KR101270088B1 - Naturally occurring antimicrobial composites originated from plants against Bacillus cereus - Google Patents

Abstract

The present invention relates to a plant-derived natural antimicrobial composition having an antimicrobial activity against food poisoning bacteria, and more specifically, Hongjingo fern (Dryopteris erythrosora Kuntze) Or zombie sedge (Carex pumila ThunbIt relates to a natural antimicrobial composition against Bacillus cereus containing the extract as an active ingredient.
According to the present invention, the natural antimicrobial composition has an effective antimicrobial activity against Bacillus cereus, and the composition containing the two extracts in combination shows higher antimicrobial activity than when each extract is used alone. Therefore, the use of the natural antimicrobial composition of the present invention may effectively inhibit food poisoning bacteria even in a small amount, and may contribute greatly to securing food safety.

Description

Naturally occurring antimicrobial composites originated from plants against Bacillus cereus}

The present invention relates to a plant-derived natural antimicrobial composition having an antimicrobial activity against food poisoning bacteria, and more specifically, Hongjingo fern ( Dryopteris) erythrosora Kuntze or Zombie Sedge ( Carex pumila Thunb ) relates to a natural antimicrobial composition against Bacillus cereus containing the extract as an active ingredient.

Food poisoning is defined as “an infectious or toxin-like disease caused by or believed to be caused by a microbe or toxic substance harmful to the human body due to the ingestion of food” [ http://fm.kfda.go.kr ], often metabolic disorders, Allergies, specific disease, microbial infectious diseases, toxin poisoning are classified. Therefore, it is very important to effectively kill food poisoning bacteria in food or food processing environment.

Bacillus cereus is a representative food poisoning bacterium that forms spores and, depending on the mechanism of disease prevention, bacteria causing emetic disease, bacteria causing dirrheal disease, or vomiting and diarrheal disease It is divided into bacteria which cause all. In particular, Bacillus cereus O38-2 is known as a highly toxic food poisoning bacterium that causes both vomiting and diarrhea. This bacterium is distributed throughout the environment, and can be inhabited by food containing plants, meat, dairy products, rice, and pasta. In addition, since it can form a resistant spores resistant to environmental stress, it is also grown in the pasteurized food, causing food poisoning. In the United States, about 84,000 food poisonings are reported to be caused by Bacillus cereus per year. In Korea, 27 cases of food poisoning and 793 patients were reported from 2002 to April 2010. In 2008, 14 cases of group food poisoning and 376 patients were reported, showing the highest number of cases and number of cases reported. It is known that the number of food poisoning patients caused by the infection of Bacillus cereus occurs about 10 to 20 times more than the reported number of patients.

Therefore, many attempts have been made to control food poisoning bacteria using chemically synthesized antimicrobial agents. However, in order to add such synthetic preservatives directly to food, it has to be proved that it is not toxic to the human body through a risk assessment process. There are many restrictions on their use because they do not favor foods containing synthetic preservatives. For this reason, many researches are being conducted to develop antimicrobial agents of natural materials.

Accordingly, the present inventors have made diligent research efforts to overcome the problems of the prior art, and when selecting the plant-derived extracts treated with Bacillus cereus, it was confirmed that exhibits an effective antimicrobial effect against the bacteria, the present invention It was completed.

Therefore, the main object of the present invention is to provide a plant-derived natural antimicrobial composition having an effective antimicrobial activity against food poisoning bacteria Bacillus cereus.

According to an aspect of the present invention, the present invention is a natural antibacterial substance Hong Jin Negosari ( Dryopteris) erythrosora Kuntze Extract or Zombie Sedge ( Carex) pumila It provides a natural antibacterial composition against Bacillus cereus containing Thunb ) extract as an active ingredient.

Bacillus cereus is a representative food poisoning bacterium that forms spores and, depending on the mechanism of disease prevention, bacteria causing emetic disease, bacteria causing diarrheal disease, or vomiting and diarrheal disease It is divided into bacteria which cause all. Inhibiting the activity of Bacillus cereus using the natural antimicrobial composition of the present invention may not only prevent food poisoning, but also greatly contribute to securing food safety.

As confirmed in the embodiment of the present invention, Hong Zingo sari extract and zombie sedge extract has a concentration-dependent antibacterial effect against Bacillus cereus bacteria. That is, as shown in Figure 1, the size of the inhibitory ring was observed the largest when each extract was treated at a concentration of 50 mg / ml.

In the composition of the present invention, the Hong Zingo fern extract or zombie sedge extract may be extracted by any conventionally known extraction method, preferably polarity such as alcohol or ethyl acetate of 1 to 4 carbon atoms, such as water or ethanol It may be an organic solvent such as a solvent and a nonpolar solvent of hexane or dichloromethane, or a mixed solvent thereof, more preferably water, alcohol having 1 to 4 carbon atoms, or a mixture thereof.

Specifically, in the embodiment of the present invention, the plant raw material dried at room temperature was extracted with methanol at 50 ° C., concentrated under reduced pressure and concentrated to obtain an extract, and then dissolved in a solvent mixed with DMSO and sterilized tertiary distilled water.

The natural antimicrobial composition of the present invention is characterized in that it contains a combination of Hong Jingo fern extract and zombie sedge extract.

The natural antimicrobial composition containing a combination of the two extracts showed synergistic antimicrobial activity against Bacillus cereus than when each extract was used alone. Specifically, as confirmed in the embodiment of the present invention, when treated with a mixture of Hong Jin Negosari extract and zombie sedge extract was measured not only the size of the inhibitory ring larger than the treatment alone, fractional inhibitory concentration (fractional inhibiting concentration) FIC) was also measured at 0.5 or less to confirm the synergistic effect. Accordingly, by using a mixture of two natural extracts showing a synergistic effect on the antimicrobial activity can be seen a great antimicrobial effect in a small amount.

In the composition of the present invention, the Hong Jingo sari extract and zombie sedge extract is characterized in that it contains a weight ratio of 1: 64 to 64: 1 by weight.

In the embodiment of the present invention it was used by mixing the red zingo fern extract and zombie sedge extract 1: 1 weight%.

The term "antibacterial" of the present invention includes not only inhibiting the growth of pathogenic microorganisms or harmful microorganisms, such as bacteria, but also inhibiting their survival to sterilize.

The term "natural antibacterial substance" of the present invention refers to a substance that is recognized as a material of a preservative or a preservative enhancer formulation because of an antibacterial effect among food additives other than a chemical synthetic product. Since these natural antimicrobial substances are made of extracts of edible plants, they can overcome the shortcomings of conventional chemically synthesized antimicrobial substances, and reduce the consumer's antagonism to antimicrobial substances, as well as widely used foods. Can contribute to ensuring the safety of

The present inventors tested the antimicrobial activity against various microorganisms in 96 natural extracts, and showed that they exhibit antimicrobial activity against different microorganisms according to the types of natural extracts, and were specific for the bacterium Bacillus cereus. As a result, the present invention was found to have a red zingo fern extract and zombie sedge extract having a high antibacterial activity.

According to another aspect of the present invention, the present invention provides a food preservative containing the natural antibacterial composition as an active ingredient. The natural antimicrobial composition according to the present invention has antimicrobial activity against bacteria causing food poisoning by breeding in microorganisms or foods that cause corruption of food, and thus can be used as an active ingredient of food preservative compositions.

The preservative composition inhibits or sterilizes the growth of harmful microorganisms or decaying microorganisms such as bacteria, fungi and yeasts, and prevents the deterioration, rancidity or decay of the preservatives that may be infected or decayed by the harmful microorganisms or decayed microorganisms. Means used to maintain the state of the means, preferably means that can be used in one or more selected from the group consisting of food, feed, cosmetics, and medicines. The composition used for food among the said preservative compositions is called a food preservative composition.

The food preservative means a substance added to prevent the deterioration, rancidity or decay of the food, which should be able to control the changes over time, such as the taste, flavor of the food while inhibiting the growth of microorganisms. Therefore, the food preservative may include 0.01 to 99.9 parts by weight or 0.1 to 50 parts by weight of the natural antibacterial composition of the present invention based on 100 parts by weight of the total composition. In addition, the food preservative may further include a conventional material included in the preservative composition in addition to the natural antimicrobial composition of the present invention, for example, further adding a food-acceptable solvent or diluent for the purpose of increasing the preservation of foods. can do.

According to one aspect of the invention, the present invention provides a composition for treating or preventing food poisoning containing the natural antibacterial composition as an active ingredient.

The food poisoning is an acute or chronic disease caused by the ingestion of food containing natural poisons or harmful substances, mainly accompanied by symptoms such as fever, diarrhea and abdominal pain. The food poisoning can be classified into bacterial food poisoning, chemical food poisoning, natural poisoning food poisoning and food poisoning by microbial toxic metabolites according to the causative agent, Bacillus cereus ( Bacillus cereus ) is a representative bacteria causing the bacterial food poisoning.

In order to be used as a medicament for treating or preventing food poisoning of the present invention, it may be prepared by a method known in the pharmaceutical field, and in this case, the active ingredient includes a pharmaceutically acceptable carrier in addition to the natural antibacterial extract of the present invention. . Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, phosphorus calcium, alginate, gelatin, silicic acid Calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oils, and the like. It is not. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The composition for treating or preventing food poisoning may be administered alone or in combination with other pharmaceutically active ingredients for enhancing efficacy as a therapeutic agent for infection by the food poisoning causative agent. In this case, the content of the natural antimicrobial composition of the present invention in the composition may include 0.001 to 99% by weight. When the content of the natural antimicrobial composition is less than 0.001% by weight may require a large amount of administration for effective efficacy, when it exceeds 99% by weight may be uneconomical because the efficacy may be constant compared to the amount used. In addition, the content of the natural antimicrobial composition of the present invention can be properly adjusted according to the method of use and purpose of use.

The pharmaceutical compositions of the present invention may be prepared in unit dosage form by being formulated using a pharmaceutically acceptable carrier or excipient according to methods which can be easily carried out by those skilled in the art. It can be prepared by incorporation into a multi-dose container. The formulation may be in the form of a solution, suspension or emulsion in oil or medium, or may be in the form of extracts, powders, granules, tablets, capsules or injections, and may further comprise dispersants or stabilizers. They may be parenterally administered (eg, applied intravenously, subcutaneously, intraperitoneally or topically) or orally.

Appropriate dosages of the pharmaceutical compositions of the present invention may be appropriately determined by such factors as formulation method, mode of administration, age, weight, sex, health condition, degree of disease symptom, food, time of administration, method of administration and response to reaction. It may be selected, preferably 0.01 to 100 mg per day of adult can be administered.

According to one aspect of the invention, the present invention provides a food composition for preventing or improving food poisoning containing the natural antibacterial composition as an active ingredient.

Foods to which the natural antimicrobial composition of the present invention may be added include, for example, various foods, beverages, gums, teas, vitamin complexes, functional foods, and the like. The food composition for preventing or improving food poisoning is a special nutrition in addition to the natural antimicrobial composition. May contain foods (prepared milk, baby food, infant food), processed foods, fish meat products, tofu, jelly, noodles, health supplements, seasoned foods, sauces, confectionery, dairy products, other processed foods, pickled foods, beverages, natural seasonings have.

The functional food refers to the control of biological defense rhythms, disease prevention and recovery of food groups or food compositions that have added value to the food by using physical, biochemical, or biotechnological techniques to act and express the function of the food for a specific purpose. It refers to a food that is designed and processed to fully express the gymnastics function related to the living body. The functional food may include food acceptable food additives, and may further include appropriate carriers, excipients and diluents commonly used in the manufacture of functional foods.

In addition, the food composition for preventing or improving food poisoning is a variety of nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, such as flavoring agents, fillers and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids , Protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like, which may be used independently or in combination.

According to another aspect of the present invention, the present invention provides a cleaning composition containing the natural antimicrobial composition as an active ingredient.

The cleaner may be a detergent for clothing, dishwasher, food cleaner, household appliance cleaner, and the natural antimicrobial composition is a natural ingredient, which is not harmful to the human body. It can be used for the purpose of imparting antimicrobial properties.

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 cleaning agent may be prepared by powdering, granulating, tableting or liquefying according to the purpose and use thereof. In addition, the amount of the natural antimicrobial composition of the present invention 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. It is not. Therefore, since the natural antimicrobial composition of the present invention is not harmful to the human body, if the purpose of application can be achieved in the nature of the product, it can be applied in various amounts.

As described above, according to the present invention, a composition having an antimicrobial effect against food poisoning bacteria can be prepared using a natural antibacterial substance.

In the embodiment of the present invention, it was confirmed that the composition containing the Hong Jinego fern extract or zombie sedge extract according to the present invention has an effective antimicrobial activity against Bacillus cereus, in the case of a composition containing a combination of the two extracts It showed higher antimicrobial activity than the extract used alone. Therefore, the use of the natural antimicrobial composition of the present invention may effectively inhibit food poisoning bacteria even in a small amount, and may contribute greatly to securing food safety.

Figure 1 shows the results of measuring the size of the inhibitory ring for each concentration of the extract.

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

Example  1. Preparation of plant extract sample

1-1. Preparation of Plant Extracts

The plant extract used in the present invention was used by receiving a sample extracted from the Korea Plant Extract Bank ( http://extract.pdrc.re.kr) . Extraction method of the plant extract received as follows.

First, the plant material is divided by parts, and then dried at room temperature for about 5 days. Then the dried plants were ground and extracted with methanol (methanol) at 50 ℃. The sample was dispensed into a tube, and concentrated under reduced pressure and concentrated at 45 ° C. to obtain a final extract, which was then stored at low temperature. The final plant extract, dried under the reduced pressure, is supplied at a dose of 20 mg (within ± 0.2) per tube.

1-2. Preparation of Plant Extract Samples

20 mg of each extract obtained above was dissolved in dimethyl sulfoxide (≥99.9%), ACS reagent, Sigma-Aldirich, Co., St. Louis, MO, USA) in a ratio of 2: 8 It was dissolved using. 50 mg / ml sample was dissolved by adding 0.4 ml of total solvent to 20 mg of the obtained extract, and 20 mg / ml sample was dissolved by adding 1 ml of total solvent. And a 10 mg / ml sample was prepared by diluting a 20 mg / ml sample in a solvent. Then, the extracts dissolved in the solvent were stored in a freeze at -30 ℃ less than the amount used for 96-well plate (30096, SPL Life Sciences Co. Ltd, Seoul, Republic of Korea).

Example 2.Well diffusion assay

The antimicrobial effect against Bacillus cereus was measured by the well diffusion test using the concentration samples prepared in Example 1 (50 mg / ml, 20 mg / ml and 10 mg / ml).

First, 300 ml of Tryptic Soy Agar (TSA, BBL, Becton Dickinson, Sparks, MD, USA) to be used for the test was prepared according to the manufacturer's instructions, autoclaved at 121 ° C. for 15 minutes, and then using a water bath. The temperature was maintained at 45 ° C. Bacillus cereus was inoculated into the liquid TSA maintained at 45 ° C. to obtain a population of 10 ⁷ CFU per 17 ml of medium. The inoculated medium was dispensed in 17 petri dishes in 12 petri dishes. Petri dishes dispensed with the inoculated medium were kept at room temperature for about 15 minutes to allow the medium to harden. After the medium had hardened, eight wells were made using a sterile 6 mm biopsy punch (Stiefel Biopsy Punch Size: 6 mm, Stiefel Laboratories, Inc., FL, USA), followed by eight for each Petri dish. A total of 96 extracts were dispensed into 50 μl petri dishes. After the completion of the extract dispensing, and incubated for 24 hours in a 30 ℃ incubator, the presence and size of the inhibitory ring was measured. Of the 96 extracts, the measurement results for the two most effective extracts are shown in FIG. 1. In this case, the diameter of the inhibitory ring is the size of the pure inhibitory ring except 6 mm, the diameter of the well.

As shown in Figure 1, the size of the inhibitory ring was measured differently according to the concentration of the extract, the higher the concentration can be seen that the size of the inhibitory ring.

Example  3. Minimum inhibitory concentration of extract MIC ) And Minimum lethal concentration ( MLC ) Measure

To determine the minimum inhibitory concentration (MIC) and the minimum lethal concentration (MLC), the Broth dilution method was used.

The autoclave Tryptic Soy Broth (TSB, BBL) was mixed with the appropriate amount of each extract (Hongji Negosari and Zamboria sedge extract) to make a mixture solution of 4 mg / ml. A plurality of mixed liquids prepared by diluting 1/2 of the extract mixture prepared as described above using TSB were prepared. 20 μl of the Bacillus cereus strain diluted to about 10 ⁷ CFU / ml was inoculated into the extract mixture to obtain 10 ⁵ CFU / ml of bacteria in each tube. After inoculation, the tubes were incubated for 24 hours in an incubator at 30 ° C., and then visually observed to set the minimum concentration at which the bacteria did not grow to the minimum inhibition concentration (MIC). And, after incubating for 24 hours in a 30 ℃ incubator by streaking the tube was not grown in TSA twice, the minimum concentration in which a single colony did not grow was set to the minimum lethal concentration (MLC). The measurement results are shown in Table 1 below.

As a result, both Hongjin Negosari and Zombori sedge showed a low MIC of 0.0625 mg / ml, indicating that the antimicrobial effect of Bacillus cereus was excellent, and that the minimum lethal concentration (MLC) was higher than the minimum inhibitory concentration. It showed a high level of 4 mg / ml.

TABLE 1. MIC and MLC of Extracts

Example  4. Natural Antibacterial Compound Antibacterial Synergy  Confirm

4-1. Well diffusion assay

In the same manner as in Example 2 was measured by comparing the size of the inhibitory ring of the natural antimicrobial formulations (hongjinegosari and zombie sedge mixed extract). The measurement test sample was prepared by mixing each extract with a concentration (20 mg / ml, 10 mg / ml) and two extracts prepared with each 20 mg / ml in a 1: 1 ratio. Zinegosari 10 mg / ml, 10 mg / ml zombie sedge) was used. Each sample was dispensed 50 μl of the same amount. The measurement results are shown in Table 2 below.

As a result of the measurement, the size of the inhibitory ring was increased when the two extracts were treated in combination than when the extracts were treated alone. Therefore, it can be seen that there is a synergistic effect of the combination of the two extracts.

Table 2. Size Measurement Results of Inhibitory Ring According to Mixed Extracts]

4-2. Checkerboard assay

Checkerboard assay was used to confirm the antimicrobial activity of natural antimicrobial formulations.

50 μl each of the red zingo fern extract diluted from 2 times the concentration of MIC to 1/32 times the concentration of MIC was added to rows 1 to 7 of the 96-well plate. And, in column 1-7, 50 μl of zombie sedge extract diluted from 2 times concentration of MIC to 1/32 times concentration of MIC was added. The Bacillus cereus of 10 ⁶ CFU / ml to each well, including the combination of various concentrations were put to each 100 μl 10 ⁵ CFU / well. The plate was incubated for 24 hours in a 30 ℃ incubator, and then visually observed to determine the culture rate of the lowest concentration to inhibit the growth of bacteria. Fractional inhibiting concentration (FIC) was calculated by substituting the result of the compound concentration shown most efficiently into the following equation. If the FIC index is less than or equal to 0.5, there is a synergistic effect, an additive effect between 0.5 and 1, an indifferent between 1 and 4, and an antagonistic effect above 4. Judging The measurement results are shown in Table 3 below.

As a result of the measurement, when the mixture was treated with the Hong Zingo fern extract and the zombie sedge extract, the FIC index was decreased and the FIC index (0.2656) was smaller than 0.5 when the extract was treated alone. It can be seen that the combination of has a synergistic effect.

&Quot; (1) "

[Table 3. Antimicrobial Activity of Mixed Extracts]

Claims (8)

Natural antibacterial composition against Bacillus cereus , characterized in that it contains a combination of dryopteris erythrosora Kuntze extract and Carex pumila Thunb extract as an active ingredient.
According to claim 1, wherein the dryopteris erythrosora Kuntze extract and Carex pumila Thunb extract is at least one selected from the group consisting of water, alcohol having 1 to 4 carbon atoms, ethyl acetate, hexane and dichloromethane Natural antimicrobial composition, characterized in that extracted with a solvent.
delete The method of claim 1, wherein the natural antibacterial composition characterized in that the Hong Jin Negosari extract and zombie sedge extract containing 1: 64 to 64: 1 by weight of the mixing ratio.
A food preservative comprising the natural antibacterial composition according to claim 1 as an active ingredient.
A pharmaceutical composition for treating or preventing food poisoning, comprising the natural antibacterial composition according to claim 1 as an active ingredient.
A food composition for preventing or improving food poisoning, comprising the natural antibacterial composition according to claim 1 as an active ingredient.
Cleaner composition containing the natural antibacterial composition according to claim 1 as an active ingredient.

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