TW202014520A - Preparation method of 3'-hydroxygenistein - Google Patents

Abstract

A preparation method of 3'-hydroxygenistein includes following steps. A recombinant microorganism which expresses tyrosinase is constructed. A reaction substrate formulation step is performed, wherein the reaction substrate includes borate, ascorbic acid, and genistein. Then, the recombinant microorganism is inoculated into the reaction substrate for performing a biotransformation reaction step to obtain a biotransformation substance. Further adding a hydrochloric acid solution to the biotransformation substance for performing a termination reaction step to obtain a reaction solution including 3'-hydroxygenistein. Therefore, the preparation method of 3'-hydroxygenistein of the present disclosure can produce a high-purity 3'-hydroxygenistein in a large amount.

Description

Preparation method of 3'-hydroxygenistein

The invention relates to a method for preparing an oxygen-containing organic compound, in particular to a method for preparing 3'-hydroxygenistein.

Genistein is one of the main active factors of soybean isoflavones and has various physiological functions. Genistein is similar in structure to mammalian estrogen-estradiol, and has an estrogen active group-diphenolic hydroxyl group. Therefore, genistein has various physiological activities such as estrogen-like activity, for example, it can prevent osteoporosis, Improve menopausal syndrome, prevent cancer (eg breast, colon, lung, prostate, and skin cancer), prevent Alzheimer's disease, delay aging, improve menstrual discomfort, lower cholesterol, and regulate blood lipids.

3'-hydroxygenistein is a derivative of genistein. Changes in the structure of isoflavones often result in significant changes in the biological activity of the compounds. For example, genistein at a concentration of 30μM~60μM will promote the melanocytes to accelerate the production of melanin. However, after bioconversion, 3'-hydroxygenistein has a low-dose, highly effective skin whitening effect, before its bioconversion Repellent has the opposite characteristics. Another study pointed out that 3’-hydroxygenistein can inhibit T47D tumorigenic breast epithelial cells Growth activity, as well as inhibition of HIV-1 integrase (HIV-1 integrase), anti-inflammatory and hepatoprotective activities.

However, the low productivity of 3'-hydroxygenistein is still the main factor hindering its use in health foods or pharmaceutical products. Although there are currently methods for preparing 3'-hydroxygenistein, its yield is still limited, and the purity of the prepared 3'-hydroxygenigenin is not high. Therefore, how to develop a better method to increase the yield and purity of 3'-hydroxygenistein has become the current development direction of related industries.

One aspect of the present invention is to provide a method for preparing 3'-hydroxygenistein, which includes the following steps. Constructing a genetically recombinant microorganism, transforming a circular recombinant plastid into a microbial expression system to obtain a genetically recombinant microorganism, wherein the circular recombinant plastid contains tyrosinase as shown in sequence identification number 1 (tyrosinase) gene. A reaction matrix preparation step is performed, wherein the reaction matrix comprises borate at a final concentration of 400 mM to 600 mM, ascorbic acid at a final concentration of 10 mM to 40 mM, and genistein at a final concentration of 500 ppm to 2000 ppm. A bioconversion reaction step is carried out, in which a genetically recombinant microorganism is inoculated in the reaction matrix, and a bioconversion reaction time is cultivated at a bioconversion reaction temperature and a bioconversion reaction ventilation rate at a bioconversion reaction stirring speed to obtain a bioconversion Convert substances. A further reaction termination step is carried out by adding a hydrochloric acid solution to the bioconversion material and at a termination reaction temperature and a termination ventilation volume The reaction is performed at a termination stirring speed for a termination reaction time to obtain a reaction liquid, wherein the reaction liquid contains 3'-hydroxygenistein.

According to the aforementioned preparation method of 3'-hydroxygenistein, wherein the microorganism expression system may be Escherichia coli .

According to the aforementioned preparation method of 3'-hydroxygenistein, the preparation step of the reaction matrix may include the following steps. Dissolve genistein in dimethyl sulfoxide to obtain a genistein solution. Dissolve ascorbic acid in water to obtain a solution of ascorbic acid. After dissolving the borate in hot water, adjust the pH to pH 8 to pH 10 to obtain a borate solution. Then, the genistein solution and the ascorbic acid solution are added to the borate solution to obtain the reaction matrix.

According to the aforementioned preparation method of 3'-hydroxygenistein, the bioconversion reaction temperature may be 40°C to 60°C. The ventilation rate of the bioconversion reaction may be 2L/min to 5L/min, and the stirring rate of the bioconversion reaction may be 250rpm to 350rpm. The bioconversion reaction time can be 60 minutes to 90 minutes.

According to the aforementioned preparation method of 3'-hydroxygenistein, the final concentration of the hydrochloric acid solution may be 100 mM to 200 mM.

According to the aforementioned preparation method of 3'-hydroxygenistein, the reaction termination temperature may be 40°C to 60°C. The termination reaction ventilation volume may be 2L/min to 5L/min, and the termination reaction stirring speed may be 250rpm to 350rpm. The termination reaction time may be 5 minutes to 15 minutes.

According to the aforementioned preparation method of 3'-hydroxygenistein, an extraction step may be further included. Ethyl acetate is added to the reaction solution, and shaking is repeated at a shaking speed for 3 times.

According to the aforementioned preparation method of 3'-hydroxygenistein, the volume ratio of the reaction solution to ethyl acetate may be 1:1 to 1.5:1.

According to the aforementioned preparation method of 3'-hydroxygenistein, the shaking speed can be 200 rpm to 300 rpm, and each shaking time can be 2 minutes to 20 minutes.

In this way, the preparation method of 3'-hydroxygenistein in the present invention uses genetically engineered microorganisms to convert 3'-hydroxygenigenin, and the preparation method can be used for large-scale industrial production, because the starting product, The production processes such as bioconversion reaction conditions make the 3'-hydroxygen genistein produced with stable quality and high purity, so it can solve the problem of low productivity of 3'-hydroxygenistein.

100‧‧‧3’-hydroxygenin preparation method

110, 120, 130, 140 ‧‧‧ steps

In order to make the above and other objects, features, advantages and examples of the present invention more obvious and understandable, the description of the attached drawings is as follows: Figure 1 illustrates the preparation of 3'-hydroxygenistein in an embodiment of the present invention Flow chart of the steps of the method; Figure 2 shows a statistical chart of the mass production of mycoplasma of the genetically recombinant microorganism of the present invention; Figure 3 shows the 3'produced by the preparation method of 3'-hydroxygenistein of the present invention -Statistics of the output of hydroxygenistein formulated liquid raw materials; Figure 4 shows the analysis result of the stability of the liquid raw material containing 10% 3'-hydroxygenistein; and Figure 5 shows the analysis of the stability of the liquid raw material containing 20% 3'-hydroxygenistein Results graph.

[Preparation method of 3’-hydroxygenistein]

Please refer to FIG. 1, which is a flow chart of the steps of a method 100 for preparing 3'-hydroxygenistein according to an embodiment of the present invention. In FIG. 1, a method 100 for preparing 3'-hydroxygenistein includes step 110, step 120, step 130, and step 140.

Step 110 is to construct a genetically recombinant microorganism, which transforms a circular recombinant plastid into a microbial expression system to obtain a genetically recombinant microorganism, wherein the circular recombinant plastid contains a casein as shown in sequence identification number 1 Tyrosinase gene. Preferably, the microorganism expression system may be Escherichia coli .

Step 120 is to perform a reaction matrix preparation step, wherein the reaction matrix comprises borate at a final concentration of 400 mM to 600 mM, ascorbic acid at a final concentration of 10 mM to 40 mM, and genistein at a final concentration of 500 ppm to 2000 ppm. Preferably, the reaction matrix preparation step may include the following steps. Dissolve genistein in dimethyl sulfoxide to obtain a genistein solution. Dissolve ascorbic acid in water to obtain a solution of ascorbic acid. After dissolving the borate in hot water, adjust the pH to pH 8 to pH 10 to obtain a monoborate solution. Then, the genistein solution and the ascorbic acid solution are added to the borate solution to obtain the reaction matrix.

Step 130 is a bioconversion reaction step, in which a genetically modified microorganism is inoculated into the reaction matrix, and a bioconversion reaction time is cultivated at a bioconversion reaction temperature and a bioconversion reaction aeration rate at a bioconversion reaction stirring speed, to Obtain a biological conversion substance. Preferably, the bioconversion reaction temperature may be 40°C to 60°C. The ventilation rate of the bioconversion reaction may be 2L/min to 5L/min, and the stirring rate of the bioconversion reaction may be 250rpm to 350rpm. The bioconversion reaction time can be 60 minutes to 90 minutes.

Step 140 is to perform a termination reaction step, which is to add a hydrochloric acid solution to the bioconversion material, and react at a termination reaction temperature and a termination ventilation rate at a termination stirring speed and a termination reaction time to obtain a reaction liquid, wherein The reaction solution contains 3'-hydroxygenistein. Preferably, the final concentration of the hydrochloric acid solution may be 100 mM to 200 mM. The reaction termination temperature may be 40°C to 60°C. The termination reaction ventilation volume may be 2L/min to 5L/min, and the termination reaction stirring speed may be 250rpm to 350rpm. The termination reaction time may be 5 minutes to 15 minutes.

In addition, the preparation method 100 of 3'-hydroxygenistein can further include an extraction step, in which ethyl acetate is added to the reaction solution, and the shaking is repeated at a shaking speed for three times. Preferably, the volume ratio of the reaction solution to ethyl acetate may be 1:1 to 1.5:1. The shaking speed can be 200rpm to 300rpm, and each shaking time can be 2 minutes to 20 minutes.

The following specific test examples are used to further illustrate the present invention, in order to benefit those of ordinary knowledge in the technical field to which the present invention belongs, without excessive interpretation. The present invention is fully utilized and practiced under the circumstances, and these test examples should not be regarded as limiting the scope of the present invention, but are used to illustrate how to implement the materials and methods of the present invention.

[Test example] 1. Construct genetically recombinant microorganisms

In this test example, a circular recombinant plastid was constructed first. The expression vector used was pETDuet-1 ^TM vector (purchased from Novagen), and the insert was the tyrosinase gene shown in sequence identification number 1, which was derived from Bacillus megaterium ( Bacillus megaterium ). In the experiment, the forward primer as shown in sequence identification number 2 and the reverse primer as shown in sequence identification number 3 were used to genetically encode Bacillus megaterium BCRC 10608 strain (purchased from the Center for Biological Resources Conservation and Research of the Food Industry Development Institute) Amplification, where the forward primer is designed with the restriction enzyme Nco I recognition position ( Nco I: CCATGG) sequence, and the reverse primer is designed with the restriction enzyme Sac I recognition position ( Sac I: GAGCTC) sequence. Sequence the amplified tyrosinase gene, and clone the inserted fragment into the pETDuet-1 ^TM vector after being cut by Nco I/ Sac I to obtain the constructed circular recombinant plasmid PETDuet-BmTYR. Then, the circular recombinant plastid pETDuet-BmTYR was transformed into Escherichia coli BL21 (DE3) strain (purchased from the Center for Biological Resources Preservation and Research of the Food Industry Development Institute) to form a genetically recombinant microorganism. The method of transformation may include, but not limited to, chemical methods such as calcium chloride treatment or physical methods such as electroporation.

2. Preparation of 3’-hydroxygenistein 2.1. Preparation of genetic microorganism powder

After activating the genetically-recombinant microorganisms constructed as described above, a single colony was picked and cultured in two bottles of 100 mL LB medium (containing 100 μg/mL ampicillin) and cultured at 37°C and 180 rpm until the next day. Two bottles of a total of 200 mL of inoculum were added to a fermentation tank containing 5 L of LB medium (containing 100 μg/mL ampicillin), and cultured at 30-40° C., 100-200 rpm, and 2-5 L/min ventilation. When the bacterial density grows to an optical density value (OD ₆₀₀ ) of 0.6 to 1, 5 mL of 0.1 mM IPTG is added, and the culture is continued at 18° C. and 180 rpm for 24 hours to induce tyrosinase gene expression. Collect the fermentation broth and pour it into four centrifuge bottles, centrifuge at 4500 rpm for 15 minutes at 4°C, remove the supernatant, pour the remaining fermentation broth, and wash out the precipitating bacteria, then centrifuge at 4500 rpm Centrifuge at 4°C for 15 minutes, and repeat until the fermentation broth is completely centrifuged. A small amount of PBS was used to disperse the precipitated bacterial cells, and then filled with PBS, and centrifuged at 4500 rpm at 4°C for 15 minutes to remove the supernatant. Then add a small amount of PBS to disperse the precipitated cells, concentrate into two bottles of centrifuge bottles, refill with PBS, centrifuge at 4500 rpm at 4 ℃ for 15 minutes, remove the supernatant to obtain precipitated cell clumps. Finally, the pelleted cell clumps were washed away with 150 mL each, divided into 50 mL centrifuge tubes, placed in ice to -80°C refrigerator, and lyophilized the next day. Freeze-dry for about 4 to 5 days. After the completion of the determination, weigh the weight and deduct the empty tube weight to obtain the dry weight of the bacterial powder.

Please refer to FIG. 2 and Table 1 below, which is a statistical chart of the mass production of the genetically recombinant microorganism of the present invention, which is the statistical result of mass production of the genetic powder of the genetically recombinant microorganism of the present invention 5 times according to the foregoing steps. Results in Figure 2 and Table 1 It is shown that the yield of the bacterial powder prepared by the foregoing method can reach more than 12 grams each time, and the resulting bacterial powder of the genetically-recombinant microorganism will be subsequently used for biological conversion reaction. In addition, if you want to test the tyrosinase activity of the bacterial powder, you can take 10mg of the bacterial powder, re-dissolve with 1mL PBS, and dilute ten times to get 1mg/mL of bacterial solution. Take 100 μL of 1 mg/mL bacterial solution, add 900 μL of 2.5 mM L-Dopa (L-3,4-dihydroxyphenylalanine), shake twice, and then use ultraviolet-visible light spectrophotometer to detect dopa in the reaction at a wavelength of 470 nm In the case of pigment (dopachrome) production, the obtained data is then multiplied by 10, which is equal to the tyrosinase activity contained in 1 mg of bacterial powder for 5 minutes. The results in Table 1 show that the tyrosinase activity of the bacterial powder prepared by the foregoing method can reach 5.0 or more each time, and can be further converted into 3'-hydroxygenistein.

2.2. Examples

When preparing 3'-hydroxygenistein, the genetically-recombined microorganism used is the bacterial powder of the genetically-recombinant microorganism prepared in Test Example 2.1, followed by a reaction substrate preparation step, the reaction substrate containing boric acid at a final concentration of 400 mM to 600 mM Salt, ascorbic acid with a final concentration of 10 mM to 40 mM, and genistein with a final concentration of 500 ppm to 2000 ppm ketone. When preparing the reaction matrix, first dissolve genistein in dimethyl sulfoxide to obtain a genistein solution, and dissolve ascorbic acid in water to obtain an ascorbic acid solution. After dissolving the borate in hot water, the pH value is adjusted to pH 8 to pH 10, preferably, the pH value is adjusted to pH 9 to obtain a borate solution. Then, the genistein solution and the ascorbic acid solution are added to the borate solution to obtain the reaction matrix. There are 7 examples in this test. The final concentration of borate, the final concentration of ascorbic acid, the final concentration of genistein, and the total volume of the reaction matrix in Examples 1 to 7 are shown in Table 2.

The prepared reaction matrix is subjected to a bioconversion reaction step, inoculated with genetically recombinant microorganisms in the reaction matrix, and at a bioconversion reaction temperature of 40°C to 60°C and a bioconversion reaction ventilation volume of 2L/min to 5L/min. The bioconversion reaction time of 250 rpm to 350 rpm is stirred for 60 to 90 minutes to obtain a bioconversion material. The concentration of the added bacterial powder, the bioconversion reaction time, the bioconversion reaction stirring speed, and the bioconversion reaction ventilation rate in Example 1 to Example 7 are shown in Table 3, and the bioconversion reaction temperature is preferably 50°C.

Then, the obtained bioconversion material is subjected to a termination reaction, a hydrochloric acid solution is added to the bioconversion material, and the termination reaction temperature of 40°C to 60°C and the termination ventilation volume of 2L/min to 5L/min are at 250rpm to 350rpm. The termination stirring speed of the reaction is 5 minutes to 15 minutes to terminate the reaction time to obtain a reaction solution, wherein the reaction solution contains 3'-hydroxygenistein. Preferably, the final concentration of the hydrochloric acid solution may be 100 mM to 200 mM. The termination reaction temperature may be 50° C., the termination reaction ventilation volume may be 3 L/min, and the termination reaction stirring speed may be 280 rpm. The concentration and volume of the added hydrochloric acid solutions and the reaction termination time in Example 1 to Example 7 are shown in Table 4.

In order to obtain high-purity 3'-hydroxygenistein, the reaction solution may be further extracted by adding ethyl acetate to the reaction solution, wherein the volume ratio of the reaction solution to ethyl acetate may be 1:1 to 1.5:1, and repeated shaking at a shaking speed of 200rpm to 300rpm for 2 minutes to 20 minutes for 3 times to obtain an extract. The volume, shaking speed and shaking time of ethyl acetate added in Examples 1 to 7 are shown in Table 5.

The extract can be further concentrated under reduced pressure. The extract is concentrated with a reduced-pressure concentrator. It is first packed into a 2L eggplant-shaped concentration bottle and concentrated. When the remaining amount is about 1L, it is concentrated to dryness in a 500mL eggplant-shaped concentration bottle and scraped. Scoop down and load into 20mL sample bottle to save. The remaining residual dry powder of 3'-hydroxygenistein was dissolved in 100 mL of ethyl acetate, then placed in a serum bottle and stored at -20°C. After accumulating several times, it was concentrated.

The crude extract dry powder concentrated under reduced pressure was weighed and the yield was recorded, and 1 mg of the crude extract powder was dissolved in 1 mL of methanol, and the purity was analyzed by HPLC and recorded in the record table. Please refer to Table 6 for the yield and purity of Example 1 to Example 7.

The results in Table 6 show that the production method of 3'-hydroxygenistein prepared by the method of 3'-hydroxygenistein of the present invention is as high as 6.64g to 14.06g, and the prepared 3'-hydroxygenigenin The purity can be as high as 71.55% to 99.30%. It shows that the preparation method of 3'-hydroxygenistein of the present invention can produce 3'-hydroxygenigenin with high yield and high purity.

2.3. Comparative example

In this test example, the bacterial powder of the genetically recombinant microorganism prepared in Test Example 2.1 was used to prepare 3'-hydroxygenistein under different preparation conditions as a comparative example. The difference between the comparative example and the example lies in the total volume of the reaction substrate and the conditions of the bioconversion reaction step during cultivation, and the remaining steps, conditions, and genetically recombinant microorganisms used are the same as in the example. The preparation method of 3'-hydroxygenistein in the comparative example includes the following steps: a reaction matrix preparation step is performed, the reaction matrix comprises borate at a final concentration of 500 mM, ascorbic acid at a final concentration of 10 mM to 20 mM, and a final concentration of 1000 ppm Genistein and the reaction matrix are prepared in the same order as in the examples. There are 6 comparative examples in this test, the most of the borate in Comparative Examples 1 to 6 The final concentration, the final concentration of ascorbic acid, the final concentration of genistein and the total volume of the reaction matrix are shown in Table 7.

The prepared reaction substrate is subjected to a bioconversion reaction step, in which a genetically recombinant microorganism is inoculated into the reaction substrate, and the bioconversion reaction time is incubated at a bioconversion reaction temperature of 50° C. at a stirring speed of 220 rpm for 90 minutes. Obtain a biological conversion substance. The concentration of the added bacterial powder, the bioconversion reaction time, and the bioconversion reaction stirring speed in Comparative Examples 1 to 6 are shown in Table 8.

Then, the obtained bioconversion material is subjected to a termination reaction, a hydrochloric acid solution is added to the bioconversion material, and the reaction temperature is terminated at 50°C. The reaction was terminated at a termination stirring speed of 220 rpm for 10 minutes to obtain a reaction solution in which the reaction solution contained 3'-hydroxygenistein. The concentration and volume of the added hydrochloric acid solutions and the reaction termination time of Comparative Examples 1 to 6 are shown in Table 9.

In the comparative example, an extraction step was also performed on the reaction solution. Ethyl acetate was added to the reaction solution, and shaking was repeated at a shaking speed of 250 rpm for 2 minutes to 3 minutes for 3 times to obtain an extraction solution. Table 10 shows the volume, shaking speed and shaking time of ethyl acetate added in Comparative Examples 1 to 6.

Then, the extracts of Comparative Examples 1 to 7 were concentrated under reduced pressure, the crude extract dry powder concentrated under reduced pressure was weighed and the yield was recorded, and 1 mg of the crude extract powder was dissolved in 1 mL of methanol, and the purity was analyzed by HPLC Remember Recorded in the record sheet. Please refer to Table 11 for the yield and purity of Comparative Examples 1 to 6.

The results in Table 11 show that the 3'-hydroxygenistein prepared by the comparative example 3'-hydroxygenistein has only 0.26g to 2.29g of 3'-hydroxygenistein, and the prepared The purity of 3'-hydroxygenistein is only 21.21% to 35.11%. Compared with Test Example 2.2, the preparation method of 3'-hydroxygenistein of the present invention can not only significantly increase the yield from 6.64g to 14.06g, but also can significantly improve the purity of the prepared 3'-hydroxygenistein. 71.55% to 99.30%.

2.4. Preparation of liquid raw materials

In this test example, the 3'-hydroxygenistein prepared by the method for preparing 3'-hydroxygenigenin of the present invention is further prepared as a liquid raw material, which can be used as a cosmetic in the future. In the experiment, liquid raw materials containing 10% of 3'-hydroxygenistein and 20% of 3'-hydroxygenistein were prepared. The liquid raw material containing 10% of 3'-hydroxygenistein is 10g of 3'-hydroxygenistein dissolved in 90g of propanediol, and the liquid raw material containing 20% of 3'-hydroxygenigenin is 20% 20g of 3'-hydroxygenistein is dissolved in 80g of propylene glycol. system When preparing liquid raw materials, first heat propylene glycol to 80°C, add 3'-hydroxygenistein, add and stir for 5 minutes to 10 minutes until the 3'-hydroxygenigenin is completely dissolved, and the preparation of liquid raw materials is completed. After the prepared liquid raw material is filtered and vacuum defoamed, it is stored at room temperature in the dark.

Please refer to FIG. 3, which is a statistical graph of the yield of 3'-hydroxygenistein formulated liquid raw materials prepared by the method for preparing 3'-hydroxygenigenin of the present invention, which is the statistical result of 5 trials. The results in Figure 3 show that the 3'-hydroxygenistein prepared by the method for preparing 3'-hydroxygenigenin of the present invention can be used as a liquid raw material, and the output can reach more than 100 grams each time. The preparation method of 3'-hydroxygenistein has the potential to prepare high-yield liquid raw materials.

This test example further tests the stability of the prepared liquid raw material at different temperatures. The test will respectively contain 10% of 3'-hydroxygenistein liquid and 20% of 3'-hydroxygenigenin. The liquid raw materials were placed in an environment of -10°C to 45°C, and the contents of 3'-hydroxygenistein in the liquid raw materials were measured on days 1, 3, 7, 22, 42 and 57 respectively.

Please refer to Figure 4 and Figure 5, Figure 4 shows the analysis results of the stability of the liquid raw material containing 10% 3'-hydroxygenistein, Figure 5 shows the 3'-hydroxy dye containing 20% The analysis result diagram of the stability of the liquid raw material of wood flavonoids, in which the temperature change is placed at high temperature, room temperature and low temperature alternately. The results show that the liquid raw material containing 20% of 3'-hydroxygenistein is observed to be more stable than the 57'-hydroxygenistein in the liquid raw material on day 57 regardless of the environment at -10°C to 45°C. 100%, showing 20% The liquid raw material of 3'-hydroxygenistein can be stably stored in an environment of -10°C to 45°C. The liquid raw material containing 10% 3'-hydroxygenistein has a stability of less than 100% at 45°C to day 57, but the stability is still higher than 80%. However, under the environment of -10℃ to 25℃, the stability of the 3'-hydroxygenistein in the liquid raw material was still more than 100% on the 42nd day, showing that the liquid raw material containing 10% of the 3'-hydroxygenigenin It can be stably stored in the environment of -10℃ to 25℃.

In summary, the preparation method of 3'-hydroxygenistein in the present invention uses genetically engineered microorganisms to convert 3'-hydroxygenigenin, and its preparation method can be used for large-scale industrial production because it can control the start Production processes such as products and bioconversion reaction conditions make the 3'-hydroxygen genistein prepared with stable quality and high purity, so it can solve the problem of low productivity of 3'-hydroxygenistein. In addition, the prepared 3'-hydroxygenistein can be further formulated into high-yield liquid raw materials, and the prepared liquid raw material has high stability, which is prepared by the preparation method of the 3'-hydroxygenistein of the present invention The obtained 3'-hydroxygenistein can be applied to cosmetic compositions, biopharmaceutical compositions, feed compositions, beverage compositions, nutritional supplement compositions, edible compositions and health food compositions, and has application in biomedical health care The potential of the market.

Although the present invention has been disclosed as above by way of implementation, it is not intended to limit the present invention. Any person who is familiar with this art can make various modifications and retouching without departing from the spirit and scope of the present invention, so the protection of the present invention The scope shall be determined by the scope of the attached patent application.

<110> Zodenis International Co., Ltd.

<120> Preparation method of 3’-hydroxygenistein

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<210> 1

<211> 894

<212> DNA

<213> Artificial sequence

<223> Tyrosinase gene

<400>

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<211> 30

<212> DNA

<213> Artificial sequence

<223> Forward primer

<400>

<210> 3

<211> 32

<212> DNA

<213> Artificial sequence

<223> Reverse primer

<400>

100‧‧‧3’-hydroxygenin preparation method

110, 120, 130, 140 ‧‧‧ steps

Claims (13)

A method for preparing 3'-hydroxygenistein includes: constructing a genetically recombinant microorganism, transforming a circular recombinant plastid into a microbial expression system to obtain the genetic recombinant microorganism, wherein the circular recombinant substance The body contains the tyrosinase gene as shown in sequence identification number 1; a reaction matrix preparation step is performed, wherein the reaction matrix comprises borate at a final concentration of 400 mM to 600 mM, ascorbic acid at a final concentration of 10 mM to 40 mM, and Genistein at a final concentration of 500 ppm to 2000 ppm; performing a bioconversion reaction step, inoculating the genetically recombinant microorganism in the reaction matrix, and performing a bioconversion reaction at a bioconversion reaction temperature and a bioconversion reaction ventilation Stirring speed for culturing a bioconversion reaction time to obtain a bioconversion material; and performing a termination reaction step by adding a hydrochloric acid solution to the bioconversion material and terminating at a termination reaction temperature and a termination ventilation rate The reaction speed is stirred for a reaction time to obtain a reaction liquid, wherein the reaction liquid contains the 3'-hydroxygenistein. The method for preparing 3'-hydroxygenistein as described in item 1 of the patent application scope, wherein the microorganism expression system is Escherichia coli . The preparation method of 3'-hydroxygenistein as described in item 1 of the patent application scope, wherein the step of preparing the reaction matrix comprises: dissolving the genistein in dimethyl sulfoxide to obtain a genistein solution; Dissolve the ascorbic acid in water to obtain an ascorbic acid solution; after dissolving the borate in a hot water, adjust the pH to pH 8 to pH 10 to obtain a borate solution; and the genistein solution and the The ascorbic acid solution is added to the borate solution to obtain the reaction matrix. The method for preparing 3'-hydroxygenistein as described in item 1 of the patent application scope, wherein the bioconversion reaction temperature is 40°C to 60°C. The preparation method of 3'-hydroxygenistein as described in item 1 of the patent application scope, wherein the ventilation rate of the bioconversion reaction is 2L/min to 5L/min, and the stirring rate of the bioconversion reaction is 250rpm to 350rpm. The preparation method of 3'-hydroxygenistein as described in item 1 of the patent application scope, wherein the bioconversion reaction time is 60 minutes to 90 minutes. The method for preparing 3'-hydroxygenistein as described in item 1 of the patent application scope, wherein the final concentration of the hydrochloric acid solution is 100 mM to 200 mM. The preparation method of 3'-hydroxygenistein as described in item 1 of the patent application scope, wherein the termination reaction temperature is 40°C to 60°C. The method for preparing 3'-hydroxygenistein as described in item 1 of the patent application scope, wherein the termination reaction aeration is 2L/min to 5L/min, and the termination reaction stirring speed is 250rpm to 350rpm. The preparation method of 3'-hydroxygenistein as described in item 1 of the patent application scope, wherein the termination reaction time is 5 minutes to 15 minutes. The preparation method of 3'-hydroxygenistein as described in item 1 of the scope of the patent application further includes an extraction step, in which ethyl acetate is added to the reaction solution, and repeated shaking at a shaking speed for a shaking time of 3 times . The preparation method of 3'-hydroxygenistein as described in item 11 of the patent application scope, wherein the volume ratio of the reaction solution to the ethyl acetate is 1:1 to 1.5:1. The preparation method of 3'-hydroxygenistein as described in item 11 of the patent application scope, wherein the shaking speed is 200 rpm to 300 rpm, and the shaking time is 2 minutes to 20 minutes.

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