CN108828086A - A kind of artificial bear gall powder and its method for evaluating quality - Google Patents

A kind of artificial bear gall powder and its method for evaluating quality Download PDF

Info

Publication number
CN108828086A
CN108828086A CN201810384741.2A CN201810384741A CN108828086A CN 108828086 A CN108828086 A CN 108828086A CN 201810384741 A CN201810384741 A CN 201810384741A CN 108828086 A CN108828086 A CN 108828086A
Authority
CN
China
Prior art keywords
bear gall
artificial bear
gall powder
artificial
powder
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201810384741.2A
Other languages
Chinese (zh)
Inventor
赵淑娟
王峥涛
许盈芃
杨莉
周吉燕
胡之璧
张金家
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai University of Traditional Chinese Medicine
Original Assignee
Shanghai University of Traditional Chinese Medicine
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai University of Traditional Chinese Medicine filed Critical Shanghai University of Traditional Chinese Medicine
Priority to CN201810384741.2A priority Critical patent/CN108828086A/en
Publication of CN108828086A publication Critical patent/CN108828086A/en
Pending legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/57—Birds; Materials from birds, e.g. eggs, feathers, egg white, egg yolk or endothelium corneum gigeriae galli

Landscapes

  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

The present invention provides a kind of artificial bear gall powder and its method for evaluating quality, belongs to field of biotechnology.The preparation process is simply controllable, prepares quality of finished qualification, and raw material is cheap and easy to get, is the breakthrough that artificial bear gall powder is prepared by biotechnological method.

Description

A kind of artificial bear gall powder and its method for evaluating quality
Technical field
The present invention relates to a kind of artificial bear gall powder and its method for evaluating quality.It specifically, is being sent out using recombination engineering Scale fermented and cultured is carried out in fermentation tank and purifies the technique of preparation artificial bear gall powder, and to the artificial of technique production preparation Bear gall powder carries out quality evaluation, belongs to field of biotechnology.
Background technique
Bear gall powder is the pulverulent product of ursidae animal black bear or brown bear gall-bladder, has heat-clearing, calming the liver, improving eyesight, cholagogue, solution Convulsion, dissolve stone and other effects.Clinic is usually used in excessive heat of infant infantile convulsion, epilepsy, twitch, jaundice；Carbuncle swells, hemorrhoid, hot eyes cloudiness are controlled in external application Deng.Bear gall powder mainly contains bile acid, and principle active component is Tauro ursodesoxy cholic acid (TUDCA), Chinese Pharmacopoeia regulation TUDCA content is not less than 23%.
Currently, the bear gall powder of domestic medical market relies primarily on the method self-control of import and " bear living takes gallbladder ".Chemical synthesis Method preparation TUDCA because complex steps are at high cost and environmental pollution is not used widely greatly.Using biotechnology by source Extensive poultry bladder juice product bioconversion not only solves bear gall powder resource scarcity at the alternate resources with natural bear gall powder, but also has Conducive to promotion traditional Chinese medicine technology modernization.
Poultry bile and bear gall juice in chemical composition main difference is that：Poultry bile without containing TUDCA but with Taurochenodeoxycholic Acid (TCDCA) is main component.It is the epimer of 7 hydroxyls in TUDCA and TCDCA structure.Research Show that TCDCA can pass through 7alpha-Hydroxysteroid dehydrogenase (7 α-HSDH) and 7beta-Hydroxysteroid dehydrogenase (7 β-HSDH) Be catalytically converted into TUDCA.
Summary of the invention
The technical problem to be solved in the present invention is that a kind of artificial bear gall powder and its method for evaluating quality
The present invention relates to following technical solutions：
A kind of artificial bear gall powder.
A kind of artificial bear gall powder, it is characterised in that：TUDCA content is not less than 23%, and TUDCA and TCDCA
For ratio between 1~1.5, intermediate product T-7-KLCA content is lower than 5%.
A kind of artificial bear gall powder, it is characterised in that：Moisture content is lower than 6%.
A kind of artificial bear gall powder, it is characterised in that：Endotoxic content is lower than 0.015EU/ml.
A kind of extracting method of artificial bear gall powder：It is characterized in that：
It collects fermentation liquid 8000rpm centrifugation 20min and takes supernatant liquor, cross D101 macroreticular resin, an applied sample amount and resin In equal volume, flow control be not linked to be line at dripping to fall, and first uses H2O rushes colourless to eluent, then is rushed with 95% ethyl alcohol to elution Liquid is colourless and collects the elution fractions, and 50 DEG C of vacuum rotary steams, concentrate crosses 0.45 μm of organic filter membrane, and filtrate water-bath is waved to medicinal extract Shape, 50 DEG C of vacuum ovens are dry to constant weight, and taking-up is smashed to pieces up to artificial bear gall powder；
The source of fermentation liquid includes but not limited to artificial bear ball fermentation liquid.
A kind of analysis method of artificial bear gall powder：It is characterized in that：
The monitoring of artificial bear ball powder content
The solution of artificial bear gall powder is sampled, 12000rpm is centrifuged 1.5min, takes supernatant and by 1:5 ratio is added The n-butanol being saturated with water extracts it, after mixing layering, takes out supernatant liquor and with being dried with nitrogen or 70 DEG C are waved Dry, the isometric HPLC grade methanol of the supernatant taken after addition and centrifugation re-dissolves, and crosses 0.22 μm of filter membrane；
The source of the solution of artificial bear gall powder includes but not limited to the first of the ethanol solution of artificial bear gall powder, artificial bear gall powder Alcoholic solution；
With 120 EC-C18 column of reverse chromatograms column Agilent Poroshell, 2.7 μm, 4.6mm × 150mm makes With CAD detector, flow velocity 0.6ml/min, sample volume is 1 μ l, 40 DEG C of column temperature, frequency acquisition 10Hz, 45 DEG C of atomization temperature, is flowed Dynamic is mutually the 5mmol/L ammonium acetate aqueous solution of acetonitrile and 0.3% formic acid, carries out gradient elution；
A kind of analysis method of artificial bear gall powder：It is characterized in that：
The monitoring of various Determination of Bile Acids
Fermentation liquid is sampled, 12000rpm is centrifuged 1.5min, takes supernatant and by 1:What 5 ratio addition was saturated with water N-butanol extracts it, after mixing layering, takes out supernatant liquor and with being dried with nitrogen or 70 DEG C volatilize, be added and from The isometric HPLC grade methanol of the supernatant taken after the heart re-dissolves, and crosses 0.22 μm of filter membrane；
The source of fermentation liquid includes but not limited to artificial bear ball fermentation liquid；
With 120 EC-C18 column of reverse chromatograms column Agilent Poroshell, 2.7 μm, 4.6mm × 150mm makes With CAD detector, flow velocity 0.6ml/min, sample volume is 1 μ l, 40 DEG C of column temperature, frequency acquisition 10Hz, 45 DEG C of atomization temperature, is flowed Dynamic is mutually the 5mmol/L ammonium acetate aqueous solution of acetonitrile and 0.3% formic acid, carries out gradient elution；
In order to solve the above technical problems, the technical solution adopted by the present invention is as follows：
Optimum combination engineering bacteria carries out scale fermented and cultured using fermentor, with poultry bile that is inexpensive and being easily obtained Product is fermentation substrate, and by Taurochenodeoxycholic Acid in substrate (TCDCA) component portion converted in-situ is Tauroursodeoxycholic Acid Sour (TUDCA), and to the preparation process that main component in fermentation liquid is isolated and purified.
It is a kind of to use recombination engineering BL21*-α1β2Scale fermented and cultured prepares the technique of artificial bear gall powder, feature It is：The process optimization for the high density fermentation that recombination engineering carries out in the fermenter, optimization including strain preculture condition, The optimization of engineering bacteria inducing culturing condition, the optimization of fermentation medium, fermented and cultured process substrate transformation time and zymotechnique The optimization of process and product preparation.
The engineering bacteria BL21*-α1β2The Escherichia coli of 7 α-HSDH and 7 β-HSDH genes are expressed while building for us (Escherichia coli, E.coli) BL21*-α1β2(number of patent application 201610191921.X)；
The substrate is poultry bladder juice product, including highly finished product and semifinished product；Chicken gallbladder powder and crude chicken gallbladder powder are such as refined, such as Duck gallbladder powder and crude duck gallbladder powder are refined, goose gallbladder powder and crude goose gallbladder powder are such as refined.
The technique of recombination engineering scale fermented and cultured preparation artificial bear gall powder of the present invention, which is characterized in that It comprises the following specific steps that：
A. the culture of recombination engineering
1. takes out engineering bacteria from -80 DEG C of refrigerators, it is being attached with 50~100mg/L (preferably 80~100mg/L) ammonia benzyl Penicillin (Amp+) streak inoculation in LB solid plate culture medium, in 20~40 DEG C of (preferably 25~37 DEG C) activation cultures 9~ 36 hours (preferably 10~16 hours) (preferably 25~37 DEG C, preferably 37 DEG C)；
2. first order seed：From picking single colonie on LB solid plate culture medium, it is inoculated in additional 50~100mg/L (preferably 80~100mg/L) ampicillin (Amp+) in LB liquid medium, 75~225rpm (preferably 180~225rpm), 20~ Preculture 8~24 hours (preferably 8~16 hours) under 40 DEG C (preferably 25~37 DEG C)；
3. secondary seed：Above-mentioned culture is pressed 1:20 ratios are inoculated into additional 50~100mg/L (preferably 80~100mg/ L) ampicillin (Amp+) in 2-YT fluid nutrient medium, 75~225rpm (preferably 180~225rpm), 20~37 DEG C it is (excellent Select 25~37 DEG C) under shake culture 3~12 hours (preferably 3~9 hours)；
4. secondary seed is pressed 1:20 ratios are inoculated into additional 50~100mg/L (preferably 80~100mg/L) ammonia benzyl mould Element (Amp+) in M9-GY Optimal Medium, 1~6L/min of ventilatory capacity (preferably 2~4L/min), batch phase pH value is maintained at 5.0~8.0 (preferably 6.0~7.0), 20~37 DEG C of temperature (preferably 30~37 DEG C), 400~800rpm of stirring rate (preferably 600 ~800rpm), culture 3-8 hours (preferably 4-6 hours) to OD600 is 3.0~7.0 (preferably 4.0~6.0), adds isopropyl β-D- Thiogalactopyranoside (IPTG) carries out Fiber differentiation to final concentration of 0.1~2mM (preferably 0.1~1mM)：Induction training The stage of supporting makes pH value be maintained at 5.0~8.0 (preferably 6.0~7.0) by Feeding ammonia water and orthophosphoric acid, and 20~37 DEG C of temperature (excellent Change 25~30 DEG C), cultivate 2~6 hours (preferably 3~5 hours).
A) fermenting substrate converts
1. added into M9-GY Optimal Medium appropriate quality it is thick/purification chicken gallbladder powder, 20~37 DEG C of temperature (preferably 20~ 25 DEG C), so that pH value is maintained at 5.0~8.0 (preferably 6.0~7.0), 0~6L/min of ventilatory capacity by Feeding ammonia water and orthophosphoric acid (preferably 0~4L/min), 400~800rpm of stirring rate (preferably 400~600rpm) cultivate 1 hour~10 hours (preferably 1~ 5 hours), stop ventilation, do not adjust pH value, do not stir, 5~18h is stood at 16 DEG C~37 DEG C.
B) fermentation liquid converted product is analyzed
1. fermentation process terminates, fermentation liquid is collected, 12000rpm is centrifuged 1.5min, takes supernatant and by 1:5 ratio is added The n-butanol being saturated with water extracts it, after mixing layering, takes out supernatant liquor and (or 70 DEG C are waved with being dried with nitrogen It is dry), the isometric HPLC grade methanol of the supernatant taken after addition and centrifugation re-dissolves, and crosses 0.22 μm of filter membrane, reverse chromatograms column 120 EC-C18 column of Agilent Poroshell (2.7 μm, 4.6mm × 150mm) produces conversion using CAD detector Object is analyzed.
C) preparation of artificial bear gall powder
It collects fermentation liquid 8000rpm centrifugation 20min and takes supernatant liquor, cross D101 macroreticular resin, an applied sample amount and resin In equal volume, flow control be not linked to be line at dripping to fall.First use H2O rushes colourless to eluent, then is rushed with 95% ethyl alcohol to elution Liquid is colourless and collects the elution fractions.50 DEG C of vacuum rotary steams, concentrate cross 0.45 μm of organic filter membrane, and filtrate water-bath is waved to medicinal extract Shape, 50 DEG C of vacuum ovens are dry to constant weight, and taking-up is smashed to pieces up to artificial bear gall powder.
The technique using engineering bacteria scale fermented and cultured preparation artificial bear gall powder, it is characterised in that：Described The composition of LB solid medium is as follows：Tryptone (Tryptone) 10g/L, yeast extract (Yeast Extract) 5g/L, Sodium chloride (NaCl) 10g/L, agarose 15.0g/L.
The technique using engineering bacteria scale fermented and cultured preparation artificial bear gall powder, which is characterized in that described The composition of LB liquid medium is as follows：Tryptone 10g/L, yeast extract 5g/L, sodium chloride 1g/L.
The technique using engineering bacteria scale fermented and cultured preparation artificial bear gall powder, it is characterised in that：Described The composition of 2-YT fluid nutrient medium is as follows：Peptone 16g/L, yeast powder 10g/L, NaCl 5g/L.
The technique using engineering bacteria scale fermented and cultured preparation artificial bear gall powder, it is characterised in that：Described M9-GY Optimal Medium preparation method is as follows：5~8g/L Na2HPO4, 2~5g/L KH2PO4, 2~5g/L (NH4)2HPO4,0.1 ~1.0g/L NaCl, 1~3g/L NH4Cl, 10~30ml/L glycerol, 10~30g/L yeast extract, 1.0~3.0mM MgSO4, 0.1~1.0mM CaCl2
The technique using engineering bacteria scale fermented and cultured preparation artificial bear gall powder, it is characterised in that：Step e) In substrate be added in powder form.
The technique using engineering bacteria scale fermented and cultured preparation artificial bear gall powder, it is characterised in that：The bottom Object concentration is 2~20g/L, makes Taurochenodeoxycholic Acid in substrate (TCDCA) final concentration of 2~20g/L (preferably 2~10g/L).
The technique using engineering bacteria scale fermented and cultured preparation artificial bear gall powder, it is characterised in that：Step a) Middle first order seed is in 50~100mg/L (preferably 80~100mg/L) ampicillin (Amp+) culture 9 in LB liquid medium~ 24 hours (preferably 9~16 hours), secondary seed culture was in 50~100mg/L (preferably 80~100mg/L) ampicillin (Amp+) cultivate 3~12 hours (preferably 4~9 hours) in 2-YT fluid nutrient medium.
The technique using engineering bacteria scale fermented and cultured preparation artificial bear gall powder, it is characterised in that：Step a) Middle first order seed presses 1:3~6 hours (preferably 3~5 hours) to secondary seed OD600=2~5 are cultivated after 20 inoculations；
The technique using engineering bacteria scale fermented and cultured preparation artificial bear gall powder, it is characterised in that：Step a) Before middle addition IPTG cultivation temperature be 20~37 DEG C (preferably 25~37 DEG C), the time be 3~6 hours (preferably 3~4 hours) extremely OD600=1.5~7；
The technique using engineering bacteria scale fermented and cultured preparation artificial bear gall powder, it is characterised in that：Step a) Middle addition IPTG concentration is 0.1~2mM (preferably 0.1~0.5mM)；Fiber differentiation temperature is 20~37 DEG C (preferably 25~35 DEG C), the Fiber differentiation time is 2~6 hours (preferably 2~4 hours)；
The technique using engineering bacteria scale fermented and cultured preparation artificial bear gall powder, it is characterised in that：Step b) In directly thick/purification chicken gallbladder powder is added in the engineering bacterium solution of Liquid Culture；Fermentation temperature is 16~37 DEG C (preferably 20~28 DEG C), 400~800rpm of stirring rate (preferably 400~600rpm) makes pH value be maintained at 5.0 by Feeding ammonia water and orthophosphoric acid ~8.0 (preferably 6.0~7.0), 0~6L/min of ventilatory capacity (preferably 0~4L/min) are cultivated 1 hour~10 hours (preferably 1~5 Hour), then stop ventilation, do not adjust pH value, do not stir, 5~18h is stood at 16 DEG C~37 DEG C；
The technique using engineering bacteria scale fermented and cultured preparation artificial bear gall powder, it is characterised in that：Step c) TUDCA and TCDCA content ratio is between 1~1.5 in the fermentation liquid of middle removal thallus, and T-7K-LCA content is lower than bile acid The 8% of ingredient total amount；
The technique using engineering bacteria scale fermented and cultured preparation artificial bear gall powder, it is characterised in that：Step d) Resin used is D101 macroreticular resin, and an applied sample amount and resin are isometric, and flow control be not linked to be line at dripping to fall, and first use H2O rushes colourless to eluent, then rushes colourless to eluent with 95% ethyl alcohol and collects the elution fractions.
The present invention relates to a kind of techniques using engineered strain scale fermented and cultured preparation artificial bear gall powder.
Engineered strain BL21 is used the present invention relates to a kind of*-α1β2The work of scale fermented and cultured preparation artificial bear gall powder Skill, it is characterised in that:
Processing step is as follows：
A. engineered strain BL21*-α1β2The preculture of seed liquor
1. engineering bacteria is inoculated into the LB solid plate culture medium for be attached with 50~100mg/L ampicillin and activates by, In 37 DEG C activation culture 12 hours；
The composition of the LB solid medium is as follows：Tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/ L, agarose 15.0g/L；
2. is inoculated in the LB liquid for being attached with 100mg/L ampicillin from picking single colonie on LB solid plate culture medium In body culture medium, the shake culture 9~12 hours at 225rpm, 37 DEG C；
The composition of the LB liquid medium is as follows：Tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/ L；
3. 2. middle bacterium solution is seeded to the 2-YT liquid training for being attached with 100mg/L ampicillin by by 2~10% inoculum concentrations It supports in base, the shake culture 3~12 hours at 225rpm, 37 DEG C；
The composition of the 2-YT fluid nutrient medium is as follows：Tryptone 16g/L, yeast powder extracts 10g/L, sodium chloride 5g/L；
B. the ferment tank technique of thick/purification chicken gallbladder powder；
1. is by 3. gained bacterium solution is seeded in M9-GY Optimal Medium by 2~10% inoculum concentrations in A, 1~6L/ of ventilatory capacity Min, 37 DEG C, pH value maintains 7.0, stirring rate 600rpm by orthophosphoric acid and ammonium hydroxide, and culture 3~6 hours to OD600 is 1.5~6；
2. the isopropyl-beta D-thio galactopyranoside of final concentration of 0.3mmol/L, 1~6L/ of ventilatory capacity is added in Min, temperature are down to 30 DEG C, and pH value is adjusted to 6.5, stirring rate 600rpm, and Fiber differentiation 1~3 hour；
3. appropriate thick/purification chicken gallbladder powder is added in, 25 DEG C of temperature, pH value is maintained at 6.5,1~6L/min of ventilatory capacity, stirring After fermenting 1~5 hour under the conditions of rate 600rpm, stopping ventilation, temperature is still 25 DEG C, pH value do not adjusted, is not stirred, 16 DEG C~ 5~18h is stood at 37 DEG C；
C. in fermentation liquid various Determination of Bile Acids monitoring
1. samples fermentation liquid, 12000rpm is centrifuged 1.5min, takes supernatant and by 1:5 ratio addition is full with water The n-butanol of sum extracts it, after mixing layering, takes out supernatant liquor and with being dried with nitrogen (or 70 DEG C volatilize), addition Isometric HPLC grade methanol re-dissolves with the supernatant that takes after centrifugation, crosses 0.22 μm of filter membrane；
2. 120 EC-C18 column of reverse chromatograms column Agilent Poroshell, 2.7 μm, 4.6mm × 150mm, using CAD detector, flow velocity 0.6ml/min, sample volume is 1 μ l, and 40 DEG C of column temperature, frequency acquisition 10Hz, atomization is warm 45 DEG C of degree, mobile phase are the 5mmol/L ammonium acetate aqueous solution of acetonitrile and 0.3% formic acid, carry out gradient elution；
Time (min) Flow velocity (mL/min) Acetonitrile The 5mmol/L ammonium acetate aqueous solution of 0.3% formic acid
0 0.6 20 80
15 0.6 27 73
25 0.6 35 65
35 0.6 37 63
45 0.6 90 10
D. bile acid isolating and purifying and prepares in fermentation liquid
It collects fermentation liquid 8000rpm centrifugation 20min and takes supernatant liquor, cross D101 macroreticular resin, an applied sample amount and resin In equal volume, flow control be not linked to be line at dripping to fall.First use H2O rushes colourless to eluent, then is rushed with 95% ethyl alcohol to elution Liquid is colourless and collects the elution fractions.50 DEG C of vacuum rotary steams, concentrate cross 0.45 μm of organic filter membrane, and filtrate water-bath is waved to medicinal extract Shape, 50 DEG C of vacuum ovens are dry to constant weight, and taking-up is smashed to pieces up to artificial bear gall powder.
The present invention relates to a kind of technique using engineered strain scale fermented and cultured preparation artificial bear gall powder, feature exists In：The M9-GY Optimal Medium 1. used in step B is：6.78g/L Na2HPO4, 3g/L KH2PO4, 3.3g/L (NH4)2HPO4, 0.5g/L NaCl, 1g/L NH4Cl, 28ml/L glycerol, 10~30g/L yeast extract, 2mM MgSO4, 0.1mM CaCl2。
The present invention relates to a kind of technique using engineered strain scale fermented and cultured preparation artificial bear gall powder, feature exists In：If it is 25 DEG C of temperature that crude chicken gallbladder powder zymotechnique is 3. added in step B, pH value is maintained at 6.5,1~6L/min of ventilatory capacity, After fermenting 1~5 hour under the conditions of stirring rate 600rpm, stopping ventilation, temperature is still 25 DEG C, pH value do not adjusted, is not stirred, 16 5~18h is stood at DEG C；If it is 25 DEG C of temperature that purification chicken gallbladder powder zymotechnique, which is added, pH value is maintained at 6.5,1~6L/ of ventilatory capacity After fermenting 5 hours under the conditions of min, stirring rate 600rpm, stopping ventilation, temperature is still 25 DEG C, pH value do not adjusted, is not stirred, 5~18h is stood at 25 DEG C.
The present invention relates to a kind of technique using engineered strain scale fermented and cultured preparation artificial bear gall powder, feature exists In：Gained artificial bear gall powder is detected with method in step C in step D, TUDCA and TCDCA ratio is between 1~1.5 and T-7- KLCA content is lower than 5%, and moisture content is lower than 6%, and endotoxic content is lower than 0.015EU/ml.
Compared with prior art, the present invention has the advantages that：
1) non-pathogenic Escherichia coli (Escherichia coli, the E.coli) BL21star (DE3) selected is used as work Journey bacterium host strain, is easy to cultivate, fermentation process is easily controllable.
2) M9-GY Optimal Medium is full of nutrition is conducive to thalli growth and protein expression, and fermentation process is simple, is easy to real Existing industrialized production.
3) scale fermented incubation time is short, technical process control is simple, conveniently, it is economical.
4) Taurochenodeoxycholic Acid (TCDCA) conversion rate is fast in substrate, can obtain proportion-controllable within a short period of time TUDCA, and TUDCA content is not less than 23%, TUDCA and TCDCA ratio between 1~1.5 in the artificial bear gall powder prepared, in Between product T-7-KLCA content be lower than 5%.
5) fermentation process is pollution-free, and fermentation liquid post-processing and product preparation process are easy, and efficiency of pcr product is high, preparation it is artificial Bear gall powder quality is stablized, and makes it possible large-scale production artificial bear gall powder, theoretical research and medicinal valence to artificial bear gall powder Extensive utilize of value has significant contribution.
Detailed description of the invention
Fig. 1 optimum induction cultivation results figure (influence of the a.IPTG concentration to conversion ratio；B. inductive condition combination is excellent Change)
Fig. 2 software optimization culture medium result figure
(1 is TUCA to the HPLC map of Fig. 3 primary bile acid standard items of the present invention, and 2 be TUDCA, and 3 be TCA, and 4 be T-7- KLCA, 5 be TCDCA)
Substrate used in Fig. 4 present invention refines, (a. refines chicken gallbladder powder HPLC map to the HPLC map of crude chicken gallbladder powder；B. thick Chicken gallbladder powder HPLC map processed) (3 be TCA, and 5 be TCDCA)
(a. is to refine chicken gallbladder powder as substrate optimization of fermentation conditions for the HPLC map of gained sample under the conditions of Fig. 5 different fermentations The HPLC map of post-fermentation liquid；B. to refine the HPLC map of the artificial bear gall powder finished product that chicken gallbladder powder is prepared as substrate；C. with thick Chicken gallbladder powder processed is the HPLC map of substrate optimization of fermentation conditions post-fermentation liquid；D. the artificial bear prepared using crude chicken gallbladder powder as substrate The HPLC map of gallbladder powder finished product)
(1 is TUCA, and 2 be TUDCA, and 3 be TCA, and 4 be T-7-KLCA, and 5 be TCDCA)
The stage is stood in Fig. 6 zymotechnique of the present invention and stands phase temperature investigates result (shadow of a. standing to conversion ratio It rings；B. dwell temperature investigates purification chicken gallbladder powder conversion situation；C. dwell temperature investigates crude chicken gallbladder powder conversion situation)
(a. is prepared using refining chicken gallbladder powder as substrate the artificial bear gall powder finished figure that Fig. 7 uses technique of the present invention to prepare Artificial bear gall powder finished figure；B. the artificial bear gall powder finished figure prepared using crude chicken gallbladder powder as substrate)
Specific embodiment
The present invention is described in detail with reference to the accompanying drawings and examples, these examples and attached drawing only play explanation Property effect, the content being not intended to limit the present invention.Therefore, all modification and deformation done without prejudice to spirit of that invention, should all It is included in the scope of the present invention.
Embodiment 1
Influence of 1 secondary seed medium to growth curve
1) engineering bacteria that -80 DEG C of refrigerators save is applied to the LB solid containing 100mg/L ampicillin with method of scoring In plating medium, in 12 hours of 37 DEG C of activation cultures；
The composition of the LB solid medium is as follows：Tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/ L, agarose 15.0g/L.
2) from picking single colonie on LB solid plate culture medium, it is inoculated in the LB liquid of additional 100mg/L ampicillin In culture medium, first order seed is obtained within shake culture 9 hours in 225rpm, 37 DEG C；
The composition of the LB liquid medium is as follows：Tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/ L；
3) above-mentioned culture is pressed 1:20 ratios be inoculated into respectively 2-YT fluid nutrient medium and with 2) described in consistent LB In fluid nutrient medium, the shake culture at 225rpm/min, 37 DEG C surveys the two growth curve respectively；
The composition of the 2-YT fluid nutrient medium is as follows：Tryptone 16g/L, yeast extract 10g/L, sodium chloride 5g/L；
Influence of the 2 seed inoculum concentrations to growth curve
1) and 2) by operation carries out obtaining first order seed in 1, first order seed is pressed 1 respectively:10,1:20,1:50 ratios connect Kind is into 2-YT fluid nutrient medium, the shake culture at 225rpm, 37 DEG C, surveys three's growth curve respectively；
1) and 2) influence of the 3 secondary seed incubation times to growth curve after inoculation is by operation carries out obtaining level-one kind in 1 First order seed is pressed 1 by son respectively:20 ratios are inoculated into 2-YT fluid nutrient medium, the shake culture at 225rpm, 37 DEG C, point 1 is not pressed in 2h (logarithm early period), 5h (mid-log phase), 9h (late log phase):20 ratios are inoculated into M9 fluid nutrient medium, 225rpm, shake culture at 37 DEG C, survey three's growth curve respectively；
Embodiment 2
The optimization of M9-GY Optimal Medium is tested
1) using the design method of Box-Behnken Design in Design-Expert.V8.0.6.1 software, with M9 base Based on basal culture medium constituent, design is the test of four factors with glucose, glycerol, yeast extract and tryptone, Glucose, yeast extract, tryptone select 0,10, tri- level of 20g/L, and glycerol selects 0,15, tri- level of 30ml/L, with OD600 and conversion ratio are used as inspection target, the 29 groups of schemes and its result such as following table that software design goes out simultaneously：
2) specific fermentation process is as follows：First order seed is obtained by operation in embodiment 1, first order seed is pressed 1:20 ratios It is inoculated into liquid 2-YT culture medium, shake culture 4 hours is at 225rpm, 37 DEG C up to secondary seed；Secondary seed is pressed 1: 20 ratios are inoculated into above-mentioned various fluid nutrient mediums, and fluid nutrient medium presses above-mentioned table based on M9 minimal medium formula Lattice formula adds remaining four kinds of ingredients, in triplicate, the shake culture 5 hours at 225rpm, 37 DEG C；IPTG is added to final concentration For 0.3mM, the shake culture 3 hours at 225rpm, 30 DEG C；Adding purification chicken gallbladder powder by final concentration of 5g/L is substrate, 225rpm, it ferments within shake culture 6 hours at 25 DEG C, collects sample, survey OD600 and the substrate transformation rate.
M9 minimal medium composition is as follows：6.78g/L Na2HPO4, 3g/L KH2PO4, 0.5g/L NaCl, 1g/L NH4Cl, 2mM MgSO4, 0.1mM CaCl2。
The PBS-M9 optimization culture based formulas for calculating through Design-Expert.V8.0.6.1 software and being obtained before integrating It is formed with the liquid in conventional fermentation process fluid infusion stage, obtains optimal medium i.e. M9-GY Optimal Medium, be formulated as follows： 6.78g/L Na2HPO4, 3g/L KH2PO4, 0.5g/L NaCl, 1g/L NH4Cl, 2mM MgSO4, 0.1mM CaCl2, 4g/L (NH4)2HPO4, 20g/L yeast extract, 28ml/L glycerol.Software optimization culture medium result is as shown in Fig. 2.
Embodiment 3
The optimization in Fiber differentiation stage
Influence of the 1IPTG concentration to conversion ratio
1) first order seed is obtained by operation in embodiment 1, first order seed is pressed 1:20 ratios are inoculated into liquid 2-YT culture Base, shake culture 4 hours is at 225rpm, 37 DEG C up to secondary seed；
2) secondary seed is pressed 1:20 ratios are inoculated into M9-GY Optimal Medium fluid nutrient medium, in triplicate, 225rpm, shake culture 5 hours at 37 DEG C；
3) be added IPTG to final concentration be respectively 0.1,0.3,0.6,0.8,1.0mM, the shake culture at 225rpm, 30 DEG C 3 hours；Adding purification chicken gallbladder powder by final concentration of 5g/L is substrate, is sent out within shake culture 6 hours at 225rpm, 25 DEG C Ferment；As a result as shown in Figure 1a：
2 inductive condition Combinatorial Optimizations
Choose respectively recombination engineering in M9-GY Optimal Medium logarithm early period (4 hours) of growth curve, in logarithm The time point of phase (5.5 hours), late log phase (7.5 hours) as addition IPTG, induction duration select 3h, 4h, 5h group two-by-two It closes, nine groups of experimental programs are as shown in the table, as a result as shown in Figure 1 b：
Embodiment 4
5 liters of fermentor high density fermentations
1) by first order seed is operated to obtain in embodiment 1, first order seed is pressed 1:20 ratios are inoculated into liquid 2-YT culture medium, Shake culture 4 hours is at 225rpm, 37 DEG C up to secondary seed；
2) 3 liters of fluid nutrient mediums are prepared by M9-GY optimization culture based formulas in embodiment 2 and (MgSO is not added4And CaCl2) dress Emerging biological 5 are protected to Shanghai and lifts away from a disinfection fermentation tank, add ampicillin in autoclave after sterilizing again to final concentration of 100mg/L and by formula in concentration add MgSO4And CaCl2；
3) secondary seed is pressed 1:20 ratios are inoculated into fermentor, ventilatory capacity 2L/min, and stirring rate 600rpm passes through Feeding ammonia water and orthophosphoric acid make pH value be maintained at 7.0, and 37 DEG C of temperature are cultivated 4 hours；
4) IPTG to final concentration 0.3mM, ventilatory capacity 2L/min, stirring rate 600rpm are added, pH value is maintained at 6.5, temperature 30 DEG C are spent to cultivate 3 hours；
5) weighing purification chicken gallbladder powder by final concentration 10g/L is that fermentor, ventilatory capacity 2L/min, stirring rate is added in substrate 600rpm makes pH value be maintained at 6.5, after 25 DEG C of temperature are cultivated 5 hours, stops ventilation by Feeding ammonia water and orthophosphoric acid, uncomfortable PH value is saved, is not stirred, is detected after 12h is stood at 25 DEG C by 5 the method for embodiment, HPLC map is as shown in attached drawing 5a；
6) weighing crude chicken gallbladder powder by final concentration 11.5g/L is that fermentor, ventilatory capacity 2L/min, stirring rate is added in substrate 600rpm, pH value are maintained at 6.5, after 25 DEG C of temperature are cultivated 3.5 hours, stop ventilation, do not adjust pH value, do not stir, at 16 DEG C It is detected after standing 12h by 5 the method for embodiment, HPLC map is as shown in attached drawing 5c；
Embodiment 5
The HPLC of product is tested and analyzed
1) chromatographic column：120 EC-C18 column of Agilent Poroshell (2.7 μm, 4.6mm × 150mm)
Thermo LPG-3400SD high performance liquid chromatograph, Corona Veo Charged Aerosol Detector electricity Spraying detector；
40 DEG C of column temperature, frequency acquisition 10Hz, 45 DEG C of column temperatures of atomization temperature：40℃；Flow velocity：0.6ml/min；1 μ l of sample volume；
Mobile phase：A- acetonitrile；B- contains the 5mmol/L ammonium acetate aqueous solution of 0.3% formic acid
Gradient condition：
Time (min) Flow velocity (mL/min) Acetonitrile The 5mmol/L ammonium acetate aqueous solution of 0.3% formic acid
0 0.6 20 80
15 0.6 27 73
25 0.6 35 65
35 0.6 37 63
45 0.6 90 10
Fermentation broth sample 12000rpm is centrifuged 1.5min, is centrifuged with the water saturated extracting n-butyl alcohol 100ul fermentation liquid of 500ul Supernatant afterwards, supernatant is fully transferred to another centrifuge tube after layering, is dissolved in HPLC grades of methanol solutions after being dried with nitrogen again, uses 0.22 μm of membrane filtration is the processing of sample to be tested.
2) the HPLC map of bile acid standard items is as shown in Fig. 3
3) essence/crude chicken gallbladder powder HPLC map is as shown in Fig. 4.
Embodiment 6
The investigation for whether being conducive to conversion is stood in fermentation technology process
1) by first order seed is operated to obtain in embodiment 1, first order seed is pressed 1:20 ratios are inoculated into liquid 2-YT culture medium, Shake culture 4 hours is at 225rpm, 37 DEG C up to secondary seed；
2) 3 liters of fluid nutrient mediums are prepared by M9-GY optimization culture based formulas in embodiment 2 and (MgSO is not added4And CaCl2) dress Emerging biological 5 are protected to Shanghai and lifts away from a disinfection fermentation tank, add ampicillin in autoclave after sterilizing again to final concentration of 100mg/L and by formula in concentration add MgSO4And CaCl2；
3) secondary seed is pressed 1:20 ratios are inoculated into fermentor, ventilatory capacity 2L/min, and stirring rate 600rpm passes through Feeding ammonia water and orthophosphoric acid make pH value be maintained at 7.0, and 37 DEG C of temperature are cultivated 4 hours；
4) IPTG to final concentration 0.3mM, ventilatory capacity 2L/min, stirring rate 600rpm are added, pH value is maintained at 6.5, temperature 30 DEG C are spent to cultivate 3 hours；
5) weighing purification chicken gallbladder powder by final concentration 10g/L is that fermentor, ventilatory capacity 2L/min, stirring rate is added in substrate 600rpm makes pH value be maintained at 6.5 by Feeding ammonia water and orthophosphoric acid, and 25 DEG C of temperature are cultivated, the 0h after substrate is added, 6h, 17h taking-up 9ml fermentation liquid are loaded in 15ml centrifuge tube (volume ratio in simulation fermentor) and stand in 25 DEG C of incubators It is detected simultaneously by 5 the method for embodiment after 29h, 23h, 12h, as a result as shown in fig. 6.
Embodiment 7
Refine the investigation that chicken gallbladder powder stands process optimum temperature
1) by first order seed is operated to obtain in embodiment 1, first order seed is pressed 1:20 ratios are inoculated into liquid 2-YT culture medium, Shake culture 4 hours is at 225rpm, 37 DEG C up to secondary seed；；
2) 3 liters of fluid nutrient mediums are prepared by M9-GY optimization culture based formulas in embodiment 2 and (MgSO is not added4And CaCl2) dress Emerging biological 5 are protected to Shanghai and lifts away from a disinfection fermentation tank, add ampicillin in autoclave after sterilizing again to final concentration of 100mg/L and by formula in concentration add MgSO4And CaCl2；
3) secondary seed is pressed 1:20 ratios are inoculated into fermentor, ventilatory capacity 2L/min, and stirring rate 600rpm passes through Feeding ammonia water and orthophosphoric acid make pH value be maintained at 7.0, and 37 DEG C of temperature are cultivated 4 hours；
4) IPTG to final concentration 0.3mM, ventilatory capacity 2L/min, stirring rate 600rpm are added, by Feeding ammonia water and just Phosphoric acid makes pH value be maintained at 6.5, and 30 DEG C of temperature are cultivated 3 hours；
5) weighing purification chicken gallbladder powder by final concentration 10g/L is that fermentor, ventilatory capacity 2L/min, stirring rate is added in substrate 600rpm makes pH value be maintained at 6.5, after 25 DEG C of temperature are cultivated 5 hours, stops ventilation by Feeding ammonia water and orthophosphoric acid, uncomfortable PH value is saved, is not stirred, is detected after standing 12h at 16 DEG C, 25 DEG C, 37 DEG C by 5 the method for embodiment, HPLC map is such as Shown in attached drawing 5a.
Embodiment 8
Crude chicken gallbladder powder stands the investigation of process optimum temperature
1) by first order seed is operated to obtain in embodiment 1, first order seed is pressed 1:20 ratios are inoculated into liquid 2-YT culture medium, Shake culture 4 hours is at 225rpm, 37 DEG C up to secondary seed；；
2) 3 liters of fluid nutrient mediums are prepared by M9-GY optimization culture based formulas in embodiment 2 and (MgSO is not added4And CaCl2) dress Emerging biological 5 are protected to Shanghai and lifts away from a disinfection fermentation tank, add ampicillin in autoclave after sterilizing again to final concentration of 100mg/L and by formula in concentration add MgSO4And CaCl2；
3) secondary seed is pressed 1:20 ratios are inoculated into fermentor, ventilatory capacity 2L/min, and stirring rate 600rpm passes through Feeding ammonia water and orthophosphoric acid make pH value be maintained at 7.0, and 37 DEG C of temperature are cultivated 4 hours；
4) IPTG to final concentration 0.3mM, ventilatory capacity 2L/min, stirring rate 600rpm are added, by Feeding ammonia water and just Phosphoric acid makes pH value be maintained at 6.5, and 30 DEG C of temperature are cultivated 3 hours；
5) weighing crude chicken gallbladder powder by final concentration 11.5g/L is that fermentor, ventilatory capacity 2L/min, stirring rate is added in substrate 600rpm makes pH value be maintained at 6.5, after 25 DEG C of temperature are cultivated 3.5 hours, stops ventilation, no by Feeding ammonia water and orthophosphoric acid PH value is adjusted, is not stirred, is detected after standing 12h at 16 DEG C, 25 DEG C, 37 DEG C by 5 the method for embodiment, HPLC map As shown in attached drawing 5c.
Embodiment 9
The preparation and purification of tunning
1) it collects fermentation liquid 5000xg and is centrifuged 20min, collect supernatant liquor.
2) supernatant liquor crosses D101 macroreticular resin, and an applied sample amount and resin are isometric, and flow control is fallen not at dripping It is linked to be line.First use H2O rushes colourless to eluent, then rushes colourless to eluent with 95% ethyl alcohol and collects the elution fractions.
3) 50 DEG C of vacuum rotary steams, concentrate cross 0.45 μm of organic filter membrane, and filtrate water-bath is waved to medicinal extract shape.
4) 50 DEG C of vacuum ovens are dry to constant weight, and taking-up is smashed to pieces up to artificial bear gall powder.
Gained finished product is as shown in Fig. 7.Finished product is according to 5 the method for embodiment, according to established standard curve product, Measure TUDCA, TCDCA and T-7K-LCA content in tunning.The typical HPLC map of artificial bear gall powder finished product such as attached drawing 5b, Shown in 5d.
Embodiment 10
Determination of moisture in artificial bear gall powder
1) weighing bottle takes out to be placed in drier after drying half an hour after 105 DEG C of baking ovens dry 1 hour and weigh, and is denoted as m1, 105 DEG C of baking ovens continue to dry 1 hour, and drier is weighed after dry half an hour, is denoted as m2, and error is no more than between weighing twice 0.3mg is constant weight.
2) after constant weight, about 300mg samples weighing is taken out, is denoted as m3, drier dry half is small after 105 DEG C of baking ovens dry 5 hours When weigh m4.
3) 105 DEG C of baking ovens continue to dry 1 hour, and drier is weighed m5 after dry half an hour, and error does not surpass between weighing twice Crossing 0.3mg is constant weight.
4)
Embodiment 11
Endotoxin assay in artificial bear gall powder
It is tested using chromogenic substrate tachypleus amebocyte lysate box (Xiamen BioEndo Technology , Co.Ltd) of the present invention using this Endotoxic content in the artificial bear gall powder of preparation.
1) preparation of bacterial endotoxin standard solution：It is diluted to by mother liquor according to the form below of the endotoxin solution of 1.0EU/ml 0.01,0.025,0.05,0.1EU/ml concentration gradient；
2) preparation of reagent：It is configured shown in reagents, chromogenic substrate, the equal by specification of idol diazotizing reagent 1-3；
3) measuring endotoxin：Apyrogeneity test tube is taken, 100 μ l baterial endotoxin test water, endotoxin mark are separately added into Quasi- solution, artificial bear gall powder test sample；100 μ l reagents solution are added, are mixed, 37 DEG C incubate 8 minutes；Incubation terminates, and adds Enter 100 μ l color-developing substrate solutions, mix, 37 DEG C incubate 6 minutes；Incubation terminates, and 500 μ l idol diazotizing reagent, 1 solution is added, and mixes It is even, 500 μ l idol diazotizing reagent, 2 solution is added, mixes, 500 μ l idol diazotizing reagent, 3 solution is added, mixes, stands 5 minutes, in Absorbance value is read at 545nm wavelength.
4) endotoxin standard curve is y=1.2578x+0.0295, R2=0.9966.Using the people of preparation of the present invention Endotoxic content is lower than 0.015EU/ml in work bear gall powder.

Claims (7)

1. a kind of artificial bear gall powder.
2. a kind of artificial bear gall powder according to claim 1, it is characterised in that：TUDCA content be higher than 23%, and TUDCA with For TCDCA ratio between 1~1.5, intermediate product T-7-KLCA content is lower than 5%.
3. a kind of artificial bear gall powder according to claim 1, it is characterised in that：Moisture content is lower than 6%.
4. a kind of artificial bear gall powder according to claim 3, it is characterised in that：Endotoxic content is lower than 0.015EU/ml.
5. a kind of extracting method of artificial bear gall powder according to claim 1：It is characterized in that：
It collects fermentation liquid 8000rpm centrifugation 20min and takes supernatant liquor, cross D101 macroreticular resin volume, the resinites such as loading Long-pending fermentation liquid, flow control be not linked to be line at dripping to fall, are first using H2O rush it is colourless to eluent, then with 95% ethyl alcohol rush to Eluent is colourless and collects the elution fractions, 50 DEG C of vacuum rotary steams, and concentrate crosses 0.45 μm of organic filter membrane, filtrate water-bath wave to Medicinal extract shape, 50 DEG C of vacuum ovens are dry to constant weight, and taking-up is smashed to pieces up to artificial bear gall powder；
The source of fermentation liquid includes but not limited to artificial bear ball fermentation liquid.
6. a kind of analysis method of artificial bear gall powder according to claim 1：It is characterized in that：
The monitoring of artificial bear ball powder content
The solution of artificial bear gall powder is sampled, 12000rpm is centrifuged 1.5min, takes supernatant and by 1:Water is used in 5 ratio addition The n-butanol of saturation extracts it, after mixing layering, takes out supernatant liquor and with being dried with nitrogen or 70 DEG C volatilize, adds Enter the HPLC grade methanol isometric with the supernatant that takes after centrifugation to re-dissolve, 0.22 μm of filter membrane of mistake；
The source of the solution of artificial bear gall powder includes but not limited to that the ethanol solution of artificial bear gall powder, the methanol of artificial bear gall powder are molten Liquid；
With 120 EC-C18 column of reverse chromatograms column Agilent Poroshell, 2.7 μm, 4.6mm × 150mm, use CAD detector, flow velocity 0.6ml/min, sample volume are 1 μ l, 40 DEG C of column temperature, frequency acquisition 10Hz, 45 DEG C of atomization temperature, are flowed Mutually it is the 5mmol/L ammonium acetate aqueous solution of acetonitrile and 0.3% formic acid, carries out gradient elution；
7. a kind of analysis method of artificial bear gall powder according to claim 1：It is characterized in that：
The monitoring of various Determination of Bile Acids
Fermentation liquid is sampled, 12000rpm is centrifuged 1.5min, takes supernatant and by 1:The positive fourth being saturated with water is added in 5 ratio Alcohol extracts it, after mixing layering, takes out supernatant liquor and with being dried with nitrogen or 70 DEG C volatilize, be added be centrifuged after The isometric HPLC grade methanol of the supernatant taken re-dissolves, and crosses 0.22 μm of filter membrane；
The source of fermentation liquid includes but not limited to artificial bear ball fermentation liquid；
With 120 EC-C18 column of reverse chromatograms column Agilent Poroshell, 2.7 μm, 4.6mm × 150mm, use CAD detector, flow velocity 0.6ml/min, sample volume are 1 μ l, 40 DEG C of column temperature, frequency acquisition 10Hz, 45 DEG C of atomization temperature, are flowed Mutually it is the 5mmol/L ammonium acetate aqueous solution of acetonitrile and 0.3% formic acid, carries out gradient elution；
CN201810384741.2A 2018-04-26 2018-04-26 A kind of artificial bear gall powder and its method for evaluating quality Pending CN108828086A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810384741.2A CN108828086A (en) 2018-04-26 2018-04-26 A kind of artificial bear gall powder and its method for evaluating quality

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810384741.2A CN108828086A (en) 2018-04-26 2018-04-26 A kind of artificial bear gall powder and its method for evaluating quality

Publications (1)

Publication Number Publication Date
CN108828086A true CN108828086A (en) 2018-11-16

Family

ID=64155534

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810384741.2A Pending CN108828086A (en) 2018-04-26 2018-04-26 A kind of artificial bear gall powder and its method for evaluating quality

Country Status (1)

Country Link
CN (1) CN108828086A (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210015874A1 (en) * 2019-07-17 2021-01-21 Shanghai Kaibao Pharmaceutical Co., Ltd. Use of bio-transformed bear bile powder in preparation of anti-inflammatory drugs
CN112903836A (en) * 2021-01-13 2021-06-04 上海凯宝药业股份有限公司 Method for determining isopropyl-beta-D-thiogalactopyranoside in-vitro cultured bear gall powder

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101062057A (en) * 2006-04-30 2007-10-31 成都尚科药业有限公司 Method for preparing lyophiled powder injection of bear bile for cooling and the function thereof
CN106367465A (en) * 2016-09-26 2017-02-01 上海中医药大学 Method for producing artificial bear gall powder through engineering bacteria fermentation
CN107312054A (en) * 2017-07-25 2017-11-03 贵州慧静生物科技有限公司 A kind of method that urso and Tauro ursodesoxy cholic acid are synthesized from pig's bile

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101062057A (en) * 2006-04-30 2007-10-31 成都尚科药业有限公司 Method for preparing lyophiled powder injection of bear bile for cooling and the function thereof
CN106367465A (en) * 2016-09-26 2017-02-01 上海中医药大学 Method for producing artificial bear gall powder through engineering bacteria fermentation
CN107312054A (en) * 2017-07-25 2017-11-03 贵州慧静生物科技有限公司 A kind of method that urso and Tauro ursodesoxy cholic acid are synthesized from pig's bile

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
XIAOYUAN NIU ET AL.: "Analytical methods for characterization of bile acids and its application in quality control of cow-bezoar and bear bile powder", 《AMERICAN JOURNAL OF APPLIED CHEMISTRY》 *
王一博 等: "高效液相色谱-电雾式检测器同时测定猪、牛、羊、熊胆粉中5种胆汁酸的含量", 《分析化学研究简报》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210015874A1 (en) * 2019-07-17 2021-01-21 Shanghai Kaibao Pharmaceutical Co., Ltd. Use of bio-transformed bear bile powder in preparation of anti-inflammatory drugs
CN112903836A (en) * 2021-01-13 2021-06-04 上海凯宝药业股份有限公司 Method for determining isopropyl-beta-D-thiogalactopyranoside in-vitro cultured bear gall powder

Similar Documents

Publication Publication Date Title
CN101731568B (en) Method for preparing high-salt dilute soy by adopting immobilized cell fermentation
JP6113808B2 (en) Cordyceps culture method
CN108330075B (en) High-yield ethyl acetate yeast and application thereof in mechanized fen-flavor Xiaoqu liquor
CN106834140B (en) A kind of anaerobic fungi and the method with its wheat stalk production ethyl alcohol that ferments
CN109251951A (en) A kind of method that semicontinuous Liquid Culture efficiently produces ganoderma lucidum polysaccharide
CN108588162A (en) A kind of technique that artificial bear gall powder is prepared using engineering bacteria scale fermented and cultured
CN106834141B (en) A kind of anaerobic fungi and the method with its rice straw production formic acid that ferments
AU2020103076A4 (en) Method of preparing Lepista sordida culture spawn with mushroom residue
CN106978355A (en) The coculture of a kind of anaerobic fungi and methane backeria and the application in the feeding native enzyme of milk cow
CN103468463A (en) Production method of monascus liquid state fermentation yeast
CN108828086A (en) A kind of artificial bear gall powder and its method for evaluating quality
CN113980828A (en) Bacillus badius mutant strain with high chlorogenic acid yield
CN101928671B (en) Alternaria spp and method thereof for preparing ginsenoside Rg3 from fermented ginseng stem-leaf total saponin
CN109055241A (en) A method of preparing mortierella Diding culture
CN101849613A (en) Biological detoxification technology for rape-seed meal
CN113046253B (en) Culture method for improving heat resistance of kluyveromyces marxianus
CN109182226A (en) A kind of the lactic acid bacteria method of mutagenesis and screening technique of highly producing gamma-aminobutyric acid
CN111909881B (en) Bacillus pumilus capable of producing feruloyl esterase and application thereof
CN110904156B (en) Method for increasing yield of triterpenoids in phellinus igniarius liquid state fermentation
CN103805520B (en) One plant height acid protease rhizopus chinensis and cultural method and application
CN110106210A (en) Epiphysin is promoting the application in Haematococcus pluvialis production γ-aminobutyric acid
CN104450670B (en) A method of improving host cell inulinase-producing activity
CN104342377A (en) Zygosaccharomyces rouxii strain for producing D-arabitol and application thereof
CN107446968A (en) A kind of fermented and cultured accelerator and its application method
CN105219811B (en) Enzymatic fatty acid mixed synthesizes human milk fat substituted product and preparation method thereof in a kind of microwave

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20181116

RJ01 Rejection of invention patent application after publication