Method for preparing artificial bear gall powder by using black-bone chicken gall

Abstract

The invention discloses a method for preparing artificial bear gall powder by using black-bone chicken gall, according to the method, black-bone chicken bile refined liquid is used as a substrate, mixed enzyme catalysis is performed to obtain the artificial bear gall powder, according to the preparation method, the black-bone chicken gall is used as a raw material, the black-bone chicken bile is catalyzed by enzyme, and a one-pot reaction is performed to obtain the artificial bear gall powder with high tauroursodeoxycholic acid content and high yield. And the iron and zinc contents of the artificial bear gall powder are obviously higher than those of the existing artificial bear gall powder.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine, and particularly relates to a method for preparing artificial bear bile powder by using black chicken gall. Background Art

[0002] Bear bile powder has been clinically certified in the treatment of acute and chronic hepatitis and cholelithiasis, and has good therapeutic effects. However, due to the small number of bear farming companies in the country, the production of tubeless drainage is not high and cannot meet the growing market demand.

[0003] The main components of bear bile powder include bile acids and non-bile acids, among which the main active ingredients are in bile acids, including tauroursodeoxycholic acid (TUDCA), taurochenodeoxycholic acid (TCDCA), unconjugated tauroursodeoxycholic acid (CDCA), bilirubin, deoxycholic acid (DCA) and cholic acid (CA). Among them, tauroursodeoxycholic acid (TUDCA) is used to distinguish the main components of other animal bile.

[0004] Chinese patent CN201210162695.4 "Artificial Bear Bile" discloses an artificial bear bile, which is obtained by mixing sodium tauroursodeoxycholate with mixed bile, amino acids and trace elements.

[0005] Chinese patent CN201510996540.4 "A method for preparing artificial bear bile powder for poultry" discloses that taurochenodeoxycholic acid (TCDCA) is oxidized into 3α-hydroxy-7-carbonyl-5β-cholanoyl-N-taurine (T7-ketoLCA) using sodium peroxide as an oxidant, and the oxide T7-ketoLCA is reduced into tauroursodeoxycholic acid (TUDCA) and tauroursodeoxycholic acid (TCDCA) using metallic sodium as a reducing agent. After the reaction is completed, the bile acid components change to TUDCA, TCDCA and TCA, and the ratio of the three is close to 47:36:17. The product is similar to the bile acid components of natural bear bile. The above method is a chemical synthesis method.

[0006] Chinese patent CN201410588581.5 "An artificial bear bile powder and its preparation method" discloses that taurochenodeoxycholic acid (TCDCA) in livestock bile powder is converted into tauroursodeoxycholic acid (TUDCA) by 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase to obtain artificial bear bile powder, wherein TUDCA accounts for 23-33% of the mass percentage of artificial bear bile powder, and TCDCA accounts for 12-30% of the mass percentage of artificial bear bile powder. The total bile acid content is 40- 65%. The coenzyme in the above patents uses coenzyme I (NAD+) or coenzyme II (NADPH), but the bile powder of poultry and livestock is used instead of fresh bile of poultry and livestock as the raw material. Although the components of poultry and livestock bile are generally the same, the contents of essential trace elements and essential trace elements vary greatly. Choosing appropriate fresh bile of poultry and livestock as the raw material can further increase the trace elements beneficial to the human body in artificial bear bile powder, thereby improving the quality of artificial bear bile powder.

[0007] Therefore, it is particularly important to design an artificial bear bile powder with high content of trace elements necessary for the human body. Summary of the invention

[0008] The object of the present invention is to provide a method for preparing artificial bear bile powder using black chicken bile. The preparation method uses black chicken bile as a raw material, catalyzes the black chicken bile through an enzyme, and adopts a one-pot reaction to obtain a product with high tauroursodeoxycholic acid content, golden color, and significantly higher iron and zinc contents than the existing artificial bear bile powder.

[0009] In order to achieve the above object, the present invention provides the following:

[0010] A method for preparing artificial bear bile powder using black chicken gallbladder, characterized in that:

[0011] 1) preparing substrate: preparing a purified black-bone chicken bile solution, and detecting the content of TCDCA in the purified black-bone chicken bile solution; the content of TCDCA in the purified black-bone chicken bile solution is not less than 20%;

[0012] 2) preparing a mixed enzyme and coenzyme: the mixed enzyme includes LDH-7α-hydroxysteroid dehydrogenase, GDH-7β-hydroxysteroid dehydrogenase and biliverdin reductase, the coenzyme includes NAD+ and NADP+, the enzyme activity of LDH-7α-hydroxysteroid dehydrogenase is 200-220 U / mL, and the enzyme activity of GDH-7β-hydroxysteroid dehydrogenase is 30-40 U / mL;

[0013] 3) Substrate-enzyme conversion: The purified black-bone chicken bile solution is mixed with the mixed enzyme and coenzyme for reaction for 0.5-1.5h, the added amount of LDH-7α-hydroxysteroid dehydrogenase is 10% of the volume of the reaction system; the added amount of GDH-7β-hydroxysteroid dehydrogenase is 40% of the volume of the reaction system; the purified black-bone chicken bile solution is 5.6% of the volume of the reaction system.

[0014] Preferably, the step 1) is specifically:

[0015] Selecting black-bone chicken gallbladder, breaking the gallbladder and obtaining bile to obtain black-bone chicken bile; concentrating the black-bone chicken bile to 50% of the original volume to obtain concentrated black-bone chicken bile, and the concentration temperature is 70° C.;

[0016] The concentrated black-bone chicken bile is subjected to alcohol extraction, the concentrated black-bone chicken bile is cooled to 30°C, 3 times the volume of 95% ethanol is added to the concentrated black-bone chicken bile, and then stirred for 200 minutes; the temperature of the concentrated black-bone chicken bile after stirring is maintained at 4°C, and the concentrated black-bone chicken bile is centrifuged at a speed of 3500rpm for 25 minutes, each centrifugation is 5 minutes, and the centrifugation is divided into 5 times; after the centrifugation is completed, the supernatant is taken, and the supernatant is secondary concentrated, the concentration time is 40 minutes, and the concentration temperature is 45°C, so as to obtain a refined black-bone chicken bile liquid.

[0017] Preferably, in step 2), in the LDH-7α-hydroxysteroid dehydrogenase, 7α-hydroxysteroid dehydrogenase is derived from Escherichia coli, and lactate dehydrogenase is derived from Lactobacillus; in the GDH-7β-hydroxysteroid dehydrogenase, 7β-hydroxysteroid dehydrogenase is derived from Ruminococcus active, glucose dehydrogenase is derived from Bacillus, and biliverdin reductase is derived from Synechocystis sp. PCC6803.

[0018] Preferably, the step 3) is specifically as follows: taking the refined black chicken bile liquid and adjusting the pH to 7, then adding sodium pyruvate and glucose monohydrate, mixing enzymes, coenzymes and water to form a reaction system, and adjusting the pH of the reaction system to 7; the molar ratio of sodium pyruvate to TCDCA in the refined black chicken bile liquid is 1.7:1, the molar ratio of glucose monohydrate to TCDCA in the refined black chicken bile liquid is 2.4:1, and the remainder is water.

[0019] Beneficial Effects

[0020] The invention adopts black chicken gallbladder as raw material, and the iron and zinc contents of the finally obtained artificial bear bile powder are obviously higher than those of the existing bear bile powder and the artificial bear bile powder prepared by white-feathered chicken. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the embodiments of the present invention are summarized below to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Preparation of black chicken bile extract

[0023] Select commercially available chicken gallbladders, thaw at room temperature, break the gallbladders and extract bile to obtain black-bone chicken bile;

[0024] The black-bone chicken bile is concentrated to 50% of the original volume to obtain concentrated black-bone chicken bile, and the concentration temperature is 70° C.;

[0025] The concentrated black-bone chicken bile is subjected to alcohol extraction, the concentrated black-bone chicken bile is cooled to 30°C, 3 times the volume of 95 ethanol is added to the concentrated black-bone chicken bile, and then stirred for 200 minutes; the temperature of the concentrated black-bone chicken bile after stirring is maintained at 4°C, and the mixture is centrifuged at a speed of 3500 rpm for 25 minutes, each time for 5 minutes, and divided into 5 centrifugations; after the centrifugation is completed, the supernatant is taken, and the supernatant is concentrated for a second time, the concentration time is 40 minutes, and the concentration temperature is 45°C, so as to obtain a refined black-bone chicken bile liquid.

[0026] The pH was adjusted to 7 with NaOH and set aside; after testing, 20.55 g TCDCA was contained in every 100 g of refined black chicken bile liquid.

[0027] LDH-7α-hydroxysteroid dehydrogenase, GDH-7β-hydroxysteroid dehydrogenase and enzyme construction can be seen in the LDH-7α-hydroxysteroid dehydrogenase, GDH-7β-hydroxysteroid dehydrogenase and their construction method disclosed in Chinese patent CN202111304130.0 "A method for preparing ursodeoxycholic acid".

[0028] The above-mentioned black-bone chicken is Taihe black-bone chicken.

[0029] Enzyme preparation

[0030] Obtaining the pRSFDuet-1 vector: The Top10 strain (containing the pRSFDuet-1 vector) stored at -80°C was zoned with three lines on LB solid plate culture medium supplemented with 50 μg / ml kanamycin (Kan). After culturing overnight at 37°C, a single clone was picked out and inoculated into 5 ml LB liquid culture medium (Kan = 50 μg / ml). After culturing overnight at 37°C and 220 rpm, the plasmid was extracted using the Tiangen Plasmid Extraction Kit.

[0031] Construction of pRSF-7α recombinant vector: Since the LDH gene fragment contains NdeI restriction site, the restriction site upstream of 7α is also NdeI, so the 7α fragment is first connected when constructing the dual expression vector; pRSFDuet-1 and 7α fragment use NdeI and XhoI double enzyme 20μl system: pRSFDuet-1 plasmid or 7α fragment 5μl, 10x buffer 2μl, NdeI and XhoI 0.5μl, double distilled water 12μl. 37℃ water bath enzyme digestion for 1h, 80℃ inactivation for 15min, add 10x loading buffer 2μl, use 1% agarose gel electrophoresis to cut the gel to recover the enzyme digestion product, and the gel recovery kit used is the full gold DNA purification kit. Recover the purified plasmid large fragment and 7α ligation reaction: 7α fragment and plasmid fragment 3μl, 10x T4 buffer 1μl, T4 ligase 0.5μl, double distilled water 3.5μl. Ligate at 22℃ for 1h. Transform Top10 competent cells with 3μl ligation product, resuscitate for 1h and apply to LB solid plate containing 50μg / ml kanamycin, and culture at 37℃ overnight. Use 7α_F / R to verify the transformants by colony PCR, and transfer the verified transformants to LB liquid medium (Kan=50μg / ml), culture at 37℃ overnight to extract plasmid pRSF-7α for use.

[0032] Construction of pRSF-LDH-7α recombinant vector: pRSF-7α and LDH fragments were treated with NcoI and HindIII double enzyme 20μl system: 5μl of pRSF-7α and LDH fragments, 2μl of 10x buffer, 0.5μl of NcoI and HindIII, 12μl of double distilled water. Digestion was performed in a 37℃ water bath for 1h, inactivated at 80℃ for 15min, and 2μl of 10x loading buffer was added. The digestion products were recovered by 1% agarose gel electrophoresis. Recover the purified plasmid large fragment and LDH ligation reaction: 3μl of LDH fragment and plasmid fragment, 1μl of 10xT4 buffer, 0.5μl of T4 ligase, and 3.5μl of double distilled water. Ligation was performed at 22℃ for 1h. 3μl of ligation product was transformed into Top10 competent cells, recovered for 1h, coated with LB solid plate containing 50μg / ml kanamycin, and cultured overnight at 37℃. The transformants were verified by colony PCR using LDH_F / R, and the verified transformants were transferred to LB liquid culture medium (Kan=50 μg / ml), cultured at 37°C overnight, and the plasmid pRSF-LDH-7α was extracted for later use.

[0033] The pRSF-LDH-7α plasmid was used to transform 50 μl of BL21 (DE3) competent cells, revived at 37°C for 1 h, coated on LB solid plates containing 50 μg / ml kanamycin, and cultured at 37°C overnight.

[0034] Pick a single clone and transfer it to 5 ml LB liquid medium (Kan = 50 μg / ml), culture it at 37°C overnight, transfer it to 50 ml LB liquid medium (Kan = 50 μg / ml) at 1:100, culture it at 37°C, 220 rpm for 2-3 h, OD600 = 0.5-0.8, add 0.5 mM IPTG and induce it at 30°C for 4 h, and collect the bacteria by centrifugation at 8000 rpm for 10 min.

[0035] The bacteria were added with 50 ml of PBS buffer (100 mM, pH 8.0) and ultrasonicated at 30% power for 10 min. The supernatant was collected by centrifugation at 10,000 rpm to obtain the LDH-7α enzyme solution. The enzyme activity of the LDH-7α enzyme solution was 200 U / mL.

[0036] Construction of pRSF-7β recombinant vector: pRSFDuet-1 and 7β fragment were double-digested with NdeI and XhoI in 20μl system: 5μl of pRSFDuet-1 plasmid or 7β fragment, 2μl of 10x buffer, 0.5μl of NdeI and XhoI, 12μl of double distilled water. Digestion was carried out in a water bath at 37℃ for 1h, inactivated at 80℃ for 15min, and 2μl of 10x loading buffer was added. The digestion product was recovered by gel electrophoresis using 1% agarose gel. The gel recovery kit used was the DNA purification kit of Quanshijin. Recover the purified large plasmid fragment and 7β ligation reaction: 3μl of 7β fragment and plasmid fragment, 1μl of 10x T4 buffer, 0.5μl of T4 ligase, and 3.5μl of double distilled water. Ligation was carried out at 22℃ for 1h. 3 μl of the ligation product was used to transform Top10 competent cells, and the cells were revived for 1 hour and coated on LB solid plates containing 50 μg / ml kanamycin, and cultured overnight at 37°C. Colony PCR verification of transformants was performed using 7β_F / R, and the verified transformants were transferred to LB liquid medium (Kan=50 μg / ml), cultured overnight at 37°C, and the plasmid pRSF-7β was extracted for use.

[0037] Construction of vector pRSF-GDH-7β recombinant vector: pRSF-7β and GDH fragments were double-digested with NcoI and HindIII in 20μl system: 5μl of pRSF-7β and GDH fragments, 2μl of 10x buffer, 0.5μl of NcoI and HindIII, 12μl of double distilled water. Digestion was carried out in a water bath at 37℃ for 1h, inactivated at 80℃ for 15min, and 2μl of 10x loading buffer was added. The digestion products were recovered by 1% agarose gel electrophoresis. Recover the purified plasmid large fragment and GDH ligation reaction: 3μl of GDH fragment and plasmid fragment, 1μl of 10xT4 buffer, 0.5μl of T4 ligase, and 3.5μl of double distilled water. Ligation was carried out at 22℃ for 1h. 3μl of ligation product was transformed into Top10 competent cells, recovered for 1h, coated with LB solid plate containing 50μg / ml kanamycin, and cultured at 37℃ overnight. The transformants were verified by colony PCR using GDH_F / R, and the verified transformants were transferred to LB liquid culture medium (Kan=50 μg / ml), cultured at 37°C overnight, and the plasmid pRSF-GDH-7β was extracted for later use.

[0038] The pRSF-GDH-7β plasmid was used to transform 50 μl of BL21 (DE3) competent cells, revived at 37°C for 1 h, coated on LB solid plates containing 50 μg / ml kanamycin, and cultured at 37°C overnight.

[0039] Pick a single clone and transfer it to 5 ml LB liquid medium (Kan = 50 μg / ml), culture it at 37°C overnight, transfer it to 50 ml LB liquid medium (Kan = 50 μg / ml) at 1:100, culture it at 37°C, 220 rpm for 2-3 h, OD600 = 0.5-0.8, add 0.5 mM IPTG and induce it at 30°C for 4 h, and collect the bacteria by centrifugation at 8000 rpm for 10 min.

[0040] The bacteria were added to 50 ml of PBS buffer (100 mM, pH 8.0) and ultrasonicated at 30% power for 10 min. The supernatant was collected by centrifugation at 10,000 rpm to obtain the GDH-7β enzyme solution with an enzyme activity of 40 U / mL.

[0041] Sodium pyruvate, D-glucose monohydrate, NAD+, NADP+ and NaOH are all commercially available products. The molecular weight of sodium pyruvate is 110.04. The CAS number of D-glucose monohydrate is 14431-43-7 and the molecular weight is 240.2509.

[0042] NAD+ is coenzyme I, the full name of which is nicotinamide adenine dinucleotide, also known as nicotinamide diphosphate. It exists in every cell and participates in thousands of reactions. NAD+ is an important coenzyme in the tricarboxylic acid cycle, promoting the metabolism of sugar, fat, and amino acids, and participating in the synthesis of energy; NAD+ is also the only substrate for coenzyme I consuming enzymes (the only substrate for DNA repair enzyme PARP, the only substrate for longevity protein Sirtuins, and the only substrate for cyclic ADP ribose synthetase CD38 / 157).

[0043] NADP+ is coenzyme II. The Chinese name of NADP+ is nicotinamide adenine dinucleoside phosphate. It is the oxidized form of reduced coenzyme II (NADPH), which means it has lost an electron and has a positive charge.

[0044] NAD+ and NADP+, as coenzymes of dehydrogenase, act as hydrogen donors in enzymatic reactions and are single hydrogen donors.

[0045] Example 1

[0046] Preparation of artificial bear bile powder, 81ml small test reaction:

[0047] Take 20g of spare black chicken bile refined liquid (TCDCA content is about 4.11g), add 1.5g of sodium pyruvate (industrial grade), 4.7g of D-glucose (industrial grade), 0.96g of NAD+ (10mg / mL), 0.8g of NADP+ (10mg / mL), and add 8mL of LDH-7α-hydroxysteroid dehydrogenase, 32mL of GDH-7β-hydroxysteroid dehydrogenase, add them together in a stirrer, add appropriate amount of water to form 80ml reaction system, adjust the pH of the reaction system to 7, the enzyme activity of LDH-7α-hydroxysteroid dehydrogenase is 200U / mL, the enzyme activity of GDH-7β-hydroxysteroid dehydrogenase is 40U / mL, the enzyme activity of biliverdin reductase is 0.2U / g, and react for 90min. The TUDCA content is 52.51% and the TCDCA content is 31.25% after testing.

[0048] Example 2

[0049] Preparation of artificial bear bile powder, 50L reaction:

[0050] Take 12.5 kg of spare black chicken bile refined liquid (TCDCA content is about 2.569 kg), add 0.9375 kg of sodium pyruvate (industrial grade), 2.93 kg of D-glucose monohydrate (industrial grade), 0.6 kg (10 mg / mL) of NAD+, 0.5 kg (10 mg / mL) of NADP+, 5 L of LDH-7α-hydroxysteroid dehydrogenase, 20 L of GDH-7β-hydroxysteroid dehydrogenase, add appropriate amount of water to form a 50 L reaction system, and react for 90 min.

[0051] After the reaction, the mixture was concentrated under reduced pressure and then extracted with alcohol to obtain artificial bear bile powder. The TUDCA content was 49.29% and the TCDCA content was 29.25%.

[0052] Comparative Example 1

[0053] The only difference between Comparative Example 1 and Example 1 is that 20 g of white-feathered chicken bile refined liquid is taken, and the only difference between the preparation of white-feathered chicken bile refined liquid and the preparation of black-bone chicken bile refined liquid is that the raw material is white-feathered chicken gallbladder, and the white-feathered chicken bile refined liquid contains 20.27 g TCDCA.

[0054] Comparative Example 2

[0055] Comparative Example 2 is commercially available bear bile powder.

[0056] Comparative Example 3

[0057] Comparative Example 3 is black chicken gallbladder powder. Fresh black chicken gallbladder is broken to obtain bile, the bile is placed in a refrigerator at -40°C for 24 hours, and then placed in a freeze dryer for 48 hours to obtain black chicken gallbladder powder.

[0058] The trace elements of Examples 1-2 and Comparative Examples 1-3 were detected, and the results are shown in Table 1 below.

[0059]

[0060] It can be seen from Table 1 that the black chicken gall powder of this application contains more Fe and Zn, but the content of Cd and Pd is relatively high. The artificial bear bile powder prepared from black chicken gall not only ensures the content of essential trace elements Fe and Zn, but also greatly reduces the content of toxic heavy metals Cd and Pd. By comparing Examples 1-2 with Comparative Example 2, it can be seen that the content of essential trace elements Fe and Zn is significantly improved, that is, the artificial bear bile powder prepared using black chicken gall as raw material has better quality.

Claims

1. A method for preparing artificial bear bile powder using black chicken gallbladder, characterized in that: 1) preparing a substrate: preparing a purified black-bone chicken bile solution, and detecting the content of TCDCA in the purified black-bone chicken bile solution; the content of TCDCA in the purified black-bone chicken bile solution is not less than 20%; 2) preparing a mixed enzyme and coenzyme: the mixed enzyme includes LDH-7α-hydroxysteroid dehydrogenase, GDH-7β-hydroxysteroid dehydrogenase and biliverdin reductase, the coenzyme includes NAD+ and NADP+, the enzyme activity of LDH-7α-hydroxysteroid dehydrogenase is 200-220 U / mL, and the enzyme activity of GDH-7β-hydroxysteroid dehydrogenase is 30-40 U / mL; 3) Substrate-enzyme conversion: The purified black-bone chicken bile solution is mixed with the mixed enzyme and coenzyme for reaction for 0.5-1.5h, the added amount of LDH-7α-hydroxysteroid dehydrogenase is 10% of the volume of the reaction system; the added amount of GDH-7β-hydroxysteroid dehydrogenase is 40% of the volume of the reaction system; the purified black-bone chicken bile solution is 5.6% of the volume of the reaction system.

2. The preparation method according to claim 1, characterized in that: The step 1) is specifically as follows: Selecting black-bone chicken gallbladder, breaking the gallbladder and obtaining bile to obtain black-bone chicken bile; concentrating the black-bone chicken bile to 50% of the original volume to obtain concentrated black-bone chicken bile, and the concentration temperature is 70° C.; The concentrated black-bone chicken bile is subjected to alcohol extraction, the concentrated black-bone chicken bile is cooled to 30° C., 3 times the volume of 95% ethanol is added to the concentrated black-bone chicken bile, and then stirred for 200 minutes; the temperature of the concentrated black-bone chicken bile after stirring is maintained at 4° C., and the concentrated black-bone chicken bile is centrifuged at a speed of 3500 rpm for 25 minutes, each time for 5 minutes, divided into 5 centrifugations; After the centrifugation is completed, the supernatant is taken and concentrated twice, the concentration time is 40 minutes, and the concentration temperature is 45° C. to obtain the black chicken bile refined liquid.

3. The preparation method according to claim 1, characterized in that: In step 2), in the LDH-7α-hydroxysteroid dehydrogenase, 7α-hydroxysteroid dehydrogenase is derived from Escherichia coli, and lactate dehydrogenase is derived from Lactobacillus; in the GDH-7β-hydroxysteroid dehydrogenase, 7β-hydroxysteroid dehydrogenase is derived from Ruminococcus active, glucose dehydrogenase is derived from Bacillus, and biliverdin reductase is derived from Synechocystis sp. PCC6803.

4. The preparation method according to claim 3, characterized in that: The step 3) is specifically as follows: taking the black chicken bile purified liquid and adjusting the pH to 7, then adding sodium pyruvate, glucose monohydrate, mixed enzyme, coenzyme and water to form a reaction system, The pH of the reaction system was adjusted to 7; the molar ratio of sodium pyruvate to TCDCA in the purified black chicken bile solution was 1.7:1, the molar ratio of glucose monohydrate to TCDCA in the purified black chicken bile solution was 2.4:1, and the remainder was water.

Images