Preparation process of artificial bear gall powder

A production process, technology of bear bile powder, applied in the field of biomedicine, can solve problems such as high cost, difficulty in maintaining enzyme activity, complicated enzyme purification steps, etc., and achieve the effects of increasing content, improving efficiency, and low viscosity of the system

Pending Publication Date: 2020-07-14
CHONGQING KINBEAR BIOTECHNOLOGY CO LTD
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The present invention intends to provide a production process of artificial bear bile powder, by using the bacterial suspension or supernatant containing two kinds of enzymes as the transformation liquid, it solves the technology that the purification steps of the enzyme are complicated, the cost is high and the enzyme activity is not easy to maintain question

Method used

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  • Preparation process of artificial bear gall powder
  • Preparation process of artificial bear gall powder
  • Preparation process of artificial bear gall powder

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Preparation of engineering bacteria

[0036] The 7α-hydroxysteroid dehydrogenase gene S1-a-1 and the 7β-hydroxysteroid dehydrogenase gene Y1-b-1 were codon-optimized for expression in E. coli, and affinity tags were added, and the whole gene was synthesized. The optimized 7α-hydroxysteroid dehydrogenase gene S1-a-1 is abbreviated as 7α-steroid dehydrogenase gene in this paper, and denoted as 7α-HSDH (SEQ ID NO: 1); the optimized 7β-hydroxysteroid dehydrogenase The gene Y1-b-1 is abbreviated as 7β-steroid dehydrogenase gene herein, and is denoted as 7β-HSDH (SEQ ID NO: 3).

[0037] 1. Construction of Expression Vectors

[0038] a) Preparation of recombinant plasmid pET28a-7α-HSDH containing 7α-steroid dehydrogenase gene

[0039] 7α-HSDH (DNA sequence: SEQ ID NO: 1, encoded protein sequence: SEQ ID NO: 2) was prepared with primer pairs 5′-GGAATTCCATATGGGCAGCAGCCATCATCA-3′ (SEQ ID NO: 5) and 5′-TCCCTCGAGTTAACGGCTGCGCTCCATCAT-3 ' (SEQ ID NO: 6) was amplified b...

Embodiment 2

[0047] Example 2: Fermentation expression of engineered bacteria

[0048] (1) Preparation of strains

[0049] The bacterial solutions of engineering bacteria A and engineering bacteria B are respectively coated with kana-resistant LB plates, and the bacterial solutions of engineering bacteria C are coated with ampicillin-resistant LB plates. In LB medium of kana or ampicillin, 37 ° C, 220 rpm, culture, when the OD value is 0.8-1.2, add 1 mM IPTG to induce 2 h, detect the expression level by SDS-PAGE, and select clones with high expression levels as intermediate strains (Intermediate strain A, intermediate strain B and intermediate strain C) are preserved.

[0050] Take 20 μL of Escherichia coli BL21 (DE3) strains (three intermediate strains: intermediate strain A, intermediate strain B and intermediate strain C) containing the recombinant plasmid, inoculated into 200 mL of kana- or ampicillin-resistant LB medium, respectively Medium, 37°C, 220rpm, overnight culture, OD 600 ...

Embodiment 3

[0054] Example 3: Preparation of poultry bile powder

[0055] The technical route for the preparation of poultry gall powder is: cut fresh poultry gall bladder or thawed poultry gall bladder with a meat grinder, pass through a 100-mesh sieve to get the liquid phase part (mainly bile), and add 90-95 g of the liquid phase part. % ethanol to a final concentration of 60-85% ethanol, centrifugation or filtration to remove the precipitate, and the supernatant is concentrated under reduced pressure and vacuum-dried to obtain avian gall powder.

[0056]Specifically in this embodiment: take 20Kg of thawed chicken gallbladder, cut it with a meat grinder, filter with a 100-mesh stainless steel filter screen and a multi-layer 100-mesh nylon mesh, take the liquid phase, and decompress the liquid phase to remove water to paste (density controlled at 1.10±0.05g / ml), add 25L of 95% ethanol to the paste and mix it overnight, centrifuge at 3800rpm for 5min to remove the precipitation, the super...

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Abstract

The invention relates to the field of biological medicine, relates to a preparation process of bear gall powder, and in particular relates to a preparation process of artificial bear gall powder. According to the scheme, a conversion solution containing cell active substances is used, so that the technical problems that the purification step of enzyme is complex, the cost is high and the enzyme activity is not easy to maintain are solved. According to the preparation process, after bioconversion is performed on livestock gall powder, the ratio of tauroursodeoxycholic acid to taurochenodeoxycholic acid of the obtained artificial bear gall powder is similar to that of natural bear gall powder. The artificial bear gall powder in the scheme can be used as a substitute of the natural bear gallpowder for deep processing and further application. The artificial bear gall powder product produced by adopting the preparation process is small in batch difference, the production process can be standardized, the conversion efficiency is improved, and industrial large-scale production can be achieved.

Description

technical field [0001] The invention relates to the field of biomedicine, relates to a production process of bear bile powder, and in particular relates to a production process of artificial bear bile powder. Background technique [0002] Bear bile powder is obtained by drying the gallbladder bile of black bears or brown bears. Bear bile powder has antispasmodic, anticonvulsant, anti-inflammatory and gallstone-dissolving effects, and is a good medicine for protecting the liver and gallbladder. Various modern clinical practice applications also show that bear bile can treat a variety of hepatobiliary diseases. Due to the limited resources of wild bears, artificial breeding is now commonly used to produce bear bile powder (that is, natural bear bile powder). At present, the annual output of natural bear bile powder in my country is about 30 tons, which cannot meet people's growing health needs. [0003] In order to solve the problem of the shortage of natural bear bile powd...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/413A61K9/14
CPCA61K9/14A61K35/413
Inventor 赵志斌王丹丹秦松柏程雷丁峰罗德彬张苏敏
Owner CHONGQING KINBEAR BIOTECHNOLOGY CO LTD
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