KR101372885B1 - Method for the production of regioselective 7, 8, 4'- trihydroxyisoflavone by fermentation of Aspergillus oryzae - Google Patents

Abstract

The present invention relates to a method for preparing 7, 8, 4'-trihydroxyisoflavone (7, 8, 4'-trihydoxyisoflavone, and more particularly, has an antioxidant effect, a whitening effect and an inhibitory effect on tyrosinase activity. In order to efficiently produce 7, 8, and 4'-trihydroxyisoflavones, the soybean extract containing daidzin was bioconverted into Aspergillus oryzae to produce 7, 8, 4 '. It relates to a process for producing trihydroxyisoflavones.

Description

Method for the production of regioselective 7, 8, 4'- trihydroxyisoflavone by fermentation of Aspergillus oryzae}

The present invention utilizes a biotransformation system of Aspergillus oryzae to convert daidzin, which is present in soybean, into daidzein and in daidzein. , 8,4'-trihydroxyisoflavone (7, 8,4'- trihydoxyisoflavone) of the present invention relates to a method for the production of filamentous fungi Aspergillus oryzae ( biotransformation system) To hydrolyze glucose of carbon number 7 of the substrate diadzin and to specifically hydroxylate at position 8 carbon to finally produce 7, 8, 4'-trihydroxyisoflavone It is about.

Soy isoflavones are plant compounds that have similar properties to female estrogens. It is present in the form of aglycons, such as daidzein, genistein, and glycidin. Isoflavones have preventive effects on hormone-related diseases such as osteoporosis, cardiovascular disease, breast cancer, prostate cancer and colorectal cancer. , Chelating agents and antioxidant effects have been reported. It has the effect of lowering cholesterol and inhibiting platelet production and is associated with increasing immune regulation as well as preventing and treating menopausal disorders such as headache, dementia, inflammation and alcohol abuse. In addition, UV protection, skin aging inhibition, skin elastic fiber degrading enzyme inhibition and anti-inflammatory effect.

Soy isoflavones are present in most glycosides. It is reported that these glycoside-type isoflavones have 6 times or less bioabsorption with respect to the nonglycoside type, and are mainly absorbed in the form of the nonglycoside in the body.

Hydroxylated isoflavones have cholesterol lowering, cardiovascular, anticancer and anticarcinogenic abilities, and have anti-inflammatory and anti-allergic activity. It also inhibits protein tyrosine kinase and acts as an inhibitor of lipoxygenase. Hydroxide isoflavones are very valuable compounds that inhibit the incidence of cancer-related diseases. Since these hydroxylated compounds are difficult to synthesize by organic chemical methods, they can only be produced in biosynthesis or natural product extraction.

As described above, the isoflavone in the hydrated form can be separated and purified only through extraction from natural products or biosynthesis using microorganisms. Aspergillus Cytoids saitoi ) produced soy extract medium with 23 mg / l of 7, 8, 4'-trihydroxyisoflavone at 86 hours of incubation time, which is the highest yield and productivity in the literature (Hideo, E .; Ryoko, W). Hiromichi, O .; Shunro, K .; Toshihiko, O., Formation Mechanism for Potent Antiocidative o -Dihydroxyisoflavones in Soybeans Fermented with Aspergillus saitoi . Biosci Biotechnol Biochem 1999,63 (5), 851-858) However, industrial production requires higher output and higher productivity.

As a related prior patent, Korean Patent Publication No. 1020090100893, 'Luconostock Pseudomecentroides Ｋ-AP3 and high isoflavone concentrates using the same with high activity of α-galactosidase and β-glucosidase' As related to the stock pseudometheroides K-AP3 (Leuconostoc pseudomesenteroides K-AP3, accession no. As a strain having high activity of β-glucosidase and β-gluactidase, which degrades the indigestible oligosaccharides such as stachyose and raffinose into monosaccharides, It is described as having excellent value as a raw material for health functional food or medicine using the invention,

As a related prior patent, Korean Patent Publication No. 1020020032078, 'Method for producing high purity isoflavone aglycone by fermentation from soybean embryos, relates to a method for producing high purity isoflavone aglycone through fermentation from soybean embryos. Fermented soybean embryos using Bacillus licheniformis CK 11-4, a fermentation strain, and agglomerate that is predominantly absorbed with isoflavones in the form of glycosides in soybean embryos. By converting to aglycones, there is no objection to the human body, unlike the method of acid / alkali to convert to the conventional aglycone form, it is economical at a much lower process cost than the conventional enzyme treatment method, and the biological fermentation Extracted soybean embryos with a specific extraction solvent and then styrene-based porous By purification through recrystallization, it is described that high purity and a significantly improved recovery of the production method of the functional isoflavone aglycone.

The present invention has been made by the above necessity, and an object of the present invention is to provide a method for efficiently preparing 7, 8, 4'-trihydroxyisoflavone (7, 8,4'-trihydoxyiso flavone) from soybean extract. It is.

Another object of the present invention is to provide a composition for preparing 7, 8, 4'-trihydroxyisoflavone (7, 8,4 '-trihydoxyiso flavone).

In order to achieve the above object, the present invention provides a method for producing 7, 8, 4'-trihydroxy isoflavone comprising bioconverting the soybean extract into a microorganism derived from filamentous fungi.

In one embodiment of the present invention, the filamentous fungi-derived microorganism is preferably Aspergillus olease, and the Aspergillus olease is more preferably Aspergillus olease KACC40247, but is not limited thereto.

In another aspect, the present invention provides a method for producing 7, 8, 4'-trihydroxy isoflavone comprising the step of converting dydazine into microorganisms derived from filamentous fungi.

In another aspect, the present invention provides a method for producing 7, 8, 4'-trihydroxy isoflavone comprising the bioconversion of dyedzein to a microorganism derived from filamentous fungi.

In another aspect, the present invention provides a composition for preparing 7, 8, 4'-trihydroxyisoflavones containing Aspergillus olease as an active ingredient.

Hereinafter, the present invention will be described.

The present invention utilizes a biotransformation system of Aspergillus oryzae to position-specific 7, 8, and 4 'to daidzein and to daidzein. Provides a method for the efficient preparation of trihydroxyisoflavones (7, 8,4'-trihydoxyiso flavone).

The present invention provides a method for efficiently preparing 7, 8, 4'-trihydroxyisoflavone having excellent antioxidant function by bioconverting soybean extract to Aspergillus oryzae KACC 40247.

In addition, the present invention increases the production of 7, 8, 4'-trihydroxyisoflavones by adding the substrates Dydzin and Dyzezein during the biotransformation.

The present invention screened microorganisms capable of producing 7, 8, and 4'-trihydroxyisoflavones via Dyzedine using Dydazine of soybean extract as a substrate and selected the most productive strains.

As the screening strain is Aspergillus mate with (Aspergillus), A tricot deolma (Trichoderma) genus Fusarium (Fusarium), A penny room Solarium (Penicillium), An access Tallahassee Playa bus (Talaromyces flavus ), neurospora Chrysler Corporation (Neurospora crassa), Pseudomonas coarse pearl pure mouse (Monascus purpureus ), Klastosporium Cloud FIG sports riohyi to scan (Cladosporium cladosporioides), Rhizopus crispus come now to (Rhizopus oryzae), stearyl reeum Hi suteom (Stereum hirsutum), Lin is the hetero chuseu mousse (Zygorhynchus heterogamus) and Sepharose Ars coarse Cephaloascus fragrans (Table 1). Of these strains Aspergillus Highest in oryase (Aspergillus oryzae) The productivity of 7, 8, 4'-trihydroxyisoflavone was shown.

What is not indicated by the numbers in Table 1 is that there is no productivity of 7, 8, 4'-trihydroxyisoflavones, the microorganisms listed in Table 1, respectively, the Korean Agricultural Microbiological Resources Center, Rural Development Administration, Seodun-dong, Gwonseon-gu, Suwon-si, Gyeonggi-do, Korea Korean Agricultural Culture Collection, Suwon, Republic of Korea), and the Korea Institute of Biotechnology and Biotechnology Center (KCTC: Korean Collection for Type Culture, Daejeon, Republic of Korea) located in Yuseong-gu, Daejeon, Korea.

Also the Aspergillus Come now by the strain number (KACC44847, KACC44967, KACC40244, KACC41860, KACC40233, KACC40250, KACC41403, KACC40232, KACC44824, KACC40247, KACC44836) was screened, Aspergillus come now KACC40247 the highest 7,8, 4'-tree Hydroxyisoflavone productivity was confirmed.

In addition, the present invention is the step of culturing the potato solid medium using the KACC40247 strain, immersing domestic soybean, hot water extraction and high temperature and high pressure sterilization, soybean extract as a culture medium, culturing KACC40247 strain, at the culture time It is characterized in that it comprises the step of adding a substrate dyed zin and dyed zein.

The strain is preferably incubated in the range of about 30 ℃ the optimum temperature for producing 7, 8, 4'-trihydroxyisoflavones in soybean extract. In addition, the optimum pH for 7, 8, 4'-trihydroxyisoflavone production is preferably cultured in the range of about 6.0.

As can be seen from the present invention, the present inventors investigated a strain capable of converting a diazine, an isoflavone contained in soybean, into 7, 8, 4'-trihydroxyisoflavone via Dyzedine. A microorganism capable of producing high yields of 7, 8, 4'-trihydroxyisoflavones without any by-products was selected to produce 7, 8, 4'-trihydroxyisoflavones from soybean extracts.

1 is a result of HPLC analysis of a material converting soybean extract of the present invention into microorganisms. (Peak (1), Daidzin; Peak (2), Glycitin; Peak (3), 7, 8, 4'-trihydroxyisoflavone (7, 8, 4'- trihydroxyisoflavone; Peak (4), Genistin; Peak (5), Daidzein; Peak (6), Glytein; Peak (7), Genistein
Figure 2a shows the effect of production with temperature of microorganisms in 7, 8, 4'-trihydroxyisoflavone production. (●: 22 hours ○: 26 hours ■: 30 hours □: 34 hours)
Figure 2b shows the effect of pH-dependent production of microorganisms on 7, 8, 4'-trihydroxyisoflavone production. (●: pH 4 ○: pH 5 ■: pH 6 □: pH 7)
Figure 2c shows the effect of the production according to the rpm of the microorganisms in 7, 8, 4'-trihydroxyisoflavone production (●: 100 rpm ○: 200 rpm ■: 300 rpm □: 400 rpm).
Figure 3a shows the effect of the addition of dydazine over time in 7, 8, 4'-trihydroxyisoflavone production. (●: control ○: 0 hours ■: 3 hours □: 6 hours ▲: 9 hours △: 12 hours ◆: 15 hours)
Figure 3b is the result of the addition of didzin by concentration at the fixed time of Figure 3a in 7, 8, 4'-trihydroxyisoflavone production. (●: control ○: 208 mg / l ■: 416 mg / l □: 1248 mg / l ▲: 1664 mg / l)
Figure 4a shows the effect of the addition of dydzein over time in 7, 8, 4'-trihydroxyisoflavone production. (●: control ○: 6 hours ■: 9 hours □: 12 hours ▲ : 15 hours)
Figure 4b is the result of the addition of dydzein by concentration in the fixed time of Figure 4a in 7, 8, 4'-trihydroxyisoflavone production. (●: control ○: 127 mg / l ■: 254 mg / l □: 381 mg / l)
5 is dyed. Figure showing the structure and reaction of Dyzedine and 7, 8, 4'-trihydroxyisoflavone

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 examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Preparation Example 1. Preparation of Soybean Extract

In order to prepare a soybean extract containing dyed jin soaked for 12 hours at 26 ℃ soybean, and then hot water extraction for 1 hour at 105 ℃. After cooling at room temperature for about 1 hour and centrifugation at 3000rpm, the supernatant was filtered using Whatman filter paper. After recovering the filtrate, the soybean extract was prepared by sterilization at 121 ℃ for 15 minutes.

Example  1.7, 8, 4'- Trihydroxyisoflavones  Strain Screening to Produce

First, we identified the highest productivity of 7, 8, and 4'-trihydroxyisoflavones in flasks with fungi of different genus, and fungi of different species of the same genus. Screened with

In the present invention, all the strains were screened for 5 days at 28 ° C. in Potato dextrose agar medium (PDA 27 g / l) for screening strains producing 7, 8, 4'-trihydroxyisoflavones and then The soybean extract was recovered and used as a medium, and passaged to confirm production of 7, 8, 4'-trihydroxyisoflavone. As a culture tank for screening, a 100 mL baffle flask was used.

Strains Used 7 8 4'- trihydroxyisoflavone  ( mg / l) Aspergillus oryzae KACC 44847 19.54 Aspergillus oryzae KACC 44967 39.67 Aspergillus oryzae KACC 40244 51.77 Aspergillus oryzae KACC 41860 14.61 Aspergillus oryzae KACC 40233 54.19 Aspergillus oryzae KACC 40250 45.22 Aspergillus oryzae KACC 41403 51.38 Aspergillus oryzae KACC 40232 46.47 Aspergillus oryzae KACC 44824 23.29 Aspergillus oryzae KACC 44836 28.92 Aspergillus oryzae KACC 40247 61.57

Table 2 Aspergillus 7, 8, 4'-trihydroxyisoflavone production comparison table for each strain.

Example  2. Method of quantifying isoflavones

Quantification of the isoflavone component was performed by HPLC analysis. The culture supernatant was taken by time, extracted with ethyl acetate, centrifuged at 13000 rpm, the supernatant was evaporated using a vacuum centrifuge, dissolved in DMSO (dimethyl sulfoxide), and filtered with a 0.45 syringe filter. Used as a sample. HPLC analyzed isoflavones at 254 nm using Aglient series 1100. The column used for analysis was YMC C18 column, inject volume 5µl, flow rate 1.5ml / min. HPLC solvents and analytical conditions were analyzed for isoflavones under the conditions of Table 3.

Items Conditions Instrument  Agilent 1100 series / YMC C18 (50 * 4.6mm) Mobile phase  A: water (3% acetic acid), B: 50% acetonitrile (3% acetic acid (75:25 40:60 (12 min), 40:60 75:25 (3 min)) Detector UV (254 nm) Flow rate 1.5 ml / min Injection volume 5 μl '

Example  3. Temperature on microbial isoflavone production pH , And Stirring speed  Investigation of effects

In the present invention, in order to investigate the effects of temperature, pH, and stirring speed change on the isoflavone production of microorganisms, productivity was compared using soybean extract as a medium under various temperature, pH, and stirring speed conditions.

(A) Effect of temperature

First, in order to investigate the effect of temperature on the isoflavone production of the microorganism, it was incubated for 2 days at 22, 26, 30, 34 ℃ using a fermentor (fermentor). The culture solution was sampled every 6 hours during the cultivation, and the resulting product was quantified by the analysis method of Example 2. As a result (Fig. 2a) it was confirmed the maximum production at 30 ℃.

(B) Effect of pH

In addition, in order to investigate the effect of pH on the production of isoflavones of microorganisms, the culture was incubated for 2 days at pH 4, 5, 6, and 7 using the fermenter and the optimum temperature of (A). Sampling and analysis were carried out by the method (A) above. As a result (Fig. 2b), it was confirmed that the optimum pH is 6. This is equal to the initial pH of soybean extract.

(C) Influence of stirring speed

In the present invention, in order to investigate the effect of isoflavone growth on the stirring speed by using a fermenter, the stirring speed at 200, 300, 400, 500 rpm for 2 days at the optimum conditions identified in (a) and (b) Incubated. Sampling and analysis were carried out by the method (A) above. As a result (as shown in Figure 2c) it was confirmed that the optimum stirring speed is 300 rpm.

Example  4. During incubation DIDJIN  Productivity check by addition time and amount

Through the above process, the microorganism converted 100% of the didazine of the soybean extract, and the productivity of 7, 8, 4'-trihydroxyisoflavone was 61 mg / l. To increase the productivity, 416 mg / l of substrate Dydzin was added every 0, 3, 6, 9, 12, 15 hours. The result was 139 mg / l when didzin was added at 12 hours as shown in FIG. 3a.

In addition, 208, 416, 832, 1248, and 1664 mg / l of didazine were added to the concentration at the time of the experiment. When 1248 mg / l was added, 160 mg / l of 7, 8, 4'-trihydroxyisoflavone was produced and similar productivity was confirmed at higher concentrations (FIG. 3b).

Example  5. During incubation Dyed Jane  Productivity check by addition time and amount

As can be seen from the formula (1), 7, 8, 4'- trihydroxy isoflavones hydrolyze glucose of carbon number 7 of the dydazine and specifically hydroxylates the position at carbon number 8. When glucose of the seventh carbon is hydrolyzed, it becomes a died agent. This dyed agent is also used as a substrate for 7, 8, 4'-trihydroxyisoflavones.

Through the above process, Dydazine is converted to 100% to form Dyzedine, and Dydazine is converted to 100% to produce 7, 8, 4'-trihydroxyisoflavone.

In order to increase the productivity, 127 mg / l, which is a substrate Dyzede, was added every 6, 9, 12, and 15 hours. As a result (as shown in Figure 4a) was confirmed that the productivity of 79 mg / l when added Dyzein at 12 hours.

In addition, 127, 254, 381 mg / l of dyzezein was added to each concentration at the addition time obtained in the above experiment. Addition of 254 mg / l produced 160 mg / l of 7, 8, 4'-trihydroxyisoflavones (FIG. 4B).

Having described specific portions of the present invention in detail, those skilled in the art will appreciate that these specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (10)

7, 8, 4'-trihydroxyiso, comprising the step of inoculating Aspergillus oriase KACC40247 to a medium containing soybean extract and fermenting at a pH of 6, and adding dydazine 12 hours after the start of fermentation. Method for preparing flavones. delete The method according to claim 1, wherein the didazine is added to 1248 mg / l, 7, 8, 4'- trihydroxy isoflavones manufacturing method. The method of claim 1, wherein the preparation method comprises adding didzein at 12 hours after the start of fermentation. 7, 8, 4'-trihydroxyisoflavone manufacturing method. 7. The method according to claim 4, wherein the diezein is added at 254 mg / l. The method of claim 1, wherein the method is performed at 25-35 ° C. 7, 8,4'-trihydroxyisoflavone production method. delete delete delete delete

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