside and scarlet is scarlet like camphor. It is cultivated in India, China, Southeast Asia and so on. Korea is cultivated in Jindo Jeonnam, Haenam in Jeonnam, Buan in Jeonbuk, Siheung in Gyeonggi and Cheongyang in Chungnam. Stems and roots are used for edible and medicinal purposes. Root stems are often used as herbal medicines. It is a spicy, bitter, yellow medicinal product, effective for pain relief and menstrual irregularities. In India, it is used as a medicine for bruises and sprains, and also as a spice for curry powder.

In one embodiment of the present invention, " rice bran " is also called rice bran or simply bran. It is a fine puke that is separated from the process of removing rice hulls from rice and then turning brown rice into white rice. There are some starches, but they are mainly used for livestock feed because they are rich in fat and protein, and there are many ashes such as phosphorus. Most of the rice bran contains about 15% protein and 17% fat, depending on the ratio. Because it contains a high fat content, hexane extraction can be used to obtain rice bran oil. Although crude oil of rice bran oil is reddish yellow or greenish yellow, it can be produced from edible oil through treatment such as dehydration, deoxidation, deodorization, discoloration. Protein is similar to protein in rice, carbohydrate is 40% starch, 30% pentanoic acid. The fat is prone to sowing because of the strong lipase. Vitamin B1 is more than 2mg%.

In one embodiment of the present invention, " Sancho " is a deciduous shrub with a dicotyledonous mushroom, and grows in a mountainous area. It is 3m high and twigs are spiny and reddish brown. Leaves are alternate phyllotaxis of 13-21 small leaves. The small leaf is a wide basaloid with a length of 1-5cm, narrow both ends, with wavy sawtooth on the edge, and a transparent oil spot. Pickled leaves are used as raw leaves, dried or peeled, and then dried or concentrated. Sancho fruit is effective for indigestion, diarrhea, gastric warts, gastric enlargement, vomiting, diarrhea, diarrhea, nervous breakdown, cough, and redness. Cooking is used in a similar way to pepper.

In one embodiment of the present invention, " bamboo shoot " is a deciduous tree belonging to Quercus variabilis, the leaves are oval-shaped, and the ends are suddenly narrowed and sharpened. Length 5 ~ 16㎝, width 2 ~ 7㎝, there are few hairs on both sides, there are sharp sawtooth on edge. In Korea, it is used to read and write Chinese characters, but China and Japan are written as 杜 冲. It has a plate-like appearance and gray or dark gray on the outside. There are many cell wrinkles, small pores in the skin, and smooth inside and dark brown color on the inside. It is sometimes called cotton because it produces a thin, white fiber yarn with tenacity. The taste of this medicine is very sweet and its quality is warm. Antihypertensive action, anti-aging, cholesterol lowering, anti-inflammation, sedation, pain relief, immune control, blood clotting, uterine contraction, antiallergic and antibacterial activity have been reported. There are two kinds of tinnitus: 仲 中, 四), 四 仙, 木 cotton, cotton, masseon, and 肌 皮.

In one embodiment of the present invention, " Omija " is a fruit of an alfalfa tree, having a ball shape with a diameter of about 1 cm and a deep red color. It contains 1-2 drops of red juice and reddish brown seeds. There are five flavors of sweetness, sourness, bitter taste, salty taste and spicy taste, among which the sour taste is strong. There are two types of omija (north omija), south omija, and black omija. Omiza grows mainly in the area of Taebaek Mountain, Namimiza grows in the southern island region, and black omija grows in Jejudo. It is produced in Korea, Japan, Sakhalin, and China. It contains ingredients such as thiazide, thymidine, citrulline, malic acid, and citric acid, which strengthens the heart, lowers the blood pressure, increases immunity, and is used as a tonic. It strengthens the pulmonary function, and it helps to cure the cough and thirst by the deep sea bream and the geodam function. It puts dried fruit in cold water and puts honey and sugar in the water which is reddish, and drinks it with a drink, and it makes it to eat a leaf or a starch piece. Chestnuts, jujube, and mushrooms are put together and boiled to make tea or soak.

In one embodiment of the present invention, " basidiomycetes " is a fungus that forms a cell as a result of sexual reproduction of a fungus as a result of sexual reproduction. There are many things known as mushrooms which are parasitic to other organisms because they can not make their own nutrients. Typical examples are mushroom, mushroom, shiitake, mushroom, mushroom, and mushroom. On the freshly corrugated surface of mature mushrooms, a thick layer of volcanic ash is formed. Most of the mushrooms live as secondary organisms in the fluid of higher plants. In particular, they decompose cellulose and lignin and play an important role in the biological circulation of the material. In addition, they form mycorrhiza in the roots of trees, There are many things to do. There are other kinds that are important as edible bacillus or should be cautious with poisoning.

In one embodiment of the present invention, " mycelium " is a nutritive cell of a filamentous fungus (mold). Functional differentiation such as nutritional function or physiological function can also be seen. Morphologically, the presence or absence of the bulkhead is used for classification, and it develops in germination tube of spore and elongates by tip growth. The group of branched mycelium is called mycelium. Depending on the physiological function, it is distinguished into basal hypha that grows on the surface or inside of the pouch or host, and parasitic bacteria which grows in the air. Parasitoids depend on the transfer of nutrients from basal hyphae, some of which differentiate into spore forming organs and the like. If there are organic matter and suitable environmental conditions such as appropriate humidity and temperature, various mycelial tissues are formed. The mycelial membrane of mycelium of fungus is simple, but it can be seen that it forms a braid connection. In the fungi, it branches into several branches and is entangled like a thread. It does not have chlorophyll and does not have photosynthesis. It attaches to the host organism and absorbs nutrients. In the case of the mushroom-bearing bacterium, the mycelium germinated in the spore has one nucleus, which when fused with another mycelium has two nuclei in one cell. Mycelium with one nucleus is called mycelium, and mycelium with two nuclei is called secondary mycelium. The cell wall of the mycelium is generally composed of chitin or hemicellulose, which is sometimes combined.

In the present specification, the mycelia of mycelia can be selected from mycelium of mushroom, chaga mushroom, shiitake mushroom, mushroom mushroom, mushroom mushroom, The mycelia of these mycelia were collected from the KCCM, KCTC, KCLB, KACC, ATCC, And the like.

In one embodiment of the present invention, " bioconversion " refers to the conversion of a substance added using a physiological function of an organism into a chemically modified form.

In one embodiment of the present invention, " fermentation " refers to a process in which a microorganism decomposes an organic substance using an enzyme contained therein. Fermentation is a phenomenon in which organic matter changes into a simple compound by enzymatic action and releases free energy. In general, microorganisms decompose organic matter and accumulate metabolites. That is, fermentation of alcohol by anaerobic digestion of sugars by anaerobic digestion of yeast, fermentation of lactic acid by lactic acid bacteria decomposing sugar nonoxiously into lactic acid is a typical fermentation, but now, A phenomenon in which alcohol is oxidized to acetic acid by use of oxygen, and a phenomenon in which mold oxidizes glucose to gluconic acid by using oxygen in the air are also referred to as acetic acid fermentation and gluconic acid fermentation. These anaerobic fermentation can be divided into anaerobic fermentation and aerobic fermentation (oxidation fermentation), and other anaerobic fermentation furnaces include glycerol fermentation, acetone butanol fermentation, 2, 3-butylene glycol fermentation, butyric acid fermentation, Fermentation, and methane fermentation. Examples of aerobic fermentation include citric acid fermentation, itaconic acid fermentation, yeast acid fermentation, 2-keto glutaric acid fermentation, oxalic acid fermentation, fumaric acid fermentation and sorbose fermentation. In addition, production of amino acids, vitamins and antibiotics by microorganisms is also called glutamic acid fermentation, riboflavin fermentation, penicillin fermentation and the like, which is not a good title. The name of the fermentation is usually called the product, but sometimes the name of the substrate is given by cellulosic fermentation, pectin fermentation and so on.

In one embodiment of the present invention, the term " poultry " means a bird raised by a person in order to obtain livestock products useful for human beings such as meat, eggs, hairs and feathers. Ducks, turkeys, geese, ancestors, pigeons and quails. In particular, broiler chickens have the characteristics of hatching in other birds and eggs of poultry, the characteristics of having wings and beaks, the features of the body being covered by feathers, Will be effective for other poultry. In this specification, feed for poultry refers to edible material supplied to change the quantitative and qualitative size of poultry. The fermentation composition contained therein includes morphological changes such as a concentrate or powder.

In one embodiment of the present invention, " sepsis " refers to a condition in which a microorganism is infected and a serious inflammatory reaction occurs in the whole body. The number of breaths increased more than 24 times per minute (ventilation), heart rate more than 90 times per minute (tachycardia), increase in leukocyte count in blood test, or significant decrease in respiratory rate If you have symptoms that are more than a branch, it is called systemic inflammatory response syndrome (SIRS). This systemic inflammatory response syndrome is called sepsis when it is caused by microbial infection. The source of the infection can be any organs of the body. Pneumonia, pyelonephritis, meningitis, cellulitis, infective endocarditis, peritonitis, pressure ulcer, cholecystitis, and cholangitis may cause sepsis. If such infection occurs, the causative microorganism may invade into the blood and cause sepsis. However, even if microorganisms do not penetrate into the blood, systemic sepsis may occur due to the inflammation reaction of some body parts and the production of inflammatory substances. Early symptoms of sepsis may include respiratory arrest, neurological disturbances such as loss of locomotion (time, place, cognitive ability) or delirium. The skin may appear visually opaque due to lowering of blood pressure and lowering of the amount of blood supplied to the distal end of the body. Bacteremia (a symptom of germs circulating in the blood) can cause germs to move around the bloodstream, to position themselves in certain parts of the body and cause pathological changes in those areas. As the skin changes specific to the causative bacteria, it may help to diagnose the cause of sepsis. Symptoms of the digestive system include nausea, vomiting, diarrhea and intestinal paralysis, and severe gastrointestinal bleeding may also occur.

&Quot; Pharmaceutical composition " in one embodiment of the present invention means a composition to be administered for a specific purpose. For the purpose of the present invention, the pharmaceutical composition of the present invention is used for improving the survival rate or inhibiting the inflammatory active substance in a group infected with Salmonella or sepsis, and a composition therefor and a pharmaceutically acceptable carrier, excipient or diluent . ≪ / RTI > The pharmaceutical composition according to the present invention comprises 0.1 to 50% by weight of the active ingredient corresponding to the pharmaceutical composition of the present invention, based on the total weight of the composition. Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In one embodiment of the present invention, the term " food composition " is used variously as a food composition for improving salmonella infection or sepsis disease. The food composition comprising the composition of the present invention as an active ingredient may be used in various foods, , Beverages, gums, tea, vitamin complexes, powders, granules, tablets, capsules, confectionery, rice cakes and breads. Since the food composition of the present invention is composed of a natural food and its fermented product which have little toxicity and side effects, it can be safely used for prolonged use even for prophylactic purposes. When the composition of the present invention is contained in the food composition, the amount thereof may be added in a proportion of 0.1 to 50% of the total weight. Here, when the food composition is prepared in a beverage form, there are no particular limitations other than those containing the food composition at the indicated ratios and may contain various flavors or natural carbohydrates such as ordinary beverages as an additional ingredient. That is, natural carbohydrates include monosaccharides such as glucose, disaccharides such as fructose, sucrose and the like, and sugar sugars such as polysaccharide, dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol can do. Examples of the flavors include natural flavors (such as tau martin, stevia extract (for example, rebaudioside A and glycyrrhizin), and synthetic flavors (for example, saccharin and aspartame). The food composition of the present invention can be used as a food composition containing various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants, pectic acid and its salts, alginic acid and its salts, Stabilizers, antiseptics, glycerin, alcohols, carbonating agents used in carbonated beverages, etc. These components may be used independently or in combination. The ratio of these additives is not so important, Is generally selected in the range of 0.1 to about 50 parts by weight per part.

In one embodiment of the present invention, the term " feed composition " includes all commercially available general diets, including, but not limited to, sancho, corn, soybean meal, soybean, sorghum, rice, barley, Corn bran, starch powder, coffee starch, cloves, clam, seaweed, cottonseed, seeds, canola wheat, chestnut, wheat, barley, Corn gluten meal, corn germ meal, red pepper seed meal, lupine seed meal, fish meal, oyster meal and meat meal, and these feeds may be subjected to fermentation ≪ / RTI > compositions. Feeds of the present invention may include feed additives for general purpose such as general feed additives such as salt, bone meal, calcium phosphate, mineral compound, vitamin, amino acid, antibiotic, hormone and the like.

In one embodiment of the present invention, there is provided a method for producing an amino acid composition comprising the steps of: (a) hydrolyzing an acid plant with an enzyme of a cellulase and hemicellulase system and an enzyme of an amylase system; (b) inoculating the hydrolyzate of step (a) with mycelia of mycelia and culturing to produce a fermented product; And (c) treating the fermented product produced by the culturing step in the step (b) with a fibrinolytic enzyme, wherein the mycelium of bacillus mycelia contains a mushroom, a car Wherein the cellulolytic enzyme in step (c) comprises at least one member selected from the group consisting of mushrooms, shiitake mushrooms, mushrooms, mushrooms, snow mushrooms, Wherein the cellulolytic enzyme is at least one selected from the group consisting of a cellulase, a hemicellulase, a pectinase, and a glucanase, wherein the cellulolytic enzyme is an enzyme capable of decomposing ≪ / RTI >

In another embodiment of the present invention, an acid-hydrolysis product obtained by hydrolyzing an acidulant with an enzyme of a cellulase and hemicellulase system and an enzyme of an amylase system, and inoculating the mycelium with a mycelium, Wherein said pharmaceutical composition provides a pharmaceutical composition for the prevention or treatment of Salmonella infection, said pharmaceutical composition providing a pharmaceutical composition for enhancing the phagocytosis and phagocytosis of macrophages against Salmonella, , The pharmaceutical composition provides a pharmaceutical composition for improving the induction of Th1 helper T cell immune response upon Salmonella infection, wherein the pharmaceutical composition provides a pharmaceutical composition for the prevention or treatment of sepsis.

In still another embodiment of the present invention, a method of hydrolyzing an acidophilus with an enzyme of a cellulase and hemicellulase system and an enzyme of an amylase system and inoculating the mycelia with a mycelium, and cultivating the fermented product, ≪ / RTI >

In still another embodiment of the present invention, a method of hydrolyzing an acidophilus with an enzyme of a cellulase and hemicellulase system and an enzyme of an amylase system and inoculating the mycelia with a mycelium, and cultivating the fermented product, Wherein the feed composition provides feed compositions for cattle such as cows, pigs, poultry and the like, wherein the poultry is selected from the group consisting of chicken, duck, turkey, goose, At least one selected feed composition is provided.

Hereinafter, the present invention will be described in detail.

Fermentation and Enzymatic Treatment of Microorganisms (Mycobacterium mycelium) Biodegradation of Biotransformed Sancho Bioconversion Products through Bioconversion Process has a great effect on the reduction of salmonella infection and sepsis in livestock including cattle, pigs, and poultry, It is expected to contribute.

FIG. 1 is a schematic view showing a biological conversion process of a raw material of turmeric, rice bran, or anthraquinone and a process of obtaining a biotransformation product powder according to an embodiment of the present invention.
FIG. 2 is a graph showing the results of salmonella sputum smearing and killing enhancement in macrophages treated with turmeric, rice bran, or sancho bioconversion products according to an embodiment of the present invention.
FIG. 3 is a graph showing the results of improving the macrophage Salmonella sputum production ability of raw or biotransformed products according to one embodiment of the present invention.
FIG. 4 is a graph showing the results of improving the viability of macrophages Salmonella in native or bioconverted products according to one embodiment of the present invention.
FIG. 5 is a graph showing the results of evaluating the effect of improving the survival rate of mice in the peritoneal administration of biotransformation products in a mouse model of lethal Salmonella infection, according to an embodiment of the present invention.
FIG. 6 is a graph showing the results of evaluating the effect of improving the survival rate of mice when the biotransformation product is orally administered in a mouse model of lethal Salmonella infection according to an embodiment of the present invention.
FIG. 7 is a graph showing the results of evaluating the effect of improving the survival rate of mice in the case of oral administration of the biotransformation product in the lethal dose Salmonella-infected mouse model according to an embodiment of the present invention.
FIG. 8 is a graph showing the results of measurement of Salmonella colonies in mouse abdominal cavity during administration of a biotransfer product in a mouse model of asthma salmonella infection according to an embodiment of the present invention.
FIG. 9 is a graph showing the results of measurement of lymphocyte count in mouse spleen upon administration of a biotransfer product in a mouse model of asthma Salmonella infection according to an embodiment of the present invention. FIG.
Figure 10 is a graph showing changes in expression levels of IFN-y, IL-l [beta], IL-2 and IL-5 cytokines upon administration of biotransformation products in a mouse model of asthma Salmonella infection in accordance with an embodiment of the present invention .
IL-1, IL-2, IL-4, IL-5, IL-6, IL-6, and IL-6, respectively, upon administration of a turmeric biotransformation product in a mouse model of infection with Salmonella typhimurium according to an embodiment of the present invention. IL-10 < / RTI > and < RTI ID = 0.0 > IL-12 < / RTI > cytokines.
IL-4, IL-5, IL-6, IL-6, and IL-10 in the mouse model of infectious salmonellosis infection according to an embodiment of the present invention. IL-10 < / RTI > and < RTI ID = 0.0 > IL-12 < / RTI > cytokines.
FIG. 13 is a graph showing an effect of inhibiting damage to liver tissues upon administration of a biotransfer product of turmeric or rice bran in a mouse model of saliva Salmonella infection in accordance with an embodiment of the present invention.
FIG. 14 is a graph showing the results of evaluating the effect of improving the survival rate of mice according to the concentration of the bioconversion product in the LPS-induced sepsis model according to an embodiment of the present invention.
15 is a graph showing the results of evaluating the effect of improving the survival rate of mice according to the route of biotransformation products in the LPS-induced sepsis model according to an embodiment of the present invention.
FIG. 16 is a graph showing the mouse survival rate difference according to the same administration dose and administration method based on the biotransformation product in the LPS-induced sepsis model according to an embodiment of the present invention.
FIG. 17 is a graph showing an immunostimulatory effect on macrophages of anthrax biotransformation product according to an embodiment of the present invention as a result of NO (Nitric Oxide) analysis.
FIG. 18 is a graph showing the immunostimulatory effect of the bivalent biotransformation product on macrophages according to one embodiment of the present invention as a result of NO (Nitric Oxide) analysis. FIG.
FIG. 19 is a graph showing the immunostimulatory effect of Omija bioconversion product on macrophages according to one embodiment of the present invention as a result of NO (Nitric Oxide) analysis.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example 1 Bioconversion Process of Fermentation and Enzyme Treatment

Example 1-1. Pretreatment of raw materials

In order to pre-treat the raw materials of purchased ganghwang (Jindo, Indo), rice gruel, and sancho, the mold contamination degree of the raw material was measured and then washing work was performed to remove foreign matters and contaminants of the natural raw materials. (1) air washing step to remove foreign matters and mold spores, (2) water washing step to remove residual pesticides, (3) alcohol (Et-OH) to remove contaminants such as microorganisms, The washing of the raw materials was carried out through the washing step. Each of the raw materials after washing and cutting was dried in a hot air drier capable of mass drying, and subjected to pulverization of raw materials of raw materials through crude pulverization and pulverization.

Examples 1-2. Obtain bioconversion products of dry materials

Each of turmeric, rice bran, and acidophilus, which removed foreign and fungal contamination, was subjected to enzymatic treatment and heat treatment sterilization for liquid culture culture. The enzymes used were cellulase and hemicellulase hydrolytic enzymes (enzyme A) and amylase hydrolytic enzymes (enzyme B) capable of decomposing bark, muscle, roots and leaves. After the addition, the reaction was carried out at 60 DEG C for 1 hour and sterilized at a high temperature for 30 minutes to carry out culture medium culture of each material.

Then, the mycelium of mycelia cultivated separately in the culture medium was added. When the concentration of the residual carbon source is depleted to a certain concentration or less under the conditions of 28 to 30 ° C and pH 4.5 to 7, inoculated with a 10% (v / v) culture of mycelia of mycelia, a concentrated liquid turmeric medium, Or an oil-in-water process in which liquid soy sauce medium was added for 7 to 10 days. The above mycelium of mycelia may be selected from mycelia such as mushroom, chaga mushroom, shiitake mushroom, mushroom mushroom, mushroom mushroom, snow mushroom mushroom, and skirting mushroom, which are medicinal and edible bacillus species, Ryu Myung-sa is a member of the Korean Society for Microbiology (KCCM), Korea Biotechnology Research Institute (KCTC), Korea Cell Line Bank (KCLB), Korea Agricultural Microbiology Resource Center (KACC) It is possible to use the strain in an institution.

The enzymatic treatment of the biotransformation process for the above fermented product is carried out by adding various enzymes such as cellulose decomposing enzyme, cellulase, hemicellulase, pectinase and glucanase after hot water extraction to an appropriate amount (The optimum amount indicated in the product manual of each enzyme preparation). Examples of the enzymes include commercially available enzymes such as a cellulase (a compound derived from Aspergillus niger), a filtrate (a compound derived from Filtrase (Tricoderma reesei)), a rapidase (a compound derived from a fibrinolytic enzyme, Viscozyme (Complex containing Aspergillus niger) and Sumizyme (a complex containing Aspergillus niger-derived pectinase) was added at the recommended concentration (10% v / v) given in the product manual of each enzyme preparation, The enzyme / substrate reaction was performed by rotating at 250 rpm for 1 to 3 hours. The bioconversion products produced by the fermentation and enzyme-treated bioconversion process were lyophilized and pulverized after enzyme inactivation and sterilization at 90 ° C for 1 hour. The culture medium, biotransformation process and biotransformation product powder of the above-described turmeric, rice bran, or sancho cultivation are shown in Fig.

Example  2. Salmonella in macrophages Pickling  And after digestion Kill ability  Evaluation of improvement efficacy

RAW264.7 cell line (mouse macrophage line) was purchased from the World Cell Line Bank (ATCC) to confirm Salmonella robbing and killing ability of macrophages and 15% complement inactivated under conditions of 37 ° C, 5% CO ₂ , The cells were cultured in RPMI-1640 medium containing heat-inactivated fetal bovine serum (FBS), 100 units of penicillin, and 100 μg / ml of streptomycin.

First, RAW264.7 cells were inoculated in a 96-well culture dish at a concentration of 1 x 10 ⁴ cells / well, and cultured overnight. For the salmonellosis test of macrophages, the mycelium fermented broth was treated for 4 hours, Lt; / RTI > Salmonella typhimurium (1 × 10 ⁶ CFU / ml) was treated with 10 μl (final 1 × 10 ⁴ CFU) and reacted for 30 minutes and 60 minutes, followed by further washing with a medium . The cells were treated with 1 ml of distilled water (or 0.1% triton X-100 in PBS 1), and the cells were washed with PBS and treated with trypsin. The cells were treated with 30 ml of gentamycin for 30 minutes to remove the remaining Salmonella in the medium. Ml) and the mixture was stirred at room temperature for 30 minutes to dissolve the cells. Subsequently, the cells were inoculated in series with a nutrient agar and cultured for 16 hours to determine the number of colonies of Salmonella.

In order to test the viability after phagocytosis of macrophages, RAW264.7 cells cultured in the same manner as above were inoculated in a 96-well culture dish at a concentration of 1 x 10 ⁴ cells / well and cultured overnight. Treated with time and washed once with medium. Salmonella typhimurium (1 × 10 ⁶ CFU / ml) was treated with 10 μl (final 1 × 10 ⁴ CFU) at 1 × 10 ⁶ CFU / ml for 1 hour and further washed with the medium. The remaining Salmonella in the medium was removed by treatment with 30 μg / ml of gentamycin for 2 hours, 4 hours or 8 hours, washed with PBS, treated with trypsin to obtain cells, and then 1 ml of distilled water (or 0.1% triton X-100 in PBS (1 ml)) and the mixture was stirred at room temperature for 30 minutes to dissolve the cells. Subsequently, the cells were inoculated in series with a nutrient agar and cultured for 16 hours to determine the number of colonies of Salmonella.

As a result, it was confirmed that the salmonellosis of macrophages was improved as the treatment time was increased or the treatment concentration was increased in all bioconversion products of turmeric, rice bran, or sancho. In the case of salmonellosis after macrophage salmonellosis, as the concentration of treated bioconverted products increased, the number of colonies of Salmonella increased at 2 hours after treatment in all bioconversion products compared to the control (group not treated with bioconversion products) In 4 hours and 8 hours, the salmonella colonies decreased rapidly as the concentration of treated bioconverted products increased. As a result, the numbers of Salmonella colonies were lower than those of the control group. In other words, it was confirmed that the biodegradation product treatment improves the kill ability after phagocytosis in addition to the salmonella attack ability of macrophages. The results are shown in Fig.

In the experiment in which RAW264.7 cell line was treated with Jindo jinhodang or Ganoderma lucidum, the colonization efficiency of macrophage Salmonella largely improved as the concentration of the bioconversion product increased in both Jindo and India . Especially, when macrophages were treated with 10 ㎍ / ㎖ or more of bioconversion products, the phagocytosis of macrophages was more than 2 times higher than that of the control. In the evaluation of viability after salmonellosis of macrophages, salmonella colonies were significantly decreased regardless of the origin of turmeric when the bioconversion product of 10 ㎍ / ㎖ or more was treated for 4 hours or more. Fig. 3 and Fig. 4 show the results of the macrophage salmonellosis and the salmonella kill ability of the raw or biotransformed product of each turmeric origin.

Example  3. Lethal dose Salmonella-infected mouse model (in vivo ) Efficacy evaluation

Biotransformation products of turmeric, rice bran, or sancho were administered to mice by intraperitoneal injection or oral administration at a concentration of 10 mg / kg. In the case of intraperitoneal injection, 200 ㎕ of intraperitoneal injection was made at a dose of 10 mg / kg using a 1 ㎖ syringe and 10 mg / kg of the compound was orally administered to the diets. Later, a lethal dose of Salmonella (1 x 10 ⁵ CFU) was injected to induce a mouse model of lethal Salmonella infection. The survival rate of the lethal Salmonella-infected model mouse was observed until 30 days. As a result, all of the mice in the control group (the group not treated with the biotransformation products) died within 6 days in both the peritoneal administration (FIG. 5) and the oral administration (FIG. 6) In contrast, the survival time of mice in the group treated with the bioconversion product was much longer. According to the biotransformation products, it was shown to be effective in order of turmeric, rice gruel and sancho.

Compared with the above-mentioned peritoneal administration and oral administration, overall survival time of the lethal Salmonella-infected model mouse was increased when the bioconversion product was administered to the abdominal cavity more than in the case of oral administration. The results are shown in FIG.

Example  4. Abalone  In a Salmonella-Infected Mouse Model (in vivo ) Efficacy evaluation

Example 4-1. Mouse model of infection with saliva Salmonella

Biotransformation products of turmeric, rice bran, or sancho were administered to mice by intraperitoneal injection or oral administration at a concentration of 10 mg / kg. In the case of intraperitoneal injection, 200 ㎕ of intraperitoneal injection was made at a dose of 10 mg / kg using a 1 ㎖ syringe and 10 mg / kg of the compound was orally administered to the diets. Subsequently, saline (1 x 10 ⁴ CFU) of saliva was injected to induce mouse model of saliva Salmonella infection.

Example 4-2. Measurement of Salmonella colony count in the abdominal cavity of mice infected with saliva Salmonella infection

Mice were sacrificed 48 hours after the Salmonella infection, and the peritoneum was sacrificed. Then, 10 ml of PBS was injected, and PBS was recovered after peritoneal massage. The recovered PBS was centrifuged at 3500 rpm for 20 minutes, washed twice with fresh PBS, resuspended in 1 ml of PBS, and inoculated on a solid medium (LB plate) for microorganism. After culturing for 24 hours, the number of Salmonella colonies was counted. The results showed that the Salmonella colony counts were significantly reduced in all the bioconversion product treatment groups of turmeric, rice bran, or sancho, as compared to the control (the group not treated with bioconversion product). Comparisons by bioconversion product dosing showed that Salmonella colonies decreased more in all bioconversion product groups when administered intraperitoneally than oral doses. The results are shown in FIG.

Example 4-3. Measuring the number of lymphocytes in mouse spleen salmonella-infected mice

Mice were sacrificed 48 hours after the salmonella infection in a mouse model of saliva Salmonella infected by the method of Example 4-1, and the total spleen cells (total splenocytes) were isolated using a stainless steel mesh Respectively. Separated spleen cells were inoculated into 96-well culture dishes at a concentration of 2 x 10 ⁵ cells / well, treated with 0.5 μg / ml of Concanavalin A and 1 μg / ml of LPS, Proliferation was induced. After 72 hours, the number of cells was measured using an MTT assay, and the amount of increase was calculated by setting the total number of splenocytes in healthy mice not infected with Salmonella to 1. Experimental results showed that the spleen lymphocytes of the mice in the control group (the group not treated with the biotransformation product) increased about 1.5 times as compared with the healthy mice, Mice were approximately 2-fold increased compared to mice. Especially, when the bioconversion product was administered intraperitoneally, the number of mouse spleen lymphocytes was increased more than that of oral administration. The results are shown in Fig.

Example 4-4. Measuring cytokine in mouse spleen infected with saliva Salmonella infection

Mice were sacrificed 48 hours after infection with Salmonella and the spleen was sacrificed. The spleen was pulverized with a homogenizer, and then subjected to ELISA IL-2 (R & D system, M2000) and Th2 immunoreactivity, which are related to the inflammatory response, IL-1β (R & D system, MLB00C), the cytokine IFN-γ And cytokine levels of IL-5 (R & D system, M5000), a cytokine associated with IL-5. Measurement of the cytokine was carried out according to the manufacturer's recommended protocol. As a result, the IFN-γ and IL-2, which are cytokines related to the Th1 immune response together with IL-1β, which is a cytokine related to the inflammatory response, were observed in all the bioconversion product treatment groups of turmeric, Compared with the control group (group not treated with the bioconversion product). Overall, the cytokine levels were also increased more when peritoneal administration of the bioconversion product was administered than by oral administration. However, in the case of IL-5, there was no significant difference between the bioconversion product treatment group and the control group (the untreated bioconversion product group). The results are shown in FIG.

In addition to IL-1β, IFN-γ, IL-2 and IL-5, IL-4 (R & D system, M4000B), IL-6 (R & D system, M12000), IL-10 (R & D system, M1000) and IL-12 (R & D system, M1270) The measurement of added cytokines was carried out according to the manufacturer's recommended protocol as with IFN-y, IL-1 beta, IL-2 and IL-5. The expression levels of IFN-γ, IL-2, and IL-12, which are cytokines related to the Th1 immune response in combination with the IL-1β and IL-6 cytokines related to the inflammatory response, In addition, IL-4 and IL-5, which are cytokines related to Th2 immune response, and IL-5, which are related to Treg immunoreactivity, were significantly increased in IL- 10 was not significantly different from the control group of Salmonella infected mice. In other words, the cytokine production associated with the Th2 immune response was not changed when the biotransformation product of turmeric or rice bacilli was administered in the immune response after the salmonella infection, but the amount of the cytokine related to the Th1 immune response was increased Considering that Th1 immune response is important for cellular immunity related to the defense of viruses and intracellular infecting microorganisms (bacteria, protozoa), and also because Salmonella is an intracellular infecting microorganism causing infection in macrophages, , Or all bioconversion products of Sancho are expected to be able to effectively cope based on immunological evidence. The above results of turmeric or corona are shown in Fig. 11 and Fig. 12, respectively.

Example 4-5. Identification of liver injury inhibition effect of mice infected with saliva Salmonella

To determine if tissue damage caused by the systemic inflammatory response caused by Salmonella infection could be inhibited by the administration of an immunomodulatory agent, 10 mg / kg of turmeric (bioconversion) material was administered via diet for 2 weeks and 1 x Infection was induced by intraperitoneal injection of ATCC14028 salmonella strain of 10⁴ CFU. At 2 days after the infection, liver tissue was isolated and sacrificed at the expense of systemic inflammatory response, and it was observed that the liver tissue damage was suppressed in the mice treated with the carbohydrate (bioconversion) material compared with the control. The results are shown in Fig.

Example 5. Evaluation of in vivo efficacy in LPS-induced sepsis model

Example 5-1. Assessment of efficacy by bio-conversion product dose

Biotransformation products of turmeric, rice gruel, or sancho were administered to mice in a dose of 1, 10, and 100 mg / kg by intraperitoneal injection of 200 ㎕ for 2 weeks using a 1 ㎖ syringe, and lethal dose of LPS (20 ㎍ / kg) Sepsis was induced by injecting galactosamine (700 mg / kg). The survival rate over time of the sepsis model mice was observed for up to 60 hours. Experimental results showed that the survival time of the sepsis model mice was increased as the concentration of bioconverted products treated in all bioconversion products of turmeric, rice bran, or sancho was increased. The results are shown in Fig.

Example 5-2. Evaluation of efficacy according to route of biotransformation product

Biotransformation products of turmeric, rice bran, or sancho were administered to mice by intraperitoneal injection or oral administration at a concentration of 10 mg / kg. In the case of intraperitoneal injection, 200 ㎕ of intraperitoneal injection was made at a dose of 10 mg / kg using a 1 ㎖ syringe and 10 mg / kg of the compound was orally administered to the diets. Thereafter, sepsis was induced by injecting LPS (20 / / kg) and galactosamine (700 mg / kg) at a lethal dose and the survival rate over time of the sepsis model mice was observed for 60 hours. Experimental results showed that the survival time of septicemic mice was increased in all bioconversion products of turmeric, rice bran, and sancho. In addition, regardless of the method of administration, the survival time was increased in the administration group as compared to the control (the group in which the biotransformation product was not treated). The results are shown in Fig.

The results of the above experiments were compared according to the biological conversion products of the same administration method. In the case of 10 mg / kg administration of both peritoneal administration and oral administration, the efficacy was shown to be in the order of turmeric, rice gruel, and sancho, and the control group had the lowest effect. The results are shown in Fig.

Example  6. Comparison of immunostimulatory effects of Sancho, Gichi and Omiza

In order to compare the immunostimulatory effects of the biotransformation products of Sancho, Mitochondria, and Omija, which are known to have antimicrobial properties, culture of macrophages (macrophages) and NO (nitric oxide) analysis were carried out using 5% DMEM medium . For measurement of immunological activity against macrophages (macrophages), macrophages were used either as a cell line or 5 ml of 1X PBS was injected into mouse abdominal cavity, the mice were sufficiently shaken, and then intraperitoneal macrophages were infected with mouse Cells were subcultured in a 96-well culture dish using peritoneal macrophages at a concentration of 1 x 10 ⁶ cells / ml per well in 0.2 ml portions. After culturing for 24 hours, the culture conditions of the cells were checked with a microscope, and the medium was completely removed, followed by washing with a medium containing no serum for 2-3 times. Then, each well was filled with 0.5 ml of 5% FBS medium supplemented with a sample of a biotransformation product of Sancho, Mitochondrion or Omija, and the concentration of the sample was 1/256, 1/64, 1/16, 1/4, , 16, 64, 256, and 1024 占 퐂 / ml. As a positive control, the final concentration was treated with 0.1 μg / ml of LPS (200 μg / mL stock). After culturing the 24-well culture dishes treated with the samples for 20 to 24 hours, 100 μl of the culture solution of each well was centrifuged (12,000 rpm, 5 minutes), and the supernatant was taken for NO analysis.

The prepared samples were put into each well of a 96-well culture dish and 100 μl of a grease solution (① N- (1-naphtyl) ethylene diamine dihydrochloride ②sulfanilamide) was dispensed and reacted for 1 minute. Then, the absorbance values were measured at a wavelength of 540 to 550 nm using an ELISA reader, and the immobilized activity titers of various raw materials and bioconversion fermentation products were measured. The results are shown in Fig. 17 to Fig.

As a result, nitric oxide (NO) was produced at a concentration of 1/4 ㎍ / ㎖ or higher in the group treated with the sancho bioconversion product and in the group treated with 4 ㎍ / A high concentration of NO (nitric oxide) production was measured, and it was confirmed that the bioaccumulation product of Sancho was remarkably effective in immunostimulation.

However, the group treated with the biotrans and omija bioconversion products showed very low nitric oxide (NO) at the negative control level at all concentrations except 1024 ㎍ / ㎖ irrespective of the place of production, and the immunostimulating effect . This suggests that not all of the bioconverted bioconverted products activate the immune response through microbial fermentation and enzymatic bioconversion.

Claims (13)

(a) hydrolyzing anthocyanins with carbohydrate (polysaccharide) degrading enzymes cellulase, hemicellulase, and amylase enzyme;
(b) inoculating the hydrolyzate of step (a) with mycelia of mycelia and culturing to produce a fermented product; And
(c) treating the fermentation product produced by the culturing step in the step (b) with cellulose decomposing enzyme,
The bacterium mycelia in step (b) are selected from the group consisting of mushroom, chaga mushroom, shiitake mushroom, mushroom mushroom, mushroom mushroom, snow mushroom mushroom, and skirting mushroom,
Wherein the cellulolytic enzyme in step (c) is selected from the group consisting of cellulase, hemicellulase, pectinase, and glucanase.
delete delete delete A pharmaceutical composition for preventing or treating Salmonella infectious disease or sepsis, which comprises as an active ingredient an organozotocin conversion product produced by the production method of claim 1.
delete 6. The method of claim 5,
The pharmaceutical composition is a pharmaceutical composition for preventing or treating Salmonella infection or sepsis, which enhances the phagocytic ability and phagocytosis of macrophages against Salmonella.
6. The method of claim 5,
The pharmaceutical composition is a pharmaceutical composition for preventing or treating Salmonella infection or sepsis, which improves the induction of Th1 helper T cell immune response upon Salmonella infection.
delete A food composition comprising the sanchoic biotransformation product produced by the method of claim 1 as an active ingredient.
A feed composition comprising as an active ingredient an organozotocin conversion product produced by the method of claim 1.
12. The method of claim 11,
The feed composition is for feeding cattle, pigs, and poultry.
13. The method of claim 12,
Wherein the poultry is any one or more selected from the group consisting of chickens, ducks, turkeys, geese, birds, pigeons, and quails.

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