KR100852744B1 - A antibacterial composition comprising an extract of ulva lactuca - Google Patents

Abstract

The present invention relates to antimicrobial compositions comprising galparae extract as an active ingredient, in particular so for antimicrobial composition methicillin-resistant Staphylococcus aureus (Methicillin-Resistant Staphylococcus aureus, MRSA), including galparae extract according to the present invention as an active ingredient It relates to an antimicrobial composition having excellent antibacterial activity.

Brown seaweed, MRSA, antimicrobial composition

Description

An antimicrobial composition comprising a green onion extract {A ANTIBACTERIAL COMPOSITION COMPRISING AN EXTRACT OF ULVA LACTUCA}

1 is a photograph showing the antimicrobial activity of gram-positive bacteria of the extract of brown lees according to an embodiment of the present invention, Figure 1 A shows the antimicrobial activity against B. subtilis PM 125, Figure 1 B shows the antimicrobial activity against M. luteus KCTC 1056, Figure 1 C shows the antimicrobial activity against S. aureus KCTC 1916, Figures A to C a to c of the green ether ethyl ether fraction Divided by the quantity, a is 5 μl, b is 10 μl, c is 15 μl,

Figure 2 is a photograph showing the antimicrobial activity of MRSA (Methicillin-Resistant Staphylococcus aureus ) of the extract of brown lees according to an embodiment of the present invention, A of Figure 2 shows the antimicrobial activity against MRSA CCARM 3115, Figure 2B Figure 2 shows the antimicrobial activity against MRSA CCARM 3089, Figure 2 C shows the antimicrobial activity against MRSA CCARM 3661, a to c of Figure 2 A to C are divided according to the amount of brown ether ethyl ether fraction , a is 5 μl, b is 10 μl, c is 15 μl,

Figure 3 is a photograph showing the antimicrobial activity against gram-negative bacteria of gram-negative bacteria of the extract according to an embodiment of the present invention A of Figure 3 shows the antimicrobial activity against E. coli D 31, Figure 3 B shows the antimicrobial activity against E. aerogenes KCTC 2190, Figure 3 C shows the antimicrobial activity against E. tarda NUF 251, Figures A to C a to c of the brown ether ethyl ether fraction It is classified according to the quantity, where a is 5 μl, b is 10 μl, and c is 15 μl.

[Industrial use]

The invention antimicrobial agent compositions and as it relates to a process for the preparation, and more particularly, the antimicrobial composition has an antimicrobial activity against gram-positive and gram-negative bacteria, particularly MRSA (Methicillin-Resistant Staphylococcus aureus), including galparae extract as an active ingredient It relates to an antimicrobial composition comprising as an active ingredient the extract of brown seaweed having a very good antimicrobial activity against.

[Private Technology]

An antibiotic is a generic term for an antimicrobial agent. In particular, an antimicrobial agent, particularly a substance that has excellent antimicrobial activity by inhibiting a system for synthesizing a cell wall or protein, or a product prepared from such an antimicrobial agent it means. Antibiotic components are mainly extracted from fungi, and are used today to treat diseases caused by bacterial infection. The most representative antibiotic is penicillin, manufactured in 1928 by British physician Alexander Fleming. Penicillin was the first antibiotic manufactured by humans to respond to bacteria in earnest. A typical antibiotic developed after penicillin is methicillin, which is considered to be superior to penicillin. Methicillin is made by making some modifications to the chemical structure of penicillin.

The methicillin is classified as a beta-lactam class of antibiotics, and the beta-lactam antibiotic is formed after binding to penicillin-binding proteins (PBPs). By removing the drug is to exercise.

Antibiotic-resistant bacteria are bacteria that are resistant to certain antibiotics and do not work. For example, the penicillin-resistant Staphylococcus aureus which does not hear the above mentioned effects of the penicillin is equivalent to this. In addition, there is Methicillin-Resistant Staphylococcus aureus ( MRSA), which was first reported to academia in 1961 and has since become the world's leading hospital infection. The MRSA was found to have a unique gene that can be resistant to antibiotics of penicillin or methicillin. The MRSA is mainly not infected to healthy people, but mainly to patients with weak immunity or to patients who have undergone surgery, but is reported to cause sepsis or pneumonia when infected.

In recent years, healthy people have been increasingly infected with MRSA. The US medical community and the public health community are very concerned about the increase in infections caused by pathogens that do not work with many antibiotics, such as MRSA. For example, the spread of Staphylococcus aureus in indigenous people in Alaska in 1984 and the spread of skin infections in its population in August 2000. This confirms that MRSA can be infected not only from patients in hospitals but also from public life in the community, as is known in the art.

In Korea, 40 children in Gyeongnam Province have been reported to be infected with MRSA in the last two years. Children who have been treated with Staphylococcus aureus skin syndrome, which is a symptom of infection, were re-examined by Seoul National University's Department of Diagnostic Testing and Medicine. It has been shown that they have never been hospitalized or taken antibiotics, and are suspected to be infected with MRSA from the community's surroundings. In addition, according to the results announced by the Korea Food and Drug Administration and the Centers for Disease Control, antibiotic resistance monitoring at 10 university hospitals, including Seoul National University, was 10% higher than the previous year, and among the staphylococcus aureus detected in the blood. In addition, it is reported that the ratio of MRSA is 71.6%, which is 10% higher than 61.2% in 2004.

On the other hand, the recent increase in the amount of marine dumping of food waste, including food processing debris has become a problem of infection of diseases caused by Vibrio bacteria. Typically, pathogenic microorganisms such as bacteria and viruses contained in the sewage discharged from the home are diluted or killed while flowing into the sea, and then diluted again in the sea, which is not a big problem. In particular, in the case of sea water, not all pathogens disappear in a short time, but high salts are not suitable for survival or reproduction of pathogenic microorganisms that cause disease in humans.

However, the situation has changed with the recent increase in the amount of ocean dumping of food waste. The amount of marine dumping of domestic food waste increased from 552 tons in 1988 to 4,170 tons in 1995, and along with the introduction of large amounts of pathogenic microorganisms mixed with manure into the sea, fish cholera caused by pathogenic microorganisms , Typhoid, dysentery, hepatitis, botulism is reported to occur. In particular, pathogenic microorganisms in living sewage have been reported to survive for several days to several months in the gut of fish, the epidermis of fish or shellfish, gills, digestive tract of crustaceans, and sediments. In addition, the infection by the pathogenic microorganisms can be moved not only in the vicinity of the living sewage flow or where the ocean dumping, but also contaminants can be moved to a long distance by algae, pathogenic microorganisms caused by these pollutants are pollutants It is reported that it can survive during this migration and can occur in very large areas.

Pathogenic microorganisms introduced into marine ecosystems by marine dumping of food wastes cause diseases in marine ecosystems such as fish, which create conditions for pathogenic microorganisms to grow on the surface or body of animals and cause disease or By producing toxic substances that can grow and cause disease, or by changing their habitat, animals can no longer survive in a healthy state. Such conditions often occur when an imbalance between groups in a microbial community is formed. As a result of environmental pollution, a change in microbial community is caused, and pathogenic microorganisms multiply in large quantities.

Due to the increase in the number of marine organisms, such as fish, shellfish and the like caused by the pathogenic microorganisms, in particular fish or shellfish that are the target of aquaculture, a countermeasure is urgently required.

On the other hand, seaweeds, which have not received much attention because they contain a large amount of non-digestible polysaccharides, have been found to contain a large amount of vitamins and minerals, especially magnesium, calcium, iodine and iron, compared to land plants. Interest in this is increasing.

In addition, recently, natural seafood, in particular, seaweed has been reported to have a natural life, and interest in seaweed, which is known as a physiologically active substance due to its unique structural properties, has also increased, and various drugs or antibiotics using seaweed are used. Attempts to develop are actively underway.

The present invention has an antimicrobial activity against Gram-positive bacteria and Gram-negative bacteria, and in particular, an object of the present invention is to provide an antimicrobial composition comprising a blueberry extract having an effective antimicrobial activity against MRSA (Methicillin-Resistant Staphylococcus aureus ).

In order to achieve the above object, the present invention provides an antimicrobial composition comprising the green rind extract as an active ingredient.

In addition, in order to achieve the above object, the present invention provides a composition for the prevention or treatment of diseases caused by methicillin resistant Staphylococcus aureus infection, including an antimicrobial composition comprising a blueberry extract as an active ingredient.

In addition, in order to achieve the above object, the present invention provides a composition for animal feed comprising an antimicrobial composition comprising a green lard extract as an active ingredient.

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

Brown seaweed ( Ulva lactuca ) is an algae belonging to the green algae Ullotrix algae family, usually 15 to 30 cm long, and 5 to 10 cm long, and is attached to rocks at the bottom of the intertidal zone. They grow mostly in winter and summer. It is a species distributed all over the world, inhabits rocks and piles in intertidal zones affected by waves, and grows in estuaries with lots of organic matter and dirt. It has been used as a feed for livestock rather than for food because it has no taste, but it has recently been spotlighted as a raw material for cosmetics because it contains a large amount of vitamins.

The extraction solvent of the present invention may be water or an organic solvent such as a polar solvent such as alcohol having 1 to 4 carbon atoms such as methanol or ethanol, ethyl acetate, and a nonpolar solvent of hexane or dichloromethane, or a mixed solvent thereof. 50 to 99% of methanol, ethanol and the like, alcohol dilution of 1 to 4 carbon atoms, more preferably 50 to 99% ethanol dilution water.

Brown seaweed extract of the present invention may be prepared according to a conventional method for preparing seaweed extract, specifically, may be cold extraction, hot extraction or heat extraction.

In addition, the extract extracted with the solvent may be further fractionated with a solvent selected from the group consisting of hexane, methylene chloride, acetone, ethyl acetate, ethyl ether, chloroform, water and mixtures thereof. Preferably the fractional solvent may be ethyl ether. One or more solvents may be used in the fractionation. Extract preparation temperature may be 4 to 120 ℃, but is not limited thereto.

The extraction time is not particularly limited, but may be 10 minutes to 300 minutes, and a conventional extraction device, an ultrasonic grinding extractor, or a fractionator may be used. The prepared extract can then be filtered or concentrated or dried to remove the solvent, it can be carried out both filtration, concentration and drying. Specifically, the filtration may be performed using a filter paper or a reduced pressure filter, the concentration may be concentrated under reduced pressure using a reduced pressure concentrator, for example, a rotary evaporator, and the drying may be performed by, for example, a lyophilization method.

As an example of the present invention, the brown leek extract is dried after removing the salt and impurities of the brown lees, to prepare a dry sample, to obtain a crude extract by adding ethanol dilution to the dried sample, the crude extract is concentrated under reduced pressure extract Can be prepared. At this time, the removal of the salt and impurities of the brown sea may be carried out by washing with running water, the dried sample may be prepared by grinding after drying the brown sea from which the salt and impurities are removed. The drying may preferably be carried out by lyophilization.

The obtained blueberry extract may be stored in a deep freezer until use.

In addition, the brown leek extract may be one obtained by completely removing the moisture through the concentration and freeze-drying the obtained extract, the brown leek extract completely removed the water is used in the form of powder or dissolved in distilled water or a common solvent Can be.

The present invention provides an antimicrobial composition comprising a green onion extract.

In the present invention, the antimicrobial composition may have the same meaning as an antibiotic which is a generic term for an antimicrobial agent, and may have the same meaning as an antimicrobial agent, a fungicide, a preservative, a preservative or an antimicrobial agent, and preferably the development of Gram-positive bacteria and Gram-negative bacteria. Means a substance that can inhibit or inhibit life and life functions.

The antimicrobial composition of the present invention may include 0.001 to 99.99% by weight, preferably 0.1 to 99% by weight, based on the total weight of the composition, depending on the method of use and purpose of use of the antimicrobial composition. The content can be adjusted appropriately.

The antimicrobial composition may be an antimicrobial composition having antimicrobial activity against pathogenic microorganisms, specifically Gram-positive bacteria or Gram-negative bacteria, and more specifically, methicillin-resistant Staphylococcus aureus (MRSA), Edwards Paper. Ella bacteria (Edwardsiella. sp), Vibrio bacteria (Vibrio. sp), Bacillus bacterium (Bacillus. sp), micro larger carcass bacteria (Micrococcus. sp), stearyl Philo Cocos bacteria (Staphylococcus. sp), Enterobacter bacteria (Enterobacter. sp), Klebsiella bacteria (Klebsiella. sp), Pseudomonas bacterium (Pseudomonas. sp), an antimicrobial composition having antibacterial activity against Salmonella (Salmonella. sp) or Escherichia coli (E.coli), preferably a methicillin ampicillin-resistant Staphylococcus aureus, Edward tar is not Ella (Edwardsiella tarda, E. tarda), Vibrio parahaemolyticus (Vibrio parahemolyticus, V. parahemolyticus), Bacillus sub-blocks bus (Bacillus subtilis, B. subtilis), Mai Locker Curse Lou Proteus (Micrococcus leuteus, M. luteus), Stephen Philo Cocos Auray Earth (Staphylococcus aureus, S. aureus), Ness's children as Enterobacter (Enterobacter aerogenes, E. aerogenes), Klebsiella pneumoniae (Klebsiella. Pnermoniae, K . pnermoniae), Pseudomonas aeruginosa (Pseudomonas. aerogigosa, P. aerogigosa ), Salmonella tie blood force and Titanium (Salmonella. typhimurium, the antimicrobial composition having an antimicrobial activity against S. typhimurium) or Escherichia coli (E.coli), more preferably It is an antimicrobial composition having an antimicrobial activity against methicillin resistant Staphylococcus aureus, Edward Giella tarda or enteritis vibrio bacteria, and most preferably an antimicrobial composition having an antimicrobial activity against methicillin resistant Staphylococcus aureus.

The methicillin-resistant Staphylococcus aureus is a pathogenic bacterium belonging to Gram-positive cocci, and refers to a bacterium that exhibits resistance to penicillin antibiotics, such as methicillin, oxacillin, or nafcillin. . The methicillin resistant Staphylococcus aureus is resistant to most beta-lactam class of antibiotics, including cephalosporins and imipenem, and aminoglycosides and quinolone antibiotics. It is also common to be resistant to most antibiotics.

The methicillin-resistant Staphylococcus aureus has been reported as a causative agent of not only skin infections such as abscesses and wound infections, but also serious infections such as pneumonia and sepsis, and vancomycin or teicoplanin ( It is reported that extremely limited antibiotics such as teicoplanin can be used.

Infection with methicillin resistant Staphylococcus aureus has been reported to cause pneumonia, pneumonia, respiratory infections such as empyema, sepsis, bacteremia, infective endocarditis, intestinal infection, intractable bedsores or wound infections.

Since the methicillin-resistant Staphylococcus aureus can settle in any part of the human body and cause infection, not only infection by contact transmission, but also infection by airborne transmission, in particular, the skin's protective device It has been reported that it can settle well in injured patients or burn patients with fatal infections and cause fatal infections. In the case of medical workers who have been working for a long time in hospitals, it is reported that infection by air propagation is possible because of high intranasal fixation rate.

The enteritis Vibrio bacillus is a bacteriostatic bacillus that grows well in the presence of salts, including salt, and the like. Vibrio parahaemoliticus causing enteritis and sepsis vibrio (Vibrio vulnificus) causing sepsis are typical.

The enteritis vibrio bacteria are the main causative agents of food poisoning and are mainly found in seawater, sedimentary soil, and invertebrate organisms in coastal and salt estuaries, from June to October. Infect your back. Enteritis vibrio bacteria attached to the fish and shellfish may contaminate other foods through cooking utensils such as refrigerators, cutting boards, hangers, knives and cookers, and food poisoning may be caused by humans eating these contaminated foods. .

Edward G. gill is a Gram-negative bacterium that is a bacterium of the intestinal bacteria and is a causative agent of disorders of the digestive tract in fish. In particular, Edward G. tar is a causative agent of Edward disease. The Edward disease occurs in large fish over two years at high temperature, and the external symptoms include ulceration of the head, bloating or redness of the abdomen, ocular turbidity or protrusion, etc. Redness and hardening of the ovary are characterized, and the cumulative mortality from the disease reaches 20 to 30%.

The antimicrobial composition of the present invention may be directly applied to animals including humans, and may exhibit antimicrobial activity, and may be applied to marine organisms such as fish or shellfish to lower the mortality rate of fish or shellfish and increase productivity, as well as to marine organisms such as humans. Can prevent secondary infection of other animals that consume

The animal is a biological group corresponding to plants, mainly ingesting organic matter as nutrients, and means that digestion, embryogenicity, and respiratory organs are differentiated, specifically, echinoderm, crustaceans, molluscs, fish, amphibians, reptiles, birds, It may be a mammal, preferably mollusks such as shellfish such as sea urchins or sea cucumbers, crustaceans such as crabs, shrimps, and lobsters, mollusks such as cephalopods, gastropoda or bivalve shells, red snapper, sea bream, cod, flounder, It may be a bird including a fish such as a flounder, a poultry such as a pheasant or a chicken, or a mammal such as a human, a pig or a cow goat.

The antimicrobial composition of the present invention may include sugars, proteins, lipids, vitamins, and minerals in addition to the green onion extract.

The sugar may be appropriately selected according to the purpose and use thereof, and may be, for example, honey, dextrin, sucrose, palatinose, glucose, fructose, syrup, sugar alcohol, sorbitol, xylitol, maltitol, It is not particularly limited thereto. The protein may be appropriately selected depending on the purpose and use thereof. For example, animal-derived enzymes such as milk derived proteins such as casein and whey protein, soy protein, trypsin of these proteins, and pepsin. And hydrolysates by neutrases and alkalases, but are not particularly limited thereto. The lipid may be appropriately selected according to the purpose and use thereof. For example, the monohydric saturated fatty acid, sunflower oil containing polyunsaturated fatty acid, rapeseed oil, olive oil, safflower oil, corn oil Oil, soybean oil, palm oil (palm oil), palm oil and other plant-derived fats, such as middle-chain fatty acids, EPA, DHA, soybean-derived phospholipids, milk-derived phospholipids, but is not particularly limited thereto. The vitamins are not particularly limited, and the minerals may be appropriately selected depending on the purpose and use thereof. For example, potassium phosphate, potassium carbonate, potassium chloride, sodium chloride, calcium lactate, calcium gluconate, calcium pantothenate, and casein calcium , Magnesium chloride, ferrous sulfate, sodium hydrogen carbonate, but is not particularly limited thereto.

For any of the above saccharides, proteins, lipids, vitamins and minerals, as long as the antimicrobial properties of the composition are maintained, not limited to the above embodiments and may include other ingredients, each content is not limited as long as the antimicrobial properties are maintained Preferably, it may be 0.1 to 15% by weight, preferably 0.5 to 10% by weight relative to the total composition.

The antimicrobial composition of the present invention can be used for various purposes and uses requiring antimicrobial activity, specifically, the composition for the prevention or treatment of diseases caused by pathogenic microbial infection, the composition for animal feed, additives for animal feed, food, food additives, It can be used for the use of beverages, cosmetic compositions, cosmetic additives or detergents.

The present invention also provides a composition for the prevention or treatment of diseases caused by infection of methicillin resistant Staphylococcus aureus comprising the antimicrobial composition.

The disease caused by the methicillin-resistant Staphylococcus aureus infection means various infectious diseases that can be caused by infection of Staphylococcus aureus, and preferably, respiratory infections such as pneumonia, pneumonia, empyema, sepsis, bacteremia, and infectious disease. Endocarditis, enteritis, intestinal infections, intractable bedsores or wound infections, and the like, more preferably, any one selected from the group consisting of pneumonia, sepsis, bacteremia, infective endocarditis, and enteritis.

The composition for the prevention or treatment of diseases caused by methicillin resistant Staphylococcus aureus infection of the present invention may further comprise a carrier or excipient that can be used pharmacologically according to the formulation and method of use, in addition to the extract of brown seaweed, the composition of the present invention alone Or in combination with other pharmaceutically active ingredients for enhanced efficacy as a therapeutic agent for infection by methicillin resistant Staphylococcus aureus. In this case, the content of brown leek extract in the composition for the prevention or treatment of diseases caused by methicillin resistant Staphylococcus aureus infection may be 0.001 to 99% by weight. If the content of the active ingredient in the composition is less than 0.001% by weight may require a large amount of administration for effective efficacy, if the amount exceeds 99% by weight may be constant compared to the amount may be uneconomical. In addition, the composition for the prevention or treatment of diseases caused by methicillin-resistant Staphylococcus aureus infection according to the method of use and purpose of use can be appropriately adjusted the content of the brown leek extract.

Composition for the prevention or treatment of diseases caused by methicillin resistant Staphylococcus aureus infection of the present invention may be administered orally or parenterally, preferably orally, but the formulation thereof may vary depending on the method of use It is not limited to what was described. Examples of formulations include PLASTERS, GRANULES, LOTIONS, POWDERS, SYRUPS, LIQUIDS AND SOLUTIONS, AEROSOLS, OINTMENTS, FLOWERS FLUIDEXTRACTS, EMULSIONS, SUSPENSIONS, INFUSIONS, TABLETS, INJECTIONS, CAPSULES and PILLS.

Further compositions for the prevention or treatment of diseases caused by methicillin resistant Staphylococcus aureus infections can be prepared using appropriate methods known in the art or by Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Formulated preferably using the method disclosed in Easton PA).

Excipients such as pharmacologically acceptable carriers or diluents, preservatives, stabilizers, wetting agents, emulsifiers, solubilizers, sweeteners, colorants, osmotic pressure regulators, antioxidants, etc. may use conventional materials according to the formulation, and when formulated, fillers , Extenders, wetting agents, disintegrating agents or surfactants may be used. Representative diluents or excipients include water, dextrin, calcium carbonate, lactose, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyloleate, liquid paratin and saline.

Specifically, in the case of oral administration depending on the formulation, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used can be used, and in the case of parenteral administration, sterilized aqueous solution, non-aqueous solvent Suspensions, emulsions, lyophilized preparations, suppositories, and the like can be used. The non-aqueous solvent or suspending agent may be, for example, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like, and suppositories may include, for example, utopsol, macrogol, and tween. 61, cacao butter, laurin butter or glycerogelatin and the like.

In addition, the composition for the prevention or treatment of diseases caused by methicillin resistant Staphylococcus aureus infection of the present invention can be used in combination with various carriers accepted as a medicament, such as physiological saline or organic solvent, to increase the stability or absorption For use with carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, small molecule proteins or other stabilizers Can be.

Subjects of the composition for the prevention or treatment of diseases caused by methicillin resistant Staphylococcus aureus infection of the present invention may be echinoderm, crustacean, molluscs, fish, amphibians, reptiles, birds, mammals, preferably echinoderm, Marine life including arthropods, mollusks and fish and mammals that can ingest the marine life, more preferably the marine life belongs to echinoderm, arthropods such as sea urchins or sea cucumbers, Can be aquaculture such as shellfish such as shrimp, crayfish, cephalopods, gastropods or bivalve, fish such as sea bream, sea bream, cod, flounder, flounder, etc. It may be a livestock such as pigs, cattle, goats, etc. can be ingested and poultry such as pheasants, chickens.

Dose or amount of the composition for the prevention or treatment of diseases caused by methicillin resistant Staphylococcus aureus infection of the present invention is the weight, age, sex, health status, diet, time of administration, method of administration, excretion rate and severity of the disease, etc. The effective daily dosage is typically from 10 to 5000 mg / kg and preferably from 100 to 3000 mg / kg for adults (70 kg), to be administered once or several times a day. It may be administered, preferably 1 to 3 times a day.

Since the dosage may vary depending on various conditions, it will be apparent to those skilled in the art that the dosage may be added or subtracted, and thus the dosage does not limit the scope of the present invention in any aspect. The frequency of administration can be administered once a day or divided into several times within the desired range, the administration period is not particularly limited. In addition, the composition for preventing or treating a disease caused by methicillin-resistant Staphylococcus aureus infection of the present invention may be added to any food and consumed in addition to daily administration orally.

The composition for the prevention or treatment of diseases caused by methicillin-resistant Staphylococcus aureus infection of the present invention not only provides a prophylactic or therapeutic effect of the infection by the antimicrobial effect against methicillin-resistant Staphylococcus aureus, but also the green seaweed extract which is a natural seaweed Because it contains as an active ingredient can be taken for a long time.

The present invention also provides a composition for animal feed comprising the antimicrobial composition.

The animal is a biological group corresponding to plants, mainly ingesting organic matter as nutrients, and means that digestion, excretion and respiratory organs are differentiated, specifically, echinoderm, crustaceans, molluscs, fish, amphibians, reptiles, birds, It may be a mammal, preferably mollusks such as arthropods, cephalopods, gastropods or bivalve shells, including shellfish such as sea urchins or sea cucumbers, crustaceans such as crabs, shrimps, and lobsters, red snapper, sea bream, cod, and flounder , Birds such as flounder, birds including poultry such as pheasants or chickens, or mammals such as pigs, cattle, goats.

The animal feed composition may further comprise grains, vegetable protein feed, animal protein feed, sugar or dairy products in the antimicrobial composition comprising the brown leek extract as an active ingredient. The grains may be specifically milled or crushed wheat, oats, barley, corn and rice, and the vegetable protein feed may be specifically based on rapeseed, soybean and sunflower, the animal protein feed specifically May be blood meal, meat meal, bone meal and fish meal, and the sugar or dairy product may specifically be a dry ingredient consisting of various powdered milk and whey powder.

The animal feed composition may further be used with ingredients such as nutritional supplements, digestion and absorption enhancers, sex stimulants or disease prevention agents.

The antimicrobial composition included in the composition for animal feed of the present invention may vary depending on the purpose and conditions of use of the feed. For example, the antimicrobial composition may be included in an amount of 0.1g to 100g based on 1kg of the final produced feed.

In addition, the feed composition may be made of a viscous granulated or granular material depending on the degree of grinding of the components, the composition may be supplied in a mesh or formed into a desired separated shape for further processing and packaging. It may be subjected to a pelletizing, expanding or extruding process for storage, and preferably excess water may be dried off for ease of storage.

The present invention also provides a cleaning agent comprising the antimicrobial composition. The detergent may be preferably a detergent for clothes, a dish cleaner, a food cleaner, a household appliance cleaner. Since the antimicrobial composition is not harmful to the human body as a natural ingredient, more preferably, it may be used for the purpose of washing or providing antimicrobial properties of various household goods including kitchenware or food washing.

The cleaning agent may further include an appropriately selected additive according to the purpose of use and use thereof, and may be used in admixture with other active ingredients such as commonly used cleaning agents or additives such as colorants, surfactants, and preservatives. The detergent may be prepared by powdering, granulating, tableting or liquefying according to the purpose and use thereof, and a conventional method may be used for packaging for commercialization.

In addition, the amount of the antimicrobial composition used in the cleaning agent may be suitably used according to the purpose of use, application form, application site, object to be applied, and the like, and may include, for example, 0.01 to 50 parts by weight based on the total composition. The content is not limited thereto. Since the antimicrobial composition of the present invention is a natural ingredient, which is not harmful to the human body, it can be used in various amounts as long as it can achieve an application purpose due to the characteristics of the product.

Hereinafter, preferred embodiments of the present invention are described. The following examples are only for illustrating the present invention and the present invention is not limited by the following examples.

EXAMPLE

Example 1 Preparation of Brown Sea Extract

Brown seagrass collected from the waters of Busan, Korea was washed 7 times in running water to remove impurities and salts on the surface, and then lyophilized and ground to prepare a dry sample. To 100 g of the dry sample, 2L of distilled water of ethanol mixed with purified water and ethanol 1: 5 (purified water: ethanol, w / w) was added and extracted at 20 ° C. for 20 hours, followed by filtration using a filter paper and a rotary evaporator (rotary). A crude ethanol extract was prepared by the method of vacuum concentration using an evaporator. The crude extract was partitioned sequentially using ethyl ether, ethyl acetate, hexane and water as solvents, depending on the polarity. Each fraction was concentrated under reduced pressure using a rotary evaporator and then lyophilized to prepare a powdery sample. The extract was stored at −70 ° C. until use.

Example 2: Determination of Antimicrobial Activity

2-1. Target strain, culture medium and sample

Strains used for the determination of antimicrobial activity were each published strains distributed from the corresponding deposit institution. Specifically, the deposit number of the strain measuring the antimicrobial activity is as follows.

As Gram-positive bacteria, the accession number of Bacillus subtilis is PM 125, the accession number of MicroCaucus Luteus is KCTC 1056, the accession number of Staphylococcus orreas is KCTC 1916, and the deposition of methicillin resistant Staphylococcus aureus. The numbers are CCARM 3089, CCARM 3115 and CCARM 3561. For Gram-negative bacteria, E. coli accession number is D 31, Enterobacter airogenes accession number is KCTC 2190, Pneumococcal bacterium accession number is KCTC 2208, Pseudomonas aeruginosa accession number is KCTC 2004, Salmonella typhimurium Um's accession number is KCTC 1925, enteritis vibrio accession number is KCTC 2471, Edward Giella tarda accession number is NUF 251, and the fungal bacteria Candida. Albicance accession number is KCTC 7965.

Strains beginning with accession number KCTC were purchased from Korea Gene Bank (Daejeon, Korea), and methicillin-resistant Staphylococcus aureus was purchased from Korea Antibiotic Resistant Bacteria (Seoul, Korea).

Tryptic soy broth (TSB) medium, Muller-Hinton broth (MHB) medium, and Luria-Bertani broth (LB) medium used as the medium used in the initial culture of the strain, the medium used for the radial diffusion assay method. All were purchased from Merk (Germany), and Melittin, EEO agar and streptomycin sulfate, which were used as controls when measuring minimum inhibitory concentration (MIC), were purchased from Sigma (St. Louis, USA). .

During the culturing in the medium, basophilic bacteria Bacterial Vibrio (KCTC 2471) and Edward Giella tarda (NUF 251) were cultured in the medium containing 1% NaCl.

2-2. Antibacterial activity measurement by radioactivity method

The antimicrobial activity was measured using a radiation diffusion assay (Edward, T et. al ., N-thiolated β-lactams; novel antibacterial agents for methicillin-resistant staphylococcus aureus. Bioorg. Med. Chem. Lett. 12: 2229-22 (2002). Measurement of the antimicrobial activity using the radioactivity method was carried out specifically as follows.

Each of the subject strains was incubated in a mid-logarithmic phase at 37 ℃ using TSB medium. After the incubation, washed three times with 1.0 M phosphate buffer solution (1.0M phosphate buffere solution, pH 6.7) and diluted. The bacterial dilution was measured by absorbance at ₅₇₀ nm (OD ₅₇₀ ) and the concentration was adjusted so that the bacterial count was 0.1, that is, the cell count was 5 × 10 ⁷ CFU / ml.

1 ml of the diluted bacterial dilution was mixed with 100 ml of LB medium and poured onto the plate to harden. The LB medium was mixed with 20.7 ml of 5 x LB broth, 20.7 ml of 1.0 M PBS (pH 6.7), 58.6 ml of distilled water, 0.5 g of NaCl, and 1.57 g of low EEO agar based on 100 ml of the total liquid medium. Aseptic conditions were prepared using autoclave, and the mixing with the bacterial dilution was performed by cooling the LB medium to 50 ° C. and then mixing with the bacterial dilution, followed by a sterile petri dish. The mixture is poured into the dishe and hardened.

The mixed solution poured into the sterile culture dish was incubated at 18 ° C. for overnight to confirm whether the strain was cultured in the medium.

Wells of about 2 to 3 mm were made in the medium where it was confirmed that each strain was cultured. After injecting the sample obtained by dissolving the crude extract prepared in Example 1 in 0.1% acetic acid solution, incubated for 18 hours at 37 ℃, the activity was measured by the size of the inhibitory (clear zone or inhibition zone). . The samples were prepared at different concentrations, namely 5 μl (0.2 mg dry weigh / ml), 10 μl (0.3 mg dry weigh / ml) and 15 μl (0.4 mg dry weigh / ml). As a result of experiments with E. coli D31, it was confirmed that the ethyl ether fraction had the best antimicrobial activity in the extract, and the antimicrobial activity was measured using ethyl ethyr fraction for each strain. As shown in FIG. 3, the experiment was performed twice, and the average value of the results is shown in Table 1.

TABLE 1

Microorganisms Inhibitory ring (mm) 5 μl 10 μl 15 μl Gram-positive bacteria B. subtilis PM 125 5 10 15 M. luteus KCTC 1056 8 10 12 S. aureus KCTC 1916 11 15 18 MRSA CCARM 3561 24 25 27 MRSA CCARM 3115 18 24 26 MRSA CCARM 3089 18 20 24 Gram-negative bacteria E.coli D 31 11 14 17 E. aerogenes KCTC 2190 8 15 20 K. pnermoniae KCTC 2208 NA 6 8 P. aerogigosa KCTC 2004 5 6 8 S. typhimurium KCTC 1925 10 15 18 V. parahemolyticus KCTC 2471 10 15 18 E. tarda NUF 251 10 15 18 Fungus C. albicance KCTC 7965 NA NA NA

In Table 1, NA means no antimicrobial activity at all, the size of the inhibitory ring is 1 to 8 mm antimicrobial activity is weak (week activities), 8 to 15 mm moderate antibacterial activity (moderate) activity, and the antimicrobial activity was evaluated to be greater than 15 mm in size.

As shown in FIG. 2 and Table 1, the brown green extract of the present invention showed a strong antimicrobial activity against MRSA bacteria. As shown in A and Table 1 of FIG. 2 showing antimicrobial activity against MRSA CCARM 3561, when the brown extract was used at a concentration of 15 μl, the inhibitory ring size was 27 mm, indicating the strongest antimicrobial activity among the target strains. As shown in B and C of FIG. 2 and Table 1 showing antimicrobial activity against CCARM 3115 and MRSA CCARM 3089, the size of the low ringlets was 26 mm and 24 mm, respectively. It showed strong antimicrobial activity. In particular, even when using the green rind extract at the lowest concentration of 5 μl compared to other strains showed a strong antimicrobial activity against all three kinds of MRSA bacteria.

As shown in FIG. 1 and Table 1, the brown green extract of the present invention showed a strong antimicrobial activity against most other Gram-positive bacteria other than MRSA. In the case of using the green rind extract at the concentration of 15 μl, the inhibitory ring size was 15 mm in the case of Bacillus subtilis PM 125, and the inhibitory ring size was 12 mm in the case of MicroCcus Luteus KCTC 1056. In the case of Staphylococcus Oreas KCTC 1916, the inhibitory ring had a strong antimicrobial activity of 18 mm in size.

As shown in FIG. 3 and Table 1, the brown leek extract of the present invention showed a strong antimicrobial activity mostly against Gram-negative bacteria. In the case of using the green lard extract at the concentration of 15 μl, the inhibitory ring size of the Enterobacter airogenes KCTC 2190 showed the strongest activity of 20 mm, and showed the activity of Edward Geella tarda NUF 251 and Escherichia coli D 31. Inhibitory ring size was 18 mm and 17 mm, respectively.

In addition, as shown in Table 1, the brown green extract of the present invention showed excellent antimicrobial activity against other Gram-negative bacteria. In the case of Salmonella typhimurium KCTC 1925, the inhibitory ring size was 18 mm, and in the case of enteritis vibriovirus KCTC 2471, the inhibitory ring size was 18 mm. However, in the case of pneumococcal KCTC 2208 and Pseudomonas aeruginosa KCTC 2004, the size of the inhibitory ring was 8 mm, and the extract of Brown Algae of the present invention showed lower antimicrobial activity than the above bacteria.

On the other hand, antibacterial activity was not recognized at all against the fungal Candida albican KCTC 7965.

2-3. Measurement of Minimal Inhibitor Concentration (MIC)

Minimal inhibitory concentration (MIC) was measured using liquid growth inhibitors for Gram-positive and Gram-negative bacteria (Lehre, RI, et al, Ultrasensitive assays for endogenous antimicrobial polypeptides. J. Immunol. Methods. 137: 167-173 (1991).), Fungus, ie C. ablicance , was measured for C. ablicance using the standard serial broth micro-dilution assay (Zasloff, M Magainins a class of antimicrobial peptides from Xenopus skin: Isolation characterization of two active forms, and partial cDNA sequence of a precursor.Proc. Natl. Acad. Sci. USA 84: 5449-5453 (1987). and Zasloff, M., et al Antimicrobial activity of synthetic magainin peptides and several analogues. Proc. Natl. Acad. Sci. USA 85: 910-913 (1988).

Specifically, the measurement of the minimum inhibitory concentration was carried out on a bacterial dilution of which the concentration was adjusted so that the cell number was 5 x 10 ⁷ CFU / ml in the same manner as in Example 2-2 for each of the target strains. However, unlike Example 2-2, the strain for measuring the minimum inhibitory concentration was used Muller-Hinton broth (Muller-Hinton broth, MHB). In order to measure the minimum inhibitory concentration, 100 μl of the bacterial dilution was dispensed into a 96 well plate, and the browned ethyl ether fraction of the present invention prepared in Example 1 was diluted twice and adjusted to various concentrations. Then 11 μl each was added. After adding the fraction of the present invention to the bacterial diluent, and incubated for 20 hours at 37 ℃, by measuring the absorbance at 570nm to measure the minimum inhibitory concentration to the sample concentration without turbidity is shown in Table 2 below. Among the target strains, Enteritis Vibrio KCTC 2471, Edward Giella Tarda NUF 251, and Kanderda Albican KCTC 7965 were carried out in the same manner as other strains except incubation at 30 ℃.

TABLE 2

Microorganisms MIC (μg / ml) Ethyl ether extract melittin Gram-positive bacteria B. subtilis PM 125 12.5 0.75 M. luteus KCTC 1056 > 200 1.56 S. aureus KCTC 1916 100 3.13 MRSA CCARM 3561 12.5 > 200 MRSA CCARM 3115 12.5 > 200 MRSA CCARM 3089 50 > 200 Gram-negative bacteria E.coli D 31 12.5 0.78 E. aerogenes KCTC 2190 > 200 1.56 K. pnermoniae KCTC 2208 200 0.78 P. aerogigosa KCTC 2004 > 200 25 S. typhimurium KCTC 1925 200 25 V. parahemolyticus KCTC 2471 25 1.56 E. tarda NUF 251 50 0.78 Fungus C. albicance KCTC 7965 NA > 200

Melittin of Table 2 has been reported to cause strong anti-inflammatory action as the most representative component of bee venom, was used as a control in the experiment to measure the minimum inhibitory concentration.

As shown in Table 2, the brown ether ethyl ether fraction of the present invention showed a strong antimicrobial activity against MRSA bacteria. The minimum inhibitory concentrations of MRSA CCARM 3561 and MRSA CCARM 3115 were 12.5 μg / ml as shown in Table 2, and the minimum inhibitory concentration of MRSA CCARM 3089 was 50 μg / ml, which is much lower than that of melittin. As shown in Table 1 and Figure 2, the brown green extract of the present invention was confirmed to exhibit a very high antimicrobial activity against methicillin resistant staphylococcus.

On the other hand, for other Gram-positive bacteria and Gram-negative bacteria, the minimum inhibitory concentration was found to be higher than that of melittin. As shown in Table 2, the minimum inhibitory concentrations were 12.5 μg / ml for Bacillus subtilis PM 125 and 100 μg / ml for Staphylococcus Oreas KCTC 1916, respectively. For Gram-negative bacteria, the minimum inhibitory concentration was 200 μg / ml for Salmonella typhimurium KCTC 1925 and pneumococcal KCTC 2208.

On the other hand, among Gram-negative bacteria, the minimum inhibitory concentration was 25 μg / ml for enteritis vibriovirus KCTC 2471, and the minimum inhibitory concentration was 50 μg / ml for Edward Geella tarda NUF 251.

In addition, for E. coli D 31, the minimum inhibitory concentration was found to be 12.5 μg / ml. The E. coli D 31 is a mutant that is defective in lipopolysaccharide, and the strain is reported to be more sensitive to membrane attack antibiotics than wild-type E. coli . The results suggest that the extracts of brown lees of the present invention may be effective against bacteria susceptible to membrane attack antibiotics.

Based on the results of the experiment, the brown leek extract of the present invention indicates that the bacterium, particularly antibiotic resistant bacteria, may act as an alternative antibiotic against methicillin resistant Staphylococcus aureus.

The antimicrobial composition comprising the extract of brown lees of the present invention is against methicillin-resistant Staphylococcus aureus, Edward G.ella, enteritis Vibrio, Bacillus, Microcuscus, Staphylococcus, Enterobacter, Salmonella or Escherichia coli. Antimicrobial Activity Not only does it have antimicrobial activity against methicillin-resistant Staphylococcus aureus, but it is extracted from natural algae, so there is no concern about side effects, so it is very valuable as a therapeutic agent for diseases caused by natural antibacterial and methicillin-resistant Staphylococcus aureus infections. In addition, the antimicrobial activity against enteritis vibrio and Edward G. tarda is considered to be very useful as a feed composition that can effectively prevent diseases caused by bacteria that threaten farmed fish.

Claims (7)

To the crude extract prepared by adding alcohol having 1 to 4 carbon atoms or diluting water thereof to the extract of Ulva lactuca , ethyl acetate or ethyl ether was added to the crude extract, and the extract was prepared by fractionation. Antimicrobial composition having antimicrobial activity against at least one selected from the group consisting of E. coli, Edwardsiella tarda, E. tarda, and Vibrio parahemolyticus, V. parahemolyticus. . delete delete delete delete delete An animal feed composition comprising the composition according to claim 1 and used in at least one animal selected from the group consisting of echinoderm, crustaceans, molluscs and fishes if it has a prophylactic or ameliorating effect on enteritis or ward disease.

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