KR101291082B1 - Method for extraction of anti bacterial in ginger - Google Patents

Abstract

The invention more specifically relates to a method for the threshold to extract the ginger seconds to extract the antibacterial active ingredient, and ginger and supercritical extraction under 200 to 300bar, 30 to 40 ℃ antibacterial active ingredient for in Listeria (Listeria) The method comprising: The ginger extracting method according to the present invention can effectively extract an effective ingredient having an antibacterial component according to supercritical extraction conditions.

Description

METHOD FOR EXTRACTION OF ANTI BACTERIAL IN GINGER [0002]

The present invention relates to a method for extracting an antimicrobial active ingredient by supercritical extraction of ginger.

Ginger (Zingiber officinale Roscoe) is a Zingiberaceae plant belonging to the genus Zingiberaceae. It is widely distributed in Southeast Asia and has been used as a folk remedy. Examples of the use of bamboo shrubs as folk remedies include those in Malaysia where the roots of several bamboo shrubs are used for the treatment of diseases such as stomach pain, nausea, vomiting, epilepsy, sore throat, coughing, bruises, wounds, seizures, eye diseases, liver diseases, rheumatism, Asthma, fever, malignant tumors, and boils. Ginger contains 40 ~ 60% of whole starch, and contains oriental sincere ingredient, resin protein, fibrin, pentanoic acid and minerals. The spicy taste of ginger is gingerone, gingerol, Shogaol, dihydrogingerol, and oriental components such as citral and camphene. Also, curcuminoid, flavonoid glycoside, sesquiterpenoid, and polyphenol have been reported as the main components of ginger root. Ginger roots have been used as traditional medicines in Indonesia to treat abdominal pain, diarrhea, insect pains, pain, rheumatism, and uterine disease (Ozaki et al., Chem. Pharm. Bull., 39, pp2353-2356, 1991). (Jitoe et al., 1994, pp. 981-984, 1994), anti-inflammatory activity (Ozaki et al., Chem. Lett. (Nugroho et al., Phytochemistry, 41, < RTI ID = 0.0 > In the study on the components of root of ginger, phenylbutenoid and cucurbituric acid were investigated in the study of ginger root components (Kanzawa et al., 1996; pp129-132, 1996) and uterine relaxation (Kanjanapothi et al., Planta Med., 53, pp 329-332, 1987) Curcuminoid has been reported (Masuda and Jitoe, Phytochemistry, 39, pp 459-461, 1995; Tuntiwachwuttikul et al., Phytochemistry, 20, 1164-1165, 1981; Masuda et al., Chem. Lett., 1, -192, 1993; Jitoe et al., Tetrahedron Lett., 35, pp981-984, 1994).

Especially, it is required to develop an effective extraction method to commercialize ginger oil and oleoresin extract of domestic ginger in order to secure domestic ginger and ensure the shipment throughout the year as Chinese ginger is imported. .

It is an object of the present invention to provide an optimum extraction condition for effectively extracting ginger with an active ingredient having antioxidant, antibacterial and perfume ingredients.

In order to achieve the above object, the present invention provides a method for extracting an antimicrobial active ingredient against Listeria by supercritical extraction of ginger at a temperature of 200 to 300 bar and 30 to 40 ° C. In another embodiment, the ginger is any one selected from the group consisting of sulfur, ginger, and salted ginger. In another embodiment, there is provided a method of extracting an antimicrobial active ingredient characterized in that the Listeria genus is L. monocytogenes .

In one embodiment, there is provided a method for extracting an antimicrobial active ingredient against Listeria sp . By extracellular extraction of ginger at a temperature of 200 to 300 bar and 60 to 70 ° C. In another embodiment, the ginger is any one selected from the group consisting of sulfur, ginger, and salted ginger. In another embodiment, there is provided a method of extracting an antimicrobial active ingredient characterized in that the Listeria genus is L. monocytogenes .

In one embodiment, the present invention provides a method for preparing an antimicrobial ginger extract against Listeria by supercritical extraction of ginger with under 200 to 300 bar and 30 to 40 캜. In another embodiment, the ginger is any one selected from the group consisting of sulfur, ginger and salted ginger. In yet another embodiment, the Listeria monocytogenes (Listeria) genus provides a process for preparing an antimicrobial extract of ginger, wherein the Listeria monocytogenes (L. monocytogenes).

In one embodiment, the present invention provides a method for preparing an antimicrobial ginger extract against Listeria by supercritical extraction of ginger at a temperature of 200 to 300 bar at 60 to 70 ° C. In another embodiment, the ginger is any one selected from the group consisting of sulfur, ginger and salted ginger. In yet another embodiment, the Listeria monocytogenes (Listeria) genus provides a process for preparing an antimicrobial extract of ginger, wherein the Listeria monocytogenes (L. monocytogenes).

The spice extract "oleoresin" consists of a complex of volatile oil, triglyceride and resin material. Each of these ingredients is widely used in the food industry because it acts as a unique functional substance such as flavor and flavor.

In the present invention, "supercritical extraction" is a method in which supercritical extraction is performed by setting the pressure and temperature above the critical point, which is the vapor phase and liquid phase position of the substance, so as to have the characteristics of liquid phase dissolution power and gas phase diffusion coefficient and viscosity, Method. Since the supercritical fluid is mainly formed of carbon dioxide or a small amount of co-solvent, there is no risk of residual harmful solvent compared with the solvent extraction method. In addition, since the extraction operation can be performed at a room temperature, It is a useful method for extraction.

"Gingerol" accounts for about one-third of the oleoresin and exists as a mixture of several homologs of different lengths of the aldehyde unit (side chain). Connell et al. reported that gingerol was separated from ginger and named 6-, 8-, and 10-gingerol according to the length of the aldehyde unit, and they were present at a ratio of 53:17:30. Masada et al. Later discovered the presence of 3-, 4-, and 5-gingerol, and chen first confirmed the presence of 14-gingerol. Thus, gingerol is a mixture of 3-, 4-, 5-, 6-, 8-, 10-, 11-, 12-, and 14-gingerol homologues and is generally 6-gingerol, - It is known to contain small amounts of gingerol and it is known that there is a slight difference in irritability depending on the length of the side chain.

On the other hand, shogaol and zingerone are not originally present in fresh ginger but are artifacts caused by dehydration and retro-aldol reaction during preparation, processing and storage of ginger, and they are known to be less irritating than gingerol.

Among the essential components of ginger, 6-Gingerol, which is the main component of the ginger pungent taste, is characterized by antioxidant and anti-inflammatory properties.

The ginger extract method according to the present invention can effectively extract an active ingredient having antioxidant, antibacterial and perfume ingredients according to supercritical extraction conditions.

Fig. 1 is a result of an experiment evaluating the antimicrobial activity of an ethanol solvent ginger extract.
FIG. 2 shows the results of an evaluation of antimicrobial activity of the ginger extract by supercritical extraction.
FIG. 3 is a chromatogram of the fragrance components of a 100 bar supercritical extract at 35 ° C.
FIG. 4 is a chromatogram of fragrance components of a 100 bar supercritical extract at 65 ° C.
FIG. 5 is a chromatogram illustrating the aroma component of a 500 bar supercritical extract at 35 ° C.
FIG. 6 is a chromatogram of the aroma component of a 500 bar supercritical extract at 65 ° C.
FIG. 7 is a chromatogram of the fragrance component of the ethanolic ginger extract.
8 and 9 are graphs showing DPPH radical scavenging activity of ginger extract.
10 is a graph showing the inhibitory effect of ginger extract on ATP reduction due to H ₂ O ₂ in L6 cells.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example  One. Oleoresin's  Establish optimal extraction conditions

1-1. Using ethanol Oleoresin  Establish optimal extraction conditions

Extraction was carried out using ethanol as a solvent in order to extract oleoresin components in ginger. Extraction conditions were 1: 2, 1: 4, 1: 6 and 1: 8 ratio of ginger powder to ethanol. Extraction temperature was 50 ℃ and extraction time was 90min. Extraction yields were determined by extracting the extracting temperature at 35 ℃, 50 ℃, 65 ℃ and 80 ℃ based on the ratio of ginger powder and ethanol. The ginger powder was extracted at 1, 3, 5, and 7 hours at the extraction temperature, which was the highest extraction yield. After extracting ginger powder according to each condition, a filter paper (whatman No. 42) was placed on a Buhner funnel, and the extract was suction filtered. The filtrate was concentrated under reduced pressure at 50 ° C to obtain a ginger concentrate as a solvent extraction sample .

1-2. Supercritical  Using an extraction device Oleoresin  Establish optimal extraction conditions

Extraction was carried out using a supercritical carbon dioxide extraction device (Greentek21 Co., Ltd., Anyang, Korea) for the extraction of oleoresin components in ginger. 200 g of ginger powder was poured into the extraction tank at a volume of 500 mL and sealed with an energized seal. The head portion and the main body of the extractor were clamped. CO ₂ from the CO ₂ cylinder is liquefied through the condenser is put twos extracted after being compressed in the CO ₂ pump. The carbon dioxide and extracts from the extraction tank were lowered to normal pressure and the carbon dioxide was blown. The extract collected in the receiver was used as a ginger supercritical extract. Extraction pressure (100 bar, 250 bar, 500 bar) and extraction temperature (35 ℃, 50 ℃, 65 ℃) were extracted in order to establish optimal extraction conditions of oleoresin.

Example  2. Evaluation of ginger extract

2-1. Evaluation of antimicrobial activity

The antimicrobial activity of Gram negative and Gram positive bacteria, which are known as food spoilage and pathogenic microorganisms, were purchased from the BRC and cultured 3 times. The test strains, the working medium and the culture temperature are shown in Table 1.

division Bacterium Badge Incubation temperature
(° C) Gram (-) Pseudomonas aeruginosa KCTC 2004 Nutrient agar 37 Escherichia coli KCTC 1039 Nutrient agar 37 Gram (+) Staphylococcus aureus KCTC 1621 Nutrient agar 37 Bacillus subtilis KCTC 1022 Nutrient agar 30 Listeria monocytogenes KCTC 3569 Brain Heart Infusion agar 37

Ethanol and supercritical ginger extract were diluted in DMSO (dimethyl sulfoxide) at concentrations of 0.1%, 1% and 5%. The antimicrobial activity of ginger extract was examined by agar diffusion method (paper disc agar diffusion). The bacterial stocks stored at -70 ° C were inoculated into the growth media of Nutrient (Difco, USA) and Brain Heart Infusion (BHI: Difco, USA) for each strain and cultured for 24 hours. 100 μl of each strain was cultured on Nutrient agar and Brain Heart Infusion agar. After spreading, the strain was absorbed into the medium, and the sterilized 8 mm paper disc (Tokyo Roshi Kaisha) was placed on the culture medium, Of ginger extract solution was absorbed. After culturing for 24 hours at the culture temperature suitable for each strain, the size of inhibition of growth around the disc was measured to confirm the antibacterial activity.

2-2. Flavor component analysis of ginger extract

Separation and analysis of volatile odor components of ginger extracts were carried out using a GC-mass spectrometry (Agilent 5972MSD, Palo Alto, USA) and a gas chromatograph (GC) (Agilent 6890, Palo Alto, USA) USA), and the components of peaks separated by GC-Mass were identified based on the mass spectrum library.

The operating conditions of GC and GC-Mass for the analysis of volatile components are as follows. GC was a Agilent 6890 (USA) equipped with a Supelcowax 10 ^™ (0.25 mm × 60 m, film thickness 0.25 μm) column. The oven temperature was raised at a rate of 5 ° C / min starting at 50 ° C, maintained at 280 ° C for 4 minutes, and carrier gas was used at a flow rate of 1.1 mL / min of helium (He). The GC-Mass was an Agilent 5972MSD equipped with Supelcowax 10 ^™ (0.25 mm × 60 m, film thickness 0.25 μm) column.

2-3. Changes in quality characteristics during storage

The ginger extracts were stored in vials at 4 ℃ and 30 ℃ for 30 days. The ethanol solvent extracts were examined for the storage stability of the extracts at the ratio of 1: 6, extraction temperature of 50 ℃ and extraction time of 5 hours, which were the highest extraction yields. In the case of supercritical extracts, The storage stability test was performed on the whole sample.

2-3-1. Chromaticity measurement

The L , a , b , and ΔE values of ginger were measured for each treatment using color and color difference meter. For chromaticity measurement, the sample was dissolved in 10 mL of DMSO (dimethyl sulfoxide) in 0.1 g of the extract. The L , a , and b values of the standard white plate used were L : 100.01, a : -0.02, and b : -0.00, respectively.

2-3-2. HPLC Analysis of useful components during storage of extracts

The contents of 6-gingerol, 8-gingerol, 10-gingerol, 6-sucrose and curcumin in ginger extracts were determined by HPLC. HPLC was performed by Jasco Co. (65:35, v / v) was used for the analytical liquid chromatography (Japan). The column was a Waters symmetry C-8 reversed phase column (150 × 3.9 mm, Cat. No. WATO 54235) ) Was eluted at a rate of 1 ml / min and the detection of the sample was carried out at 282 nm. Analytical standards were purchased from 6-gingerol, 8-gingerol, 10-gingerol and 6-Chromadex, and curcumin was purchased from Sigma-Aldrich. The sample solution was prepared by dissolving ginger extract at 5 mg / ml in methanol and filtering with 0.45 um syringe filter (Millipore).

2-4. Evaluation of Antioxidant and Activity of Ginger Extract

2-4-1. DPPH radical  Measure erasure effect

The DPPH radical scavenging activity of the ginger extract samples was measured. For the measurement, change the method of Blois (1958), add 0.2 mL of each ginger extract diluted with 80% ethanol to 3 mL of ethanol, add 4 mL of 4 × 10 ^-4 M DPPH (2,2- diphenyl-1-picrylhydrazyl, Sigma-Aldrich Inc.) was added and the mixture was shaken for 10 seconds. After incubation for 10 minutes at room temperature, the absorbance was measured at 517 nm. The DPPH radical scavenging activity was expressed as a percentage by measuring the absorbance of the sample supplement and the negative control, and the positive control was vitamin C.

2-4-2. L6  Cell ATP  Production amount measurement

ATP bioluminescence assay kit HS II (Roche, Germany) was used to measure ATP production in L6 cells. L6 cells were seeded in a 96-well plate at a concentration of 1 × 10 ⁵ cells / ml, cultured for 24 hours, treated with ginger samples at a concentration of 2 mM H ₂ O ₂ at the same time, cultured again for 24 hours After the cells were digested, ATP bioluminescence assay was performed and the absorbance was measured using a measuring device (Molecular Devices, USA).

Example  3. Extraction yield of ginger extract

3-1. Extraction with solvent

3-1-1. The ratio of ginger powder to ethanol Extraction yield

The yields of the extracts were 1: 2, 1: 4, 1: 6 and 1: 8 ratio of ginger powder to ethanol. The extraction solvent ratio of 1: 2 was slightly lower than other treatments (9.73%). It was concluded that the amount of ethanol did not affect the extraction yield. In addition, the extraction yields of ethanol extracts of 1: 6 and 1: 8 were not significantly different from 10.91% and 10.96%, respectively. In the extraction of oleoresin, the ratio of ginger powder to ethanol was decreased to 1: 6 Respectively.

Extraction solvent ratio (ginger powder: EtOH) Yield (%) 1: 2 9.73 1: 4 10.72 1: 6 10.91 1: 8 10.96

3-1-2. Of ginger powder according to extraction temperature Extraction yield

Table 3 shows the yield of ginger extracts extracted with ethanol and ginger powder at a ratio of 1: 6 and extracting temperature of 35 ℃, 50 ℃, 65 ℃ and 80 ℃, respectively. The higher the extraction temperature, the smaller the yield. Especially, at 65 ℃ and 80 ℃, 12.08% and 12.56% were high, respectively. This is because the resin which is difficult to recover other than oleoresin, which was not found in extracts at 35 ℃ and 50 ℃, The results were similar to those reported by Jeong, who reported that the yield was affected. As a result, it was determined that extraction at a high temperature was not suitable and that the extraction of oleoresin was performed at 50 ° C.

Extraction temperature (캜) Yield (%) 35 10.05 50 10.84 65 12.08 80 12.56

3-1-3. The extraction time of ginger powder Extraction yield

Table 4 shows the yields of the extracts obtained by setting the ratio of the solvent of ginger and ethanol determined at the above experiment to 1: 6, the extraction temperature at 50 ° C, and the extraction times of 1, 3, 5 and 7 hours. As shown in Table 4, the extraction time did not significantly affect the yield.

Extraction time (hrs) Yield (%) One 10.53 3 10.64 5 10.64 7 10.43

3-2. Supercritical  Of ginger powder by extraction Oleoresin  Extraction yield

Table 5 shows the extraction results of nine treatments with different pressure and temperature using supercritical fluid for the extraction of active ingredients in ginger. Unlike the yields of the solvent - extracted extracts, there was a clear difference in the yields depending on the pressure and temperature of the supercritical system. Unlike the pressure conditions of 250 bar and 500 bar at the extraction pressure of 100 bar, the higher the extraction temperature, the lower the yield. That is, the extraction yield was 6.08% at extraction pressure 100 bar 35 ℃, 4.17% at 50 ℃ and 3.77% at 65 ℃. At extraction pressure of 250 bar, extraction yields were 7.36%, 7.60% and 8.90% at the extraction temperature of 35 ℃, 50 ℃ and 65 ℃, respectively. The extraction yield was found to be 8.96% at the extraction pressure of 500 bar and the extraction temperature of 65 ℃ under supercritical fluid extraction conditions. As a result, it was found that the supercritical extraction yield of ginger increased with increasing extraction pressure and extraction temperature.

Extraction condition Extraction yield (%) Extraction pressure (bar) Extraction temperature (캜) 100 35 6.08 100 50 4.17 100 65 3.77 250 35 7.36 250 50 7.60 250 65 8.09 500 35 7.80 500 50 7.90 500 65 8.96

Example  4. Evaluation results of ginger extract

4-1. Evaluation of antimicrobial activity

The strains used to verify the antimicrobial activity of oleoresin for each treatment were Pseudomanas aeruginosa), Bacillus subtilis (Bacillus subtilis), Listeria monocytogenes (Listeria monocytogenes), Staphylococcus aureus (Staphylococcus aureus ) and Escherichia coli O157 ( Escherichia coli O157). Spoilage organisms include, but are used as a decay cause Pseudomonas aeruginosa (Pseudomanas aeruginosa) and gram-positive aerobic bacteria to form a heat-resistant spores are of animal products in the fermentation of food bupaegyun of Bacillus subtilis (Bacillus subtilis ) was used. It is an anaerobic bacterium which is an indicator of hygiene status and causes food poisoning. It is a well-developed anaerobic bacterium that grows well at refrigeration temperature and produces listeriosis bacteria ( Listeria monocytogenes ) , a thermophilic anaerobic microorganism called Staphylococcus, is a thermostable, poisonous food poisoning causative organism ( Staphylococcus aureus ) , a major food poisoning incident in the United States and Japan, and Escherichia coli 0157 ( Escherichia), which causes symptoms such as hemorrhagic nephrotic syndrome and hemorrhagic enteritis upon infection coli O157) was used to investigate the antimicrobial activity of ginger extracts against each strain. The extracts of 12 treatments extracted with alcohol and 9 treatments extracted with supercritical system were diluted to 0.1%, 1% and 5%, respectively. 7 and Figs. 1 and 2.

As a result of the antimicrobial activity of the extracts, the size of the clear zone showing the antimicrobial activity increased proportionally with the concentration of the other strains except for P. aeruginosa. In the case of L. monocytogenes, the clear zone was 22 mm under the condition of supercritical extraction at 250 ° C and 5% treatment at 35 ° C, showing strong antibacterial activity compared with other extracts.

In case of 0.1% diluted ginger extract, the clear zone of Pseudomonas aeruginosa showed a stronger antimicrobial activity than that of the other strains. In the case of supercritical extracts, 0.1%, 1% and 5% Listeria monocytogenes Of the antimicrobial activity. In the case of Staphylococcus aureus, no clear zone was found at 0.1% concentration of solvent and supercritical extraction, and E. coli did not show clear zone in the supercritical extract, indicating that it had little antimicrobial activity at low concentration .

The clear zone of Listeria monocytogenes and Pseudomonas aeruginosa strains in the solvent extraction samples of 1% dilution of ginger extract showed a stronger and stronger antimicrobial activity than the other three strains, and supercritical extract In the case of Listeria monocytogenes, the antimicrobial activity was the strongest and the activity was the weakest in E. coli .

In case of 5% diluted ginger extract, the antimicrobial activity of Listeria monocytogenes was the highest in solvent extraction samples as in 0.1% and 1% concentration. clear zone) of 16.5 mm showed the strongest antimicrobial activity compared to other treatments. Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus showed similar antimicrobial activities according to extraction conditions. In E. coli , there was no significant difference in antimicrobial activity regardless of extraction conditions and extract concentration.

The supercritical extract showed the highest antimicrobial activity in Listeria monochytogenes. The clear zone of extracts at 250 and 65 ℃ and at 100 bar and 35 ℃ showed the highest antimicrobial activity. In the case of B. subtilis , the clear zone was increased to 16.5 mm under the extraction conditions of 14 mm, 500 bar and 65 ℃ at 100 bar 35 ℃, showing higher antimicrobial activity than other extraction conditions. E. coli showed stronger antimicrobial activity than extracts obtained under different pressure conditions at extraction pressure of 100 bar. P. aeruginosa , S. aureus The strains showed lower antimicrobial activity than the other strains.

In order to investigate the antimicrobial activity of each ginger extract according to solvent and supercritical extraction conditions, Listeria monocytogenes The antimicrobial activity of the extracts was the highest. In addition, the extractive conditions which showed the strongest antibacterial activity were solvent extract extracts at 1: 6 concentration and supercritical extract showed strong antibacterial activity at 100 bar 35 ℃, 250 bar 35 ℃, 250 bar 65 ℃.

Extraction condition Clear zone (mm) L. monocytogenes < RTI ID = 0.0 & gt; P. aeruginosa
(P. aeruginosa ) Bacillus
(B. subtilis ) Staphylococcus
(S. aureus ) Escherichia coli
(E. coli ) density(%) 0.1 One 5 0.1 One 5 0.1 One 5 0.1 One 5 0.1 One 5 Control group ^{1 )} - ²⁾ - - + ³⁾ + + - - - - - - - - - 1: 2 12.0 12.5 15.5 14.0 ++ ⁴⁾ ++ 9.0 11.0 12.0 ++ 10.0 ++ 9.0 9.0 9.0 1: 4 11.0 14.0 15.5 12.5 12.5 ++ 9.0 12.0 11.0 ++ 10.0 11.0 8.0 9.0 9.0 1: 6 11.5 12.5 16.5 12.0 12.0 11.5 9.0 12.0 13.0 ++ 10.0 12.0 8.0 9.0 9.0 1: 8 11.5 13.0 16.0 11.5 12.0 12.0 9.0 10.0 13.0 ++ 10.0 11.0 8.0 8.0 8.0 35 ℃ 8.0 10.0 13.0 11.5 12.0 12.5 9.0 11.0 11.0 ++ 10.0 12.0 8.0 8.0 8.0 50 ℃ 8.0 11.0 13.0 12.0 11.5 11.5 8.0 10.0 12.0 ++ 11.0 12.0 8.0 8.0 9.0 65 ℃ 8.0 11.0 13.0 12.0 ++ 12.5 9.0 11.0 12.0 ++ ++ 11.5 8.0 9.0 9.0 80 ℃ 11.0 12.0 15.0 11.5 12.0 12.0 9.0 11.0 13.0 ++ ++ 12.0 9.0 9.0 9.0 1 hr 8.0 11.5 14.0 12.0 ++ ++ 9.0 12.0 11.0 ++ 11.0 12.0 9.0 9.0 9.0 3 hrs 10.0 11.0 14.0 11.0 12.0 12.0 9.0 11.0 12.0 ++ 10.0 11.0 9.0 9.0 9.0 5 hrs 10.0 12.0 15.0 ++ 11.5 11.5 9.0 11.0 11.0 ++ 10.0 11.0 9.0 9.0 9.0 7 hrs 11.0 13.0 15.0 12.0 11.5 12.0 9.0 11.0 11.0 ++ ++ 11.0 8.0 9.0 10.0 ¹⁾ DMSO (dimethyl sulfoxide).
²⁾ Not detected.
³⁾ Slight growth.
⁴⁾ Less than 8mm.

Extraction condition Clear zone (mm) L. monocytogenes < RTI ID = 0.0 & gt;
P. aeruginosa
(P. aeruginosa ) Bacillus
(B. subtilis ) Staphylococcus
(S. aureus ) Escherichia coli
(E. coli ) density(%) 0.1 One 5 0.1 One 5 0.1 One 5 0.1 One 5 0.1 One 5 Control ^{1 )} - ²⁾ - - + ³⁾ + + - - - - - - - - - 100/35 12.0 11.5 20.0 10.0 11.0 11.0 9.0 11.0 14.0 ++ ⁴⁾ 11.0 12.0 ++ ++ 13.0 100/50 12.0 16.5 17.0 11.0 11.0 11.0 9.0 10.5 11.0 ++ 11.0 12.0 ++ 11.0 11.0 100/65 16.0 15.0 16.0 10.5 11.5 11.0 8.0 9.0 10.0 ++ 8.0 10.0 ++ 11.0 12.0 250/35 11.5 15.0 22.0 10.0 10.0 10.0 9.0 9.0 13.0 ++ ++ 10.0 ++ ++ 11.0 250/50 12.5 15.0 18.0 11.0 11.0 11.0 8.0 9.0 12.0 ++ 11.0 11.5 ++ ++ 10.0 250/65 12.0 15.0 23.0 10.0 11.0 11.0 9.0 10.0 12.0 ++ 11.0 11.0 ++ ++ 11.0 500/35 11.0 11.0 16.5 10.0 10.0 10.0 8.0 9.0 11.0 ++ 10.0 10.0 ++ ++ 9.0 500/50 12.5 15.0 16.5 10.0 11.0 11.0 8.0 10.0 15.0 ++ 10.0 10.0 ++ ++ 10.0 500/65 11.5 15.0 17.0 11.0 11.0 11.0 10.0 10.0 16.5 ++ 10.0 11.0 ++ ++ 10.0 ¹⁾ DMSO (dimethyl sulfoxide).
²⁾ Not detected.
³⁾ Slight growth.
⁴⁾ Less than 8mm.
* It can be seen that there is a critical significance because the zona pellucida seems to be less than 20 mm at 250 bar / 20 ° C and 250 bar / 80 ° C and the antimicrobial activity against Listeria monokitogen is the best at 250 bar / 35 ° C and 250 bar / 65 ° C.

4-2. Investigation of flavor components and quality characteristics of ginger extract

4-2-1. Flavor component analysis of ginger extract

The volatile essential oil of the ginger consists of very complex components, such as sesquiterpene hydrocarbons (50 to 65%) and oxygenated sesquiterpenoids (according to Connell and Chen et al. It is a complex compound of a terpenoid composed of a monoterpenoid compound and a miscellaneous compound. Hydrocarbons of sesquiterpene in ginger essential oils have (-) - (-) - (-) - β-bisabolene, (+) - αγ-curcumin based on the bisabolene carbon skeleton , And oxygenated sesquiterpenoids include jinjirol and monoterpenoids include camphene, geraniol, 1,8-cineol, and the like.

The fractions of the extracts at 100 bar, 35 ℃, 100 bar, 65 ℃ and 500 bar, respectively, were analyzed by GC / MS. The volatile fractions The compositions and contents of the components are shown in Table 8 and plotted in Figures 3, 4, 5, 6 and 7 as chromatograms.

Among the supercritical extracts, the main volatile flavor components of the 100 bar 35 ° C extract were 31.18%, 24.40% of the total fragrance components identified as 2,3-butanediol, jinjirolin, αγ-curcumin, β-sesquiphellandre, , 9.41%, 8.92% and 4.66%, respectively, and the content of 2,3-butanediol was higher than other supercritical extraction conditions. Supercritical extraction 100 bar The most highly detected 2,3-butanediol at 35 ° C is a volatile low-boiling fraction with acetaldehyde and ethyl acetate. It is a volatile component that is difficult to separate by the simultaneous distillation extraction (SDE) It is known as a substance.

Supercritical extraction conditions The major volatile fragrance components of 100 bar and 65 ℃ extract were 31.60%, 13.91%, 12.32%, 11.56% for jinjielin, 2,3-butanediol, αγ-curcumin, β-sesquiphellandre, and β- , And 6.26% respectively. Among them, Jinji Berin was the highest among other supercritical extracts. Jinji berin has been widely used in cosmetics and fragrances, and recent studies have reported that antiviral, anti-ulcerative and contraceptive effects have been confirmed. It is also possible to use neral, geranial, alpha-terpineol, vonyl acetate, ginseng berlin, [beta] -iodesomal, [beta] -Sesquipelandol sesquiphellandrol). The contents of α-terpineol, vinyl acetate and genipiperin were 1.34%, 0.64% and 31.60%, respectively, which were highest in supercritical extraction conditions at 100 bar and 65 ℃ .

500 bar The main aroma composition of the extract at 35 ° C was found to be 29.05% for jinjielin, 14.10% for 2,3-butanediol, 10.92% for αγ-curcumin, 9.79% for β-sesquiphellandre and 5.09% for β-bisabrene β-pelendrene and α-pelendrene were found to be 8.20% and 6.90%, respectively, compared to other extraction conditions.

500 bar The main aroma composition of the extract at 65 ° C was 25.48%, 24.34%, 9.58%, 8.66%, and 6.52% of jinjielin, 2,3-butanediol, αγ-curcumin, β-sesquiphellandre, and β- And the content of acetic acid was 5.32% higher than other extraction conditions.

Peak No. Retention time Compounds 1 ¹⁾ 2 ²⁾ 3 ³⁾ 4 ⁴⁾ One 7.26 acetic acid 3.66 2.19 4.95 5.32 2 13.25 2,3-butanediol 31.18 13.91 14.10 24.34 3 18.11 6-methyl-5-hepten-2-one 0.49 0.74 0.98 0.81 4 20.39 β-phellandrene 4.66 6.26 8.20 6.52 5 20.49 Eucalyptol 0.95 1.27 1.53 1.31 6 23.12 terpinolene 0.43 0.66 0.68 0.54 7 23.39 cumic alcohol 0.32 0.47 0.57 0.48 8 24.24 borneol 0.08 0.11 0.12 0.09 9 28.33 alpha-terpineol 0.64 1.34 1.13 0.83 10 32.60 bornyl acetate 0.43 0.64 0.49 0.43 11 32.72 2-undecanone 0.22 0.39 0.29 0.24 12 36.47 cycloisosativene 0.87 1.09 1.08 0.92 13 36.84 α-cubebene 1.25 1.52 1.17 1.36 14 37.47 2,4-diisopropenyl-1-methyl-1-vinyl cyclohexane 0.95 1.30 1.16 0.97 15 39.20 γ-elemene 0.84 1.13 1.04 0.87 16 39.94 β-farnesene 0.76 0.79 0.75 0.72 17 41.42 αγ-curcumene 9.41 12.32 10.92 9.58 18 42.16 zingiberene 24.40 31.60 29.05 25.48 19 42.48 alpha-farnesene 5.45 5.35 6.90 6.13 20 42.65 beta-bisabolene 4.09 5.34 5.09 4.39 21 43.29 β-sesquiphellandre 8.92 11.56 9.79 8.66 ¹⁾ Supercritical extraction 100 bar, 35 ℃ extract.
²⁾ Supercritical extraction 100 bar, 65 ℃ extract.
³⁾ Supercritical extraction 500 bar, 35 ℃ extract.
⁴⁾ Supercritical extraction 500 bar, 65 ℃ extract.
* Extraction yield of jinjirolin is expected to be less than 30.00% at 100 bar / 95 ℃, and the extraction yield of jinjirolin at 500 bar / 5 ℃ is expected to be less than 25%. Therefore, it is found that there is a critical meaning at 100 bar / 65 ℃ and 500 bar / .

4-2-2. Changes in quality characteristics of ginger extract during storage

(A) Chromaticity

In order to investigate the change of color during storage of ginger extract, the color values were compared with the initial values after storage for 4 weeks at 4 ℃ and 30 ℃. Table 9 shows the initial chromaticity results of storage of ginger extracts, and there were differences in supercritical and ethanol extraction treatments. The solvent extracted extract showed 12.48 in the redness value and 30.98 in the b value indicating yellowness, indicating that the color was darker than that of the supercritical extract. In the case of supercritical extracts, L value, a value, and b value were not significantly different from each other at extraction pressure of 250 bar and 500 bar. On the other hand, at the extraction pressure of 100 bar, as the extraction temperature increased, the L value and the yellow value b decreased and the red value a showed a tendency to increase. For the overall chromaticity (ΔE), the extracts at 100 bar and 65 ℃ showed the highest chroma at 89.12 and the extracts at 500 bar and 65 ℃ showed the lowest chroma at 81.60.

Extraction condition L a b ΔE Solvent ^{1 )} 19.67 12.48 30.98 84.01 100 bar, 35 DEG C 17.78 -5.87 12.73 84.54 100 bar, 50 ° C 10.88 -2.35 4.25 88.89 100 bar, 65 ° C 10.48 -1.60 2.65 89.12 250 bar, 35 ° C 17.66 -6.15 19.33 84.77 250 bar, 50 ° C 18.26 -6.14 19.29 84.36 250 bar, 65 ° C 17.38 -5.86 18.42 84.93 500 bar, 35 ° C 20.18 -6.68 21.98 83.12 500 bar, 50 ° C 19.82 -6.35 21.67 83.35 500 bar, 65 ° C 22.39 -6.91 23.31 81.60 ¹⁾ Extraction at 1: 6, 50 캜 for 5 hours.

Table 4 shows the color results of the ginger extracts extracted at 4 ℃ for 4 weeks. The L value indicating brightness, the a value indicating redness, and the b value indicating yellowness were both slightly decreased during storage for 4 weeks when compared with the chromaticity of initial ginger extract of Table 9. Especially, the L value indicating the color case brightness of the supercritical extract at 100 bar at 35 ° C and 250 bar at 35 ° C was slightly larger than the other treatments. It was also found that the supercritical extract was not significantly changed after 4 weeks of storage compared to the initial chromaticity of extracts treated with 500 bar of extraction pressure. After 4 weeks of storage of ethanol extract, the L value was 10.30, a value was 5.78 and b value was 15.83, which was significantly decreased compared to the initial value chromaticity L value 19.67, a value 12.48 and b value 30.98 there was.

Extraction condition L a b ΔE Solvent ^{1 )} 10.30 5.78 15.83 89.50 100/35 12.44 -4.14 9.93 88.00 100/50 10.76 -2.34 3.93 88.96 100/65 10.06 -1.38 2.08 89.38 250/35 13.70 -4.98 14.29 87.28 250/50 18.02 -6.22 17.20 84.48 250/65 15.16 -5.31 15.74 86.36 500/35 17.94 -5.86 18.96 84.57 500/50 17.48 -5.44 19.27 84.88 500/65 20.13 -6.01 21.31 83.13 ¹⁾ Extraction at 1: 6, 50 캜 for 5 hours.

The ginger extracts were stored at 30 ℃ for 4 weeks. The decrease and increase of chromaticity were slightly larger when compared with the treatments stored at 4 ℃. The b value of Supercritical Water Extract (250 bar) at 35 ℃ and 500 bar at 65 ℃ decreased in the case of 4 ℃ storage extract but increased in 30 ℃ storage extract. It was also found that the chromaticity of the supercritical extraction condition was significantly changed from 250 bar at 35 ℃ to 500 bar at 65 ℃, and the overall chromaticity (ΔE) was 77.54 and 73.86, respectively. Especially, when the color of extracts at 500 bar and 65 ℃ was compared with the initial values (L: 22.39, a: -6.91, b: 23.31) of L: 33.12, a: -5.21 and b: It was judged that the stability was lower than other extracts during storage.

Extraction condition L a b ΔE Solvent ^{1 )} 12.78 9.12 20.46 88.14 100/35 12.90 -4.10 9.01 87.69 100/50 10.30 -2.12 3.45 89.24 100/65 13.46 -1.91 1.70 87.22 250/35 27.86 -6.56 22.30 77.54 250/50 15.73 -4.69 16.45 85.98 250/65 16.30 -4.96 16.59 85.61 500/35 17.89 -4.70 19.77 84.60 500/50 20.08 -5.20 21.53 83.15 500/65 33.12 -5.21 31.27 73.86 ¹⁾ Extraction at 1: 6, 50 캜 for 5 hours.

(I) HPLC Of Ginger Extracts during Storage Gingerol  Component analysis

6-Gingerol (1-4'-hydroxy-3'-methoxyphenyl-5-hydroxy-3-decanone) is an antioxidant with a molecular weight of 294. It is known as an antioxidant that prevents oxidation of phospholipids by the FeCl ₃ -ascorbate system. In particular, xanthine oxidase, which is a substance that affects the generation of reactive oxygen species such as superoxide anion ). ≪ / RTI >

Table 12 shows the results of analysis of 6-gingerol, 8-gingerol, 10-gingerol, 6-showol and curcumin, which are the main useful components of supercritical and solvent extracts. In the case of supercritical extracts, the content of useful components tended to decrease slightly as the extraction temperature was increased under the same pressure conditions. The higher the extraction pressure, the higher the contents of useful components. Extracts containing 6 - gingerol, 8 - gingerol, 10 - gingerol, 6 - sucrose and curcumin were the most abundant extractables. The extracts of ethanol extracts were lower than those of supercritical extracts. In order to investigate the storage stability of ginger extracts, the ginger extracts were stored at 4 ° C and 30 ° C for 30 days, and the changes of useful components were measured by HPLC. The results are shown in Tables 13 and 14. The ginger extracts stored at 4 ℃ showed a tendency to remain unchanged compared with the initial contents, indicating that the extract of the present invention was stable at low temperatures. Also, the extracts stored at 30 ° C were somewhat less than the initial contents, but the decrease was not significant, indicating that they were somewhat stable even at high temperatures.

(%) Supercritical extract (pressure / temperature: bar / ℃) Solvent extract 100/35 100/50 100/65 250/35 250/50 250/65 500/35 500/50 500/65 6- Gingerol 18.76 10.88 5.35 18.38 18.95 14.35 13.76 14.98 11.35 10.46 8-Gingerol 5.34 2.69 1.30 5.35 5.46 4.06 3.93 4.30 3.35 2.90 10-Ginger roll 4.31 1.79 0.64 4.60 4.79 3.52 3.39 3.84 2.96 2.41 6- Shaw will come 2.42 2.99 1.76 2.50 2.42 1.91 1.81 1.98 1.54 1.40 curcumin 0.63 0.00 0.00 1.24 1.56 1.17 1.07 1.19 1.10 0.99

(%) Supercritical extract (pressure / temperature: bar / ℃) Solvent extract 100/35 100/50 100/65 250/35 250/50 250/65 500/35 500/50 500/65 6- Gingerol 17.24 9.47 4.91 17.64 16.91 12.44 12.45 13.66 10.79 8.15 8-Gingerol 4.95 2.83 1.25 4.94 4.89 3.71 3.61 3.92 3.05 2.56 10-Ginger roll 4.01 1.43 0.56 4.30 4.52 3.24 3.06 3.69 2.76 2.10 6- Shaw will come 2.16 2.68 1.60 2.16 2.71 1.63 1.51 0.98 1.24 1.18 curcumin 0.44 0.00 0.00 1.01 1.45 0.96 0.93 0.80 0.79 0.75

(%) Supercritical extract (pressure / temperature: bar / ℃) Solvent extract 100/35 100/50 100/65 250/35 250/50 250/65 500/35 500/50 500/65 6- Gingerol 14.83 8.26 4.72 13.41 12.23 12.78 11.81 12.38 10.49 8.86 8-Gingerol 3.75 2.03 0.92 3.94 3.47 3.68 3.42 3.61 2.98 2.61 10-Ginger roll 2.92 1.20 0.40 3.37 3.00 3.15 2.95 3.12 2.56 2.16 6- Shaw will come 1.70 2.37 1.34 1.78 1.59 1.66 1.61 1.59 1.38 1.37 curcumin 0.00 0.00 0.00 1.03 1.18 1.25 1.12 1.19 1.04 0.81

4-3. Evaluation of Antioxidant and Activity of Ginger Extract

4-3-1. Changes of physiological activity by ginger extraction method

(end) Ginger sample

Extracted ginger was extracted according to various solvent ratio (samples 1-4), extraction temperature (samples 5-8), extraction time (samples 9-12), supercritical extraction samples prepared according to extraction pressure and extraction temperature (sample 13 -21) were used for the test as shown in Table 15. < tb > < TABLE >

Sample number Extraction method, solvent, condition One Extraction ratio (ginger powder: EtOH = 1: 2), extraction temperature 50 캜, extraction for 3 hours 2 Extraction ratio (ginger powder: EtOH = 1: 4), extraction temperature 50 캜, extraction for 3 hours 3 Extraction ratio (ginger powder: EtOH = 1: 6), extraction temperature 50 캜, extraction for 3 hours 4 Extraction ratio (ginger powder: EtOH = 1: 8), extraction temperature 50 캜, extraction for 3 hours 5 Extraction ratio (ginger powder: EtOH = 1: 6), extraction temperature 35 ° C, extraction for 3 hours 6 Extraction ratio (ginger powder: EtOH = 1: 6), extraction temperature 50 캜, extraction for 3 hours 7 Extraction ratio (ginger powder: EtOH = 1: 6), extraction temperature 65 캜, extraction for 3 hours 8 Extraction ratio (ginger powder: EtOH = 1: 6), extraction temperature 80 캜, extraction for 3 hours 9 Extraction ratio (ginger powder: EtOH = 1: 6), extraction temperature 50 캜, 1 hour extraction 10 Extraction ratio (ginger powder: EtOH = 1: 6), extraction temperature 50 캜, extraction for 3 hours 11 Extraction ratio (ginger powder: EtOH = 1: 6), extraction temperature 50 캜, extraction for 5 hours 12 Extraction ratio (ginger powder: EtOH = 1: 6), extraction temperature 50 캜, extraction for 7 hours 13 Supercritical carbon dioxide extraction, extraction pressure 100 bar, extraction temperature 35 ℃ 14 Supercritical carbon dioxide extraction, extraction pressure 100 bar, extraction temperature 50 ℃ 15 Supercritical carbon dioxide extraction, extraction pressure 100 bar, extraction temperature 65 ℃ 16 Supercritical carbon dioxide extraction, extraction pressure 250 bar, extraction temperature 35 ℃ 17 Supercritical carbon dioxide extraction, extraction pressure 250 bar, extraction temperature 50 ℃ 18 Supercritical carbon dioxide extraction, extraction pressure 250 bar, extraction temperature 65 ℃ 19 Supercritical carbon dioxide extraction, extraction pressure 500 bar, extraction temperature 35 ℃ 20 Supercritical carbon dioxide extraction, extraction pressure 500 bar, extraction temperature 50 ℃ 21 Supercritical carbon dioxide extraction, extraction pressure 500 bar, extraction temperature 65 ℃ * Suppression of DPPH at supercritical carbon dioxide extraction, extraction pressure of 500 bar and extraction temperature of 80 ℃ seems to be less than 70% (100ppm), so it can be seen that there is a critical significance at 500 bar / 65 ℃.

(I) Radical  Measure erasure effect

The results of measuring the DPPH radical scavenging activity of the manufactured ginger extract samples are shown in FIGS. 8 and 9. The method of measurement was modified by Blois (1958), and 0.2 ml of each ginger extract sample diluted with 80% ethanol was added to 3 ml of ethanol at each concentration, and 4 × 10 ^-4 M DPPH (2,2 -diphenyl-1-picrylhydrazyl, Sigma-Aldrich Inc.) was added and the mixture was vortexed for 10 seconds. After incubation for 10 minutes at room temperature, the absorbance was measured at 517 nm. The DPPH radical scavenging ability was expressed as a percentage of the absorbance of the sample supplement and the negative control, while the positive control was Vit. C was used.

The DPPH radical scavenging activity was not significantly different according to the extraction method, but supercritical extract was higher than solvent extract.

(All) L6  Cell ATP  Effect on yield

ATP bioluminescence assay kit HS II (Roche, Germany) was used to measure ATP production in L6 cells. L6 cells were seeded in a 96-well plate at a concentration of 1 × 10 ⁵ cells / ml, cultured for 24 hours, treated with ginger samples at a concentration of 2 mM H ₂ O ₂ , cultured for 24 hours, ATP bioluminescence assay was performed and the absorbance was measured using a luminometer (Molecular Devices, USA). As shown in FIG. 10, it was confirmed that the ginger supercritical extract (sample 21) effectively increased the ATP production of L6 cells reduced by hydrogen peroxide compared to the general extract (sample 5).

While the present invention has been described with reference to exemplary embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. You will know. In addition, many modifications may be made to adapt a particular situation and material to the teachings of the invention without departing from the essential scope thereof. Accordingly, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention be construed as including all embodiments falling within the scope of the appended claims.

Claims (12)

(%) Of ginger supercritical extract prepared by supercritical extraction of ginger at a temperature of 200 to 300 bar and 30 to 40 ° C to extract an antimicrobial active ingredient for the genus Listeria . delete The method according to claim 1,
Wherein the Listeria (Listeria) genus extract the antimicrobial active ingredient, characterized in that to L. monocytogenes Quito Ness (L.monocytogenes). (%) Of ginger supercritical extract prepared by supercritical extraction of ginger at a temperature of 200 to 300 bar and a temperature of 60 to 70 ° C to extract an antimicrobial active ingredient for the genus Listeria . delete 5. The method of claim 4,
Wherein the Listeria (Listeria) genus extract the antimicrobial active ingredient, characterized in that to L. monocytogenes Quito Ness (L.monocytogenes). A method for preparing an antimicrobial ginger extract of Listeria with 5% v / v of ginger supercritical extract prepared by supercritical extraction of ginger at a temperature of 200 to 300 bar and 30 to 40 캜. delete 8. The method of claim 7,
Wherein the Listeria genus is L. monocytogenes. ≪ RTI ID = 0.0 > 21. < / RTI > A method for preparing an antimicrobial ginger extract of Listeria with 5% v / v of ginger supercritical extract prepared by supercritical extraction of ginger at a temperature of 200 to 300 bar and 60 to 70 캜. delete 11. The method of claim 10,
The Listeria (Listeria) genus process for preparing an antimicrobial extract of ginger, wherein the Listeria monocytogenes (L. monocytogenes).

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