JPH0551561B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to an industrial antibacterial agent (hereinafter simply referred to as an antibacterial agent) used to prevent quality deterioration due to microbial contamination of various industrial products such as foods and cosmetics. The industrial antibacterial agent of the present invention exhibits antibacterial activity against bacteria such as Bacillus, Clostridium, and Pseudomonas. Therefore, the industrial antibacterial agent of the present invention is used to prevent quality deterioration due to such microbial contamination, particularly to prevent food quality deterioration. Conventional technical background 2-methoxy-4,4'-dihydroxy-5-α,α-dimethylallyl chalcone, which is the active ingredient of the antibacterial agent according to the present invention, is commonly called licochalcone A and is present in licorice roots. It is a type of flavonoid, and its synthesis method has also been reported. [T.
Saitoh & S. Shibata: “Tetrahedron Letters” 4461, (1975)],
Expelled students et al.: Kagaku Gakuho (China) 37 , 289 (1979)]. However, there are currently no reports regarding the antibacterial properties of 2-methoxy-4,4'-dihydroxy-5-α,α-dimethylallyl chalcone (hereinafter referred to as licochalcone A). Although it has been known for a long time that licorice root has antibacterial properties, there are few reports that directly mention the relationship between antibacterial properties and components, and the antibacterial components of licorice root have not yet been fully elucidated. Incidentally, licorice root extract alone has almost no antibacterial properties. In the process of investigating the physiological activities of various flavonoids, the present inventors discovered that licochalcone A exhibits strong antibacterial activity against Bacillus subtilis, a type of bacteria, and also As a result of investigating the antibacterial properties against microorganisms, Bacillus stearothermophilus, Bacillus stearothermophilus,
Bacillus brevis, Clostridium sporogenes
It was also found that the growth of Pseudomonas aeroginosa was significantly suppressed. Problems to be Solved by the Invention The present invention was made based on the above-mentioned knowledge that licochalcone A exhibits excellent antibacterial activity against various microorganisms. The purpose of the present invention is to provide an antibacterial agent containing licochalcone A as an active ingredient, which can be used to prevent quality deterioration of industrial products due to microbial contamination. The present invention will be explained in detail below. Means for Solving the Problems Although licochalcone A used as an active ingredient in the present invention can be synthesized by various methods, the method of the above-mentioned Hansei et al. is advantageous because it can be obtained in high yield. In addition, as mentioned above, licochalcone A is present in licorice roots, so licorice roots are extracted with an organic solvent such as ethyl alcohol, ethyl acetate, diethyl ether, or dichloromethane, and the resulting extract is used with a synthetic adsorbent ( For example, Diaion HP-2MG)
or ion exchange resin (e.g. Dowex 1x
It can also be obtained by purification using 2). In addition, since licochalcone A is sparingly soluble in water, it is contained in a large amount in the residue obtained by extracting glycyrrhizin as a sweet ingredient from licorice root. Therefore, using this residue as a raw material is a simple method for purifying the extract. This is a good idea from the standpoint of environmental conservation and effective use of resources. Next, a method for synthesizing licochalcone A and a method for preparing it from licorice root will be illustrated. Synthesis of licochalcone A: Using 25 g of β-resorcyl aldehyde as a starting material, the method of SAEED AHMAD KHAN et al. [Indian Journal of Chemistry 22B ,
276 (1983)], 2-methoxy-4-(o-
prenyl)-benzaldehyde (compound)
Got 10g. Next, 5 g of 2-methoxy-4-hydroxy-5-α,α-dimethylallylbenzaldehyde (compound) was obtained by using 10 g of the above compound in accordance with the method of the above-mentioned student. 5 g of the compound obtained above and 4 g of 4-hydroxyacetophenone were subjected to aldol condensation to obtain 7 g of crude licochalcone A, which was subjected to silica gel column chromatography (column: 4 cm x 100 cm, silica gel: Fujigel).
BW-127, developing agent: benzene-ethyl acetate (80-20)], and then recrystallized in methanol solution, lycochalcone
Obtained 4.5g of A. Lycochalcone A obtained by this synthesis method has a melting point of
The temperature was 101°C, and the mass spectrometry, ultraviolet absorption spectrum, infrared absorption spectrum, and proton nuclear magnetic resonance spectrum data agreed with literature values. Preparation of licochalcone A from licorice root: After immersing licorice root in warm water at 60°C overnight, the solid content was drained and air-dried at a temperature below 60°C. Collect 2 kg of this dry solid content, add 15 ml of ethyl alcohol (95% V/V),
After soaking at 50°C overnight, solid-liquid separation was performed. After concentrating the obtained solution to 1400 ml, 600 ml of water was added and mixed. 10 g of activated carbon powder was added to this mixed solution, stirred for a while, and then left overnight. This product was then centrifuged to remove the precipitate, yielding 1900 ml of a clear brown liquid (this product is referred to as the component). Next, 1,600 ml of the above brown liquid was poured into a column with an inner diameter of 4 cm, which had been filled with acetic acid type Diaion WA-30 washed with 70% ethyl alcohol to a height of 90 cm, at an SV≒0.5 (space velocity). The liquid was passed through. Then fill the column with 70% ethyl alcohol.
After washing with 2000ml 95% ethyl alcohol
Lycochalcone A was eluted by flowing 4000ml. This eluate was concentrated and dried to give 12 g of a reddish brown solid.
I got it. (This thing is called an ingredient). 10g of the solid obtained as described above, 50%
Suspended in 200ml of ethyl alcohol, the inner diameter
Amberlite
After washing with 3,000 ml of 80% ethyl alcohol, pour 3,000 ml of 80% ethyl alcohol, and remove licochalcone A.
An eluate was obtained, which was concentrated and dried to give 5 g of a yellowish brown solid (this material is referred to as a component). 4 g of the yellowish brown solid obtained as described above was subjected to silica gel column chromatography [Column: 4 cm x 100 cm, silica gel: Fujigel]
BW-127, developer: benzene-ethyl acetate (80-20)] and Sephadex LH-20 (column: 2.2 cm x 145 cm, developer: methanol) and purified by molecular sieve chromatography.
Recrystallize licochalcone in methanol aqueous solution
Obtained 1.3g of A. Table 1 shows the content of licochalcone A in each component obtained in the extraction and purification process of licochalcone A thus obtained.

[Table] The measurement of licochalcone A was carried out as follows. Absolute calibration curve method using high performance liquid chromatography Equipment: Shimadzu Lc4A Column: Zolbax ODS φ4.6mm x 200mm
Temperature: 50°C Mobile phase: 70% methanol Flow rate: 1.5 ml/min Detection: Absorption at 295 nm The antibacterial agent according to the present invention contains the above-mentioned licochalcone A as an active ingredient, and may be used alone or in other forms. It may be used in combination with preservatives, etc. When using the antibacterial agent of the present invention, since it hardly dissolves even if added directly to water, it may be made into an emulsion using, for example, sucrose fatty acid ester, polyglycerin fatty acid ester, Quillaja saponin, or gelatin. It is preferable to use it by dissolving it in alcohol. Further, the appropriate amount to be used varies depending on the contents and pH of the target product, but it may be in the range of approximately 1 to 200 ppm as licochalcone A. For example, in the case of soup (liquid seasoning) products such as udon soup,
As licochalcone A, practical storage stability is observed at 5 to 15 ppm. In addition, for noodles such as boiled soba and raw soba, 10 to 30 ppm should be mixed in, or 15 to 30 ppm should be mixed in.
A method of immersion in a 50 ppm solution is appropriate. The antibacterial agent of the present invention exhibits particularly strong antibacterial activity against bacteria that form heat-resistant spores such as Bacillus and Clostridium, and is effective against many yeasts and filamentous fungi in a practical addition amount range. No antibacterial activity was observed. For foods containing these microorganisms, it is desirable to use treatments and additives to suppress or kill the growth of yeast and filamentous fungi. For example, heating treatment at 70 to 80°C is exemplified.
In addition to this treatment, heating (heating) treatment necessary for manufacturing and processing the product can of course be used. Therefore,
It becomes possible to manufacture high-quality products without the need for intense high-temperature heating that adversely affects the quality of products, which is done to kill heat-resistant spores. The antibacterial activity of licochalcone A, which is the active ingredient of the present invention, will be specifically explained below with reference to Examples. Example 1 The antibacterial activity of licochalcone A obtained by the above-mentioned synthesis method and preparation method from licorice root was investigated by the following test method. Test method: Apply the liquid medium dilution method and test microorganisms at 2.5~
It was added to each medium in an amount of 5×10 ³ and the presence or absence of proliferation after culturing for 7 days was determined by the turbidity of the medium. The determination results are expressed as the minimum amount added (as solid content) that can suppress the growth of microorganisms. The culture medium and culture conditions of the test microorganisms are shown in Table 2, and the test results are shown in Table 3. For comparison, Table 3 also shows the results of similar tests for each of the above components obtained in the preparation method from licorice root.

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[Table]
Licochalcone A and the components were dissolved in ethyl alcohol and added. As shown in Table 3, licochalcone A, whether synthetic or natural, exhibits strong antibacterial activity against Bacillus species such as Bacillus subtilis, Clostridium sporogenes, and Pseudomonas aeruginosa, and therefore cannot be used in foods contaminated with these microorganisms. It is effectively used to prevent quality deterioration of industrial products such as cosmetics. Example 2 Licorice roots from Xinjiang were thoroughly washed with warm water, and the solid matter was dried at a temperature of 60°C or lower. Add 50 kg of 80% ethyl alcohol to 10 kg of this dry solid,
Extraction was performed at 40-45°C for 20 hours with gentle stirring, followed by solid-liquid separation. The solid matter was further washed with 20 portions of 80% ethyl alcohol to obtain a total of approximately 65 portions of extract. Add 40% of water to this extract while stirring to create a suspension.
Column packed with Diaion HP-2MG approx. 25% in 50% ethyl alcohol (inner diameter 15cm, height 150
cm) at SV≒1. Next, 25% of 50% ethyl alcohol and 100% of 65% ethyl alcohol were flowed at the same rate, and the portion of the effluent in which the ratio of licochalcone A to solid content was 30% or more was collected. The effluent was concentrated to obtain about 200 g of licochalcone A with a purity of 35%. Add 10g of this licochalcone A (purity 35%) to Quillayan saponin (Quillayanin C-
100 (Maruzen Kasei Co., Ltd.) and 5 g of sucrose fatty acid ester (DK-SS, Daiichi Kogyo Seiyaku Co., Ltd.) were added, and the volume was made up to 500 ml with 70% ethyl alcohol to obtain antibacterial agent A. Furthermore, 10 g of the above-mentioned licochalcone A (35% purity) was repeatedly recrystallized in aqueous ethyl alcohol to obtain about 2 g of licochalcone A with a purity of 93%. 1g of this product contains 1g of Quillaja saponin and sucrose fatty acid ester DK
-1 g of SS was added and dissolved in 100 ml of 70% ethyl alcohol to obtain antibacterial agent B. Antibacterial agent A containing licochalcone A according to the present invention obtained as described above as an active ingredient
and Antibacterial Agent B, a preservation test of udon soup stock was conducted as follows. Extract 1500g of dried bonito flakes and 200g of kelp with 100% hot water for 30 minutes, then add 5% light soy sauce to the white soup stock filtered through cotton cloth.
, 1 mirin, 1000 g of sugar and 500 g of salt were added to obtain udon soup. Prepare 1 ml or 3 ml of antibacterial agent A and antibacterial agent B, 3 ml of ethyl alcohol, and no additives for 1 part of this udon soup, and add 100 ml each to a polypropylene bag (160 ml) while keeping warm at 80°C. × 110 mm) was filled and sealed, placed in a steam-insulated room at 80°C for 10 minutes, and then cooled with water. No macroscopic or sensory differences were observed in the udon soup to which antibacterial agent A and antibacterial agent B were added compared to those without the addition. 20 bags each of the above udon noodles
The bags were stored in a constant temperature room at 37°C for up to 21 days, and the development of turbidity was periodically observed, and each bag was sampled aseptically to measure the number of bacteria. The results are shown in Table 4 along with the number of bacteria as the decay rate for those with strong turbidity. The udon soup stock with and without ethyl alcohol all spoiled after 3 days, and a strong turbidity and a large increase in the number of bacteria were observed. On the other hand, when antibacterial agent A and antibacterial agent B were added, no turbidity or increase in the number of bacteria was observed. In addition, all samples from the antibacterial agent-added area were subjected to bacterial count, pH, and sensory tests after 21 days, but no abnormalities were observed. In addition, a rapid decrease in the number of bacteria was observed in the plots in which 3 ml of antibacterial agent A and antibacterial agent B were added, indicating that the antibacterial agent of the present invention also has a bactericidal effect.

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Claims (1)

[Claims] 1 2-methoxy-4,4'-dihydroxy-5-
An industrial antibacterial agent characterized by containing (α,α-dimethylallyl)chalcone as an active ingredient. 2. The antibacterial agent according to claim 1, wherein the industrial antibacterial agent is a food-grade antibacterial agent.