KR100652490B1 - Method for extracting high concentrated antimicrobial substance from Japanese Horseradish by distilled extraction process - Google Patents

Abstract

The present invention relates to a method for extracting an essential oil component including allyl isothiocyanate, an antimicrobial component, from horseradish using distillation extraction. Distilled water, an oil bath and a concentrator are used to extract essential oil components present in the pretreated horseradish raw materials to extract allyl isothiocyanate, which is an antimicrobial component.

Wasabi, Allyl Isothiocyanate (AIT), Antibacterial, Distillation Extraction

Description

Method for extracting high concentrated antimicrobial substance from Japanese Horseradish by distilled extraction process

Figures 1a to 1d shows the AIT content of the extract for each part of wasabi by organic solvent extraction method using ether.

2 is a diagram analyzing the AIT content of horseradish extract by distillation extraction method according to the present invention by gas chromatography.

Figure 3 is a view showing the antimicrobial activity of pathogenic bacteria of volatile substances in horseradish root extract.

The present invention relates to a method for extracting an antimicrobial substance from wasabi, and more specifically, allyl isothiocyanate (C ₃ H ₅ CSN) from wasabi using a distilled extraction process. ) (Hereinafter also referred to as AIT) relates to a method of extracting high concentration and high yield by an environmentally friendly method.

Distillation extraction technology has been widely used in the past, but in the fields of food industry, chemical industry, pharmaceutical industry, material industry, environmental industry, energy consumption industry, such as purification, extraction, fractionation and recrystallization of various materials. In addition to producing high quality products and improving efficiency, researches on environmental conservation and energy saving, which are emerging as a social problem, are being actively conducted, and their application range is continuously expanding.

Generally, food additives include preservatives, flavors, coloring agents, preservatives, and chemical seasonings. Most of these are artificial synthetic additives, which are harmful to humans and cause cancer. However, natural additives are nutritious and functional, and there is little need to indicate the amount added to food.

On the other hand, conventionally known extraction methods when extracting the useful components using the organic solvent extraction method, there is a problem about the quality of the product due to the incorporation of impurities and the remaining of the organic solvent of harmful substances, Because of the process, not only the process is complicated, but also useful components can be lost (이), foreign substances by chemicals may be mixed, and the purity of the extract also has a problem.

Wasabi, a seasoning essential for sashimi or fish sushi, is often called "wasabi", which is Japanese. Wasabi uses stems and roots, which are divided into Asian and Japanese species. Eastern species are used to grind fresh raw materials on a grater, while Western species are dried and then ground and kneaded with water. Like mustard, the substance called sinigrin [C ₁₀ H ₁₆ KNO ₉ S ₂ · H ₂ O] is decomposed by the enzyme myrosinase to produce allyl isothiocyanate, which is both irritating and pungent. Indicates. Wasabi powder and soft wasabi on the market are yellowish green due to the addition of food coloring to enhance the texture, but the wasabi powder has almost no color.

Mustard contains about 30% of oil and glycoside glycine green, and oil is composed of palmitic acid, oleic acid, linoleic acid, eicosenoic acid, and erric acid. . Therefore, because it is cultivated a lot of oil, such as rapeseed (油菜), may be treated as rapeseed.

Seeds contain glycoside cinigrin, which gives a spicy taste, but cinigrin itself does not have an aesthetic effect, but when the seeds are crushed, the enzyme, myrosinase, acts on them, resulting in allyl isothiocyanate and glucose. Hydrolyzed, the volatile allyl isothiocyanate exhibits a strong irritating sensation.

Wasabia ( wasabia japonica Matsum ) is a high-quality spice crop used for sashimi, sushi, noodles, etc., which is a Japanese root, ripening, perennial, and half-colored plant. The root of the plant part of wasabi contains the most spicy taste. The main ingredient of the spicy flavor is allylic isothiocyanate, which is a volatile fragrance component, which makes up about 80% of essential oil extracted from wasabi, and 20 other volatile components. This was confirmed (Lee Sung-woo et al., 1997). Allyl isothiocyanate is also present in mustard radish, mustard, mustard, rapeseed, radish, cabbage and horseradish. have.

In the conventional organic solvent extraction method of extracting useful components using organic solvents, problems with the quality of the product are caused due to the incorporation of impurities and the presence of harmful organic solvents. Since the process is not only complicated, but also useful components may be lost, foreign substances (異 物質) by chemicals may be mixed, and the purity of the extract is also low.

Therefore, the present invention is to separate and recover the antibacterial material AIT and the like from wasabi in a high concentration and high yield by an environmentally friendly method.

In order to solve the above problems, the present inventors extract the useful components such as AIT from wasabi by using a distillation extraction technology, it is possible to extract the useful components of the extract at high concentration without loss, and also using an existing ether extraction method The present invention has been completed by finding out that it is a higher yield, lower cost, and environmentally friendly method.

Therefore, according to the present invention, there is provided a method for extracting an antimicrobial substance from horseradish using a distillation extraction method characterized in that the wasabi in powder form in the extraction container, and extracted with distilled water.

In addition, the method of the present invention is mixed 200g horseradish powder sample with 550mL of distilled water, and reacted for 80 to 100 minutes at 37 ℃, and then distilled by heating in an oil bath at 120 ℃ to produce more AIT components Characterized in that.

In addition, the method of the present invention is characterized by condensing and extracting 50 mL of the volatiles which are distilled for the first 50 to 70 minutes after distillation extraction.

In addition, the method of the present invention is characterized by separating, extracting and purifying antimicrobial components such as AIT at high concentration from horseradish.

Hereinafter, the present invention will be described in more detail.

In the distillation extraction process of the present invention, horseradish as a sample is prepared by pretreatment of stems, leaves and roots of south-eastern European wasabi (Horseradish, Armoracia rusticana ) in powder form of Korean wasabi (Japanese horseradish, Wasabia japonica ). .

Powder wasabi was experimented by adjusting experimental variables (temperature, time) by distillation extraction process (continuous type).

Powdered wasabi contains antibacterial ingredients. Powdered wasabi was charged to the extraction vessel, and in order to produce more AIT components, the reaction was carried out at 37 ° C. for 80 to 100 minutes, and then condensed volatiles evaporated while heating in an oil bath at 120 ° C. in the raw material. Extract specific substances present. The collected extract will contain a large amount of antimicrobial substances such as AIT. At this time, the extraction yield depends on the temperature, extraction time, the amount of distilled water used.

Distilled water used for extraction of wasabi is not toxic, it can be called an environmentally friendly separation process.

In the distillation extraction process of the present invention, it is preferable that the conditions of the reactor at the time of extraction are a temperature range of 120 ° C to 130 ° C, and an extraction time of 50 minutes to 70 minutes.

Hereinafter, the present invention is compared with the distillation extraction process according to the present invention by comparing the concentration, yield, cost, etc. of AIT by ether extraction, which is a representative method known in the art, among the extraction methods by organic solvents through the examples. Although described in detail, it should not be considered that the present invention is limited by the following examples.

Example 1

Extraction by Conventional Ethers

200 g of horseradish dried mixture is homogenized by mixing with 550 mL of distilled water, reacted at 37 ° C. for 100 minutes, and then shaken well with 1.5 times ether. Separate the aqueous layer and the ether layer using a separatory funnel, take only the ether layer, and then use a vacuum rotary evaporator (Tokyo Rikakikai Co., Ltd.), model name. EYELA}], and the residue remaining in the receiver flask was dissolved in 50 mL of ethyl alcohol.

According to the ether extraction method, the AIT concentration of the extract extracted from wasabi was analyzed by gas chromatography.

The allyl isothiocyanate (AIT) concentration of the extract was determined by a gas chromatograph with a flame ionization detector (FID) (Hewlett-Packard Development Company, California, USA). ) Product, model name HP 6890 series}, the column (column) was used as a 5% phenyl siloxane capillary column (HP-5, 0.32mm id × 30m). The temperature of the injection port was maintained at 220 ° C., helium gas was used as a carrier gas, and the split ratio was 1: 21.5. As a standard, AIT (Fluka Chemie AG, Switzerland Books) having a purity of 99.8% was used.

As a result, the AIT concentration of horseradish root extract by ether extraction method was 7,593ppm, the AIT concentration of Korean wasabi leaf extract was 2,956ppm, and the AIT concentration of stem extract was 21,658ppm, which was the total AIT content in 50mL of the extract. Indication, horseradish root extract was 380 mg, Korean wasabi leaf extract was 148 mg, stem extract was 108 mg [see Table 1], and retention time was about 6-7 minutes [Fig. 1].

Example 2

Extraction by distillation

200 g of horseradish dried was homogenized by mixing with 550 mL of distilled water, and reacted at 37 ° C. for 100 minutes, followed by a water bath at a temperature of 90 ° C., 100 ° C., 110 ° C., 120 ° C., 130 ° C., and 140 ° C. And distilled with a vacuum rotary evaporator (Tokyo Rikakikai Co., Ltd., model name EYELA}) while heating with an oil bath and collecting the extracts in 10 mL portions, and AIT concentration was collected. Analysis by gas chromatography determined the optimum extraction conditions.

Allyl isothiocyanate (AIT) concentration of the extract was measured by a flame ionization detector (FID-attached gas chromatographic meter (Hewlett-Packard Development Company, California, model name HP 6890 series), The column was a 5% phenylsiloxane capillary column (HP-5, 0.32 mm id × 30m), the inlet temperature was maintained at 220 ° C., helium gas was used as the carrier gas, and the split ratio was 1: As a standard product, AIT (Pulka Hemiage Co., Switzerland Books) having a purity of 99.8% was used.

As a result, as shown in Table 2, in the case of 90 ° C, only a small amount of AIT was detected in 50 ml of distilled extract in Western wasabi root, but AIT was not detected in Korean leaves and stems. At 100 ° C, small amounts of AIT were detected in 30 ml of distilled extract of horseradish roots and 40 ml of distilled extract of Korean leaves and stems. The most extracts containing AIT were horseradish roots, horseradish leaves and stems from Korea, which were 30mL distilled extracts at 120 ° C (11,256ppm), and 8,519ppm of AIT was extracted from 50mL distilled extracts and distilled at 120 ° C. Collecting 50 mL (average concentration, horseradish root, 9,398ppm; Korean horseradish leaves, 3,337ppm; Korean wasabi stem, 2,564ppm) is an efficient way to obtain the highest concentration of extract containing AIT Could know. In addition, in the case of 110 ° C and 130 ° C, although the concentration and the efficiency were lower than those of 120 ° C, a distillation extract having a high AIT concentration was obtained compared to other conditions (90 ° C, 100 ° C, 140 ° C). In the case of 140 ° C., a high concentration of AIT extract was distilled at the beginning of distillation, but due to the high temperature, wasabi extract was pressed into the flask container, and as the distillation time increased, the extraction efficiency decreased, and AIT was not extracted at 60 mL. The retention time of AIT in gas chromatography was 7-8 minutes [see FIG. 2].

On the other hand, the average concentration, content, yield, and unit cost of 50 mL of AIT distilled at 120 ° C., which is an optimal condition for distillation extraction, were described in organic solvent extraction methods and Etoh et al. (Etoh H, Nishimura A, Takasawa R, Yagi). A, Saito K, Sakata K, Kishima I, Ina K. ω-methylsulfinylalkyl isothiocyanates in wasabi, Wasabia japonica Matsum. Agric. Biol. Chem. 54: 1587-1589 (1990), and the results compared with the method according to Table 3 are shown.

According to Eto et al., 100g of horseradish roots contain 96.6mg of AIT, 22.8mg of leaves and 18.6mg of stems. And it was reported that the water content of horseradish general ingredient is about 80%.

Therefore, it can be said that 200 g of the dry powder used in the present invention corresponds to 1 kg of the raw material. In addition, it can be calculated that 1 kg of raw material contains 966 mg of horseradish root, 228 mg of leaves, and 186 mg of stems.

The average AIT concentration of the extract extracted by distillation according to the present invention was 9,398 ppm of horseradish root extract, 3,337 ppm of horseradish leaf extract of Korea, and 2,564 ppm of horseradish stem extract of Korea, which were 0.9398% and 0.3337%, respectively. Corresponding to 0.2564% and from this, the AIT content of 50 mL of the distillate extracted according to the distillation extraction method of the present invention can be obtained as follows.

1. AIT Contents of Horseradish Root Extract

50mL × 0.9398% ÷ 100 × 1,000 ≒ 470mg

The reason for multiplying 1,000 is because 1g is 1,000mg.

2. AIT Contents of Korean Horseradish Leaf Extract

   50mL × 0.3337% ÷ 100 × 1,000 ≒ 167mg

  3. AIT Contents of Korean Horseradish Stem Extract

  50 mL × 0.2564% ÷ 100 × 1,000 ≒ 128 mg.

Since the AIT content contained in 50 mL of the distillate extract is extracted from 200 g of powder, the yield is 48.7% for horseradish root, 48.7% for Korean horseradish leaves, compared with data such as Etoh. 73.2%, Korean wasabi stem, 68.8%, it can be seen that from this, the distillation extraction method according to the present invention is not only higher AIT concentration, higher yield, higher production cost, compared to the organic solvent extraction method using ether It was confirmed that it was inexpensive. And it is well known that the distillation extraction method according to the present invention using water compared to the organic solvent extraction method using an organic solvent is an environmentally friendly production method.

 Example 3

The antimicrobial effect of the horseradish extract extracted in Example 1 was measured against bacteria, yeast and mold, and the results are shown in Tables 4 and 5.

In each well of a 96-well flat-bottom microplate (Greiner bio-one GmbH, Freikenhausen, Germany) the bacteria were transferred to BHI broth. ), YM broth for yeast, 6% (w / v) malt extract broth sterilized with fungus {BD Diagnostic Systems, Difco, Sfax, Midland, USA} 50 microliters were dispensed, and 50 microliters of the preculture (24-48 hours, 25-37 degreeC, about 105 cfu / mL) of each strain was dispensed. 50 µl aliquots of horseradish extract diluted in 2-fold dilutions were added thereto, and bacteria were incubated at 37 ° C. for 48 hours, and yeast and mold at 25 ° C. for 48 hours. After the incubation was terminated, the growth of bacteria and yeast was checked by a microplate reader (OD; 560 nm) (Bio-Tek Instrument Inc., Winuskey, Vermont, USA). Product, model name EL 800}, and the fungus was visually confirmed the presence of the mycelia by the presence of the proliferation was judged. Minimum bactericidal concentrations (MBC) are described in Bamba et al., Am. J. Gastroenterol. 1997 Apr .; 92 (4): 659-62. ] Was measured using the method of. In this experiment, an inoculating loop of culture medium without growth was confirmed, and the bacterium was subjected to a standard plate count agar (PCA, Difco, Midland, USA), yeast. YM agar plate, the fungus streaked on the PDA plate was confirmed the presence of growth.

The minimum bactericidal concentration of horseradish root extract (24,654 ppm) against pathogenic and perishable microorganisms was anhydrous weight 0.05 to 0.20 g / mL, Salmonella typhimurium , Listeria monocytogenes , Vibrio parahaemolyticus , The bactericidal activity (MBC: anhydrous weight 0.20 g / mL) was shown against food poisoning bacteria and perishable bacteria such as Bacillus licheniformis . In addition, it showed strong bactericidal activity (MBC: anhydrous weight 0.05 g / mL) against Saccharomyces cerevisiae , a bread rot yeast. MBC for A. niger, P. roquefortii showed the strongest bactericidal activity with anhydrous weight of 0.02 g / mL (see Table 4).

Table 5 shows the minimum bactericidal concentrations of Korean horseradish leaf extract (9,057ppm) and stem extract (4,525ppm) against pathogenic and decaying microorganisms.

MBC of Korean horseradish leaf and stem extracts against food-borne pathogenic and rot-causing bacteria were 2.5 g / mL anhydrous and 5.0 g / mL anhydrous, respectively.

Saccharomyces cerevisiae , a bread rot yeast MBC of Korean horseradish leaf extract against Saccharomycopsis fibuligera was anhydrous weight 1.3 to 2.5g / mL, and MBC of Korean wasabi stem extract was anhydrous weight 5.0g / mL.

MBC of leaf extract against bread rot fungus showed the strongest bactericidal effect among the microorganisms with anhydrous weight of 0.6g / mL, and MBC of stem extract had a strong bactericidal effect with anhydrous weight of 1.3g / mL. Bread is a typical food with low water activity (0.75 to 0.8) compared to other foods, and the bacteria and yeast are involved in the decay caused by mold decay first. Therefore, the development of natural preservatives using Korean horseradish leaves and stem extracts having a strong bactericidal effect (MBC: anhydrous weight 0.6 to 1.3 g / mL) against bread rot fungi is considered to be an effective method for extending the shelf life of bread.

Example 4

The antimicrobial activity of the volatile substance in pathogenic bacteria of horseradish root extract extracted in Example 1 is shown in FIG. 3.

1 mL (15,000 mg / L) of horseradish root extract was added dropwise. As a result, Escherichia coli O157: H7 and Listeria monocytogenes Proliferation was inhibited by at least 80%, and other strains were completely inhibited in growth (left plate).

AIT Contents of Horseradish Root, Horseradish Leaves and Stem Extracts by Organic Solvent Extraction division Horseradish root Korean horseradish leaves Korean horseradish stem AIT concentration of the extract (ppm) 7,593 2,956 2,165 AIT content (mg) in 50 mL of extract 380 148 108

Hourly AIT Contents of Horseradish Root, Horseradish Leaves and Stem Extracts by Temperature by Distillation Extraction Distillation temperature (℃) Distillation amount (mL) AIT concentration of the extract (ppm) Root leaf stem 90 10 0 0 0 20 0 0 0 30 0 0 0 40 0 0 0 50 55 0 0 60 62 0 0 70 73 0 0 100 10 0 0 0 20 0 0 0 30 562 0 0 40 852 250 65 50 1,230 612 468 60 1,520 915 756 70 2,030 1,023 932 110 10 5,326 1,253 987 20 6,235 1,532 1,265 30 7,261 2,035 1,856 40 7,023 1,732 2,013 50 6,523 1,236 1,654 60 823 521 321 70 325 82 54 120 10 8,536 2,526 2,136 20 9,025 3,256 2,695 30 11,256 3,855 3,013 40 9,654 3,624 2,550 50 8,519 3,424 2,426 60 650 356 167 70 120 75 36 130 10 7,251 2,695 2,314 20 7,834 2,321 2,018 30 9,210 1,856 1,645 40 7,620 1,156 987 50 6,130 895 653 60 550 231 178 70 230 98 56 140 10 8,210 2,932 2,532 20 6,850 2,156 1,856 30 2,120 1,562 1,023 40 659 164 56 50 215 21 0 60 0 0 0 70 0 0 0

Comparison of AIT Content, Yield and Unit Price of Horseradish Root, Horseradish Leaf and Stem Extracts by Extraction Methods Horseradish root  Korean horseradish leaves Korean horseradish stem Organic solvent Distillation extraction Reference ^a (Organic Solvent) Organic solvent Distillation extraction Reference: Organic Solvents Organic solvent Distillation extraction Reference: Organic Solvents AIT content in mg of raw material (mg) 96.6 22.8 18.6 AIT concentration of the extract (ppm) 7,593 9,398 2,956 3,337 2,165 2,564 AIT content (mg) in 50 mL of extract 380 470 148 167 108 128 AIT content in mg of powder (mg) 966 ^b 228 ^b 186 ^b yield(%) 39.6 48.7 64.9 73.2 58.1 68.8 Unit price (KRW) 30 / mL 25 / mL 3 / powder g 18 / mL 16 / mL 3 / powder g 21 / mL 17 / mL 3 / powder g

^a Reference. Etoh H, Nishimura A, Takasawa R, Yagi A, Saito K, Sakata K, Kishima I, Ina K. ω-methylsulfinylalkyl isothiocyanates in wasabi, Wasabia japonica Matsum. Agric. Biol. Chem. 54: 1587-1589 (1990)

^b The average water content of wasabi and Korean wasabi is about 80%. Therefore, 200 g of dry powder corresponds to 1 kg of raw material.

Minimum Bactericidal Concentration of Bacteria, Yeast, and Fungi from Horseradish Extract microbe Minimum sterilization concentration (anhydrous weight g / mL) Pathogenic bacteria Bacillus cereus 0.20 Staphylococcus aureus 0.20 Staphylococcus mutans 0.20 Listeria monocytogenes 0.20 Escherichia coli O157: H7 0.20 Salmonella typhimurium 0.20 Vibrio vulnificus 0.20 Vibrio parahaemolyticus 0.20 Putrefactive bacteria Bacillus subtilis 0.20 Bacillus cereus 0.20 Bacillus licheniformis 0.20 Putrefactive yeasts Saccharomyces cerevisiae 0.05 Saccharomycopsis fibuligera 0.10 Putrefactive molds Aspergillus niger 0.02 Penicillium roquefortii 0.02 Ryzopus stolonifer 0.02 Mucor plumbeus 0.02

Minimum Bactericidal Concentration of Bacteria, Yeast, and Fungi of Korean Horseradish Leaves and Stem Extracts microbe Minimum sterilization concentration Leaf (anhydrous weight g / mL) Stem (anhydrous weight g / mL) Pathogenic bacteria Bacillus cereus 2.5 5.0 Staphylococcus aureus 2.5 5.0 Staphylococcus mutans 2.5 5.0 Listeria monocytogenes 2.5 5.0 Escherichia coli O157: H7 2.5 5.0 Salmonella typhimurium 2.5 5.0 Vibrio vulnificus 2.5 5.0 Vibrio parahaemolyticus 2.5 5.0 Decaying bacteria Bacillus subtilis 2.5 5.0 Bacillus cereus 2.5 5.0 Bacillus licheniformis 2.5 5.0 Perishable Yeast Saccharomyces cerevisiae 1.3 5.0 Saccharomycopsis fibuligera 2.5 5.0 Perishable Mold Aspergillus niger 0.6 1.3 Penicillium roquefortii 0.6 1.3 Ryzopus stolonifer 0.6 1.3 Mucor plumbeus 0.6 1.3

As described above, the distillation extraction method of the present invention has the advantage of higher concentration and yield of AIT than the organic solvent extraction method, low production cost and environmentally friendly, and wasabi-derived antimicrobial material distilled by the method of the present invention is natural It is expected to be actively used in various industries such as food, medicine and cosmetics as preservatives.

Claims (5)

Horseradish in powder form was charged to an extraction vessel, reacted for 80 to 100 minutes at 37 to 40 ° C. using a distillation extraction method, and then heated and distilled in an oil bath at 120 to 130 ° C. to obtain a high concentration of antimicrobial substance from wasabi. Extracting at a reactor temperature of 120 ° C. for 40 to 45 minutes. According to claim 1, to release the horseradish is used as a raw material from Korean horseradish japonica [Wasabia japonica (Japanese horseradish)] Removing the leaves and stems, and the document production aromatic cyano ruseutikana from [Armoracia rusticana (Horseradish)] leaves, stalks Or using roots. delete delete The antimicrobial substance extracted from wasabi according to the method of Claim 1 or 2.

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