KR20080077438A - The complex extract of myristica fra grans houtt, cinnamomum cassia blume and alpinia officinarum hance, antibacterial activity fraction isolated, preparation method and uses thereof - Google Patents

Abstract

A mixed extract of Myristica fragrans Houtt, Cinnamomum cassia Blume and Alpinia officinarum Hance, or antibacterial fractions isolated from the mixed extract, and their preparation methods and uses are provided to obtain a nontoxic natural preservative for food/cosmetics and minimize the influence of the preservative on sensory properties of food. A mixed extract of Myristica fragrans Houtt, Cinnamomum cassia Blume and Alpinia officinarum Hance is prepared by using C1~C4 alcohol as a solvent. Soluble extracts are prepared by suspending the mixed extract in water, and then successively performing extraction by using hexane, ethylacetate and butanol as a solvent. Antibacterial fractions are prepared by performing silica gel chromatography of a hexane-soluble extract, wherein a developing solvent used in the chromatography is made by mixing 1~90% of ethylacetate to chloroform. The mixed extract of Myristica fragrans Houtt, Cinnamomum cassia Blume and Alpinia officinarum Hance, soluble extracts, antibacterial fractions are used as the effective ingredient of a preservative for food/cosmetics.

Description

The complex extract of Myristica fra grans Houtt, Cinnamomum cassia Blume and Alpinia officinarum Hance, antibacterial activity fraction isolated, preparation method and uses }

1 is a diagram showing the antimicrobial activity according to the concentration of the final active fraction against the Gram-negative bacteria Escherichia coli KFRI 272.

2 is a diagram showing the antimicrobial activity according to the concentration of the final active fraction for Bacillus subtilis KFRI 179.

Figure 3 is a diagram showing the antimicrobial activity according to the concentration of the final active fraction against Staphylococcus aureus KFRI 240.

Figure 4 is a diagram showing the antimicrobial activity according to the concentration of the final active fraction for Saccharomyces cerevises KCCM 50559.

The present invention relates to a nutmeg, broiler and Yanggang mixed extract or antimicrobial active fractions isolated therefrom, methods for their preparation and use thereof.

The rapid development of the food industry, the globalization of food, and the production and consumption of instant food are increasing, especially the use of food preservatives as a means to prevent food corruption and deterioration, and to extend the storage and distribution period of food. Doing.

Currently, food preservatives approved for food use include chemical compounds such as sorbic acid, benzoic acid, and butyl-p-hydrobenzoate. Various processed foods are delayed by adding food preservatives to prevent them because the microorganisms are deteriorated due to the storage period.

However, most of these food preservatives have been steadily raised by the use of chemical compounds. If these artificial preservatives continue to accumulate in the body, due to safety issues such as chronic toxicity, carcinogenicity, and mutagenesis, there are concerns about recent consumer concerns and future alternative preservatives and legal restrictions on the use of these preservatives. The trend is also strengthening.

In addition, food preservatives added for the purpose of improving the shelf life and palatability of foods may interact with food ingredients during the processing, storage and cooking of foods to generate mutations or to generate carcinogenic genetic toxic substances. Has emerged as an important issue in terms of safety.

In particular, nitrates or nitrites added to meat products, processed fish products, etc. play a role in not only color development and stabilization of meat color, but also antibacterial activity and reduced occurrence of rancidity, but nitrites remaining in foods and living bodies are toxic in themselves. Ingestion of more than a certain concentration causes various poisonings such as methemoglobin, and when reacted with amines contained in foods, it produces a carcinogenic substance nitrosamine (nitrosamine).

Currently used food preservatives can be classified into inorganic compounds, organic compounds, and natural preservatives according to their properties. Hydrogen peroxide, sulfurous acid, sodium softening and the like are used as inorganic compounds, and dehydroacetic acid (DHA), benzoic acid, ethyl alcohol, and the like are used as organic compounds.

Therefore, in recent years, interest in natural products having less concern about toxicity and excellent antimicrobial activity among ingredients used as raw materials or subsidiary materials of food has been increasing. In the United States, when people have been using food for a long time or extracting natural products and using them as food preservatives, their amounts or target foods are not included in the regulated substances unlike artificial synthetic preservatives, and they are generally recognized as safe (GRAS). Classified as

Most of the antimicrobial materials studied so far are known plant extracts, proteins and specific enzymes, organic acids such as polylysine and bacteriocin. In addition, antimicrobial active substances are known to include organic acids such as succinic acid, malic acid, tartaric acid, and benzoic acid, and flavonoids and catechins. have. In addition, antimicrobial activity is also known for fatty acids having 12 to 18 carbon atoms, lysozyme contained in eggs, transferrin and lactoferrin contained in colostrum, and nisin derived from microorganisms. In addition, edible plants having antimicrobial activity include wormwood, garlic, gat, clover and the like, as herbal medicines are reported to have antimicrobial activity, such as yellow lotus, Schisandra chinensis, grapefruit extract.

Among these ingredients, grapefruit seed extract, polylysine, lysozyme and bacteriocin, among others, are being used as food preservatives. However, research conducted by Japanese and domestic analytical organizations found that methyl ρ-Hydroxybenzoate, Propyl ρ-Hydroxybenzoate, and Benzethonium chloride, which are used as chemical synthesis preservatives in natural extract products in the market Chloride) is found to be a natural food preservative to replace these preservatives is urgently needed.

Therefore, compounds derived from specific plants have a narrow antimicrobial activity spectrum, short duration of effect, and a combination of antimicrobial active substances derived from many edible plants in order to compensate for the high cost. It is necessary to develop natural food preservatives that can be applied to foods through the development of complex food preservatives in accordance with the symbol, 3) effective in trace amounts, 4) easy to use, and 5) inexpensive.

Nutmeg ( Myristica fragrans Fruit of Houtt is 4-6 cm in length and is a nucleus. When mature, it looks like apricots, and there are seeds inside, and a reddish yellow crust is opened, and inside, you can see a split species like ribs. Seeds are nutmeg, a species called mace. Breeding is mostly seed and grows well in hot, humid climates. Nutmeg is dried and used as an aromatic dry agent, tonic, etc., and is known to have an antibacterial effect.

Also, broiler ( Cinnamomum cassia Blume) is a cinnamon we know well, a warm and nontoxic drug. Cinnamic aldehyde (Cinnamic aldehyde), Campen (Camphene), Cineol (Cineol), Linalool (Linalool), Eugenol (Eugenol). The main function of these components is to stimulate the mucous membranes of the stomach to stimulate the secretion of gastric acid, to suppress gastrointestinal spasms, and to promote the movement of the gastrointestinal tract, improving the release and absorption of gases. In addition, broilers are known to have an antiseptic effect that stimulates nerves, promotes blood circulation, warms the body, and suppresses abnormal fermentation of the intestines.

In addition, the Yanggang ( Alpinia officinarum Hance) is a perennial herb belonging to the family Ginger and dried the rhizome. The shape of the Yangtze is bent cylindrical or conical, 2-7 mm long, 1-2 mm in diameter, sometimes with several side roots. The surface is reddish brownish dark brown with gray white node, and the material is hard and not easily bent. The taste is hot and bitter, the nature is warm and acts on the rain and stomach. It is used for diarrhea, vomiting, and pain, which is a symptom of a cold caused by warming the stomach. The main components of Yanggang's essential oils are cineol, cinnamic acid and methyl ester.

Conventionally, Republic of Korea Patent No. 439845 is a natural addition from Sunhakcho extract, which is a new sterilization treatment method to prevent overheating, improve product freshness, and diversify product containers due to low temperature treatment by adding a small amount in processed foods. Food preservatives and food preservation methods using the same are described.

However, the patent has an advantage that can be diversified product containers through low temperature treatment, there is a disadvantage that the antimicrobial activity spectrum of the natural food preservatives are narrow.

Therefore, the present inventors found that the active fractions separated from the mixed extracts of nutmeg, broiler, and Yanggang selected from medicinal plants have antimicrobial activity against food rot bacteria during the implementation of the antimicrobial strategy of food rot bacteria using medicinal plants. It was confirmed to complete the present invention.

It is an object of the present invention to provide a mixed extract of nutmeg, broiler and lamb, or at least one antimicrobial active fraction obtained therefrom.

It is another object of the present invention to provide a method for preparing a mixed extract of nutmeg, broiler and Yanggang or at least one antimicrobial active fraction obtained therefrom.

Still another object of the present invention is to provide a food and cosmetic preservative comprising a mixed extract of nutmeg, broiler and lamb and one or more antimicrobial active fractions obtained therefrom.

In order to achieve the above object, the present invention provides a mixed extract of nutmeg, broiler and Yanggang extracted with alcohol.

The present invention also provides a soluble extract of a nutmeg, broiler and Yanggang mixed extract extracted with alcohol.

In addition, the present invention provides an antimicrobial active fraction obtained by performing silica gel column chromatography using hexane soluble extract of 1% to 90% of ethyl acetate in chloroform as a developing solvent among soluble extracts of nutmeg, broilers and poultry do.

The present invention also provides a food or cosmetic preservative comprising a nutmeg, broiler and Yanggang mixed extract, soluble extract or active fraction.

Hereinafter, the present invention will be described in detail.

The present invention provides a mixed extract of nutmeg, broiler and Yanggang extracted with alcohol.

The alcohol is a C ₁ ~ C ₄ alcohol, preferably methanol can be used.

The mixed extract may be prepared by a conventional extraction method. Preferably, 10 times methanol is added according to the sample volume per 100 g of the dried samples of nutmeg, broiler, and Yanggang and extracted by shaking at room temperature for 24 hours. Thereafter, this was filtered to obtain a primary extract, and the residue was extracted in the same manner as the primary extract to obtain a secondary extract. This can be concentrated under reduced pressure to produce a mixture of nutmeg, broiler and Yanggang.

The present invention also provides a soluble extract of a nutmeg, broiler and Yanggang mixed extract extracted with alcohol.

The method of fractionating and extracting the mixed extract may use a conventional extraction method, and preferably, may be fractionated using a separatory funnel, and more preferably suspended by adding water to the methanol extract of nutmeg, broiler, and Yanggang. After the addition of n-hexane to the suspension, the mixture was mixed well and separated into a distilled water layer and a hexane layer using a separatory funnel. Ethyl acetate was continuously added to the separated distilled water layer to separate the distilled water layer and the ethyl acetate layer. Butanol was added to the separated water layer to obtain a butanol layer and a final water layer, and the hexane layer, ethyl acetate, butanol and water layer were removed by using a rotary evaporator to remove solvent, thereby preparing a hexane, ethyl acetate and butanol soluble extract. have. The soluble extract is more preferably obtained with hexane.

In addition, the present invention provides an antimicrobial active fraction obtained by silica gel column chromatography using hexane soluble extract of nutmeg, broiler, and Yanggang soluble extract as a developing solvent using a solvent in which ethyl acetate is mixed with chloroform as a developing solvent. .

The soluble extract that is the fractionation target may be the above-described water, hexane, ethyl acetate and butanol soluble extract, and among these, hexane soluble extract may be selected and subjected to column chromatography to obtain an antimicrobial active fraction. The criteria for selecting the hexane soluble extract can be selected by extracting nutmeg, broiler and Yanggangang with water, hexane, ethyl acetate and butanol, respectively, by comparing the antimicrobial activity of the strains related to the decay of food and cosmetics for each extract have.

The method for preparing the antimicrobial active fraction may be prepared by a conventional chromatography method, preferably, hexane soluble extract for a mixture of nutmeg, broiler and lamb is placed in a column filled with silica gel and ethyl acetate 1 for chloroform. Silica gel column chromatography was performed using a developing solvent prepared at ˜90% to provide an antimicrobial active fraction obtained by performing a flow rate of 500 BV and a flow rate of 0.5-1.0 SV.

More preferably, from the active fractions obtained by performing silica gel column chromatography on nutmeg, broiler, and Yanggang hexane soluble extracts, a fraction having high activity and broad antimicrobial activity may be obtained by appropriately mixing the fractions showing antimicrobial activity against the decay-related strains. Can be.

In addition, the present invention provides a food or cosmetic preservative comprising the nutmeg, broiler and Yanggang mixed extract, soluble extract or antimicrobial active fraction.

In the present invention, nutmeg showed antimicrobial activity against general bacteria, but did not show antimicrobial activity against yeast, and broiler and Yanggang showed stronger antimicrobial activity against yeast than general bacteria.

In addition, the antimicrobial activity of each soluble extract of nutmeg showed excellent antimicrobial activity against gram-positive bacteria and gram-negative bacteria in hexane soluble extract, ethyl acetate soluble extract and butanol soluble extract except water soluble extract. There was no antimicrobial activity. Broilers showed weak antimicrobial activity against some Gram-positive bacteria and yeast in ethyl acetate soluble extract, while hexane soluble extract showed strong antimicrobial activity against yeast and some bacteria. Yanggang showed strong antimicrobial activity against some yeast in hexane soluble extract but weak antimicrobial activity against general bacteria.

Furthermore, the hexane soluble extracts of the nutmeg, broiler and lamb were chromatographed to obtain an active fraction, and the antimicrobial activity was evaluated. The antimicrobial activity of the active fractions showed antimicrobial activity against strains except Pseudomonas aeruginosa KCCM 11328 and Proteus vulgaris KCCM 40211 among 8 strains of Gram-negative bacteria, and Listeria food poisoning bacteria among 7 strains of Gram-positive bacteria. And Strains except Streptococcus mutans KCTC 3065 showed antimicrobial activity, and in the case of yeast Candida albicans All yeasts except KCTC 7965 showed antimicrobial activity.

In addition, the antimicrobial active fractions may be used by mixing each fraction in a predetermined mixing ratio. For example, the antimicrobial active fractions were mixed in appropriate proportions and their minimum antimicrobial activity concentration was determined.

The antimicrobial active fractions mixed in the appropriate ratio showed a high antimicrobial activity with a minimum inhibitory concentration of 6.25 ㎍ / disk except that the minimum inhibitory concentration of Proteus vulgaris KCCM 40211 was 12.5 ㎍ / disk for normal bacteria, and in the case of yeast Saccharomyces cerevisiae KCCM 50559, Zaygo Saccharomyces Bailey KCTC 7539, Candida Ripolitica KFRI 908, Candida Albicans KCTC 7965, Pichia Anomala In KCCM 50502 showed a minimum inhibitory concentration of high antibacterial activity to 6.25 ㎍ / disc, Pichia jadi you, Pichia membrane Pacific Enschede 12.5 μg / disk for KCCM 12740, Candida albicans In IFO 1594, it was found that the antimicrobial active fractions mixed at the appropriate ratio were 25.0 μg / disk and had a high antimicrobial activity while having a wide range of antimicrobial activity in a small amount.

In view of this, the nutmeg, broiler and poultry mixed extract, soluble extract or active fraction shows a high antimicrobial activity in a small amount, it can be useful as a food or cosmetic preservative.

When using a food preservative comprising the nutmeg, broiler and lamb's mixed extract, soluble extract or active fraction of the present invention as a food additive, the nutmeg, broiler and lamb's mixed extract, soluble extract or active fraction is added as is or other food or It can be used together with food ingredients and can be suitably used according to conventional methods. In the preparation of food or beverages, the nutmeg, broiler and lamb mutant extracts, soluble extracts or active fractions of the present invention may be added at 0.005 to 0.05% (or w / w, 50-500 ppm) relative to the food to be added. .

There is no particular limitation on the kind of food. Examples of the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes, and all foods in the conventional sense.

In addition, when used as a cosmetic preservative containing the nutmeg, broiler and broiler mixed extract, soluble extract or active fraction of the present invention, supple cosmetics (skin), astringent cosmetics, nutrient cosmetics (lotion), nutrition cream, massage cream, essence It can be used as a conventional cosmetic preservative, such as gel, pack, powder, skin ointment, patch, suspension, emulsion, spray, or cosmetic liquid.

Hereinafter, examples and experimental examples for explaining the present invention in more detail. However, the following Examples and Experimental Examples are only provided to explain the present invention more easily, and the contents of the present invention are not limited thereto.

<Selection of target substance according to literature investigation>

Three medicinal plants were selected by examining 191 domestic and international literature (143 domestic and 48 international) and 40 patents (15 domestic and 25 international) for edible plants and food additives. It was.

 Country  Scientific name  Used part  number  nutmeg  Myristica fragrans Houtt  Fruit  DP1  Broiler  Cinnamomum cassia Blume  skin  DP2  Yanggang  Alpinia officinarum Hance  Root  DP3

< Example  1> nutmeg Extract of Broiler, Broiler and Lamb

To obtain antimicrobial extracts of nutmeg, broiler, and Yanggang (purchased from Poongsan Pharmaceuticals Co., Ltd.), 10-fold methanol was added per 100 g of dry sample of each sample, followed by shaking at room temperature for 24 hours, followed by filtration. Whatman No. 2) to obtain a primary extract, the residue was extracted in the same manner as the primary extract to obtain a secondary extract.

 After mixing both the primary and secondary extracts, the mixed extract was concentrated and dried with a rotary vacuum concentrator to obtain a solid, and the content of the solid was calculated and used as a sample for antimicrobial activity test.

< Example  2> nutmeg , Broiler and lamb respectively Fraction  Produce

Suspended by adding purified water to the nutmeg, broiler and lamb's methanol extract prepared in Example 1, n-hexane was added to the suspension and mixed well, and separated into a distilled water layer and a hexane layer using a fraction funnel. Ethyl acetate was added to the separated distilled water layer to separate the distilled water layer and the ethyl acetate layer. Butanol was further added to the distilled water layer to obtain a butanol layer and a final distilled water layer. The solvent was removed from the hexane layer, ethyl acetate, butanol and distilled water layer using a rotary evaporator to obtain a hexane, ethyl acetate and butanol soluble extract. . After calculating the content of the soluble extract was used as a test for antimicrobial activity.

< Example  3> nutmeg , Broiler and poultry Fraction  Produce

1 kg each of nutmeg, broiler, and Yanggang were quantified and extracted with methanol at 10 times the sample volume for 24 hours at room temperature, followed by filtration (Whatman No. 2). The liquid obtained by extracting the residue in the same manner as the secondary extract. Both primary and secondary extracts were mixed and dried with a rotary evaporator to obtain a methanol extract (253.57 g).

The methanol extract was suspended in distilled water, hexane was added to the suspension, mixed well, and the distilled water layer and the hexane layer were separated using a separatory funnel. The hexane layer was removed using a rotary vacuum evaporator to obtain a hexane soluble extract (80.6g, DPHF).

Put the hexane soluble extract (DPHF, 253.57g) in a column filled with 700 g of silica hexane, and eluted with a solvent prepared from 1 to 90% ethyl acetate with respect to chloroform to obtain 7 active fractions (DPHFS1). ~ DPHFS7) was obtained.

< Example  4> nutmeg Activity of broiler, broiler and lamb Fraction  Produce

Of the seven active fractions obtained in Example 3, the active fractions 3, 4, 5 and 6th fractions were mixed in a 3: 2: 1: 1 ratio, respectively, to prepare a final active fraction.

< Experimental Example  1> nutmeg Antimicrobial Activity of Extracts from Broiler, Broiler and Lamb

In order to investigate the antimicrobial activity of the nutmeg, broiler and Yanggang methanol extract, the following experiment was conducted.

In order to determine the antimicrobial activity of the nutmeg, broiler, and Yanggang methanol extracts obtained by the method of Example 1, an agar diffusion method with the following procedure was performed. Pour the medium (hard agar) containing 1.5% agar into a petri dish and leave it in the incubator overnight, then inoculate the medium containing the agar (soft agar) containing 0.75% agar cultured with the test strain. After placing the sterilized filter paper disc, each of the nutmeg, broiler and Yanggang methanol extract was instilled in antimicrobial activity experiment.

 The results are shown in Table 2 below.

 Strain name  Control  DP1  DP2  DP3 Escherichia coli BE1182 ( Escherichia coli BE1182)  -  19 (±)  -  - KFRI 272 E. coli (Escherichia coli KFRI 272)  -  -  -  - Salmonella typhimurium SL1102  -  17 (±)  -  - Salmonella typhimurium KFRI 431  -  19 (±)  -  - Salmonella para tie blood -A ATCC11511 (Salmonella paratyphi-A ATCC11511 )  -  -  -  - Salmonella, Enterobacter Tee display (Salmonella enterotidis )  -  15 (+)  -  - Shigella Flesneri KFRI445 ( Shigella Flexnerii KFRI445)  -  -  -  - Pseudomonas fluorescein sense KCCM 11362 (Pseudomonas fluorescens KCCM 11362)  -  -  -  - Trying pseudomonas Rouge labor KCCM 11328 (Pseudomonas aeruginosa KCCM 11328)  -  -  -  - Proteus vulgaris KCCM 40211 (Proteus vulgaris KCCM 40211)  -  22 (+)  19 (±)  17 (±) Staphylococcus IFO12732 ( Staphylococcus aureus IFO12732)  -  20 (+)  -  - Staphylococcus aureus KFRI 240  -  21 (+)  -  - Streptococcus mutans KCTC 3065  -  -  - Bacillus subtilis KFRI 179 (Bacillus subtilis KFRI 179)  -  -  -  - Bacillus subtilis KCTC 1914 ( Bacillus subtilis KCTC 1914)  20 (-)  -  - Listeria food poisoning bacterium monocytogenes )  -  20 (+)  -  - Saccharomyces cerevisiae KCCM 50559  -  -  22 (++)  - Zygosaccharomyces bailli KCTC 7539  -  -  25 (+++)  - Candida albicans IFO 1594 (Candida albicans IFO 1594)  -  -  15 (±)  - Candida Li poly urticae KFRI 908 (Candida lipolytica KFRI 908)  -  -  20 (+)  17 (±) Pichia Jadini jadinii )  -  -  -  24 (++) Pichia membranaefaciens KCCM12740  -  18 (±)  18 (±)  18 (+) Pichia Anomala KCCM 50502 ( Pichia anomala KCCM 50502)  -  -  -  25 (+++)

* Is not inhibited; ± has a weak inhibitory effect; + Is an inhibitory effect;

  ++ has a strong inhibitory effect; +++ has a very strong inhibitory effect

* Diameter of growth inhibition ring (mm)

* Content of extract per disc: 2 mg / disk

As shown in Table 2, nutmeg (DP1) showed antimicrobial activity against general bacteria, but did not show antimicrobial activity against yeast, and broiler (DP2) and Yanggang (DP3) were stronger against yeast than general bacteria. It showed antimicrobial activity.

Therefore, it is expected that the effect will be greater when the antimicrobial spectrum is broadened by using a proper combination of them, and the mixture of nutmeg (DP1), broiler (DP2) and lamb (DP3) is natural food. The following experiment was conducted by selecting the final candidate group of the preservative.

< Experimental Example  2> nutmeg ( DP1 ), Broiler ( DP2 ) And Yangtze ( DP3 ) Hexane Antibacterial Activity of Soluble Extracts of Ethyl, Ethyl Acetate and Butanol

In order to determine the antibacterial activity of the nutmeg, broiler and broiler hexane, ethyl acetate and butanol soluble extract prepared in Example 2, the following experiment was carried out.

In order to determine the antimicrobial activity of the nutmeg, broiler and broiler hexane, ethyl acetate and butanol soluble extracts obtained in Example 2, the agar plate diffusion plate method was used for the Gram-positive bacteria, Gram-negative bacteria and yeast used in Experimental Example 1. The antimicrobial activity experiment was conducted.

The antimicrobial activity of each solvent fraction of nutmeg (DP1) was measured. However, there was no antimicrobial activity against yeast. Broiler (DP2) showed weak antimicrobial activity against some Gram-positive bacteria and yeast in ethyl acetate layer (DP2E), while hexane layer (DP2H) showed strong antimicrobial activity against yeast and some bacteria. Yanggang (DP3) showed strong antimicrobial activity against some yeast in hexane layer (DP3H), but weak antimicrobial activity against general bacteria.

Therefore, based on the results of each of the solvent fractions, the hexane fractions of nutmeg, broiler, and Yanggang were used as final mixtures. This is summarized in Table 3 below.

 Botanical name  Fraction  Remarks  nutmeg  DP1H  The final mixture is prepared by mixing the hexane fractions of nutmeg, broiler and lamb.  Broiler  DP2H  Yanggang  DP3H

< Experimental Example  3> nutmeg , Broiler and poultry Fraction  Antimicrobial Activity Experiment

Seven active fractions of nutmeg, broiler, and Yanggang obtained in Example 3 were prepared by diluting in methanol to a 10% (w / v) solution, and dropping the solution onto filter paper discs to 2 mg / disk. The antimicrobial activity of the active fraction was measured in the same manner as in Experimental Example 1.

The results are shown in Table 4 below.

  Name  DPHFS (2 mg / disk)  One  2  3  4  5  6  7  Escherichia coli BE1182  22  22  17  22  18  16  16  Escherichia coli KFRI 272  25  17  19  20  15  18  15  Salmonella Typhimurium SL1102  26  21  19  22  20  19  17  Salmonella Typhimurium KFRI 431  23  21  22  20  20  16  13  Salmonella Paratypy-A ATCC11511  20 (±)  20  19  24  17 (±)  18  15  Salmonella Enterotidis  25  22  21  21  17  16  18 Shigella Flesneri KFRI445  23  14  19  18  18  17  20  Pseudomonas Fluorescens KCCM 11362  25  25  22  20  15  21  18  Pseudomonas aeruginosa KCCM 11328  -  19 (±)  18  20  16  17  16  Proteus Bulgari KCCM 40211  -  17  18  21  17  15  20  Bacillus subtilis KFRI 179  21  18  18  25  18  20  19  Listeria food poisoning bacteria  -  -  20  19  -  21  21  Streptococcus mutans KCTC 3065  -  -  20  19  -  20  18  Staphylococcus aureus KFRI 240  18  -  21  19  19  18  19 Staphylococcus aureus IFO 12732  12 (±)  -  15  18  -  17  14 (±) Staphylococcus aureus R-209  11 (±)  -  17  18  15  17  14 (±)  Saccharomyces cerevisiae KCCM 50559  16  -  28  26  16  16  - Zaigo Saccharomyces Bailey KCTC 7539  16 (±)  -  18  21  17  15  16  Candida Ripolitica KFRI 908  17  21  25  27  17  21  17  Candida Albicans IFO 1594  16 (±)  -  17  20  16  13  15 Candida Albicans KCTC 7965  -  -  25  23  17  18  17  Pichia Jadini  18 (±)  -  17  19  16  13  12 Pichia Membrane Faciens KCCM 12740  19 (±)  -  17  20  17  17  18 Pichia Anomala KCCM 50502  18  -  20  22  21  18  19

* Is not inhibited; ± is a weak inhibitory effect

* Diameter of growth inhibition ring (mm)

As shown in Table 4, the antimicrobial activity of the active fraction 1 shows antimicrobial activity against strains except Pseudomonas aeruginosa KCCM 11328 and Proteus vulgaris KCCM 40211 among 8 strains of Gram-negative bacteria. And Strains except Streptococcus mutans KCTC 3065 showed antimicrobial activity, and in the case of yeast Candida albicans All yeast except KCTC 7965 showed antimicrobial activity.

In addition, active fraction 2 had antimicrobial activity against Gram-negative bacteria and some Gram-positive bacteria, but did not show antimicrobial activity against yeast.

In addition, the active fractions 3-7 showed strong antimicrobial activity against all strains, but the active fraction 7 was excluded from the final mixed fraction because the amount of solids is less than the other fractions obtained and the color is dark.

Thus, active fractions 3, 4, 5 and 6 were selected as final active fractions.

< Experimental Example  4> final active Fraction  Test of minimum inhibitory concentration

In order to determine the minimum inhibitory concentration for the final active fractions in which the active fractions 3, 4, 5 and 6 were mixed, the following experiments were carried out.

Minimal inhibition of the active fractions 3, 4, 5 and 6 selected by Experimental Example 3 at the ratio of 3: 2: 1: 1 based on the solids content with respect to the same volume at the ratio of 3: 2: 1: 1 In order to determine the concentration by using agar medium diffusion method, the solid content of the final active fraction was dissolved in alcohol so that the solid content of 10% (w / w) was diluted in stages and dropped on the filter paper disk. After culturing for 24 to 48 hours at the optimum temperature of each strain, the minimum inhibitory concentration was investigated with or without growth inhibitory rings (mm) without strains around the disk, and the results are shown in Table 5 below.

 Name  Concentration (µg / disc)  Minimum inhibitory concentration (µg / disk)  0  6.25  12.5  25.0  50.0  Escherichia coli BE1182  -  +  +  ++  ++  6.25  Escherichia coli KFRI 272  -  +  +  +  ++  6.25  Salmonella Typhimurium SL1102  -  ±  +  +  +  6.25  Salmonella Typhimurium KFRI 431  -  +  ++  ++  ++  6.25  Salmonella Paratypy-A ATCC11511  -  ±  +  +  +  6.25  Salmonella Enterotidis  -  +  +  +  ++  6.25 Shigella Flesneri KFRI445  -  +  ++  ++  +++  6.25  Pseudomonas Fluorescens KCCM 11362  -  +  +  ++  ++  6.25  Pseudomonas aeruginosa KCCM 11328  -  ±  ±  +  +  6.25  Proteus Bulgari KCCM 40211  -  -  ±  ±  +  12.5  Bacillus subtilis KFRI 179  -  +  +  +  +  6.25  Listeria food poisoning bacteria  -  ±  +  +  ++  6.25  Streptococcus mutans KCTC 3065  -  +  +  ++  ++  6.25  Staphylococcus aureus KFRI 240  -  +  +  ++  ++  6.25 Staphylococcus aureus IFO 12732  -  +  ++  ++  ++  6.25 Staphylococcus aureus R-209  -  +  +  +  ++  6.25  Saccharomyces cerevisiae KCCM 50559  -  ±  +  ++  ++  6.25 Zaigo Saccharomyces Bailey KCTC 7539  -  ±  ±  ±  +  6.25  Candida Ripolitica KFRI 908  -  +  +  ++  +++  6.25 Candida Albicans IFO 1594  -  -  -  ±  +  25.0 Candida Albicans KCTC 7965  -  ±  +  +  ++  6.25  Pichia Jadini  -  -  ±  +  +  12.5 Pichia Membrane Faciens KCCM 12740  -  -  +  +  ++  12.5 Pichia Anomala KCCM 50502  -  +  +  ++  +++  6.25

* Is not inhibited; ± has a weak inhibitory effect; + Is an inhibitory effect;

  ++ has a strong inhibitory effect; +++ has a very strong inhibitory effect

As shown in Table 5, Example 4 showed a high antibacterial activity with a minimum inhibitory concentration of 6.25 ㎍ / disk except that the minimum inhibitory concentration of Proteus vulgaris KCCM 40211 for the general bacteria is 12.5 ㎍ / disk, Yeast Saccharomyces cerevisiae KCCM 50559, Zaigo Saccharomyces Baily KCTC 7539, Candida Ripolitica KFRI 908, Candida Albicans KCTC 7965, Pichia Anomala In KCCM 50502 showed a minimum inhibitory concentration of high antibacterial activity to 6.25 ㎍ / disc, Pichia jadi you, Pichia membrane Pacific Enschede 12.5 μg / disk for KCCM 12740, Candida albicans In IFO 1594, it was found that the final active fraction of Example 4 had a high antimicrobial activity while having a wide range of antimicrobial activity in a small amount at 25.0 μg / disk.

Experimental Example 5 Antimicrobial Activity of the Final Active Fraction

In order to determine the antimicrobial activity of the final active fraction was carried out the following experiment.

Bacillus subtilis KFRI 179, Staphylococcus aureus KFRI 240, Gram-negative bacteria Escherichia coli KFRI 272 and yeast strain Saccharomyces cerevisiae KCCM 50559 were incubated at the optimum medium and temperature. To each liquid medium was added the final active fractions in which the active fractions 3, 4, 5 and 6 were mixed 3: 2: 1: 1 to a concentration of 25, 50, 100 ppm, and then 0.1 ml of the cultured strain. Inoculated and incubated for 48 hours at the optimum temperature of each strain, and compared the absorbance at 600 nm by using the UV-Vis spectrophotometer to compare the degree of microbial proliferation, and the addition of alcohol alone as a control experiment Proceeded. The results are shown in FIGS. 1 to 4.

As shown in Figure 1, the antimicrobial activity of the final active fraction against Gram-negative Escherichia coli KFRI 272 showed little growth at 50 ppm and 100 ppm.

In addition, as shown in Figure 2, Bacillus subtilis KFRI 179 did not show the growth of bacteria at all concentrations.

In addition, as shown in Figs. 3 and 4, it was found that the growth of bacteria was inhibited from 50 ppm after staphylococci KFRI 240 and Saccharomyces cerevises KCCM 50559.

Therefore, the optimal use range of the final active fraction is 0.005% to 0.05%, and the antimicrobial activity is high even in the amount less than the general natural preservative (0.05% to 0.5%). A small amount can be expected to have a high antimicrobial activity.

The extract prepared by the method according to the present invention or the active fraction separated therefrom exhibits excellent antimicrobial effect even with a small amount of additives, so that the sensory effect can be minimized when applied to foods, and a small amount can be used to lower manufacturing costs. It can be useful for food groups. In addition, since it can be applied in a minimum amount to the cosmetic, it can be usefully used for preservation of the cosmetic.

Claims (14)

Mixed extract of nutmeg, broiler and lamb pear extracted with alcohol. According to claim 1, wherein the alcohol is a mixed extract, characterized in that the C ₁ ~ C ₄ alcohol. 3. The mixed extract of claim 2, wherein the alcohol is methanol. Soluble extract of a mixture of nutmeg, broiler and lamb bran extracted with alcohol. 5. The soluble extract of nutmeg, broiler and lamb berry extract according to claim 4, wherein the alcohol is C ₁ to C ₄ alcohol. 6. 6. The soluble extract of nutmeg, broiler and lamb berry extract according to claim 5, wherein the alcohol is methanol. The soluble extract according to claim 4, wherein the mixed extract is suspended in water and extracted sequentially with hexane, ethyl acetate and butanol. The soluble extract according to claim 7, wherein the soluble extract is obtained by extracting hexane alone. An antimicrobial active fraction obtained by performing silica gel column chromatography using a hexane-soluble extract of claim 8 as a developing solvent using a solvent in which 1 to 90% of ethyl acetate is mixed with chloroform. A method for producing a mixed extract of any one of claims 1 to 3, wherein the nutmeg, broiler and lamb are extracted with alcohol. Extracting nutmeg, broiler, and Yanggang with alcohol to obtain a mixed extract thereof; And The method for producing a soluble extract according to any one of claims 4 to 8, comprising the step of soluble extraction of the mixed extract obtained in the step using hexane, ethyl acetate, butanol and water as a solvent. Extracting nutmeg, broiler and Yanggang with alcohol to obtain a mixed extract thereof (step 1); And Solubilizing the mixed extract obtained in the above step using hexane as a solvent (step 2); And A method for preparing an antimicrobial active fraction of nutmeg, broiler and lamb according to claim 9, comprising the step of obtaining the active fraction by performing silica gel chromatography on the hexane-soluble extract obtained in the step (step 3). A food preservative comprising the nutmeg, broiler and poultry mixed extract, soluble extract or antimicrobial active fraction of any one of claims 1 to 9 as an active ingredient. 10. A cosmetic preservative comprising the nutmeg, broiler and lamb mutant extract, soluble extract or antimicrobial active fraction of any one of claims 1 to 9 as an active ingredient.

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