Method for preparing artificial bear gall powder through three-enzyme conversion, artificial bear gall powder and application of method

Abstract

The invention discloses a method for preparing artificial bear gall powder through three-enzyme conversion, the artificial bear gall powder and application of the method, and belongs to the technical field of biology. According to the method, the immobilized enzymes of the alpha-type enzyme, the beta-type enzyme and the glucose dehydrogenase are adopted to convert the reaction substrate containing the taurochenodeoxycholic acid into the artificial bear gall powder through the multi-path enzymatic reaction, and the method has high application value.

Description

Technical Field

[0001] The invention relates to a method for preparing artificial bear bile powder by three-enzyme conversion, the artificial bear bile powder and the application of the method, and belongs to the field of biotechnology. Background Art

[0002] Bear bile powder is the irregular fragments, particles or powder of bile from the gallbladder of black bears or brown bears after surgical drainage. It is a precious Chinese medicinal material and ranks first among the four precious animal medicinal materials in traditional Chinese medicine. It has the effects of clearing away heat and detoxification, protecting the liver and promoting bile secretion, relieving pain and spasm, anticonvulsant, antibacterial and anti-inflammatory, and lithotripsy. It can also promote the synthesis of glycogen from sugar and lactose, enhance the action and secretion of insulin, and thus has a hypoglycemic effect. "Bile extraction from live bears" has limited sources, low yields, large batch differences, many impurities, and unstable properties. In addition, the current annual output of bear bile powder is low, which is difficult to meet the people's growing health needs.

[0003] Therefore, in order to solve the shortage of bear bile powder in the market, it is increasingly important to accelerate the research and development of artificial bear bile powder and promote the sustainable development of the bear bile powder industry. Taurochenodeoxycholic acid (TCDCA) is the main component of poultry bile. At present, poultry bile is mainly used as raw material, and biotechnology is used to catalyze and convert the main bile acid components in it in vitro to synthesize tauroursodeoxycholic acid (TUDCA), and prepare artificial bear bile powder similar to natural bear bile powder. For example, Chinese invention patent CN 201610852312.4 discloses a method for producing artificial bear bile powder by fermentation with engineered bacteria. The method constructs a double gene co-expression vector and a multi-gene co-expression vector containing 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase, respectively, uses poultry bile products as raw materials, adds substrates to the culture medium, adds them to the fermentation broth of co-expressing 7α-steroid dehydrogenase and 7-β-steroid dehydrogenase that are being fermented, and converts part of taurochenodeoxycholic acid into tauroursodeoxycholic acid while fermenting. In this method, the composition of poultry and livestock bile powder is complex, and adding it during the fermentation process will have a certain impact on the normal growth of cells, thereby affecting the activity of the enzyme. In addition, the conversion substrate concentration of this method is low, the conversion efficiency is unstable, and the content of the reaction intermediate tauro7-ketolithocholic acid is high. In addition, this method has a long conversion cycle and is not suitable for industrial scale-up production. Chinese invention patent CN201210473483.8, through a two-step enzymatic method, the bound chenodeoxycholic acid in the bile of livestock is converted into bound ursodeoxycholic acid. The content of the intermediate tauro-7-ketolithocholic acid in this method is high and uncontrollable, which affects the quality of the product. The tauro-7-ketolithocholic acid is T-7-KLCA.

[0004] Although the artificial bear bile powder prepared by the current α-type enzyme and β-type enzyme dual enzyme enzymatic conversion technology is highly similar to the natural bear bile powder and the technology is stable, it still cannot solve the problem of high content of the reaction intermediate tauro-7-ketolithocholic acid. This is determined by the dual enzyme enzymatic reaction mechanism and is difficult to solve simply by optimizing the enzymatic reaction conditions. Summary of the invention

[0005] [Technical issues]

[0006] Since the existing artificial bear bile powder preparation technology has the problem of high intermediate content, the present invention aims to provide a method for preparing artificial bear bile powder, so that artificial bear bile powder with characteristic components consistent with or close to those of natural bear bile powder can be prepared on demand.

[0007] The second technical problem to be solved by the present invention is to provide an artificial bear bile powder whose characteristic components are consistent with or close to those of natural bear bile powder.

[0008] The third technical problem to be solved by the present invention is to provide a method for applying artificial bear bile powder.

[0009] [Technical solution]

[0010] The present invention provides a method for preparing artificial bear bile powder by three-enzyme conversion, the method comprising the following steps: in the presence of coenzymes and cofactors, a reaction substrate containing taurochenodeoxycholic acid is subjected to enzymatic reaction with α-type hydroxysteroid dehydrogenase, β-type hydroxysteroid dehydrogenase and glucose dehydrogenase to prepare artificial bear bile powder. The taurochenodeoxycholic acid is TCDCA.

[0011] In certain embodiments of the present invention, the α-type enzyme, β-type enzyme, and glucose dehydrogenase are in the form of immobilized enzymes; the coenzyme is coenzyme I or coenzyme II, and the cofactor is glucose; and the reaction substrate is poultry and livestock bile powder or poultry and livestock bile concentrate.

[0012] In certain embodiments of the present invention, the immobilized enzymes of the three enzymes, α-type enzyme, β-type enzyme and glucose dehydrogenase, exist in the form of α-type enzyme, β-type enzyme and glucose dehydrogenase, which are respectively combined with immobilization resins to form α-type enzyme immobilized enzyme, β-type enzyme immobilized enzyme and glucose dehydrogenase immobilized enzyme.

[0013] In certain embodiments of the present invention, the immobilized enzymes of the α-type enzyme, β-type enzyme and glucose dehydrogenase are in the form of a community immobilized enzyme formed by combining at least two of the α-type enzyme, β-type enzyme and glucose dehydrogenase with an immobilization resin.

[0014] In certain embodiments of the present invention, the immobilized enzymes of the three enzymes, α-type enzyme, β-type enzyme and glucose dehydrogenase, exist in the form of an α-type enzyme immobilized enzyme formed by combining the α-type enzyme with an immobilization resin, and a β-type enzyme-glucose dehydrogenase immobilized enzyme formed by combining the β-type enzyme and glucose dehydrogenase with an immobilization resin.

[0015] In certain embodiments of the present invention, the poultry and livestock bile powder is a refined poultry and livestock bile powder with a mass percentage of taurochenodeoxycholic acid ≥ 63% and a mass percentage of taurochenodeoxycholic acid in the poultry and livestock bile powder ≤ 3%. The taurochenodeoxycholic acid is TCA.

[0016] In certain embodiments of the present invention, the enzymatic reaction can be carried out in one or more steps in a reaction vessel reaction system or in a multi-combination immobilized enzyme column reaction system.

[0017] In certain embodiments of the present invention, the enzymatic reaction mainly comprises the following steps:

[0018] (1) Weigh a certain amount of refined poultry and livestock bile powder, dissolve it in a buffer solution, add coenzyme I or coenzyme II and glucose, and adjust the pH to between 6.5 and 8.5 to obtain a reaction substrate solution I;

[0019] (2) The reaction substrate solution I is mixed evenly with the α-type enzyme immobilized enzyme, the β-type enzyme immobilized enzyme and the glucose dehydrogenase immobilized enzyme, and the volume is fixed. The mixture is fully contacted and reacted at 20-30° C. After the reaction is completed, the supernatant is concentrated by clarification, precipitated with alcohol, the supernatant is concentrated by clarification, and dried to obtain artificial bear bile powder.

[0020] In certain embodiments of the present invention, the enzymatic reaction mainly comprises the following steps:

[0021] (1) Weigh a certain amount of refined poultry and livestock bile powder, dissolve it in a buffer solution, add coenzyme I or coenzyme II and glucose, and adjust the pH to between 6.5 and 8.5 to obtain a reaction substrate solution I;

[0022] (2) The reaction substrate solution I is mixed evenly with a certain proportion of α-type enzyme immobilized enzyme and β-type enzyme-glucose dehydrogenase immobilized enzyme, and the volume is fixed. The mixture is fully contacted and reacted at 20-30° C. After the reaction is completed, the supernatant is concentrated by clarification, precipitated with alcohol, the supernatant is concentrated by clarification, and dried to obtain artificial bear bile powder.

[0023] In certain embodiments of the present invention, the enzymatic reaction mainly comprises the following steps:

[0024] (1) Weigh a certain amount of refined poultry and livestock bile powder, dissolve it in a buffer solution, add coenzyme I or coenzyme II, adjust the pH to between 6.5 and 8.5, and make up the volume to obtain a reaction substrate solution II;

[0025] (2) The reaction substrate solution II is mixed evenly with a certain proportion of the α-type enzyme immobilized enzyme and the β-type enzyme immobilized enzyme, and the mixture is brought into contact with each other at 20 to 30° C. for a sufficient reaction. After the reaction is completed, the supernatant is clarified and glucose is added to adjust the pH to between 6.5 and 8.5, and the mixture is mixed evenly with the β-type enzyme immobilized enzyme and the glucose dehydrogenase immobilized enzyme, and the mixture is brought into contact with each other at 20 to 30° C. for a sufficient reaction. After the reaction is completed, the supernatant is clarified and concentrated, precipitated with alcohol, and the supernatant is clarified and concentrated, and dried to obtain the artificial bear bile powder.

[0026] In certain embodiments of the present invention, the enzymatic reaction mainly comprises the following steps:

[0027] (1) Weigh a certain amount of refined poultry and livestock bile powder, dissolve it in a buffer solution, add coenzyme I or coenzyme II, and adjust the pH to between 6.5 and 8.5 to obtain a reaction substrate solution II;

[0028] (2) mixing the reaction substrate solution II with a certain proportion of α-type enzyme immobilized enzyme and β-type enzyme immobilized enzyme, fixing the volume, and fully contacting and reacting at 20-30° C., after the reaction is completed, clarifying and taking the supernatant, adding glucose, adjusting the pH to between 6.5 and 8.5, and mixing with a certain amount of β-type enzyme-glucose dehydrogenase immobilized enzyme, fixing the volume, and fully contacting and reacting at 20-30° C., after the reaction is completed, clarifying and taking the supernatant, concentrating, alcohol precipitation, clarifying and taking the supernatant, concentrating, and drying to obtain artificial bear bile powder;

[0029] In certain embodiments of the present invention, the enzymatic reaction mainly comprises the following steps:

[0030] (1) Weigh a certain amount of refined poultry and livestock bile powder, dissolve it in a buffer solution, add coenzyme I or coenzyme II, adjust the pH to between 6.5 and 8.5, and make up the volume to obtain a reaction substrate solution II;

[0031] (2) The reaction substrate solution II is mixed evenly with a certain proportion of the α-type enzyme immobilized enzyme and the β-type enzyme immobilized enzyme, and the mixture is fixed to a fixed volume. The mixture is fully contacted and reacted at 20 to 30° C. After the reaction is completed, the supernatant is clarified and taken out, glucose is added, and the pH is adjusted to between 6.5 and 8.5. The mixture is evenly mixed with the glucose dehydrogenase immobilized enzyme, and the mixture is fixed to a fixed volume. The mixture is fully contacted and reacted at 20 to 30° C. After the reaction is completed, the supernatant is clarified and concentrated, precipitated with alcohol, and the supernatant is clarified and concentrated, and dried to obtain artificial bear bile powder.

[0032] In certain embodiments of the present invention, the enzymatic reaction mainly comprises the following steps:

[0033] (1) Weigh a certain amount of refined poultry and livestock bile powder, dissolve it in a buffer solution, add coenzyme I or coenzyme II, adjust the pH to between 6.5 and 8.5, and make up the volume to obtain a reaction substrate solution II;

[0034] (2) The reaction substrate solution II is mixed evenly with a certain amount of α-type enzyme immobilized enzyme to a fixed volume, and fully contacted with the reaction at 20-30°C. After the reaction is completed, the supernatant is clarified and glucose is added to adjust the pH to between 6.5 and 8.5, and then mixed evenly with β-type enzyme immobilized enzyme and glucose dehydrogenase immobilized enzyme, and the volume is fixed. After the reaction is completed, the supernatant is clarified and concentrated, precipitated with alcohol, and the supernatant is clarified and concentrated, and dried to obtain artificial bear bile powder.

[0035] In certain embodiments of the present invention, the enzymatic reaction mainly comprises the following steps:

[0036] (1) Weigh a certain amount of refined poultry and livestock bile powder, dissolve it in a buffer solution, add coenzyme I or coenzyme II, and adjust the pH to between 6.5 and 8.5 to obtain a reaction substrate solution II;

[0037] (2) mixing the reaction substrate solution II and a certain amount of α-type enzyme immobilized enzyme uniformly, fixing the volume, and fully contacting and reacting at 20-30° C., after the reaction is completed, clarifying and taking the supernatant, adding glucose, adjusting the pH to between 6.5 and 8.5, and mixing with a certain amount of β-type enzyme-glucose dehydrogenase immobilized enzyme uniformly, fixing the volume, and fully contacting and reacting at 20-30° C., after the reaction is completed, clarifying and taking the supernatant, concentrating, alcohol precipitation, clarifying and taking the supernatant, concentrating, and drying to obtain artificial bear bile powder;

[0038] In certain embodiments of the present invention, the buffer is purified water or 10-100 mM glycine buffer; the coenzyme I or coenzyme II is NAD + or NADP + , based on the volume of the conversion system after constant volume, the final concentration of coenzyme I or coenzyme II is 0.15-0.8 g / L; the final concentration of glucose is 0.2 M based on the volume of the conversion system after constant volume.

[0039] In certain embodiments of the present invention, the mass ratio of the immobilized α-type enzyme to the immobilized β-type enzyme to the refined poultry and livestock bile powder is 1:2:20 to 1:10:50; the mass ratio of the immobilized glucose dehydrogenase to the refined poultry and livestock bile powder is 1:10 to 1:25.

[0040] In certain embodiments of the present invention, the mass ratio of glucose dehydrogenase in the β-type enzyme-glucose dehydrogenase immobilized enzyme to the β-type enzyme is 1:4-1:10; the mass ratio of the α-type enzyme immobilized enzyme to the β-type enzyme-glucose dehydrogenase immobilized enzyme to the refined poultry and livestock bile powder is 1:2:20-1:10:50.

[0041] The present invention also provides artificial bear bile powder prepared by the above method. The artificial bear bile powder refers to artificial bear bile powder whose characteristic components are consistent with or close to those of natural bear bile powder.

[0042] The invention also provides application of the method in artificial bear bile powder.

[0043] [Beneficial Effects]

[0044] (1) In the process of preparing artificial bear bile powder by the dual enzyme enzymatic method of α-type enzyme and β-type enzyme, tauro-7-keto-lithocholic acid is an inevitable intermediate product in the process of converting TCDCA to TUDCA, and its content is usually 1.7-3%. The difference between the present invention and the present invention lies in that by introducing the third enzyme glucose dehydrogenase into the reaction system, the conversion reaction is further promoted in the direction of the product, the content of the reaction intermediate is reduced to a lower level, and the intermediate tauro-7-keto-lithocholic acid is more thoroughly converted into the reaction product. The final percentage of tauro-7-keto-lithocholic acid can be reduced to within 0.35%, which is 6 to 11 times different, meeting the limit requirements of the intermediate of natural bear bile powder, and reflecting that the three-enzyme multi-path enzymatic method composed of glucose dehydrogenase, α-type enzyme and β-type enzyme can play a key role in reducing the percentage of the intermediate 7-keto-lithocholic acid.

[0045] (2) The present invention uses refined poultry and livestock bile powder as a substrate and a multi-path enzymatic method using three enzymes, α-type enzyme, β-type enzyme and glucose dehydrogenase, to prepare artificial bear bile powder with a TUDCA content of more than 35%, a TCDCA content of more than 25%, a TUA content of less than 0.8%, a TCA content of less than 1.9%, a T-7-KLCA content of less than 0.35%, a TUDCA+TCDCA content of more than 60%, and a TUDCA / TCDCA content of more than 1.35. The artificial bear bile powder has better stability and consistency between batches and can be scaled up.

[0046] (3) The present invention uses a three-enzyme reaction system of α-type enzyme, β-type enzyme and glucose dehydrogenase. This reaction system can recycle the coenzyme and reduce the usage of β-type enzyme. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is a schematic diagram of the present invention. DETAILED DESCRIPTION

[0048] The three enzymes used in the following examples are: α-type enzyme, β-type enzyme, and glucose dehydrogenase.

[0049] The enzymes used in the following examples mainly include: α-type enzyme immobilized enzyme, β-type enzyme immobilized enzyme, glucose dehydrogenase immobilized enzyme and β-type enzyme-glucose dehydrogenase immobilized enzyme.

[0050] The enzymatic reaction forms in the following embodiments mainly include: the enzymatic reaction can be carried out in one step or multiple steps in a reaction container reaction system or in a multi-combination immobilized enzyme column reaction system.

[0051] In the following examples, the characteristic product is artificial bear bile powder, or a single compound of tauroursodeoxycholic acid.

[0052] In the following examples, the buffer is purified water or glycine buffer, preferably purified water.

[0053] In the following embodiments, the coenzyme is coenzyme I or coenzyme II, preferably coenzyme I

[0054] In the following examples, the cofactor is glucose.

[0055] In the following examples, the recombinant Escherichia coli used is: refer to the Chinese application document with publication number CN109402212A.

[0056] In the following examples, TUDCA, TCDCA, the intermediate tauro-7-ketolithocholic acid, TCA, and tauro-ursocholic acid were all detected by HPLC-ELSD in accordance with the Chinese Pharmacopoeia Volume IV (2020 Edition) (General Rules for High Performance Liquid Chromatography). The tauro-ursocholic acid is TUA.

[0057] In the following examples, when each enzyme is used in the form of an immobilized enzyme, the method for determining the enzyme activity value is as follows:

[0058] The enzyme activity value (U / mg immobilized enzyme) of α-type immobilized enzyme was measured: 5mM taurochenodeoxycholic acid was used as substrate, NADP+ with a final concentration of 0.2mM and 50mg α-type immobilized enzyme were added to a 250ml pH8.0 100mM phosphate buffer reaction system, and the reaction was carried out at 25°C for 5 minutes. Samples were taken, filtered, and the change in absorbance of the clear solution was measured at 340nm, and the enzyme activity value of α-type immobilized enzyme was calculated by comparison with the blank control.

[0059] The enzyme activity value (U / mg immobilized enzyme) of the β-type immobilized enzyme was measured: 5mM tauroursodeoxycholic acid was used as the substrate, NADP+ with a final concentration of 0.2mM and 50mg of the β-type immobilized enzyme were added to a 250ml pH8.0 100mM phosphate buffer reaction system, and the reaction was carried out at 25°C for 5 minutes. The sample was taken and filtered, and the change in the absorbance value of the clear liquid was measured at 340nm, and the enzyme activity value of the β-type immobilized enzyme was calculated by comparing it with the blank control.

[0060] The enzyme activity value (U / mg immobilized enzyme) of glucose dehydrogenase immobilized enzyme was measured: 5mM glucose was used as substrate, NAD+ with a final concentration of 0.2mM and 50mg glucose dehydrogenase were added to a 250ml pH8.0 100mM phosphate buffer reaction system, and the reaction was carried out at 25°C for 5 minutes. Samples were taken and filtered, and the change of absorbance value of the clear liquid was measured at 340nm, and the enzyme activity value of glucose dehydrogenase immobilized enzyme was calculated by comparison with the blank control.

[0061] Examples 1-7: Preparation of artificial bear bile powder by enzymatic conversion using immobilized enzymes

[0062] Embodiment 1:

[0063] (1) Weigh 1000g of refined poultry and livestock bile powder, dissolve it in 2000ml of pure water, stir evenly, and add coenzyme NAD + solution to a final concentration of 0.15 g / L, add glucose solution to a final concentration of 0.2 M, adjust the pH to 8.5 with 5 M sodium hydroxide, add 780 ml of a mixed immobilized enzyme resuspension containing 40 g of α-type enzyme immobilized enzyme, 160 g of β-type enzyme immobilized enzyme and 40 g of glucose dehydrogenase immobilized enzyme, add purified water to make the reaction solution volume to 4000 ml, stir evenly, place the reaction solution in a constant temperature water bath at 25 ° C, slowly stir and react for 8 hours to obtain a conversion solution;

[0064] (2) The conversion liquid obtained in step (1) was centrifuged, and the obtained supernatant was concentrated to 1 L, 4 L of 95% ethanol was added, and the mixture was slowly stirred for 30 minutes. The mixture was placed in a constant temperature water bath at 16°C and allowed to stand for 4 hours for alcohol precipitation, and then centrifuged. The supernatant obtained by centrifugation was concentrated by rotary evaporation and vacuum dried to a dry product with a water content of less than 3%, thereby obtaining 970.01 g of artificial bear bile powder.

[0065] The artificial bear bile powder prepared in step (2) was tested using HPLC-ELSD in accordance with the "Chinese Pharmacopoeia Volume IV (2020 Edition)" (General Rules for High Performance Liquid Chromatography), wherein the content of TUDCA was 39.04%, the content of TCDCA was 26.50%, the content of TUA was 0.62%, and the content of TCA was 1.81%. The content of T-7-KLCA was 0.29%, the percentage of TUDCA+TCDCA was 66.54%, and TUDCA / TCDCA=1.42.

[0066] Embodiment 2:

[0067] (1) Weigh 1000g of refined poultry and livestock bile powder, dissolve it in 2000ml of pure water, stir evenly, and add coenzyme NAD +The solution was stirred to a final concentration of 0.15 g / L, and glucose solution was added to a final concentration of 0.2 M. The pH was adjusted to 8.5 with 5 M sodium hydroxide, and 780 ml of a mixed immobilized enzyme resuspension containing 20 g of α-type enzyme immobilized enzyme and 240 g of β-type enzyme-glucose dehydrogenase immobilized enzyme was added. The reaction solution was fixed to 4000 ml with pure water and stirred evenly. The reaction solution was placed in a constant temperature water bath at 25 ° C and slowly stirred for 8 h to obtain a conversion solution.

[0068] (2) The conversion liquid obtained in step (1) was centrifuged, and the obtained supernatant was concentrated to 1 L, 4 L of 95% ethanol was added, and the mixture was slowly stirred for 30 minutes. The mixture was placed in a constant temperature water bath at 16°C and allowed to stand for 4 hours for alcohol precipitation, and then centrifuged. The supernatant obtained by centrifugation was concentrated by rotary evaporation and vacuum dried to a dry product with a water content of less than 3%, thereby obtaining 965.87 g of artificial bear bile powder.

[0069] The artificial bear bile powder prepared in step (2) was tested using HPLC-ELSD in accordance with the "Chinese Pharmacopoeia Volume IV (2020 Edition)" (General Rules for High Performance Liquid Chromatography), wherein the content of TUDCA was 39.07%, the content of TCDCA was 28.10%, the content of TUA was 0.67%, and the content of TCA was 1.84%. The content of T-7-KLCA was 0.26%, the percentage of TUDCA+TCDCA was 67.17%, and TUDCA / TCDCA=1.39.

[0070] Embodiment 3:

[0071] (1) Weigh 1000g of refined poultry and livestock bile powder, dissolve it in 2000ml of pure water, stir evenly, and add coenzyme NAD + The solution was stirred to a final concentration of 0.15 g / L, and glucose solution was added to a final concentration of 0.2 M. The pH was adjusted to 8.5 with 5 M sodium hydroxide, and 990 ml of a mixed immobilized enzyme resuspension containing 30 g of α-type enzyme immobilized enzyme and 300 g of β-type enzyme immobilized enzyme was added. The reaction solution was fixed to 4000 ml with pure water, and stirred evenly. The reaction solution was placed in a constant temperature water bath at 25 ° C and stirred slowly for 8 h.

[0072] (2) The reaction solution obtained in step (1) was centrifuged to terminate the reaction, the supernatant was taken, the pH was adjusted to 8.5 with 5M sodium hydroxide, 1200 ml of a mixed immobilized enzyme resuspension containing 300 g of β-type enzyme immobilized enzyme and 100 g of glucose dehydrogenase immobilized enzyme was added, and the reaction solution was fixed to 5500 ml with purified water. The reaction solution was placed in a constant temperature water bath at 25°C and slowly stirred for reaction for 12 h to obtain a conversion solution;

[0073] (3) The conversion liquid obtained in step (2) was centrifuged, and the obtained supernatant was concentrated to 2 L, 8 L of 95% ethanol was added, and the mixture was slowly stirred for 30 minutes. The mixture was placed in a constant temperature water bath at 16°C and allowed to stand for 4 hours for alcohol precipitation, and then centrifuged. The supernatant obtained by centrifugation was concentrated by rotary evaporation and vacuum dried to a dry product with a water content of less than 3%, thereby obtaining 962.8 g of artificial bear bile powder.

[0074] The artificial bear bile powder prepared in step (3) was tested using HPLC-ELSD in accordance with the "Chinese Pharmacopoeia Volume IV (2020 Edition)" (General Rules for High Performance Liquid Chromatography), wherein the content of TUDCA was 39.04%, the content of TCDCA was 26.37%, the content of TUA was 0.66%, and the content of TCA was 1.73%. The content of T-7-KLCA was 0.27%, the percentage of TUDCA+TCDCA was 65.41%, and TUDCA / TCDCA=1.48.

[0075] Embodiment 4:

[0076] (1) Weigh 1000g of refined poultry and livestock bile powder, dissolve it in 2000ml of pure water, stir evenly, and add coenzyme NAD + The solution was stirred to a final concentration of 0.8 g / L, and glucose solution was added to a final concentration of 0.2 M. The pH was adjusted to 8.5 with 5 M sodium hydroxide, and 990 ml of a mixed immobilized enzyme resuspension containing 30 g of α-type enzyme immobilized enzyme and 300 g of β-type enzyme immobilized enzyme was added. The reaction solution was fixed to 4000 ml with pure water, and stirred evenly. The reaction solution was placed in a constant temperature water bath at 25 ° C and slowly stirred for 8 h.

[0077] (2) The reaction solution obtained in step (1) was centrifuged to terminate the reaction, the supernatant was taken, the pH was adjusted to 8.5 with 5M sodium hydroxide, 1200 ml of a mixed immobilized enzyme resuspension containing 400 g of β-type enzyme-glucose dehydrogenase immobilized enzyme was added, and the reaction solution was fixed to 5500 ml with purified water. The reaction solution was placed in a constant temperature water bath at 25°C and slowly stirred for reaction for 12 hours to obtain a conversion solution;

[0078] (3) The conversion liquid obtained in step (2) was centrifuged, and the obtained supernatant was concentrated to 2 L, 8 L of 95% ethanol was added, and the mixture was slowly stirred for 30 minutes. The mixture was placed in a constant temperature water bath at 16°C and allowed to stand for 4 hours for alcohol precipitation, and then centrifuged. The supernatant obtained by centrifugation was concentrated by rotary evaporation and vacuum dried to a dry product with a water content of less than 3%, thereby obtaining 965.29 g of artificial bear bile powder.

[0079] The artificial bear bile powder prepared in step (3) was tested using HPLC-ELSD in accordance with the "Chinese Pharmacopoeia Volume IV (2020 Edition)" (General Rules for High Performance Liquid Chromatography), wherein the content of TUDCA was 39.06%, the content of TCDCA was 26.94%, the content of TUA was 0.62%, and the content of TCA was 1.77%. The content of T-7-KLCA was 0.23%, the percentage of TUDCA+TCDCA was 66.00%, and TUDCA / TCDCA=1.45.

[0080] Embodiment 5:

[0081] (1) Weigh 1000 g of refined poultry and livestock bile powder, dissolve it in 2000 ml of 10 mM glycine buffer, stir evenly, and add coenzyme NAD + The solution was stirred to a final concentration of 0.5 g / L, glucose was added to a final concentration of 0.2 M, the pH was adjusted to 6.5 with 5 M sodium hydroxide, 660 ml of a mixed immobilized enzyme resuspension containing 20 g of α-type enzyme immobilized enzyme and 200 g of β-type enzyme immobilized enzyme was added, and the reaction solution was fixed to 4000 ml with pure water, stirred evenly, and the reaction solution was placed in a 25 ° C constant temperature water bath and slowly stirred for 8 h;

[0082] (2) The reaction solution obtained in step (1) was centrifuged to terminate the reaction, the supernatant was taken, the pH was adjusted to 6.5 with 5M sodium hydroxide, 120ml of immobilized enzyme resuspension containing 40g of immobilized glucose dehydrogenase was added, and the reaction solution was fixed to 4500ml with pure water. The reaction solution was placed in a constant temperature water bath at 25°C and slowly stirred for 12h to obtain a conversion solution.

[0083] (3) The conversion liquid obtained in step (2) was centrifuged, and the obtained supernatant was concentrated to 1 L, 4 L of 95% ethanol was added, and the mixture was slowly stirred for 30 minutes. The mixture was placed in a constant temperature water bath at 16°C and allowed to stand for 4 hours for alcohol precipitation, and then centrifuged. The supernatant obtained by centrifugation was concentrated by rotary evaporation and vacuum dried to a dry product with a water content of less than 3%, thereby obtaining 965.7 g of artificial bear bile powder.

[0084] The artificial bear bile powder prepared in step (3) was tested using HPLC-ELSD in accordance with the "Chinese Pharmacopoeia Volume IV (2020 Edition)" (General Rules for High Performance Liquid Chromatography), wherein the content of TUDCA was 39.01%, the content of TCDCA was 28.07%, the content of TUA was 0.54%, and the content of TCA was 1.84%. The content of T-7-KLCA was 0.33%, the percentage of TUDCA+TCDCA was 67.08%, and TUDCA / TCDCA=1.39.

[0085] Embodiment 6:

[0086] (1) Weigh 1000g of refined poultry and livestock bile powder, dissolve it in 2000ml of pure water, stir evenly, and add coenzyme NAD + The solution was adjusted to a final concentration of 0.5 g / L, and the pH was adjusted to 8.5 with 5 M sodium hydroxide. 60 ml of the immobilized enzyme resuspension containing 20 g of the α-type enzyme was added, and the reaction solution was fixed to 4000 ml with pure water. The reaction solution was stirred evenly, and the reaction solution was placed in a constant temperature water bath at 25 ° C and slowly stirred for 8 h.

[0087] (2) The reaction solution obtained in step (1) was centrifuged to terminate the reaction, and the supernatant was taken. Glucose was added to a final concentration of 0.2 M, and the pH was adjusted to 8.5 with 5 M sodium hydroxide. 720 ml of a mixed immobilized enzyme resuspension containing 200 g of β-type enzyme immobilized enzyme and 40 g of glucose dehydrogenase immobilized enzyme was added. The reaction solution was fixed to 4500 ml with pure water, and the reaction solution was placed in a constant temperature water bath at 25° C. and slowly stirred for reaction for 12 h to obtain a conversion solution.

[0088] (3) The conversion liquid obtained in step (2) was centrifuged, and the obtained supernatant was concentrated to 1 L, 4 L of 95% ethanol was added, and the mixture was slowly stirred for 30 minutes. The mixture was placed in a constant temperature water bath at 16°C and allowed to stand for 4 hours for alcohol precipitation, and then centrifuged. The supernatant obtained by centrifugation was concentrated by rotary evaporation and vacuum dried to a dry product with a water content of less than 3%, thereby obtaining 956.1 g of artificial bear bile powder.

[0089] The artificial bear bile powder prepared in step (3) was tested using HPLC-ELSD in accordance with the "Chinese Pharmacopoeia Volume IV (2020 Edition)" (General Rules for High Performance Liquid Chromatography), wherein the content of TUDCA was 38.64%, the content of TCDCA was 28.63%, the content of TUA was 0.51%, and the content of TCA was 1.88%. The content of T-7-KLCA was 0.22%, the percentage of TUDCA+TCDCA was 67.27%, and TUDCA / TCDCA=1.35.

[0090] Embodiment 7:

[0091] (1) Weigh 1000g of refined poultry and livestock bile powder, dissolve it in 5000ml of pure water, stir evenly, and add coenzyme NAD + The solution was adjusted to a final concentration of 0.8 g / L, the pH was adjusted to 8.5 with 5 M sodium hydroxide, the volume was made up to 7000 ml with pure water, and the mixture was stirred evenly. The mixture was introduced into a mixed immobilized enzyme column containing 50 g of α-type immobilized enzyme in a constant temperature water bath at 25°C at a constant flow rate. The enzymatic reaction was carried out in a contact reaction in the column in a constant flow rate cyclic loading manner for 8 hours to obtain a conversion intermediate solution.

[0092] (2) Glucose was added to the conversion intermediate solution prepared in step (1) to a final concentration of 0.8 g / L, the pH was adjusted to 8.5 with 5 M sodium hydroxide, the volume was made up to 9000 ml with purified water, and the mixture was stirred evenly. The mixture was introduced into a mixed immobilized enzyme column containing 500 g of a uniformly mixed β-type enzyme-glucose dehydrogenase immobilized enzyme in a constant temperature water bath at 25° C. at a constant flow rate, and reacted in the column for 12 h in a cyclic loading manner at a constant flow rate to obtain a conversion solution;

[0093] (3) The conversion liquid obtained in step (2) was centrifuged, and the obtained supernatant was concentrated to 2 L, 8 L of 95% ethanol was added, and the mixture was slowly stirred for 30 minutes. The mixture was placed in a constant temperature water bath at 16°C and allowed to stand for 4 hours for alcohol precipitation, and then centrifuged. The supernatant obtained by centrifugation was concentrated by rotary evaporation and vacuum dried to a dry product with a water content of less than 3%, thereby obtaining 949.2 g of artificial bear bile powder.

[0094] The artificial bear bile powder prepared in step (3) was tested using HPLC-ELSD in accordance with the "Chinese Pharmacopoeia Volume IV (2020 Edition)" (General Rules for High Performance Liquid Chromatography), wherein the content of TUDCA was 39.13%, the content of TCDCA was 25.91%, the content of TUA was 0.72%, and the content of TCA was 1.69%. The content of T-7-KLCA was 0.17%, the percentage of TUDCA+TCDCA was 65.04%, and TUDCA / TCDCA=1.51.

[0095] Comparative Example 1 (without glucose dehydrogenase) Effect of enzymatic method on conversion and preparation of artificial bear bile powder

[0096] The specific implementation of Comparative Example 1 is the same as that of Example 6, with the main difference being that no cofactor glucose is added in step (2); no glucose dehydrogenase immobilized enzyme is added; and 953.82 g of artificial bear bile powder is obtained.

[0097] Using HPLC-ELSD, according to the "Chinese Pharmacopoeia Volume 4 (2020 Edition)" (General Rules for High Performance Liquid Chromatography), the artificial bear bile powder prepared in Comparative Example 1 has a TUDCA content of 39.02%, a TCDCA content of 28.07%, a TUA content of 0.56%, and a TCA content of 1.83%. The content of T-7-KLCA is 2.03%, the percentage of TUDCA+TCDCA is 67.09%, and TUDCA / TCDCA=1.39, wherein the percentage of 2.03% T-7-KLCA is 6 to 11 times higher than that of T-7-KLCA in Example 6 and other examples.

[0098] Comparative Examples 2-5: Effects of unrefined livestock bile powder and conditions beyond the appropriate range (coenzyme concentration, temperature, pH) on the preparation of artificial bear bile powder by three-enzyme immobilized enzyme conversion

[0099] The specific implementation of Comparative Example 2 is the same as that of Example 3, except that the poultry and livestock bile powder used in step (1) is unrefined poultry and livestock bile powder, and its TCDCA mass percentage content is 45.87%, and TCA mass percentage content is 12.13%.

[0100] Using HPLC-ELSD, according to the "Chinese Pharmacopoeia Volume 4 (2020 Edition)" (General Rules for High Performance Liquid Chromatography), the artificial bear bile powder prepared in Comparative Example 2 has a TUDCA content of 23.88%, a TCDCA content of 18.95%, a TUA content of 5.08%, and a TCA content of 6.93%. The percentage of T-7-KLCA is 0.31%, the percentage of TUDCA+TCDCA is 42.83%, and TUDCA / TCDCA=1.26, wherein the contents of TUDCA and TCDCA are correspondingly low, while the contents of TUA and TCA are high.

[0101] The specific implementation of Comparative Example 3 is the same as that of Example 3, except that the NADP used in step (3) + The final concentration was 0.1 g / L.

[0102] Using HPLC-ELSD, according to the "Chinese Pharmacopoeia Volume 4 (2020 Edition)" (General Rules for High Performance Liquid Chromatography), the artificial bear bile powder prepared in Comparative Example 3 has a TUDCA content of 34.30%, a TCDCA content of 33.96%, a TUA content of 0.43%, and a TCA content of 1.97%. The percentage of T-7-KLCA is 3.32%, the percentage of TUDCA+TCDCA is 68.26%, TUDCA / TCDCA=1.07, of which the ratio of TUDCA to TCDCA is only 1.07, and the percentage of taurine 7-ketolithocholic acid is 3.32%, which is a high value.

[0103] The specific implementation of Comparative Example 4 is the same as that of Example 3, the main difference being that the use temperature in step (1) and step (2) is 35°C, rather than the suitable range of 20 to 30°C.

[0104] Using HPLC-ELSD, according to the "Chinese Pharmacopoeia Volume 4 (2020 Edition)" (General Rules for High Performance Liquid Chromatography), the artificial bear bile powder prepared in Comparative Example 3 has a TUDCA content of 32.31%, a TCDCA content of 31.99%, a TUA content of 0.30%, and a TCA content of 2.17%. The percentage of T-7-KLCA is 3.23%, the percentage of TUDCA+TCDCA is 68.49%, TUDCA / TCDCA=1.01, of which the ratio of TUDCA to TCDCA is only 1.01, and the percentage of taurine 7-ketolithocholic acid is 3.38%, which is a high value.

[0105] The specific implementation of Comparative Example 5 is the same as that of Example 3, the main difference being that the pH used in step (3) is pH 9.0, rather than the suitable range of pH 6.5 to 8.5.

[0106] Using HPLC-ELSD, according to the "Chinese Pharmacopoeia Volume 4 (2020 Edition)" (General Rules for High Performance Liquid Chromatography), the artificial bear bile powder prepared in Comparative Example 4 has a TUDCA content of 30.32%, a TCDCA content of 38.40%, a TUA content of 0.17%, and a TCA content of 2.21%. The percentage of T-7-KLCA is 3.06%, the percentage of TUDCA+TCDCA is 68.72%, and TUDCA / TCDCA=0.79, of which the ratio of TUDCA to TCDCA is only 0.79, and the percentage of taurine 7-ketolithocholic acid is 3.06%, which is a high value.

[0107] The present invention proposes a new method for preparing artificial bear bile powder. Through the refining of livestock bile powder and a three-enzyme multi-path enzymatic method, the content of the intermediate T-7-KLCA is reduced, and artificial bear bile powder with characteristic components closer to natural bear bile powder is prepared, thereby solving the problem of high intermediate T-7-KLCA content in the existing artificial bear bile powder preparation technology.

[0108] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing artificial bear bile powder by three-enzyme conversion, characterized in that: In the presence of coenzymes and cofactors, a reaction substrate containing taurochenodeoxycholic acid is subjected to an enzymatic reaction with α-hydroxysteroid dehydrogenase, β-hydroxysteroid dehydrogenase and glucose dehydrogenase to prepare artificial bear bile powder. The α-type hydroxysteroid dehydrogenase is referred to as the α-type enzyme, and the β-type hydroxysteroid dehydrogenase is referred to as the β-type enzyme.

2. The method according to claim 1, characterized in that: The three enzymes, α-type enzyme, β-type enzyme and glucose dehydrogenase, are in the form of immobilized enzymes; the coenzyme is coenzyme I or coenzyme II, and the cofactor is glucose; and the reaction substrate is livestock bile powder or livestock bile concentrate.

3. The method according to claim 2, characterized in that The immobilized enzyme forms of the three enzymes, α-type enzyme, β-type enzyme and glucose dehydrogenase, are formed by combining the α-type enzyme, β-type enzyme and glucose dehydrogenase with immobilized resins, respectively.

4. The method according to claim 2, characterized in that: The immobilized enzymes of the α-type enzyme, β-type enzyme and glucose dehydrogenase are in the form of a community immobilized enzyme formed by combining at least two of the α-type enzyme, β-type enzyme and glucose dehydrogenase with an immobilization resin.

5. The method according to claim 2 or 4, characterized in that: The immobilized enzymes of the α-type enzyme, β-type enzyme and glucose dehydrogenase exist in the form of α-type enzyme immobilized enzyme formed by combining α-type enzyme with immobilization resin, and β-type enzyme-glucose dehydrogenase immobilized enzyme formed by combining β-type enzyme and glucose dehydrogenase with immobilization resin.

6. The method according to claim 1, characterized in that The enzymatic reaction can be carried out in one step or multiple steps in a reaction container reaction system or in a multi-combination immobilized enzyme column reaction system.

7. The method according to any one of claims 1 to 3 and 6, characterized in that: The enzymatic reaction mainly comprises the following steps: (1) Weigh a certain amount of reaction substrate, dissolve it in a buffer solution, add coenzyme I or coenzyme II and glucose, and adjust the pH to between 6.5 and 8.5 to obtain a reaction substrate solution I; (2) The reaction substrate solution I is mixed evenly with the α-type enzyme immobilized enzyme, the β-type enzyme immobilized enzyme and the glucose dehydrogenase immobilized enzyme, and the volume is fixed. The mixture is fully contacted and reacted at 20-30° C. After the reaction is completed, the supernatant is concentrated by clarification, precipitated with alcohol, the supernatant is concentrated by clarification, and dried to obtain artificial bear bile powder.

8. The method according to any one of claims 1 to 2 and 4 to 6, characterized in that: The enzymatic reaction mainly comprises the following steps: (1) Weigh a certain amount of reaction substrate, dissolve it in a buffer solution, add coenzyme I or coenzyme II and glucose, and adjust the pH to between 6.5 and 8.5 to obtain a reaction substrate solution I; (2) The reaction substrate solution I is mixed evenly with a certain proportion of α-type enzyme immobilized enzyme and β-type enzyme-glucose dehydrogenase immobilized enzyme, and the volume is fixed. The mixture is fully contacted and reacted at 20-30° C. After the reaction is completed, the supernatant is concentrated by clarification, precipitated with alcohol, the supernatant is concentrated by clarification, and dried to obtain artificial bear bile powder.

9. The method according to any one of claims 1 to 3 and 6, characterized in that: The enzymatic reaction mainly comprises the following steps: (1) Weigh a certain amount of reaction substrate, dissolve it in a buffer solution, add coenzyme I or coenzyme II, adjust the pH to between 6.5 and 8.5, and make up the volume to obtain reaction substrate solution II; (2) The reaction substrate solution II is mixed evenly with a certain proportion of the α-type enzyme immobilized enzyme and the β-type enzyme immobilized enzyme, and the mixture is brought into contact with each other at 20 to 30° C. for a sufficient reaction. After the reaction is completed, the supernatant is clarified and glucose is added to adjust the pH to between 6.5 and 8.5, and the mixture is mixed evenly with the β-type enzyme immobilized enzyme and the glucose dehydrogenase immobilized enzyme, and the mixture is brought into contact with each other at 20 to 30° C. for a sufficient reaction. After the reaction is completed, the supernatant is clarified and concentrated, precipitated with alcohol, and the supernatant is clarified and concentrated, and dried to obtain the artificial bear bile powder.

10. The method according to any one of claims 1 to 2 and 4 to 6, characterized in that: The enzymatic reaction mainly comprises the following steps: (1) Weigh a certain amount of reaction substrate, dissolve it in a buffer solution, add coenzyme I or coenzyme II, and adjust the pH to between 6.5 and 8.5 to obtain a reaction substrate solution II; (2) The reaction substrate solution II is mixed evenly with a certain proportion of α-type enzyme immobilized enzyme and β-type enzyme immobilized enzyme, fixed to volume, and fully contacted to react at 20-30°C. After the reaction is completed, the supernatant is clarified and glucose is added to adjust the pH to between 6.5 and 8.5, and mixed evenly with a certain amount of β-type enzyme-glucose dehydrogenase immobilized enzyme, fixed to volume, and fully contacted to react at 20-30°C. After the reaction is completed, the supernatant is clarified and concentrated, precipitated with alcohol, and the supernatant is clarified and concentrated, and dried to obtain artificial bear bile powder.

11. The method according to any one of claims 1 to 3 and 6, characterized in that: The enzymatic reaction mainly comprises the following steps: (1) Weigh a certain amount of reaction substrate, dissolve it in a buffer solution, add coenzyme I or coenzyme II, adjust the pH to between 6.5 and 8.5, and make up the volume to obtain reaction substrate solution II; (2) The reaction substrate solution II is mixed evenly with a certain proportion of the α-type enzyme immobilized enzyme and the β-type enzyme immobilized enzyme, and the mixture is fixed to a fixed volume. The mixture is fully contacted and reacted at 20 to 30° C. After the reaction is completed, the supernatant is clarified and taken out, glucose is added, and the pH is adjusted to between 6.5 and 8.

5. The mixture is evenly mixed with the glucose dehydrogenase immobilized enzyme, and the mixture is fixed to a fixed volume. The mixture is fully contacted and reacted at 20 to 30° C. After the reaction is completed, the supernatant is clarified and concentrated, precipitated with alcohol, and the supernatant is clarified and concentrated, and dried to obtain artificial bear bile powder.

12. The method according to any one of claims 1 to 3 and 6, characterized in that: The enzymatic reaction mainly comprises the following steps: (1) Weigh a certain amount of reaction substrate, dissolve it in a buffer solution, add coenzyme I or coenzyme II, adjust the pH to between 6.5 and 8.5, and make up the volume to obtain reaction substrate solution II; (2) The reaction substrate solution II is mixed evenly with a certain amount of α-type enzyme immobilized enzyme to a fixed volume, and fully contacted with the reaction at 20-30°C. After the reaction is completed, the supernatant is clarified and glucose is added to adjust the pH to between 6.5 and 8.5, and then mixed evenly with β-type enzyme immobilized enzyme and glucose dehydrogenase immobilized enzyme, and the volume is fixed. After the reaction is completed, the supernatant is clarified and concentrated, precipitated with alcohol, and the supernatant is clarified and concentrated, and dried to obtain artificial bear bile powder.

13. The method according to any one of claims 1 to 2 and 4 to 6, characterized in that: The enzymatic reaction mainly comprises the following steps: (1) Weigh a certain amount of reaction substrate, dissolve it in a buffer solution, add coenzyme I or coenzyme II, and adjust the pH to between 6.5 and 8.5 to obtain a reaction substrate solution II; (2) The reaction substrate solution II is mixed evenly with a certain amount of α-type enzyme immobilized enzyme, fixed to volume, and fully contacted with the reaction at 20-30°C. After the reaction is completed, the supernatant is clarified and glucose is added to adjust the pH to between 6.5 and 8.5, and mixed evenly with a certain amount of β-type enzyme-glucose dehydrogenase immobilized enzyme, fixed to volume, and fully contacted with the reaction at 20-30°C. After the reaction is completed, the supernatant is clarified and concentrated, precipitated with alcohol, and the supernatant is clarified and concentrated, and dried to obtain artificial bear bile powder.

14. The method according to any one of claims 7 to 13, characterized in that: The buffer is purified water or 10-100 mM glycine buffer; the coenzyme I or coenzyme II is NAD + or NADP + , based on the volume of the conversion system after constant volume, the final concentration of coenzyme I or coenzyme II is 0.15-0.8 g / L; the final concentration of glucose is 0.2 M based on the volume of the conversion system after constant volume.

15. The method according to any one of claims 2 to 3, 7, 9, 11 to 12, characterized in that: The mass ratio of the α-type enzyme immobilized enzyme and the β-type enzyme immobilized enzyme to the reaction substrate is 1:2:20 to 1:10:50; the mass ratio of the glucose dehydrogenase immobilized enzyme to the reaction substrate is 1:10 to 1:

25.

16. The method according to any one of claims 2 and 4, 5, 8, 10, and 13, characterized in that: The mass ratio of glucose dehydrogenase in the β-type enzyme-glucose dehydrogenase immobilized enzyme to the β-type enzyme is 1:4-1:10; the mass ratio of the α-type enzyme immobilized enzyme to the β-type enzyme-glucose dehydrogenase immobilized enzyme to the reaction substrate is 1:2:20-1:10:

50.

17. The method according to claim 2, characterized in that The poultry and livestock bile powder is refined poultry and livestock bile powder with a mass percentage of taurochenodeoxycholic acid of ≥63% and a mass percentage of taurochenodeoxycholic acid in the poultry and livestock bile powder of ≤3%.

18. An artificial bear bile powder, characterized in that: Prepared by the method according to any one of claims 1 to 17.

19. Application of the method according to any one of claims 1 to 17 in artificial bear bile powder.

Images