KR101006556B1 - Anti-bacterial compositions against Streptococcus - Google Patents

Abstract

The present invention relates to an antimicrobial composition comprising at least one plant extract selected from the group consisting of red ginseng extract, spectral extract and thyme extract.

According to the present invention, by using the antimicrobial composition, it is possible to prevent and treat infections of fish of Streptococcus, which is one of the causes of bacterial diseases causing enormous economic losses to freshwater and seawater cultures in Korea. In addition, by using ginseng, spectral extract and herb thyme extract, which are widely used as herbal medicines, it is possible to replace antibiotics that are concerned about the emergence of resistant bacteria through long-term use. It is possible to provide natural antibacterial agents which are environmentally friendly and have high added value.

Antibiotics, Antibacterials, Streptococcus, Sophora flavescens, Crowd (Dryopteris crassirhizoma), Thymus vulgaris

Description

Anti-bacterial compositions against Streptococcus

The present invention relates to a natural antimicrobial composition having an antimicrobial activity against fish bacterial disease-induced streptococci, and more particularly to an antimicrobial composition comprising at least one plant extract selected from the group consisting of Gosam extract, spectral extract and thyme extract. It is about.

Streptococcus sp. , The causative agent of streptococcosis , causes huge economic losses in freshwater and seawater farming in the United States, Israel, Australia and Japan, including Korea. Streptococcus is a genus of bacteria and many species in this genus cause disease in many animals, including humans. This species is Gram-positive, ie purple when Gram-dyed. Most of the bacteria that cause disease in fish are Gram-negative bacteria, which are pink when Gram-dyed. Fish disease caused by Streptococcus is not very common, but it does involve a lot of losses once developed. Aquatic Streptococcus bacteria are rare, but they can also cause disease in fragile people. In addition to Streptococcus , species such as Lactococcus , Enterococcus and Vagococcus also cause Streptococcus-like diseases. Staphylococcal disease in fish was first identified in 1957 in farmed trout in Japan. Salmon, mullet, golden shiner, pinfish, eel, marine trout, tilapia, sturgeon and sea bass have been found to be well-infected with the disease. Many species of ornamental fish are also known to be infected. Examples of species currently causing disease in fish include Streptococcus , which has been identified in Israel. difficilis , S. milleri found in the kidneys of Koi, S. parauberis found in the turbot of northern Spain, and S. iniae found in both fish and mammals. It is also a Gram-positive bacterium, very similar to Staphylococcus aureus, and Lactococcus found in Japanese eel and defenses as a species that causes disease in fish. found in garvieae and ignorant utterance L. piscium and Vagococcus salmoninarum and the like.

In general, fish infected with streptococcal show blackening, symptoms of ocular turbidity and protrusion, and redness of the edges and fins of the eyes. Dissecting infected fish results in hyperemia, hypertrophy and shrinkage of the liver, inflammation and bleeding in the intestine, cerebral hemorrhage, and thickening inside the gill cap. In addition, infected fish rapidly reduce feed intake due to loss of appetite in the early stages of infection, so many drugs appear to be effective in drug susceptibility testing, but they are often ineffective when used in aquaculture farms. Therefore, in order to prevent damage caused by Streptococcus of cultured fish in the treatment-oriented prevention measures by existing drugs, the introduction of a fish disease prevention agent is strongly required.

In Korea, since the 1980s, fish farming has been active, various antibiotics have been used for disease prevention and treatment of farmed fish, and antibiotic-resistant bacteria in marine bacteria are feared due to the long-term use of antibiotics. Factors that determine antibiotic resistance in bacteria are easily transferred to other cells by plasmid transfer, which can be transferred to bacteria that cause human disease, and it has been reported that aquaculture fish can also act as such mediators. have.

Particularly effective drugs for streptococcosis include florfenicol, ampicillin, doxycyclin, spiramycin and the like. All of them can be cured if they are administered depending on the symptoms. However, almost all of them have antibiotic resistance or no clear effect.

In order to solve the problem of resistance to antibiotics, many attempts have been made to utilize natural materials that can replace them. Since ancient times, herbal extracts and plant essential oils have been used for edible or medicinal purposes. Recently, studies on substances having antimicrobial, antifungal and anticancer effects have been actively conducted using these resources. There are many reports on the antimicrobial effects of bacteria present in foods that harm humans, but research on the antimicrobial effects of natural products on bacteria that cause diseases in farmed fish is in the beginning stages. It is urgent to develop an antimicrobial composition to find out the efficacy on fish disease bacteria using natural plant extracts for the specific efficacy of the streptococci that occur in the farm, and to replace the antibiotics actually used for streptococci. It is true.

Ginseng ( Sophora flavescens ) is a perennial herbaceous plant belonging to the legumes. It grows wild all over Korea and is used as insecticide, painkiller, and insect repellent. Spectators ( Dryopteris crassirhizoma ) is a perennial grass that uses the root as a medicine. Insect repellent, action on the uterus, hemorrhage due to hemorrhoids, hemorrhoids, schizophrenia, it is not good for pregnant women, children, ulcer patients with gastrointestinal ulcers. Has been. Thymus vulgaris ) is a perennial deciduous shrub belonging to Lamiaceae, native to the Mediterranean coast of southern Europe, and grows from subtropical to temperate. Its effects are antiseptic, antioxidant, insecticidal, and food preservation. Recently, the demand for herbs and landscaping materials has been increasing rapidly, but mainly focused on the flavor characteristics and antioxidants.

Accordingly, the present inventors have made intensive research to overcome the problems of the prior art, when processing ginseng extract, spectral extract, thyme extract, or mixtures thereof, effective suppression against streptococci causing disease in aquaculture fish. After confirming that it can, the present invention was completed.

Accordingly, the main object of the present invention is to provide a natural antimicrobial composition having an antimicrobial activity against fish bacterial disease-causing streptococci without the risk of antibiotic resistant bacteria and resistance transfer, and having the same or more effects compared to the antimicrobial effects of conventional antibiotics. To provide.

Another object of the present invention by using a natural antimicrobial composition having an antimicrobial activity against the bacterial bacterial disease-induced streptococci against the fish of Streptococcus, which is one of the causes of bacterial diseases causing enormous economic losses to freshwater and seawater culture in Korea To prevent and treat infections.

According to one aspect of the present invention, the present invention provides an antimicrobial composition comprising one or more plant extracts selected from the group consisting of red ginseng extract, spectral extract and thyme extract. The ginseng of the present invention (Sophora flavescens) Is a perennial herbaceous plant belonging to the legume family and grows all over Korea. The main components of the ginseng extract are matrin, oxymatrine, sophoranol, anagiline, methylcytizin, barbtiphrine, sofocarpine, and allomatrine. The audience of the present invention (Dryopteris crassirhizoma) Is a perennial fern that grows in the shade and can be blown under the forests of our country. The main components of the spectral extracts are bicarin, bicarin, and wogonin. Time of the invention (Thymus vulgaris) Is native to the Mediterranean coast of southern Europe, is a perennial deciduous shrub belonging to the Lamiaceae family, and grows from subtropical to temperate. The main components of the thyme extract are thymol, carbacroll, linalool, and flavonoids. In general, the antimicrobial effect of the extract consists of phenylpropane and terpine, which are composed mainly of oxides, esters, alcohols, phenols, aldehydes, ketones, monoterpines and sesquiterpines. Determined by These components are not just molecular structure but have a very complex structure and have been shown to have antiviral, antibacterial and antifungal effects. These ingredients have a strong antiseptic effect and antibacterial properties, and about 100 other components interact with each other to have strong therapeutic power and antibacterial properties. Therefore, when mixing the plant extract of the present invention exhibits excellent antibacterial action by the synergistic effect caused by the main components of the extract.

In the antimicrobial composition of the present invention, the antimicrobial may be an antimicrobial against any bacteria that are killed by treating the natural extract of the present invention, but preferably, antimicrobial against a bacterium belonging to the Streptococcus genus. Preferably Streptococcus iniae ) is good antimicrobial against species.

In the antimicrobial composition of the present invention, the composition may be administered to an animal body for the purpose of preventing and treating a bacterial infection, but is preferably administered to fish. Examples of such administrable fish include flounder, sea bass, defense, trout, eel, carp, catfish and the like.

In the antimicrobial composition of the present invention, the plant extract is extracted with at least one solvent selected from the group consisting of water, ethanol, isopropyl alcohol, methanol, propanol, butanol, glycerin, butylene glycol and mixtures thereof. Preferably, water, lower alcohols having 1 to 5 carbon atoms, or a mixture thereof can be used as a solvent for extraction. In the embodiment of the present invention using a mixed solvent of water and ethanol extracted the ginseng and the crowd.

In the antimicrobial composition of the present invention, the selected plant extract is preferably an extract of thyme and ginseng, and the weight ratio of the thyme extract and the ginseng extract is preferably 1: 0.2 to 1: 5. In the embodiment of the present invention, the antibacterial ability of the mixture was assayed by treating the thyme extract and the ginseng extract at the above ratios. As a result, 1 (15μl): 1 (15μl), 1 (7.5μl): 3 (22.5μl), 1 (5μl): 5 (25μl), 3 (22.5μl) 17: 1% (2.6 ÷ 1.5 × 100 = 173.3) when mixed with 1: 1 (7.5µl) and 5 (25µl): 1 (5µl) treatments ) Increased effect was observed.

In the antimicrobial composition of the present invention, the selected plant extract is preferably an extract of thyme and spectators, and the weight ratio of thyme extract and spectral extract is 1: 0.2 to 1: 5. In the embodiment of the present invention, the antibacterial ability of the mixture was assayed by treating the thyme extract and the spectral extract in the above ratios. As a result, the thyme extract and the spectral extract were mixed at a ratio of 1 (15 μl): 1 (15 μl), 3 (22.5 μl): 1 (7.5 μl), and 5 (25 μl): 1 (5 μl), respectively. When treated, the effect was increased by 171.4% (2.4 ÷ 1.4 × 100 = 171.4) compared with the spectral extract (30 μl) alone, 1 (7.5 μl): 3 (22.5 μl), and 1 (5 μl). ): 5 (25 μl), when mixed and treated with extracts of spectral extract alone (30 μl in spectral), 182.1% (2.55 ÷ 1.4 × 100 = 182.1) and 178.5% (2.5 ÷ 1.4 × 100), respectively. = 178.5) increased effect was observed. Low ginseng and spectral extracts, which were the herb extracts, were significantly lower than those of thyme when mixed with thyme. These results can be used in a way that small amounts of thyme ginseng and the crowd is cheap.

Hereinafter, the production of natural antimicrobial compositions having antimicrobial activity against the bacterial bacterial disease-induced streptococci of the present invention will be described in more detail step by step for evaluation of antimicrobial activity. The present inventors assayed the antimicrobial activity against Streptococcus bacteria against 9 essential oil extracts and 6 medicinal herbs by the paper disc diffusion method. As a result, natural antibacterial composition against streptococci was found to have antibacterial ability against Streptococcus bacteria from Gosam extract, spectral extract and thyme essential oil. In addition, the present inventors compared the antibacterial ability of the ampicillin commonly used as an antimicrobial agent and the plant extracts, and verified the antimicrobial effect of the mixture by mixing the time essential oil showing excellent antibacterial ability, and the gossip extract and the spectral extract, which are less economically expensive. It was.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Example  1. Selection of natural plant material and plant essential oils with antimicrobial effect

The effects of Streptococcus growth inhibition and elimination on the treatment of 10 essential oil extracts and 6 medicinal herb extracts against the Streptococcus by the agar well diffusion method are shown. The agar well diffusion method is a method used to compare or test antimicrobial activity against bacteria. Pancreatic digest of casein 17g / L, Enzymatic digest of soybean meal 3g / L, Dextrose 2.5g / L, Sodium chloride 20g / L, Dipotassium phosphate 2.5g / L, Agar 15g / L from OD 600nm to 1.0 Make 100 μl of the Streptococci cultured for each plate, inoculate it into ST medium with agar content of 7.5 g / L, pour into ST plate prepared beforehand, wait until it dries, and then put 30 μl of natural substance extract in each hole. The reaction was confirmed for 24 hours at ℃ the antimicrobial activity. The natural substance extract used 10 essential oil extracts and 6 herbal extracts, lemon glass, cypress french, fennel, pepsi, lavender french, bergamot, tea tree, thyme, lavender, geranium, spectators, yellow lotus, yellow white, Ginseng, cloves, and gold were selected and tested. As a result, growth inhibition was observed in the medium to which red ginseng, spectators, and thyme extract were added. The main components of natural extracts Ginseng, spectators and thyme are shown in Table 1.

TABLE 1 Major Components of Natural Extracts of Red Ginseng, Species and Thyme Extracts

Kinds main ingredient Gosam
( Sophora flavesceus ) Matrine, Oxymatrine, Sophoranol, Anagiline, Methylcytizin, Barbiflin, Sopocarpine, Allomatrine crowd
( Dryopteris crassirhizoma ) Bycarin, bicarrain, wogonin time
( Thymus vulgaris ) Thymol, carbachol, linalool, flavonoids

Example  2. Preparation of natural product extract

Natural extracts of ginseng and spectators used in the present invention were extracted by immersing the roots of sun-dried ginseng and spectators in 50% ethanol each of 10-fold weight for 7 hours. Thyme essential oil was purchased from Jina Flavor Co., Ltd., which was manufactured by steam distillation.

Example  3. Pathogenicity Streptococcal  Sympathy and Subculture

To evaluate the antimicrobial effect and efficacy of Gosam and Species extract and thyme essential oil, flounder ( Paralichthys) Streptococci were isolated from olivaceus ). The isolated streptococci were brain heart digest 17.5g / L, Proteose peptone 10.0g / L, Dextrose 2.0g / L, Sodium chloride 5.0g / L, Disodium phosphate 2.50g / L, Agar 15.0g / L BHIA medium (agar) using the strain was used for each experiment while subcultured. Purely Isolated Streptococcus sp. ) Was used to identify and analyze Streptococcus sp. (Gene Bank Data homology search result: 99%) through 16S rRNA sequencing analysis.

Example  4. Agar well diffusion method Through Gosam Of antimicrobial effect by concentration of extracts, spectators and thyme extracts

 Pancreatic digest of casein 17g / L, Enzymatic digest of soybean meal 3g / L, Dextrose 2.5g / L, Sodium chloride 20g / L, Dipotassium phosphate 2.5g / L, Agar 15g / L from OD 600nm to 1.0 Make 100 μl of the streptococci cultured for each plate, inoculate it into ST medium with agar content of 7.5 g / L, pour into ST plate prepared beforehand, wait until it dries, and then extract Ampicillin (control) and natural substance extract from the stock solution ~ 1.0. After diluting to%, put 30µL in each hole, and reacted at 27 ° C. for 24 hours to confirm antibacterial activity. It was confirmed whether the antibacterial activity was increased even when diluting each extract by selecting ginseng, spectral extract, and thyme essential oil extract from Chinese herbal medicine. Dilution test was also performed to dilute the stock solution to 1% using the agar well method diffusion method to show the effects of streptococcus inhibition and elimination. Ampicillin, an antibiotic of streptococcus, was used as a control. 1 and Table 2 show the effects of streptococcal growth inhibition and elimination when treated with agar well diffusion method for each dilute concentration of ampicillin and gosam, spectral, and essence essential oil against streptococci.

TABLE 2 Antibacterial Effects of Ampicillin, Red Ginseng, Species and Thyme Essential Oil Extracts by Dilution

Example  5. Gosam , Mixed with spectral extracts Streptococcus  Inhibitory effect test method

 In Example 3, when synergistically added and mixed with red ginseng and spectral extracts based on thyme essential oil extract showed the highest inhibitory ability up to 100-fold dilution treatment, it confirmed the synergistic effect of increasing the efficacy than the treatment alone. The antibacterial activity of the mixed extract was also used in the agar well method diffusion method of Example 3. Time: Gosam or Time: Mix the crowd 1: 1 to 1: 5, put 30µL into each hole, and react for 24 hours at 27 ℃ to prevent streptococcal growth inhibition and removal. It was compared with 30 μl alone (Fig. 2 and Table 3).

TABLE 3 Inhibitory Effects of Streptococcus on the Treatment of Herbal Extracts and Time Essential Oil Extracts

As described above, according to the present invention, by using an antimicrobial composition comprising a ginseng extract, spectral extract, thyme essential oil, or a mixture thereof, one of the causes of bacterial diseases causing enormous economic loss to freshwater and seawater culture in Korea. It can prevent and treat the infection of Streptococcus fish.

In addition, by using ginseng, spectral extract and herb thyme extract, which are widely used as herbal medicines, it is possible to replace antibiotics that are concerned about the emergence of resistant bacteria through long-term use. It is possible to provide natural antibacterial agents which are environmentally friendly and have high added value. Time essential oil, ginseng extract, spectral extract, or mixtures thereof of the present invention can replace antibiotics in the fish farming industry, and because ginseng or spectators are readily available at low prices, they also have high economic added value. In addition, the present invention will use environmentally friendly natural extracts as antibiotics to solve problems such as accumulation of existing antibiotics in fish bodies and expression of resistant bacteria, and will contribute to increasing productivity of the aquaculture industry by promoting consumption by eliminating distrust in farmed fish. .

1 is a photograph evaluating the antibacterial activity of ginseng (GS), spectral (KJ), thyme oil (TM) and each dilution by the agar well diffusion method. Photos of A-1 to A-3 are photographs observed when Ampicillin (10, 8, 6, 4, 2, 1, 0.8, 0.6, 0.4, 0.2, 0.1, unit μg) was treated as a control group, respectively. B The photographs of -1 to B-3 are photographs observed when the high ginseng extract and its dilutions (100, 80, 60, 40, 20, 10, 5, 3, and 1%) were treated, respectively. The photo of -3 is the photograph observed when the spectral extract and its dilution (100, 80, 60, 40, 20, 10, 5, 3, and 1%) is treated, D-1 to D-4 is time refined The photographs were observed when the extract and its dilutions (100, 60, 40, 20, 3, 1, and 0.5%) were treated respectively.

Figure 2 is a photograph evaluating the antibacterial activity of the mixture of essence essential oil (TM) and goose (GS) or spectral (KJ) extract by the agar well diffusion method. The photographs of A-1 to A-3 are observed when time oil (TM) and ginseng (GS) are mixed and treated in a ratio of 1: 1, 1: 3, 1: 5, 3: 1, and 5: 1, respectively. Photos of B-1 to B-3 are mixed with essential oil (TM) and spectral (KJ) extract in the ratio of 1: 1, 1: 3, 1: 5, 3: 1 and 5: 1, respectively. It is a photograph observed when processing.

Claims (6)

Antimicrobial composition comprising a thyme extract, red ginseng extract or spectral extract in a 1: 1 to 1: 5 weight ratio. According to claim 1, wherein the antimicrobial is antimicrobial composition, characterized in that the antibacterial against Streptococcus. The antimicrobial composition according to claim 1, wherein the composition is administered to fish. The method of claim 1, wherein the plant extract is extracted from the plant using at least one solvent selected from the group consisting of water, ethanol, isopropyl alcohol, methanol, propanol, butanol, glycerin, butylene glycol and mixtures thereof. Antimicrobial composition. delete delete

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