KR101408004B1 - An antibacterial composition comprising ginseng by-product obtained by subcritical water extraction - Google Patents

Abstract

The present invention relates to a natural antimicrobial composition containing an extract factor obtained by the asymptomatic extraction of ginseng by-products, and provides an antimicrobial activity of an extract of Ginseng with ginseng stems and leaves discarded in the ginseng processing industry .

Description

An antibacterial composition comprising ginseng by-product obtained by subcritical water extraction,

The present invention relates to a natural antimicrobial composition containing an index of extract of ginseng derived from ginseng by means of the extraction of the asymptotic modulus.

Ginseng has been reported to contain various biochemical components such as polyacetylenes, alkaloids, vitamins, minerals, phenolics, flavonoids and triterpenes as one of the pharmacological effects widely grown in Korea (Zhu S, Zou K, Cai S, Meselhy MR, Komatsu K. Simultaneous determination of triterpene saponins in Ginseng drugs by high performance liquid chromatography. Chem. Pharm. Bull. 52: 995-998, 2004; Sun S, Wang CZ, Tong R, A, Wang Q, He TC, Du W, Yuan CS, Effects of steaming on the root of Panax notoginseng on chemical composition and anticancer activities, Food Chem 118: 307-314, 2010). Especially, stem and leaves of ginseng contain major pharmacologically active components such as kaempferol, triterpene and saponins (or ginsenosides).

The various biological effects of ginseng include anticancer activity (Yun TK, Experimental and epidemiological evidence on non-organ specific cancer preventive effect of Korean ginseng an identification of active compounds, Mutation Res. 523: 63-74, 2003) , And regulation of cellular metabolism of lipids and proteins (Wang TF, Meng MZ, Experiment for immunity effects of ginsenoside Rg _3. Zhong Guo Yao Ke Da Xue Xue Bao 2: 55-57, 1999). In addition, ginseng extract by ether extraction has been reported to have high anticancer and antifungal effect (Jee HS, Chang KH, Moon SH, Park SH, Paik HD, Anti- Helicobacter pylori , cytotoxic, and anti-inflammatory activities of white ginseng extract. Food. Sci. Biotechnol. 17: 1106-1109, 2008). In addition, many studies have shown that various components of ginseng extract inhibit the growth of microorganisms. For example, white ginseng extract has an inhibitory effect on Helicobacter pylori (Lim JK, Kang HJ, Kang SN, Lee BY, Antioxidant and antimicrobial activities of various solvent fractions of fine ginseng root, Food Sci. Biotechnol. 518, 2009). Recent studies have reported the anti- Pseudomonas aeruginosa effect in ginseng roots (Kwon JH, Belanger JMR, Pare JRJ, Yaylan V. Application of the microwave-assisted process Food Res. Int. 36: 491-498, 2003).

In general, ethanol and water are used as the solvent in the extraction process, and they have been applied to improve the extraction yield by using the reflux method and the ultrasonic wave in combination. However, recently, as interest in food extraction methods using 'Green Technology' has increased, it has been used for extracting natural substances without using chemical solvents which can cause toxicity in the food industry.

Subcritical water extraction (SWE) extracts nonpolar compounds such as flavonoids by providing low relative permittivity (1 <epsilon <25) by making water at room temperature and pressure under subcritical condition through control of pressure and temperature (Jung WG, Chung EY, Ko MJ, Cho SW, Lee JH, Chang PS, Park YS, Paik HD, Kim KT and Chung JS, Comparison of extraction efficiency and antioxidant activity of flavonoid from citrus peel by different extraction methods, Food Eng. Prog. 14: 166-172, 2010). Subcritical extraction is carried out for a very short period of time rather than a general extraction method, and is currently actively studied as a method for selectively obtaining a useful substance in a harmless and environmentally friendly process (Ra YJ, Lee YW, Kim JD , Row KH. Supercritical fluid extraction of catechin compounds from green tea. Korean J. Biotechnol. Bioeng. 16: 327-331, 2001).

However, there have been no studies on the antimicrobial activity of stem and leaf extracts, which are ginseng byproducts, using food-derived pathogens such as Bacillus cereus, Listeria monocytogenes, Escherichia coli O157: H7 and Salmonella enteritidis .

Accordingly, it is an object of the present invention to provide a method for extracting optimal indexes for effective extraction of flavonoids, which are physiologically active substances of ginseng by-products.

Another object of the present invention is to provide a natural antimicrobial composition containing an extract factor by using an index of extraction of ginseng by-product.

The above object of the present invention can be achieved by a method of extracting asphalt, ethanol and hot water from ginseng stems and leaves, Measuring the total polyphenol and flavonoid content of each ginseng by-product extract; Measuring the antibacterial activity, bacteriostatic activity and bactericidal effect of the extract of Means of the ginseng by-product using a pathogenic bacteria of food origin; The ginseng by-product was obtained through the step of observing the cell damage of the pathogenic bacteria of the food by the extract of the ash factor.

The present invention has an excellent effect of providing a natural antibacterial composition obtained by asymptomatic extraction of stem and leaves of ginseng as a by-product of ginseng.

FIG. 1 is a graph showing the correlation between the antimicrobial activity against B. cereus and the phenolic compounds contained in the 190 ° C. subcycle extract. FIG.
FIG. 2 shows the growth inhibitory effect of food-borne pathogenic fungi on the extract of ash extract by concentration of ginseng by-products.
FIG. 3 is a graph showing the bactericidal effect of a food-derived pathogenic bacterium on the extract of ash extract by concentration of ginseng by-products.
FIG. 4 is a photograph showing a cytosolic morphological change of a food-derived pathogenic fungus on a sublingual extract of ginseng by-product through a transmission electron microscope (TEM).
FIG. 5 shows the results of measuring the cell membrane inhibitory activity of a food-derived pathogenic bacterium by the subculture extract of ginseng by-products.
Fig. 6 is a graph showing the antimicrobial activity of nisin alone in milk.
FIG. 7 is a graph showing the antimicrobial activity of the ginseng by-product extract alone in milk.
FIG. 8 is a graph showing the synergistic effect of antimicrobial activity induced by simultaneous treatment of nisin and ginseng by-product extract in milk.
Hereinafter, the present invention will be described in detail with reference to preferred embodiments and experimental examples.

The test material used in the present invention was cultivated and harvested in Youngdong, Korea. The stem and leaves of ginseng were used as a sample of ginseng byproduct for extraction. The leaves and stems were dried in a dry oven (OF12GW, Jeio-Tech Co., Seoul, Korea) at 60 ° C for 10 hours until the moisture content was 4-5% (w / w) The dried samples were ground in a high-speed mixer (Blender 7012S, Waring, Torrington, USA) with a particle size of 1-10 mm and stored at 4-5 ° C. The extracts of ginseng by- .

Hereinafter, specific methods of the present invention will be described in detail with reference to Examples and Experimental Examples, but the scope of the present invention is not limited to these Examples.

Example 1: Production of hot-water extract of ginseng by-products

In the examples of the present invention, hot water extraction of ginseng by-products was carried out by Majid Precision-Roudsari method (Waterhouse AL. Polyphenolics: Determination of total phenoics. In RE Wrolstand, TE Acree, EA Decker, MH Penner, DS Reid, & SJ Indianapolis, IN: John Wiley & Sons, Inc. pp: 463-470, 2004). That is, 20 g of ginseng byproduct powder was mixed with 200 mL of water in a 500 mL flask and extracted in a water bath at 80 ° C. for 3 hours. After extraction, whatman No. After filtration through two filter paper, the filtered residue was extracted twice more under the same conditions. The extracted hot-water extract was subjected to solvent removal and concentration using a vacuum concentrator (EYELA N-1000V, Tokyo, Japan). After concentration under reduced pressure, the extract was lyophilized and stored frozen and used in the experiment of the present invention.

Example 2: Preparation of ethanol extract of ginseng by-products

Samples of ginseng byproducts were mixed with 70% ethanol and extracted at 60 ℃ for 3 hours. Whatman No. 2 filter paper, and the remaining solids were extracted twice under the same conditions. After the ethanol extraction, the solution was removed by using a rotary vacuum concentrator and then lyophilized to be used as the disclosure material of the present invention.

Example 3: Preparation of the extract of sublingual factors of ginseng by-products

Subcritical exractor system (DIONEX ASE 100, Dionex Corporation, Sunnyvale, California, USA) was used for the extraction of asymptotic coefficients of ginseng byproducts. The finely pulverized ginseng stem and leaf powder and diatomaceous earth (DE) were mixed in a stainless steel cell at a mixing ratio of 1: 3 (w / w), and then mounted on an asymptomatic extractor. The extraction temperature and time were set to 15 minutes at 110 ° C (SWE 110 ° C), 165 ° C (SWE 165 ° C) and 10 minutes at 190 ° C (SWE 190 ° C) . The limulus extract was stored at -20 ° C after lyophilization and used in the experiment of the present invention.

Experimental Example 1: Measurement of total polyphenol content of ginseng by-product extract by Folin-Ciocalteau method

Phenol content was quantitated by Folin-Denis method (Quettier-Deleu C, Gressier B, Vasseur J, Dine T, Brunet C, Luyckx M, Cazin M, Cazin J C, Bailleul F, Trotin F. Phenolic compounds and antioxidant activities of buckwheat ( Fagopyrum sculentum Moench) hulls and flour. J. Ethnopharmacol. 72: 35-42, 2000). 0.1 mL of the diluted extract and 2 mL of 2% Na ₂ CO ₃ were mixed and allowed to stand at room temperature for 3 minutes. Then, 0.1 mL of Folin-Ciocalteau reagent (Sigma Chemical Co., St. Louis, Mo., USA) . After 30 minutes of mixing, the absorbance was measured at 750 nm using a spectrophotometer. The total phenol content of the sample extract was calculated from the standard calibration curves obtained by preparing gallic acid as a standard material at various concentrations, and is shown in Table 1.

Experimental Example 2: Measurement of total flavonoid content of ginseng by-product extract

Total flavonoid content was determined by colorimetric colorimetry using the aluminum colorimetric method (NCCLS (National Committee for Clinical Laboratory Standard), Performance Standards for Antimicrobial Susceptibility Testing, 9th International Supplement, M100-S9, Wayne Pa, 1999). That is, the ginseng by-product extract was dissolved in distilled water, and 0.5 mL of the extract was mixed with 1.5 mL of 95% ethanol, 0.1 mL of 10% aluminum chloride and 0.1 mL of 1 M potassium acetate, and 2.8 mL of distilled water was added. After reacting at 25 ° C for 30 minutes, absorbance was measured at a wavelength of 415 nm using a spectrophotometer. The calibration curve was prepared using kaempferol as a standard substance, and the total flavonoid content of the sample extract was calculated and shown in Table 1 above.

Phenolic compounds, including flavonoids, are known to have potent antimicrobial activity (Denyer SP, Hugo WB. Mechanism of action of chemical biocides: their study and exploitation. Blackwell Publishing. As shown in Table 1, the highest effective component was observed at 190 ° C (SWE 190 ° C, 10 min) for ginseng by-products (98.4 mg GAE / g and 17.3 mg KE / g).

Example 4: Cultivation of pathogenic bacteria from food

For the present invention, four common strains among foodborne pathogens were selected. Bacillus cereus (KCCM 40935, KCCM 11341, KCCM 40154) and Salmonella enteritidis (KCCM 12021) were purchased from the Korean Center for Microbiological Conservation (KCCM; Seoul, Korea) and used for the experiment. Listeria monocytogenes (ScottA NADC 2045 4B, H7969 serotype 4b, H792 serotype 4b) and Escherichia coli O157: H7 (ATCC 43895, FRIK 125) were distributed at the Department of Food Science and Technology, Iowa State University. Salmonella Enteritidis ATCC 13076 was purchased from the American Type Culture Collection (ATCC) and used for experiments.

Each strain was cultured in 10 mL of tryptic soy broth supplemented with 0.6% yeast extract for 12 hours at 35 ° C and stock culture was stored at 4 ° C. To activate each strain, TSB-YE was subcultured twice and then used in the experiment. After subculturing, 100 μL of the activated strain was inoculated into 10 mL of TSB-YE and cultured for 6 hours at 35 ° C. 1 mL of the strain was transferred to a sterile eppendorf tube and incubated for 10 minutes at 4 ° C. and 10,000 × g After centrifugation, only the cells were recovered and suspended in 0.1% peptone water.

In the present invention, a cocktail bacterial culture was used by Kotzekidou et al. (Hassas-Roudsari M, Chang PR, Pegg RB, Tyler RT. Antioxidant capacity of bioactives extracted from canola meal by subcritical water, ethanolic and hot water extraction. Food Chem. 114: 717-726, 2009). Suspensions of two or three species of pathogens, respectively, were mixed together to an equal volume.

Experimental Example 3: Screening of antimicrobial activity through agar well diffusion assay

To investigate the antimicrobial activity of ginseng by-product extracts, we performed an agar well diffusion assay (Horiuchi Y, Onoe T, Noguchi M, Okumoto K, Takemura K, Fukushima H, Komoya H. Single mixing fixation using glutaraldehyde and osmium tetroxide for improved ultrastructure J. Electron Microsc. 16: 19-25, 2001).

Extracted samples of lyophilized ginseng by-products were prepared in phosphate-buffered saline (PBS, pH 7.0-7.2) at 40 mg / mL. Each strain was inoculated with TSB-YE and cultured at 35 ° C for 12 hours. The culture was suspended in 0.1% sterilized peptone water and the cells were diluted to 10 ⁵ CFU (colony-forming unit) / mL and 1 mL was inoculated into 100 mL of 0.8% soft TSB-YE agar. 20 mL of 0.8% soft agar supplemented with diluted cells was poured into the plate, and the pellet was poured into a hardened agar with a sterilized cork borer to a diameter of 8 ㎜. As a control, PBS was used in place of the extraction solution. The plate was incubated at 35 ° C for 24 hours and the clear zone formation and size were measured.

Experimental Results As shown in Table 1, among the four different food-borne pathogens, the largest effect was observed particularly in B. cereus . However, there was no significant activity for the other strains except B. cereus .

Based on the above results, it was found that the antimicrobial activity against B. cereus and the interaction between the phenolic compound contained in the subcritical extract at 190 DEG C (FIG. 1) and the antimicrobial activity against B. cereus (R ² = 0.979).

Experimental Example 4: Measurement of bacteriostatic and bactericidal effect of ginseng by-product extract on pathogenic bacteria

TSB-YE broth, a nutrient medium, was used for the bacteriostatic effect and 0.8% saline solution except for nutrients was used for germicidal efficacy.

The ginseng by-product extract was prepared so as to have final concentrations of 0, 0.5, 1.0, 2.0 and 4.0% (w / v), and the initial concentration of each strain was inoculated to about 5.0 log CFU / mL. Each sample was incubated at 35 ° C., and samples were taken at constant time intervals for 24 hours to measure changes in viable cell count. The viable cell counts were diluted in 0.1% sterile peptone water, and 100 μL of TSB-YE agar was added to each well. The plated plates were incubated at 35 ° C for 36 hours and the number of viable cells was measured.

4-1: Inhibitory effect of Ginseng by-products on growth

Based on the results of the agar well diffusion assay of Experimental Example 3, the growth inhibitory effect of the extract at 190 DEG C on the growth inhibitory effect was examined (Fig. 2) And B. cereus was the most potent antimicrobial activity against ginseng by - product. In other words, in the case of B. cereus , sterilization effect was observed at 2 hours after culturing at all concentrations except for the sample without added sample. The strains except B. cereus showed no significant change in bacterial counts until 12 hours after culture, but the number of S. enteritidis and E. coli O157: H7 live cells decreased only at the highest concentration (4%) after 12 hours . L. monocytogenes, which showed the highest resistance to ginseng byproduct extract, showed no change in bacterial counts up to 20 hours even at the highest concentration of 4% treatment.

Therefore, we observed that the resistance to ginseng by - product extract was different according to the strains, and it was confirmed that B. cereus was the least susceptible to the extract.

4-2: Sterilization effect of ginseng by-products by subchronic extract

As a result of the measurement of the bactericidal effect of the 190 ° C ash extract extract having excellent antimicrobial activity (Fig. 3), the bactericidal effect against B. cereus was the highest as in the results of growth inhibition, and it was observed that the bacteria immediately died immediately after inoculation. The bactericidal effect against S. Enteritidis was found to be 0.8 ~ 5.2 log CFU / mL at the concentration of extract except the lowest concentration of 0.5% after 12 hours of reaction. In the case of E. coli O157: H7, the ginseng byproduct extracts 2 and 4% were observed to die after 12 and 20 hours of incubation, respectively. In the case of L. monocytogenes , which showed the highest resistance to ginseng byproduct extract, the bacterial effect was not observed until 12 hours, but the bactericidal effect was observed at 1, 2 and 4% treatment after 12 hours.

Experimental Example 5: Observation of cell damage by transmission electron microscope (TEM)

Morphological changes observed by electron microscopy were performed by Horiychi et al. (Chen CZ, Cooper SL, Interactions between dendrimer biocides and bacterial membranes. Biomaterials 23: 3359-3368, 2002). The four different pathogens used in the present invention were cultured in TSB broth (10 &lt; ⁸ &gt; CFU / mL) for each strain at 35 DEG C for 12 hours. The culture broth was centrifuged at 10,000 × g for 10 min at 4 ° C., and only the cells were collected, washed with PBS three times, and treated with 0.2% of sterile 0.8% NaCl SWE ginseng by-product extract. After that, the cells were again collected by centrifugation, and the cells were fixed with 2.5% glutaraldehyde at 4 ° C overnight. The fixed cells were washed three times with 0.05 M sodium cacodylate buffer (pH 7.2) and fixed for 2 hours at 4 ° C with 1% osmium tetroxide. The cells were stained with en bloc containing 0.5% uranyl acetate for 2 h at 4 ° C, washed twice in distilled water, and stained with ethanol (30, 50, 70, 80, 90, 100% , And then passed through 100% propylene oxide for 15 minutes for 2 hours to permeate propylene oxide (Spurr's resin = 1: 1) for 2 hours. The mixture was allowed to stand in a 100% Spurr's resin for 24 hours and then polymerized in a 70 ° C dryer for 72 hours.

The polymerized samples were fractionated using Ultramicrotome (MTX 75500, RMC, USA) and observed using a Transmission Electron Microscope (JEM1010, JEOL, Tokyo, Japan). Photographs were taken at the Electron Microscopy Laboratory of the National Instrumentation Center through Environmental Management (NICEM; Seoul, Korea).

As shown in FIG. 4, the outer membrane was normally maintained in the cells not treated with the extract, and the cells were observed to have a uniform shape (FIG. 4 (i)). In the case of B. cereus and L. monocytogenes not treated with the extract, a very thick cell wall was observed as Gram - positive bacteria. However, when the extract of ginseng by-product extracted at 190 DEG C was treated with B. cereus (Fig. 4A (ii)), a bulge protruded through a small pore of the cell wall and observed that the outer membrane structure was destroyed I could. In addition, intracellular contents leaked out and abnormal morphological changes were induced.

The morphological changes of S. Enteritidis showed that after the treatment of the extract, cytoplasmic components leaked due to membrane damage and the cell wall became blurred (Fig. 4b (ii)). The morphological changes of E. coli O157: H7 are as shown in Fig. 4c (ii), as in the results of other strains preceding, the outer membrane is collapsed and irregular in shape, and a large amount of intracellular contents is withdrawn, We were able to confirm enclosure. L. monocytogenes, which did not show bactericidal effect until 12 hours, disappeared cell shape consistency and leaked contents inside the cytoplasm.

Therefore, it was concluded that the strains exposed to the asymptomatic extract of 0.2% ginseng by - product caused morphological changes such as destruction of cell wall and cell membrane.

Experimental Example 6 Measurement of Cell Membrane Damage by Ginseng Extract of Ginseng Byproduct

By observing the morphological changes of the cells by TEM in Experimental Example 5, it was observed that the asymmetry factor extract of ginseng by-products disrupted the outer membrane of all the strains and the cytoplasmic contents leaked out. Based on these results, we measured the degree of elution of intracellular substances such as nucleic acid, metabolite, protein, etc. (Kalemba D, Kunicka A. Antibacterial and antifungal properties of essential oils, Curr Med Chem. 10: 813-829, 2003). That is, cell membrane damage was confirmed by measuring the absorbance of the substance released from the plasma at 260 nm (Rauha JP, Remes S, Heinonen M, Hopia A, Khknen M, Kujala T, Pihlaja K, Vuorela H, Vuorela P. Antimicrobial effects of Finnish plant extracts containing flavonoids and other phenolic compounds, Int. J. Food. Microbiol., 56: 3-12, 2000). Each strain was cultured, washed and suspended in 0.8% NaCl solution. The final cell number of each suspension was adjusted to 10 ⁵ CFU / mL and the lyophilized extract was added to the bacterial cell suspension treated with NaCl solution to a final concentration of 0.5%. The absorbance of the suspension was measured at 260 nm using a spectrophotometer.

The amount of substance eluted in the cells for 120 minutes after treatment with 0.5% ginseng by-product was measured (FIG. 5). B. cereus was observed to increase rapidly after 20 minutes of treatment, The amount of intracellular contents was maintained constant. Similar to the results of the bacteriostatic and bactericidal effects, the extract of the Ginkgo biloba extract showed a very sensitive response to B. cereus .

Experimental Example 7: Measurement of synergistic effect of nisin and ginseng by-product extract in milk

Nisin is an antimicrobial agent widely used in the food industry, and has a disadvantage in that it exhibits an effective antibacterial activity particularly for Gram-positive bacteria but not for Gram-negative pathogenic bacteria. Recently, there have also been reports of gram-positive pathogens resistant to nisin.

To determine the antimicrobial activity against L. monocytogenes , a common contaminant of milk products, the synergistic effect of the ash extract (190 ℃, 10 min) of nisin and ginseng by - products was measured. Milk used in the present invention was whole milk milk, low-fat milk milk and skim milk milk containing different amounts of fat. The extracts of ginseng and nisin were prepared at 1%, 2%, 125, 250 IU / mL and mixed at 4 concentrations. Listeria monocytogenes (ScottA NADC 2045 4B, H7969 serotype 4b, and H792 serotype 4b) were used as the strains used in the experiment and the activated strain was inoculated to milk at an initial concentration of 5 log CFU / mL. After inoculation, the viable cell counts of L. monocytogenes were measured by staining TSA at 4 ° C for 10 days (Harris, LJ, Fleming, HP, Klaenhammer, TR Sensitivity and Resistance of Listeria monocytogenes ATCC 19115, Scott A, and UAL 500 to J. Food Prot 54: 836-840, 1991).

As shown in FIG. 6, the antimicrobial activity of skim milk was significantly higher when nisin was treated alone, but it was found that the antimicrobial activity of skim milk was decreased with increasing fat content in milk. On the other hand, in the case of ginseng by-product extract, the bacteriostatic effect was observed irrespective of the fat content of milk as shown in FIG. However, in the case of simultaneous treatment of 250 IU / mL nisin and 2% ginseng by-product extract, the total amount of L. monocytogenes , which was about 3 log CFU / mL after 10 days in the whole milk, (Fig. 8).

Therefore, it was concluded that the extract of ginseng by - product extracted at 190 ℃ could be used as a natural food preservative in the dairy industry together with nisin.

As described above, the present invention has an excellent effect of providing a natural antimicrobial composition through the extraction of the ash factor using ginseng stem and leaf, ginseng by-product, which is discarded in the processing of ginseng, will be.

Claims (5)

Ginseng stems and leaves and diatomaceous earth were mixed at a blending ratio of 1: 3 (w / w) and then mounted on an asymptomatic extractor and extracted at 60 ° C for 10 minutes at 190 ° C. Coefficient Extract.
An antimicrobial composition comprising the ginseng stem and leaf sublingual extract of claim 1 as an active ingredient.
Milk characterized in that the antimicrobial composition according to claim 2 is added as a food preservative.
An antimicrobial agent composition, which is obtained by mixing the limulus extract of claim 1 with nisin.
A food preservative containing the antibacterial composition according to claim 4 as an active ingredient.

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