CN101899485B - Benazepril hydrochloride composition with effect of reducing blood pressure and preparation method thereof - Google Patents
Benazepril hydrochloride composition with effect of reducing blood pressure and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a benazepril hydrochloride composition with effect of reducing blood pressure and a preparation method thereof. The preparation process comprises a solid fermentation method and a liquid fermentation method. Aspergillus CICC 2436 produces enzyme to hydrolyze benazepril hydrochloride, wherein the structure of part of benazepril hydrochloride is changed, namely an analog of benazepril hydrochloride. The original benazepril hydrochloride forms a composition containing the benazepril hydrochloride and the analog of the benazepril hydrochloride under the action of the enzyme, and the level of reducing the blood pressure by using the composition is superior to that of the benazepril hydrochloride with the same concentration.
Description
Technical field
The present invention relates to a kind of composition, particularly a kind of benazepril hydrochloride composition with hypotensive activity and preparation method thereof.
Background technology
At present modern medicine has 6 classes substantially to hypertensive medicine: 1. diuretic(s) 2. α receptor-blocking agent 3. beta-blockerss 4. calcium ion antagonists 5. angiotensin converting enzyme inhibitors 6. angiotensin II receptor antagonists.Along with the progress with cardiovascular molecular biology research of carrying out of extensive hypertension clinical trial, hypertensive traditional understanding has obtained renewal in recent years, and following disease medical science has become common recognition.Essential hypertension is not only the hemodynamic abnormalities disease, and follows the bad of target organ such as fat, carbohydrate metabolism disturbance and heart and brain kidney to reinvent.Therefore treatment will when effectively control blood pressure level, improve above-mentioned all metabolism disorders, prevention and the bad of reverse target organ and reinvent.This is to reduce the generation of cardiovascular complication and the key of case fatality rate.Although very much progress is all arranged on theory research and clinical efficacy than before.But because antihypertensive drugs still has arguement to the impact of metabolism and target organ damage, and all exists certain side effect mostly, part has limited its clinical application.Making hypertensive control can not use up the people is willing to.
Summary of the invention
One object of the present invention is to disclose a kind of benazepril hydrochloride composition; Another object of the present invention is to disclose a kind of preparation method with benazepril hydrochloride composition of hypotensive activity.
The present invention seeks to be achieved through the following technical solutions:
Benazepril hydrochloride composition of the present invention is prepared by following solid fermentation method:
Slant strains is cultivated: the mould CICC 2436 of selected songs carries out spawn culture, and agar is added 4 worts of spending, and makes the substratum of pH 6-8, so that every parts by volume substratum contains agar 0.02-0.1 weight part; Under 100-120 ℃ of condition, sterilize, cooling after the sterilization, ordinary method makes inclined-plane and inoculation, at 20 ℃--cultivated 3-7 days under 40 ℃ of conditions, as slant strains; Solid fermentation is cultivated: be 1 with wheat bran and wort according to weightmeasurement ratio: the ratio of 0.5-2 mixes, behind the mixing under 100-120 ℃ of condition, sterilized 20-40 minute, cooling after the sterilization, the access slant strains is so that the slant strains volume accounts for the 5-10% of culture volume, at 20-40 ℃, cultivated 1-5 days, and in cultured wheat bran, added 0.01-0.5M pH7 pbs damping fluid soaked overnight, get the solid fermentation nutrient solution; The preparation of crude enzyme liquid: add ammonium sulfate in the solid fermentation nutrient solution to saturated, stir to fully dissolving, under 4 ℃ of conditions, place and spend the night, the centrifuging and taking precipitation, dialysis, the centrifuging and taking supernatant liquor is as crude enzyme liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, the hydrochloric benazepril 0.03-0.2 of the every parts by volume of the solution of making weight part, after millipore filtration sterile filtration as substrate; Enzymatic conversion reaction: with crude enzyme liquid and substrate take volume ratio as 1: 1.5-8 mixes, and is 20-40 ℃ in invert point, and the conversion reaction time is 12-48 hour, namely gets composition.
The composition solid fermentation process of benazepril hydrochloride of the present invention is preferably:
Slant strains is cultivated: the mould CICC 2436 of selected songs carries out spawn culture, and agar is added the worts of 4 degree, makes pH and be 7 substratum, so that every parts by volume substratum contains agar 0.05 weight part; Sterilize under 110 ℃ of conditions, cooling after the sterilization makes inclined-plane and inoculation, cultivates 5 days under 30 ℃ of conditions, as slant strains; Solid fermentation is cultivated: be that 1: 1 ratio mixes with wheat bran and wort according to weightmeasurement ratio, behind the mixing under 110 ℃ of conditions, sterilized 30 minutes, cooling after the sterilization, the access slant strains is so that the slant strains volume accounts for 8% of substratum, at 30 ℃, cultivated 3 days, and in cultured wheat bran, added 0.3M pH7 pbs damping fluid soaked overnight, get the solid fermentation nutrient solution; The preparation of crude enzyme liquid: add ammonium sulfate in the solid fermentation nutrient solution to saturated, stir to fully dissolving, under 4 ℃ of conditions, place and spend the night, the centrifuging and taking precipitation, dialysis, the centrifuging and taking supernatant liquor is as crude enzyme liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, hydrochloric benazepril 0.1 weight part of the every parts by volume of the solution of making, after millipore filtration sterile filtration as substrate; Enzymatic conversion reaction: crude enzyme liquid was mixed as 1: 4 take volume ratio with substrate, was 30 ℃ in invert point, and the conversion reaction time is 24 hours, namely gets composition.
Benazepril hydrochloride composition of the present invention can also be prepared by following liquid fermentation process:
Slant strains is cultivated: the mould CICC 2436 of selected songs carries out spawn culture, and agar is added 4 worts of spending, and making pH is the substratum of 6-8, so that every parts by volume substratum contains agar 0.02-0.1 weight part; Sterilize under 100-120 ℃ of condition, cooling after the sterilization makes inclined-plane and inoculation, at 20 ℃--cultivated 3-7 days under 40 ℃ of conditions, as slant strains; Liquid fermenting: with glucose, extractum carnis, peptone, ammonium nitrate, sal epsom are added to the water and make the substratum that pH is 6-8, so that every parts by volume substratum contains glucose 0.01-0.1 weight part, extractum carnis 0.01-0.1 weight part, peptone 0.01-0.06 weight part, ammonium nitrate 0.01-0.05 weight part, sal epsom 0.01-0.08 weight part; The substratum of 20-100 parts by volume, under 100-120 ℃ of condition, sterilized 20-40 minute, cooling after the sterilization, the access slant strains is so that the slant strains volume accounts for the 5-10% of substratum, at 20-40 ℃, ferment under the rotating speed 100-250r/min condition, cultivated 1-5 days, as liquid fermentation and culture liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, the hydrochloric benazepril 0.03-0.2 of the every parts by volume of the solution of making weight part, after millipore filtration sterile filtration as substrate; Enzyme reaction: utilize liquid fermentation and culture liquid as the enzyme source, substrate being added in the liquid fermentation and culture liquid, contain substrate 0.00003-0.0001 weight part to every parts by volume fermentation culture, is 20-40 ℃ in invert point, transformation time is 5-48 hour, namely gets composition.
Benazepril hydrochloride composition liquid fermentation process of the present invention is preferably:
Slant strains is cultivated: the mould CICC 2436 of selected songs carries out spawn culture, and agar is added the worts of 4 degree, makes pH and be 7 substratum, so that every parts by volume substratum contains agar 0.05 weight part; Sterilize under 110 ℃ of conditions, cooling after the sterilization makes inclined-plane and inoculation, cultivates 5 days under 30 ℃ of conditions, as slant strains; Liquid fermenting: with glucose, extractum carnis, peptone, ammonium nitrate, sal epsom is added to the water that to make pH be 7 substratum, so that every parts by volume substratum contains glucose 0.05 weight part, and extractum carnis 0.05 weight part, peptone 0.04 weight part, ammonium nitrate 0.03 weight part, sal epsom 0.04 weight part; The substratum of 50 parts by volume under 110 ℃ of conditions, was sterilized 30 minutes, cooling after the sterilization, and the access slant strains so that the slant strains volume accounts for 8% of substratum, at 30 ℃, is fermented under the rotating speed 150r/min condition, cultivates 3 days, as liquid fermentation and culture liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, hydrochloric benazepril 0.1 weight part of the every parts by volume of the solution of making, after millipore filtration sterile filtration as substrate; Enzyme reaction: utilizing liquid fermentation and culture liquid as the enzyme source, substrate is added in the liquid fermentation and culture liquid, contain substrate 0.00008 weight part to every parts by volume fermentation culture, is 30 ℃ in invert point, and transformation time is 28 hours, namely gets composition.
The pass of above-mentioned weight part and parts by volume is the relation of gram and milliliter, i.e. g/ml.
Utilize aspergillus CICC 2436 to produce the enzymic hydrolysis benazepril hydrochloride, wherein change has occured in the structure of part benazepril hydrochloride, i.e. the analog of benazepril hydrochloride.Former benazepril hydrochloride forms the composition of hydrochloric benazepril and benazepril hydrochloride analog under the effect of enzyme, the hypotensive level of said composition is better than with the concentration benazepril hydrochloride.
Description of drawings
Fig. 1 solid fermentation reference substance one
Fig. 2 solid fermentation reference substance two
Fig. 3 solid fermentation aspergillus CICC 2436
Fig. 4 solid fermentation produces without true tumor
Fig. 5 liquid fermenting reference substance one
Fig. 6 liquid fermenting reference substance two
Fig. 7 liquid fermenting aspergillus CICC 2436
Fig. 8 liquid fermenting produces without true tumor
The below's experiment and embodiment are used for further specifying but are not limited to the present invention.
Experimental example 1 bacterial screening (solid fermentation)
Key of the present invention is to screen the bacterial classification that benazepril hydrochloride is had transformation, and the process of screening experiment is: strain screening is CICC 3093, and CICC 2203, CICC3005, and CICC 2436
Trial-product preparation: agar is added the worts of 4 degree, make pH and be 7 substratum, so that every parts by volume substratum contains agar 0.05g; Sterilize under 110 ℃ of conditions, cooling after the sterilization makes inclined-plane and inoculation, cultivates 5 days under 30 ℃ of conditions, as slant strains; Solid fermentation is cultivated: be that 1: 1 ratio mixes with wheat bran and wort according to weightmeasurement ratio, behind the mixing under 110 ℃ of conditions, sterilized 30 minutes, cooling after the sterilization, the access slant strains is so that the slant strains volume accounts for 8% of substratum, at 30 ℃, cultivated 3 days, and in cultured wheat bran, added 0.3M pH7 pbs damping fluid soaked overnight, get the solid fermentation nutrient solution; The preparation of crude enzyme liquid: add ammonium sulfate in the solid fermentation nutrient solution to saturated, stir to fully dissolving, under 4 ℃ of conditions, place and spend the night, the centrifuging and taking precipitation, dialysis, the centrifuging and taking supernatant liquor is as crude enzyme liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, the hydrochloric benazepril 0.1g of the every ml of the solution of making, after millipore filtration sterile filtration as substrate; Enzymatic conversion reaction: crude enzyme liquid was mixed as 1: 4 take volume ratio with substrate, was 30 ℃ in invert point, and the conversion reaction time is 24 hours, namely gets composition.
Reference substance one preparation: with aqueous solution dissolved salt Benazepril Hydrochloride, the hydrochloric benazepril 0.1g of the every ml of the solution of making after millipore filtration sterile filtration, gets filtrate 0.5ml, adds 0.25ml water, and 20-40 ℃ of reaction 24h namely gets in contrast product one.
Reference substance two preparations: prepare crude enzyme liquid according to the method in the trial-product preparation respectively with 4 strain screening, every kind of crude enzyme liquid 0.25ml adds 0.5ml water, 20-40 ℃ of reaction 24h.
Chromatographic column: chromatographic column: APOLLO ODS-C18 (250 * 4.6mm, 5 μ m), and detector: UV (λ=239nm)
Moving phase: A is the 0.075%HAc aqueous solution, and B is methyl alcohol.Test-results as can be known, figure one shows benazepril hydrochloride mark product; Figure two shows 4 enzyme liquid reference substances two that bacterium prepares respectively, and chromatograms shows, the enzyme liquid liquid phase figure that 4 bacterium prepare respectively is substantially identical; Figure three demonstration CICC 2436 and the enzyme digestion reaction composition of benazepril hydrochloride are that the liquid phase figure of the present composition is substantially identical, all have newly-generated thing (arrow place) to occur, occur without novel substance behind other bacterial classification and the benazepril hydrochloride enzymatic conversion reaction.
Experimental example 2 bacterial screenings (liquid fermenting)
Key of the present invention is to screen the bacterial classification that benazepril hydrochloride is had transformation, and the process of screening experiment is: strain screening is CICC 3093, and CICC 2203, CICC3005, and, CICC 2436
Trial-product preparation: slant strains is cultivated: agar is added the worts of 4 degree, make pH and be 7 substratum, so that every parts by volume substratum contains agar 0.05g; Sterilize under 110 ℃ of conditions, cooling after the sterilization makes inclined-plane and inoculation, cultivates 5 days under 30 ℃ of conditions, as slant strains; Liquid fermenting: with glucose, extractum carnis, peptone, ammonium nitrate, sal epsom are added to the water that to make pH be 7 substratum, so that every parts by volume substratum contains glucose 0.05g, and extractum carnis 0.05g, peptone 0.04g, ammonium nitrate 0.03g, sal epsom 0.04g; In the tripod bottle of 250ml, pack into the substratum of 50ml, under 110 ℃ of conditions, sterilized 30 minutes, cooling after the sterilization, the access slant strains is so that the slant strains volume accounts for 8% of substratum, at 30 ℃, carry out shake flask fermentation under the shaking flask rotating speed 150r/min condition, cultivated 3 days, as liquid fermentation and culture liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, the hydrochloric benazepril 0.1g of the every ml of the solution of making, after millipore filtration sterile filtration as substrate; Enzyme reaction: liquid fermentation and culture liquid adds substrate in the liquid fermentation and culture liquid to as the enzyme source, contains substrate 0.00008g to every ml fermentation culture, is 30 ℃ in invert point, and transformation time is 28 hours, namely gets composition.
Reference substance one preparation: with aqueous solution dissolved salt Benazepril Hydrochloride, the hydrochloric benazepril 0.1g of the every ml of the solution of making after millipore filtration sterile filtration, gets filtrate 0.5ml, adds 0.25ml water, and 20-40 ℃ of reaction 24h namely gets in contrast product one.
Reference substance two preparations: prepare crude enzyme liquid according to the method in the trial-product preparation respectively with 4 strain screening, every kind of crude enzyme liquid 0.25ml adds 0.5ml water, 20-40 ℃ of reaction 24h.
Chromatographic column: chromatographic column: APOLLO ODS-C18 (250 * 4.6mm, 5 μ m), (λ=239nm) moving phase: A is the 0.075%HAc aqueous solution to detector: UV, and B is methyl alcohol.
Test-results as can be known, figure four shows benazepril hydrochloride mark product; Fig. 6 liquid fermenting shows that 4 enzyme liquid reference substances two that bacterium prepares respectively, chromatograms show, the enzyme liquid liquid phase figure that 4 bacterium prepare respectively is substantially identical; Fig. 7 liquid fermenting demonstration CICC 2436 and the enzyme digestion reaction composition of benazepril hydrochloride are that the liquid phase figure of the present composition is substantially identical, all there is newly-generated thing (arrow place) to occur, occurs without novel substance behind other bacterial classification and the benazepril hydrochloride enzymatic conversion reaction.
Experimental example 3 pharmacodynamic experiments
Adopt the restraining effect comparison test of extracorporeal blood vessel AECI, relatively the same concentration present composition (embodiment 1 preparation) is to the contrast experiment of rat lipopenicillinase ability, the main pharmacological action of angiotensin converting enzyme inhibitor is to suppress the Zinc metallopeptidase Zace1 activity, reduce the generation of Angiotensin II, reduce the hydrolysis of bradykinin, cause vasorelaxation, the hypovolemia blood pressure drops.Test method is seen " pharmacological experiment guide-new drug is found and pharmacological evaluation " A.1.1.10
1, experimental animal male SD rat, body weight 180～220g.Experimental animal is provided by Beijing Vital River Experimental Animals Technology Co., Ltd..
2, trial drug:
A, the enzyme-added liquid of benazepril hydrochloride substrate are cultivated the mixture (embodiment 1 composition) behind the 12-48h
B, add the mixture of supporting 12-48h with the long-pending identical water planting of enzyme liquid with concentration benazepril hydrochloride substrate with A)
Experimental result such as following table: (A group B group dosage is identical, by volume calculates)
The hypotensive horizontal comparison test of table 1 A and B
Totally 5 groups of laboratory animal, every group of 10 mouse, modeling and test method are seen " pharmacological experiment guide-new drug is found and pharmacological evaluation " A.1.1.10; Upper table shows: in the restraining effect comparison test to Zinc metallopeptidase Zace1, the A group is higher than the inhibiting rate of B group, so present composition step-down ability is higher than benazepril hydrochloride.
Experimental example 4 pharmacodynamic experiments
Experimental technique with experimental example 3, trial drug A is the mixture (embodiment 6 compositions) after the enzyme-added liquid of benazepril hydrochloride substrate is cultivated 12-48h, experimental data shows: the hypotensive ability of A group will be higher than the B group, and this interior hypotensive ability of similar object that shows the benazepril hydrochloride that obtains after the enzymatic conversion benazepril hydrochloride in the A group is better than benazepril hydrochloride.
The hypotensive horizontal comparison test of table 2 A and B
The synthetic technology that using microbe enzyme or microorganism cells carry out is called as microbial transformation, they are playing the part of more and more important role at the synthetic and conversion aspect of the compound of fast development, and the microbial enzyme transformation technology generally has environmental friendliness, the advantages such as action condition gentleness; In addition, biological catalyst has the selectivity of height, comprises chemo-selective, regioselectivity, enantio-selectivity and non-enantiomer selectivity; Microbial transformation transforms than chemical means following advantage: mild condition, and equipment is simple, and public hazards are few, and speed of reaction is very fast, relatively environmental protection.
Following embodiment all can realize the effect of experimental example of the present invention.
Embodiment
Embodiment 1: the composition solid fermentation of benazepril hydrochloride
Slant strains is cultivated: the mould CICC 2436 of selected songs carries out spawn culture, and agar is added the worts of 4 degree, makes pH and be 7 substratum, so that every ml substratum contains agar 0.05g; Sterilize under 110 ℃ of conditions, cooling after the sterilization makes inclined-plane and inoculation, cultivates 5 days under 30 ℃ of conditions, as slant strains; Solid fermentation is cultivated: be that 1: 1 ratio mixes with wheat bran and wort according to weightmeasurement ratio, behind the mixing under 110 ℃ of conditions, sterilized 30 minutes, cooling after the sterilization, the access slant strains is so that the slant strains volume accounts for 8% of substratum, at 30 ℃, cultivated 3 days, and in cultured wheat bran, added 0.3M pH7 pbs damping fluid soaked overnight, get the solid fermentation nutrient solution; The preparation of crude enzyme liquid: add ammonium sulfate in the solid fermentation nutrient solution to saturated, stir to fully dissolving, under 4 ℃ of conditions, place and spend the night, the centrifuging and taking precipitation, dialysis, the centrifuging and taking supernatant liquor is as crude enzyme liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, the hydrochloric benazepril 0.1g of the every ml of the solution of making, after millipore filtration sterile filtration as substrate; Enzymatic conversion reaction: crude enzyme liquid was mixed as 1: 4 take volume ratio with substrate, was 30 ℃ in invert point, and the conversion reaction time is 24 hours, namely gets composition.
Embodiment 2: the composition solid fermentation of benazepril hydrochloride
Slant strains is cultivated: the mould CICC 2436 of selected songs carries out spawn culture, and agar is added 4 worts of spending, and makes the substratum of pH 6, so that every ml substratum contains agar 0.03g; Under 105 ℃ of conditions, sterilize, cooling after the sterilization, ordinary method makes inclined-plane and inoculation, cultivates 4 days under 35 ℃ of conditions, as slant strains; Solid fermentation is cultivated: be that 1: 1.5 ratio mixes with wheat bran and wort according to weightmeasurement ratio, behind the mixing under 105 ℃ of conditions, sterilized 35 minutes, cooling after the sterilization, the access slant strains is so that the slant strains volume accounts for 6% of culture volume, at 35 ℃, cultivated 2 days, and in cultured wheat bran, added 0.5M pH7pbs damping fluid soaked overnight, get the solid fermentation nutrient solution; The preparation of crude enzyme liquid: add ammonium sulfate in the solid fermentation nutrient solution to saturated, stir to fully dissolving, under 4 ℃ of conditions, place and spend the night, the centrifuging and taking precipitation, dialysis, the centrifuging and taking supernatant liquor is as crude enzyme liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, the hydrochloric benazepril 0.04g of the every ml of the solution of making, after millipore filtration sterile filtration as substrate; Enzymatic conversion reaction: crude enzyme liquid was mixed as 1: 7 take volume ratio with substrate, was 25 ℃ in invert point, and the conversion reaction time is 40 hours, namely gets composition.
Embodiment 3: the composition solid fermentation of benazepril hydrochloride
Slant strains is cultivated: the mould CICC 2436 of selected songs carries out spawn culture, and agar is added 4 worts of spending, and makes the substratum of pH 7, so that every ml substratum contains agar 0.08g; Under 115 ℃ of conditions, sterilize, cooling after the sterilization, ordinary method makes inclined-plane and inoculation, cultivates 6 days under 25 ℃ of conditions, as slant strains; Solid fermentation is cultivated: be that 1: 1.8 ratio mixes with wheat bran and wort according to weightmeasurement ratio, behind the mixing under 120 ℃ of conditions, sterilized 25 minutes, cooling after the sterilization, the access slant strains is so that the slant strains volume accounts for 9% of culture volume, at 25 ℃, cultivated 2 days, and in cultured wheat bran, added 0.4M pH7pbs damping fluid soaked overnight, get the solid fermentation nutrient solution; The preparation of crude enzyme liquid: add ammonium sulfate in the solid fermentation nutrient solution to saturated, stir to fully dissolving, under 4 ℃ of conditions, place and spend the night, the centrifuging and taking precipitation, dialysis, the centrifuging and taking supernatant liquor is as crude enzyme liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, the hydrochloric benazepril 0.09g of the every ml of the solution of making, after millipore filtration sterile filtration as substrate; Enzymatic conversion reaction: crude enzyme liquid was mixed as 1: 3 take volume ratio with substrate, was 25 ℃ in invert point, and the conversion reaction time is 18 hours, namely gets composition.
Embodiment 4: the composition solid fermentation of benazepril hydrochloride
Slant strains is cultivated: the mould CICC 2436 of selected songs carries out spawn culture, and agar is added 4 worts of spending, and makes the substratum of pH 8, so that every ml substratum contains agar 0.1g; Under 104 ℃ of conditions, sterilize, cooling after the sterilization, ordinary method makes inclined-plane and inoculation, cultivates 4 days under 38 ℃ of conditions, as slant strains; Solid fermentation is cultivated: be that 1: 0.7 ratio mixes with wheat bran and wort according to weightmeasurement ratio, behind the mixing under 117 ℃ of conditions, sterilized 32 minutes, cooling after the sterilization, the access slant strains is so that the slant strains volume accounts for 6% of culture volume, at 38 ℃, cultivated 3 days, and in cultured wheat bran, added 0.07M pH7 pbs damping fluid soaked overnight, get the solid fermentation nutrient solution; The preparation of crude enzyme liquid: add ammonium sulfate in the solid fermentation nutrient solution to saturated, stir to fully dissolving, under 4 ℃ of conditions, place and spend the night, the centrifuging and taking precipitation, dialysis, the centrifuging and taking supernatant liquor is as crude enzyme liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, the hydrochloric benazepril 0.1g of the every ml of the solution of making, after millipore filtration sterile filtration as substrate; Enzymatic conversion reaction: crude enzyme liquid was mixed as 1: 3 take volume ratio with substrate, was 28 ℃ in invert point, and the conversion reaction time is 38 hours, namely gets composition.
Embodiment 5: the composition solid fermentation of benazepril hydrochloride
Slant strains is cultivated: the mould CICC 2436 of selected songs carries out spawn culture, and agar is added 4 worts of spending, and makes the substratum of pH 8, so that every ml substratum contains agar 0.1g; Under 104 ℃ of conditions, sterilize, cooling after the sterilization, ordinary method makes inclined-plane and inoculation, cultivates 4 days under 38 ℃ of conditions, as slant strains; Solid fermentation is cultivated: be that 1: 0.7 ratio mixes with wheat bran and wort according to weightmeasurement ratio, behind the mixing under 117 ℃ of conditions, sterilized 32 minutes, cooling after the sterilization, the access slant strains is so that the slant strains volume accounts for 6% of culture volume, at 38 ℃, cultivated 3 days, and in cultured wheat bran, added 0.07M pH7 pbs damping fluid soaked overnight, get the solid fermentation nutrient solution; The preparation of crude enzyme liquid: add ammonium sulfate in the solid fermentation nutrient solution to saturated, stir to fully dissolving, under 4 ℃ of conditions, place and spend the night, the centrifuging and taking precipitation, dialysis, the centrifuging and taking supernatant liquor is as crude enzyme liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, the hydrochloric benazepril 0.1g of the every ml of the solution of making, after millipore filtration sterile filtration as substrate; Enzymatic conversion reaction: crude enzyme liquid was mixed as 1: 3 take volume ratio with substrate, was 28 ℃ in invert point, and the conversion reaction time is 38 hours, namely gets composition.
Embodiment 6: the composition liquid fermenting of benazepril hydrochloride
Slant strains is cultivated: the mould CICC 2436 of selected songs carries out spawn culture, and agar is added the worts of 4 degree, makes pH and be 7 substratum, so that every ml substratum contains agar 0.05g; Sterilize under 110 ℃ of conditions, cooling after the sterilization makes inclined-plane and inoculation, cultivates 5 days under 30 ℃ of conditions, as slant strains; Liquid fermenting: with glucose, extractum carnis, peptone, ammonium nitrate, sal epsom are added to the water that to make pH be 7 substratum, so that every ml substratum contains glucose 0.05g, and extractum carnis 0.05g, peptone 0.04g, ammonium nitrate 0.03g, sal epsom 0.04g; The substratum of 50ml under 110 ℃ of conditions, was sterilized 30 minutes, cooling after the sterilization, and the access slant strains so that the slant strains volume accounts for 8% of substratum, at 30 ℃, is fermented under the rotating speed 150r/min condition, cultivates 3 days, as liquid fermentation and culture liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, the hydrochloric benazepril 0.1g of the every ml of the solution of making, after millipore filtration sterile filtration as substrate; Enzyme reaction: utilizing liquid fermentation and culture liquid as the enzyme source, substrate is added in the liquid fermentation and culture liquid, contain substrate 0.00008g to every ml fermentation culture, is 30 ℃ in invert point, and transformation time is 28 hours, namely gets composition.
Embodiment 7: the composition liquid fermenting of benazepril hydrochloride
Slant strains is cultivated: the mould CICC 2436 of selected songs carries out spawn culture, and agar is added the worts of 4 degree, makes pH and be 6.5 substratum, so that every ml substratum contains agar 0.09g; Sterilize under 110 ℃ of conditions, cooling after the sterilization makes inclined-plane and inoculation, cultivates 6 days under 22 ℃ of conditions, as slant strains; Liquid fermenting: with glucose, extractum carnis, peptone, ammonium nitrate, sal epsom are added to the water that to make pH be 6.5 substratum, so that every ml substratum contains glucose 0.02g, and extractum carnis 0.9g, peptone 0.04g, ammonium nitrate 0.02g, sal epsom 0.07g; The substratum of 60ml under 102 ℃ of conditions, was sterilized 38 minutes, cooling after the sterilization, and the access slant strains so that the slant strains volume accounts for 7% of substratum, at 24 ℃, is fermented under the rotating speed 220r/min condition, cultivates 3 days, as liquid fermentation and culture liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, the hydrochloric benazepril 0.05g of the every ml of the solution of making, after millipore filtration sterile filtration as substrate; Enzyme reaction: utilizing liquid fermentation and culture liquid as the enzyme source, substrate is added in the liquid fermentation and culture liquid, contain substrate 0.00008g to every ml fermentation culture, is 29 ℃ in invert point, and transformation time is 9 hours, namely gets composition.
Embodiment 8: the composition liquid fermenting of benazepril hydrochloride
Slant strains is cultivated: the mould CICC 2436 of selected songs carries out spawn culture, and agar is added the worts of 4 degree, makes pH and be 6.5 substratum, so that every ml substratum contains agar 0.09g; Sterilize under 110 ℃ of conditions, cooling after the sterilization makes inclined-plane and inoculation, cultivates 6 days under 22 ℃ of conditions, as slant strains; Liquid fermenting: with glucose, extractum carnis, peptone, ammonium nitrate, sal epsom are added to the water that to make pH be 6.5 substratum, so that every ml substratum contains glucose 0.02g, and extractum carnis 0.9g, peptone 0.04g, ammonium nitrate 0.02g, sal epsom 0.07g; The substratum of 60ml under 102 ℃ of conditions, was sterilized 38 minutes, cooling after the sterilization, and the access slant strains so that the slant strains volume accounts for 7% of substratum, at 24 ℃, is fermented under the rotating speed 220r/min condition, cultivates 3 days, as liquid fermentation and culture liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, the hydrochloric benazepril 0.05g of the every ml of the solution of making, after millipore filtration sterile filtration as substrate; Enzyme reaction: utilizing liquid fermentation and culture liquid as the enzyme source, substrate is added in the liquid fermentation and culture liquid, contain substrate 0.00008g to every ml fermentation culture, is 29 ℃ in invert point, and transformation time is 9 hours, namely gets composition.
Embodiment 9: the composition liquid fermenting of benazepril hydrochloride
Slant strains is cultivated: the mould CICC 2436 of selected songs carries out spawn culture, and agar is added the worts of 4 degree, makes pH and be 7.5 substratum, so that every ml substratum contains agar 0.04g; Sterilize under 108 ℃ of conditions, cooling after the sterilization makes inclined-plane and inoculation, cultivates 4 days under 37 ℃ of conditions, as slant strains; Liquid fermenting: with glucose, extractum carnis, peptone, ammonium nitrate, sal epsom are added to the water that to make pH be 7.5 substratum, so that every ml substratum contains glucose 0.08g, and extractum carnis 0.05g, peptone 0.03g, ammonium nitrate 0.04g, sal epsom 0.02g; The substratum of 70ml under 117 ℃ of conditions, was sterilized 24 minutes, cooling after the sterilization, and the access slant strains so that the slant strains volume accounts for 7% of substratum, at 36 ℃, is fermented under the rotating speed 105r/min condition, cultivates 3 days, as liquid fermentation and culture liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, the hydrochloric benazepril 0.1g of the every ml of the solution of making, after millipore filtration sterile filtration as substrate; Enzyme reaction: utilizing liquid fermentation and culture liquid as the enzyme source, substrate is added in the liquid fermentation and culture liquid, contain substrate 0.00004g to every ml fermentation culture, is 34 ℃ in invert point, and transformation time is 43 hours, namely gets composition.
Embodiment 10: the composition liquid fermenting of benazepril hydrochloride
Slant strains is cultivated: the mould CICC 2436 of selected songs carries out spawn culture, and agar is added the worts of 4 degree, makes pH and be 7 substratum, so that every ml substratum contains agar 0.04g; Sterilize under 116 ℃ of conditions, cooling after the sterilization makes inclined-plane and inoculation, cultivates 3 days under 34 ℃ of conditions, as slant strains; Liquid fermenting: with glucose, extractum carnis, peptone, ammonium nitrate, sal epsom are added to the water that to make pH be 8 substratum, so that every ml substratum contains glucose 0.05g, and extractum carnis 0.01g, peptone 0.06g, ammonium nitrate 0.04g, sal epsom 0.02g; The substratum of 100ml under 108 ℃ of conditions, was sterilized 21 minutes, cooling after the sterilization, and the access slant strains so that the slant strains volume accounts for 10% of substratum, at 30 ℃, is fermented under the rotating speed 105r/min condition, cultivates 5 days, as liquid fermentation and culture liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, the hydrochloric benazepril 0.1g of the every ml of the solution of making, after millipore filtration sterile filtration as substrate; Enzyme reaction: utilizing liquid fermentation and culture liquid as the enzyme source, substrate is added in the liquid fermentation and culture liquid, contain substrate 0.00004g to every ml fermentation culture, is 40 ℃ in invert point, and transformation time is 25 hours, namely gets composition.
Claims (21)
1. benazepril hydrochloride composition with hypotensive activity is characterized in that by following solid fermentation method preparation:
Slant strains is cultivated: the mould CICC 2436 of selected songs carries out spawn culture, and agar is added 4 worts of spending, and makes the substratum of pH 6-8, so that every parts by volume substratum contains agar 0.02-0.1 weight part; Under 100-120 ℃ of condition, sterilize, cooling after the sterilization, ordinary method makes inclined-plane and inoculation, at 20 ℃--cultivated 3-7 days under 40 ℃ of conditions, as slant strains; Solid fermentation is cultivated: be 1 with wheat bran and wort according to weightmeasurement ratio: the ratio of 0.5-2 mixes, behind the mixing under 100-120 ℃ of condition, sterilized 20-40 minute, cooling after the sterilization, the access slant strains is so that the slant strains volume accounts for the 5-10% of culture volume, at 20-40 ℃, cultivated 1-5 days, and in cultured wheat bran, added 0.01-0.5M pH7pbs damping fluid soaked overnight, get the solid fermentation nutrient solution; The preparation of crude enzyme liquid: add ammonium sulfate in the solid fermentation nutrient solution to saturated, stir to fully dissolving, under 4 ℃ of conditions, place and spend the night, the centrifuging and taking precipitation, dialysis, the centrifuging and taking supernatant liquor is as crude enzyme liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, the hydrochloric benazepril 0.03-0.2 of the every parts by volume of the solution of making weight part, after millipore filtration sterile filtration as substrate; Enzymatic conversion reaction: with crude enzyme liquid and substrate take volume ratio as 1: 1.5-8 mixes, and is 20-40 ℃ in invert point, and the conversion reaction time is 12-48 hour, namely gets composition.
2. benazepril hydrochloride composition as claimed in claim 1 is characterized in that by following solid fermentation method preparation:
Slant strains is cultivated: the mould CICC 2436 of selected songs carries out spawn culture, and agar is added the worts of 4 degree, makes pH and be 7 substratum, so that every parts by volume substratum contains agar 0.05 weight part; Sterilize under 110 ℃ of conditions, cooling after the sterilization makes inclined-plane and inoculation, cultivates 5 days under 30 ℃ of conditions, as slant strains; Solid fermentation is cultivated: be that 1: 1 ratio mixes with wheat bran and wort according to weightmeasurement ratio, behind the mixing under 110 ℃ of conditions, sterilized 30 minutes, cooling after the sterilization, the access slant strains is so that the slant strains volume accounts for 8% of substratum, at 30 ℃, cultivated 3 days, and in cultured wheat bran, added 0.3M pH7pbs damping fluid soaked overnight, get the solid fermentation nutrient solution; The preparation of crude enzyme liquid: add ammonium sulfate in the solid fermentation nutrient solution to saturated, stir to fully dissolving, under 4 ℃ of conditions, place and spend the night, the centrifuging and taking precipitation, dialysis, the centrifuging and taking supernatant liquor is as crude enzyme liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, hydrochloric benazepril 0.1 weight part of the every parts by volume of the solution of making, after millipore filtration sterile filtration as substrate; Enzymatic conversion reaction: crude enzyme liquid was mixed as 1: 4 take volume ratio with substrate, was 30 ℃ in invert point, and the conversion reaction time is 24 hours, namely gets composition.
3. benazepril hydrochloride composition as claimed in claim 1 is characterized in that by following solid fermentation method preparation:
Slant strains is cultivated: the mould CICC 2436 of selected songs carries out spawn culture, and agar is added 4 worts of spending, and makes the substratum of pH 6, so that every parts by volume substratum contains agar 0.03 weight part; Under 105 ℃ of conditions, sterilize, cooling after the sterilization, ordinary method makes inclined-plane and inoculation, cultivates 4 days under 35 ℃ of conditions, as slant strains; Solid fermentation is cultivated: be that 1: 1.5 ratio mixes with wheat bran and wort according to weightmeasurement ratio, behind the mixing under 105 ℃ of conditions, sterilized 35 minutes, cooling after the sterilization, the access slant strains is so that the slant strains volume accounts for 6% of culture volume, at 35 ℃, cultivated 2 days, and in cultured wheat bran, added 0.5M pH7 pbs damping fluid soaked overnight, get the solid fermentation nutrient solution; The preparation of crude enzyme liquid: add ammonium sulfate in the solid fermentation nutrient solution to saturated, stir to fully dissolving, under 4 ℃ of conditions, place and spend the night, the centrifuging and taking precipitation, dialysis, the centrifuging and taking supernatant liquor is as crude enzyme liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, hydrochloric benazepril 0.04 weight part of the every parts by volume of the solution of making, after millipore filtration sterile filtration as substrate; Enzymatic conversion reaction: crude enzyme liquid was mixed as 1: 7 take volume ratio with substrate, was 25 ℃ in invert point, and the conversion reaction time is 40 hours, namely gets composition.
4. benazepril hydrochloride composition as claimed in claim 1 is characterized in that by following solid fermentation method preparation:
Slant strains is cultivated: the mould CICC 2436 of selected songs carries out spawn culture, and agar is added 4 worts of spending, and makes the substratum of pH 7, so that every parts by volume substratum contains agar 0.08 weight part; Under 115 ℃ of conditions, sterilize, cooling after the sterilization, ordinary method makes inclined-plane and inoculation, cultivates 6 days under 25 ℃ of conditions, as slant strains; Solid fermentation is cultivated: be that 1: 1.8 ratio mixes with wheat bran and wort according to weightmeasurement ratio, behind the mixing under 120 ℃ of conditions, sterilized 25 minutes, cooling after the sterilization, the access slant strains is so that the slant strains volume accounts for 9% of culture volume, at 25 ℃, cultivated 2 days, and in cultured wheat bran, added 0.4M pH7pbs damping fluid soaked overnight, get the solid fermentation nutrient solution; The preparation of crude enzyme liquid: add ammonium sulfate in the solid fermentation nutrient solution to saturated, stir to fully dissolving, under 4 ℃ of conditions, place and spend the night, the centrifuging and taking precipitation, dialysis, the centrifuging and taking supernatant liquor is as crude enzyme liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, hydrochloric benazepril 0.09 weight part of the every parts by volume of the solution of making, after millipore filtration sterile filtration as substrate; Enzymatic conversion reaction: crude enzyme liquid was mixed as 1: 3 take volume ratio with substrate, was 25 ℃ in invert point, and the conversion reaction time is 18 hours, namely gets composition.
5. benazepril hydrochloride composition as claimed in claim 1 is characterized in that by following solid fermentation method preparation:
Slant strains is cultivated: the mould CICC 2436 of selected songs carries out spawn culture, and agar is added 4 worts of spending, and makes the substratum of pH 8, so that every parts by volume substratum contains agar 0.1 weight part; Under 104 ℃ of conditions, sterilize, cooling after the sterilization, ordinary method makes inclined-plane and inoculation, cultivates 4 days under 38 ℃ of conditions, as slant strains; Solid fermentation is cultivated: be that 1: 0.7 ratio mixes with wheat bran and wort according to weightmeasurement ratio, behind the mixing under 117 ℃ of conditions, sterilized 32 minutes, cooling after the sterilization, the access slant strains is so that the slant strains volume accounts for 6% of culture volume, at 38 ℃, cultivated 3 days, and in cultured wheat bran, added 0.07M pH7 pbs damping fluid soaked overnight, get the solid fermentation nutrient solution; The preparation of crude enzyme liquid: add ammonium sulfate in the solid fermentation nutrient solution to saturated, stir to fully dissolving, under 4 ℃ of conditions, place and spend the night, the centrifuging and taking precipitation, dialysis, the centrifuging and taking supernatant liquor is as crude enzyme liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, hydrochloric benazepril 0.1 weight part of the every parts by volume of the solution of making, after millipore filtration sterile filtration as substrate; Enzymatic conversion reaction: crude enzyme liquid was mixed as 1: 3 take volume ratio with substrate, was 28 ℃ in invert point, and the conversion reaction time is 38 hours, namely gets composition.
6. preparation method with benazepril hydrochloride composition of hypotensive activity is characterized in that the method is:
Slant strains is cultivated: the mould CICC 2436 of selected songs carries out spawn culture, and agar is added 4 worts of spending, and makes the substratum of pH 6-8, so that every parts by volume substratum contains agar 0.02-0.1 weight part; Under 100-120 ℃ of condition, sterilize, cooling after the sterilization, ordinary method makes inclined-plane and inoculation, at 20 ℃--cultivated 3-7 days under 40 ℃ of conditions, as slant strains; Solid fermentation is cultivated: be 1 with wheat bran and wort according to weightmeasurement ratio: the ratio of 0.5-2 mixes, behind the mixing under 100-120 ℃ of condition, sterilized 20-40 minute, cooling after the sterilization, the access slant strains is so that the slant strains volume accounts for the 5-10% of culture volume, at 20-40 ℃, cultivated 1-5 days, and in cultured wheat bran, added 0.01-0.5M pH7pbs damping fluid soaked overnight, get the solid fermentation nutrient solution; The preparation of crude enzyme liquid: add ammonium sulfate in the solid fermentation nutrient solution to saturated, stir to fully dissolving, under 4 ℃ of conditions, place and spend the night, the centrifuging and taking precipitation, dialysis, the centrifuging and taking supernatant liquor is as crude enzyme liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, the hydrochloric benazepril 0.03-0.2 of the every parts by volume of the solution of making weight part, after millipore filtration sterile filtration as substrate; Enzymatic conversion reaction: with crude enzyme liquid and substrate take volume ratio as 1: 1.5-8 mixes, and is 20-40 ℃ in invert point, and the conversion reaction time is 12-48 hour, namely gets composition.
7. the preparation method of benazepril hydrochloride composition as claimed in claim 6 is characterized in that the method is:
Slant strains is cultivated: the mould CICC 2436 of selected songs carries out spawn culture, and agar is added the worts of 4 degree, makes pH and be 7 substratum, so that every parts by volume substratum contains agar 0.05 weight part; Sterilize under 110 ℃ of conditions, cooling after the sterilization makes inclined-plane and inoculation, cultivates 5 days under 30 ℃ of conditions, as slant strains; Solid fermentation is cultivated: be that 1: 1 ratio mixes with wheat bran and wort according to weightmeasurement ratio, behind the mixing under 110 ℃ of conditions, sterilized 30 minutes, cooling after the sterilization, the access slant strains is so that the slant strains volume accounts for 8% of substratum, at 30 ℃, cultivated 3 days, and in cultured wheat bran, added 0.3M pH7pbs damping fluid soaked overnight, get the solid fermentation nutrient solution; The preparation of crude enzyme liquid: add ammonium sulfate in the solid fermentation nutrient solution to saturated, stir to fully dissolving, under 4 ℃ of conditions, place and spend the night, the centrifuging and taking precipitation, dialysis, the centrifuging and taking supernatant liquor is as crude enzyme liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, hydrochloric benazepril 0.1 weight part of the every parts by volume of the solution of making, after millipore filtration sterile filtration as substrate; Enzymatic conversion reaction: crude enzyme liquid was mixed as 1: 4 take volume ratio with substrate, was 30 ℃ in invert point, and the conversion reaction time is 24 hours, namely gets composition.
8. the preparation method of benazepril hydrochloride composition as claimed in claim 6 is characterized in that the method is:
Slant strains is cultivated: the mould CICC 2436 of selected songs carries out spawn culture, and agar is added 4 worts of spending, and makes the substratum of pH 6, so that every parts by volume substratum contains agar 0.03 weight part; Under 105 ℃ of conditions, sterilize, cooling after the sterilization, ordinary method makes inclined-plane and inoculation, cultivates 4 days under 35 ℃ of conditions, as slant strains; Solid fermentation is cultivated: be that 1: 1.5 ratio mixes with wheat bran and wort according to weightmeasurement ratio, behind the mixing under 105 ℃ of conditions, sterilized 35 minutes, cooling after the sterilization, the access slant strains is so that the slant strains volume accounts for 6% of culture volume, at 35 ℃, cultivated 2 days, and in cultured wheat bran, added 0.5M pH7 pbs damping fluid soaked overnight, get the solid fermentation nutrient solution; The preparation of crude enzyme liquid: add ammonium sulfate in the solid fermentation nutrient solution to saturated, stir to fully dissolving, under 4 ℃ of conditions, place and spend the night, the centrifuging and taking precipitation, dialysis, the centrifuging and taking supernatant liquor is as crude enzyme liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, hydrochloric benazepril 0.04 weight part of the every parts by volume of the solution of making, after millipore filtration sterile filtration as substrate; Enzymatic conversion reaction: crude enzyme liquid was mixed as 1: 7 take volume ratio with substrate, was 25 ℃ in invert point, and the conversion reaction time is 40 hours, namely gets composition.
9. the preparation method of benazepril hydrochloride composition as claimed in claim 6 is characterized in that the method is:
Slant strains is cultivated: the mould CICC 2436 of selected songs carries out spawn culture, and agar is added 4 worts of spending, and makes the substratum of pH 7, so that every parts by volume substratum contains agar 0.08 weight part; Under 115 ℃ of conditions, sterilize, cooling after the sterilization, ordinary method makes inclined-plane and inoculation, cultivates 6 days under 25 ℃ of conditions, as slant strains; Solid fermentation is cultivated: be that 1: 1.8 ratio mixes with wheat bran and wort according to weightmeasurement ratio, behind the mixing under 120 ℃ of conditions, sterilized 25 minutes, cooling after the sterilization, the access slant strains is so that the slant strains volume accounts for 9% of culture volume, at 25 ℃, cultivated 2 days, and in cultured wheat bran, added 0.4M pH7 pbs damping fluid soaked overnight, get the solid fermentation nutrient solution; The preparation of crude enzyme liquid: add ammonium sulfate in the solid fermentation nutrient solution to saturated, stir to fully dissolving, under 4 ℃ of conditions, place and spend the night, the centrifuging and taking precipitation, dialysis, the centrifuging and taking supernatant liquor is as crude enzyme liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, hydrochloric benazepril 0.09 weight part of the every parts by volume of the solution of making, after millipore filtration sterile filtration as substrate; Enzymatic conversion reaction: crude enzyme liquid was mixed as 1: 3 take volume ratio with substrate, was 25 ℃ in invert point, and the conversion reaction time is 18 hours, namely gets composition.
10. the preparation method of benazepril hydrochloride composition as claimed in claim 6 is characterized in that the method is:
Slant strains is cultivated: the mould CICC 2436 of selected songs carries out spawn culture, and agar is added 4 worts of spending, and makes the substratum of pH 8, so that every parts by volume substratum contains agar 0.1 weight part; Under 104 ℃ of conditions, sterilize, cooling after the sterilization, ordinary method makes inclined-plane and inoculation, cultivates 4 days under 38 ℃ of conditions, as slant strains; Solid fermentation is cultivated: be that 1: 0.7 ratio mixes with wheat bran and wort according to weightmeasurement ratio, behind the mixing under 117 ℃ of conditions, sterilized 32 minutes, cooling after the sterilization, the access slant strains is so that the slant strains volume accounts for 6% of culture volume, at 38 ℃, cultivated 3 days, and in cultured wheat bran, added 0.07M pH7 pbs damping fluid soaked overnight, get the solid fermentation nutrient solution; The preparation of crude enzyme liquid: add ammonium sulfate in the solid fermentation nutrient solution to saturated, stir to fully dissolving, under 4 ℃ of conditions, place and spend the night, the centrifuging and taking precipitation, dialysis, the centrifuging and taking supernatant liquor is as crude enzyme liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, hydrochloric benazepril 0.1 weight part of the every parts by volume of the solution of making, after millipore filtration sterile filtration as substrate; Enzymatic conversion reaction: crude enzyme liquid was mixed as 1: 3 take volume ratio with substrate, was 28 ℃ in invert point, and the conversion reaction time is 38 hours, namely gets composition.
11. the benazepril hydrochloride composition with hypotensive activity is characterized in that by following liquid fermentation process preparation:
Slant strains is cultivated: the mould CICC 2436 of selected songs carries out spawn culture, and agar is added 4 worts of spending, and making pH is the substratum of 6-8, so that every parts by volume substratum contains agar 0.02-0.1 weight part; Sterilize under 100-120 ℃ of condition, cooling after the sterilization makes inclined-plane and inoculation, at 20 ℃--cultivated 3-7 days under 40 ℃ of conditions, as slant strains; Liquid fermenting: with glucose, extractum carnis, peptone, ammonium nitrate, sal epsom are added to the water and make the substratum that pH is 6-8, so that every parts by volume substratum contains glucose 0.01-0.1 weight part, extractum carnis 0.01-0.1 weight part, peptone 0.01-0.06 weight part, ammonium nitrate 0.01-0.05 weight part, sal epsom 0.01-0.08 weight part; The substratum of 20-100 parts by volume, under 100-120 ℃ of condition, sterilized 20-40 minute, cooling after the sterilization, the access slant strains is so that the slant strains volume accounts for the 5-10% of substratum, at 20-40 ℃, ferment under the rotating speed 100-250r/min condition, cultivated 1-5 days, as liquid fermentation and culture liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, the hydrochloric benazepril 0.03-0.2 of the every parts by volume of the solution of making weight part, after millipore filtration sterile filtration as substrate; Enzyme reaction: utilize liquid fermentation and culture liquid as the enzyme source, substrate being added in the liquid fermentation and culture liquid, contain substrate 0.00003-0.0001 weight part to every parts by volume fermentation culture, is 20-40 ℃ in invert point, transformation time is 5-48 hour, namely gets composition.
12. benazepril hydrochloride composition as claimed in claim 11 is characterized in that by following liquid fermentation process preparation:
Slant strains is cultivated: the mould CICC 2436 of selected songs carries out spawn culture, and agar is added the worts of 4 degree, makes pH and be 7 substratum, so that every parts by volume substratum contains agar 0.05 weight part; Sterilize under 110 ℃ of conditions, cooling after the sterilization makes inclined-plane and inoculation, cultivates 5 days under 30 ℃ of conditions, as slant strains; Liquid fermenting: with glucose, extractum carnis, peptone, ammonium nitrate, sal epsom is added to the water that to make pH be 7 substratum, so that every parts by volume substratum contains glucose 0.05 weight part, and extractum carnis 0.05 weight part, peptone 0.04 weight part, ammonium nitrate 0.03 weight part, sal epsom 0.04 weight part; The substratum of 50 parts by volume under 110 ℃ of conditions, was sterilized 30 minutes, cooling after the sterilization, and the access slant strains so that the slant strains volume accounts for 8% of substratum, at 30 ℃, is fermented under the rotating speed 150r/min condition, cultivates 3 days, as liquid fermentation and culture liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, hydrochloric benazepril 0.1 weight part of the every parts by volume of the solution of making, after millipore filtration sterile filtration as substrate; Enzyme reaction: utilizing liquid fermentation and culture liquid as the enzyme source, substrate is added in the liquid fermentation and culture liquid, contain substrate 0.00008 weight part to every parts by volume fermentation culture, is 30 ℃ in invert point, and transformation time is 28 hours, namely gets composition.
13. benazepril hydrochloride composition as claimed in claim 11 is characterized in that by following liquid fermentation process preparation:
Slant strains is cultivated: the mould CICC 2436 of selected songs carries out spawn culture, and agar is added the worts of 4 degree, makes pH and be 6.5 substratum, so that every parts by volume substratum contains agar 0.09 weight part; Sterilize under 110 ℃ of conditions, cooling after the sterilization makes inclined-plane and inoculation, cultivates 6 days under 22 ℃ of conditions, as slant strains; Liquid fermenting: with glucose, extractum carnis, peptone, ammonium nitrate, sal epsom is added to the water that to make pH be 6.5 substratum, so that every parts by volume substratum contains glucose 0.02 weight part, and extractum carnis 0.9 weight part, peptone 0.04 weight part, ammonium nitrate 0.02 weight part, sal epsom 0.07 weight part; The substratum of 60 parts by volume under 102 ℃ of conditions, was sterilized 38 minutes, cooling after the sterilization, and the access slant strains so that the slant strains volume accounts for 7% of substratum, at 24 ℃, is fermented under the rotating speed 220r/min condition, cultivates 3 days, as liquid fermentation and culture liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, hydrochloric benazepril 0.05 weight part of the every parts by volume of the solution of making, after millipore filtration sterile filtration as substrate; Enzyme reaction: utilizing liquid fermentation and culture liquid as the enzyme source, substrate is added in the liquid fermentation and culture liquid, contain substrate 0.00008 weight part to every parts by volume fermentation culture, is 29 ℃ in invert point, and transformation time is 9 hours, namely gets composition.
14. benazepril hydrochloride composition as claimed in claim 11 is characterized in that by following liquid fermentation process preparation:
Slant strains is cultivated: the mould CICC 2436 of selected songs carries out spawn culture, and agar is added the worts of 4 degree, makes pH and be 7.5 substratum, so that every parts by volume substratum contains agar 0.04 weight part; Sterilize under 108 ℃ of conditions, cooling after the sterilization makes inclined-plane and inoculation, cultivates 4 days under 37 ℃ of conditions, as slant strains; Liquid fermenting: with glucose, extractum carnis, peptone, ammonium nitrate, sal epsom are added to the water that to make pH be 7.5 substratum, so that every parts by volume substratum contains glucose 0.08 weight part, extractum carnis 0.05 weight part, peptone 0.03 weight part, ammonium nitrate 0.04 weight part, sal epsom 0.02 weight part; The substratum of 70 parts by volume under 117 ℃ of conditions, was sterilized 24 minutes, cooling after the sterilization, and the access slant strains so that the slant strains volume accounts for 7% of substratum, at 36 ℃, is fermented under the rotating speed 105r/min condition, cultivates 3 days, as liquid fermentation and culture liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, hydrochloric benazepril 0.1 weight part of the every parts by volume of the solution of making, after millipore filtration sterile filtration as substrate; Enzyme reaction: utilizing liquid fermentation and culture liquid as the enzyme source, substrate is added in the liquid fermentation and culture liquid, contain substrate 0.00004 weight part to every parts by volume fermentation culture, is 34 ℃ in invert point, and transformation time is 43 hours, namely gets composition.
15. benazepril hydrochloride composition as claimed in claim 11 is characterized in that by following liquid fermentation process preparation:
Slant strains is cultivated: the mould CICC 2436 of selected songs carries out spawn culture, and agar is added the worts of 4 degree, makes pH and be 7 substratum, so that every parts by volume substratum contains agar 0.04 weight part; Sterilize under 116 ℃ of conditions, cooling after the sterilization makes inclined-plane and inoculation, cultivates 3 days under 34 ℃ of conditions, as slant strains; Liquid fermenting: with glucose, extractum carnis, peptone, ammonium nitrate, sal epsom is added to the water that to make pH be 8 substratum, so that every parts by volume substratum contains glucose 0.05 weight part, and extractum carnis 0.01 weight part, peptone 0.06 weight part, ammonium nitrate 0.04 weight part, sal epsom 0.02 weight part; The substratum of 100 parts by volume under 108 ℃ of conditions, was sterilized 21 minutes, cooling after the sterilization, the access slant strains is so that the slant strains volume accounts for 10% of substratum, at 30 ℃, ferment under the rotating speed 105r/min condition, cultivated 5 days, as liquid fermentation and culture liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, hydrochloric benazepril 0.1 weight part of the every parts by volume of the solution of making, after millipore filtration sterile filtration as substrate; Enzyme reaction: utilizing liquid fermentation and culture liquid as the enzyme source, substrate is added in the liquid fermentation and culture liquid, contain substrate 0.00004 weight part to every parts by volume fermentation culture, is 40 ℃ in invert point, and transformation time is 25 hours, namely gets composition.
16. the preparation method with benazepril hydrochloride composition of hypotensive activity is characterized in that the method is:
Slant strains is cultivated: the mould CICC 2436 of selected songs carries out spawn culture, and agar is added 4 worts of spending, and making pH is the substratum of 6-8, so that every parts by volume substratum contains agar 0.02-0.1 weight part; Sterilize under 100-120 ℃ of condition, cooling after the sterilization makes inclined-plane and inoculation, at 20 ℃--cultivated 3-7 days under 40 ℃ of conditions, as slant strains; Liquid fermenting: with glucose, extractum carnis, peptone, ammonium nitrate, sal epsom are added to the water and make the substratum that pH is 6-8, so that every parts by volume substratum contains glucose 0.01-0.1 weight part, extractum carnis 0.01-0.1 weight part, peptone 0.01-0.06 weight part, ammonium nitrate 0.01-0.05 weight part, sal epsom 0.01-0.08 weight part; The substratum of 20-100 parts by volume, under 100-120 ℃ of condition, sterilized 20-40 minute, cooling after the sterilization, the access slant strains is so that the slant strains volume accounts for the 5-10% of substratum, at 20-40 ℃, ferment under the rotating speed 100-250r/min condition, cultivated 1-5 days, as liquid fermentation and culture liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, the hydrochloric benazepril 0.03-0.2 of the every parts by volume of the solution of making weight part, after millipore filtration sterile filtration as substrate; Enzyme reaction: utilize liquid fermentation and culture liquid as the enzyme source, substrate being added in the liquid fermentation and culture liquid, contain substrate 0.00003-0.0001 weight part to every parts by volume fermentation culture, is 20-40 ℃ in invert point, transformation time is 5-48 hour, namely gets composition.
17. the preparation method of benazepril hydrochloride composition as claimed in claim 16 is characterized in that the method is:
Slant strains is cultivated: the mould CICC 2436 of selected songs carries out spawn culture, and agar is added the worts of 4 degree, makes pH and be 7 substratum, so that every parts by volume substratum contains agar 0.05 weight part; Sterilize under 110 ℃ of conditions, cooling after the sterilization makes inclined-plane and inoculation, cultivates 5 days under 30 ℃ of conditions, as slant strains; Liquid fermenting: with glucose, extractum carnis, peptone, ammonium nitrate, sal epsom is added to the water that to make pH be 7 substratum, so that every parts by volume substratum contains glucose 0.05 weight part, and extractum carnis 0.05 weight part, peptone 0.04 weight part, ammonium nitrate 0.03 weight part, sal epsom 0.04 weight part; The substratum of 50 parts by volume under 110 ℃ of conditions, was sterilized 30 minutes, cooling after the sterilization, and the access slant strains so that the slant strains volume accounts for 8% of substratum, at 30 ℃, is fermented under the rotating speed 150r/min condition, cultivates 3 days, as liquid fermentation and culture liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, hydrochloric benazepril 0.1 weight part of the every parts by volume of the solution of making, after millipore filtration sterile filtration as substrate; Enzyme reaction: utilizing liquid fermentation and culture liquid as the enzyme source, substrate is added in the liquid fermentation and culture liquid, contain substrate 0.00008 weight part to every parts by volume fermentation culture, is 30 ℃ in invert point, and transformation time is 28 hours, namely gets composition.
18. the preparation method of benazepril hydrochloride composition as claimed in claim 16 is characterized in that the method is:
Slant strains is cultivated: the mould CICC 2436 of selected songs carries out spawn culture, and agar is added the worts of 4 degree, makes pH and be 6.5 substratum, so that every parts by volume substratum contains agar 0.09 weight part; Sterilize under 110 ℃ of conditions, cooling after the sterilization makes inclined-plane and inoculation, cultivates 6 days under 22 ℃ of conditions, as slant strains; Liquid fermenting: with glucose, extractum carnis, peptone, ammonium nitrate, sal epsom is added to the water that to make pH be 6.5 substratum, so that every parts by volume substratum contains glucose 0.02 weight part, and extractum carnis 0.9 weight part, peptone 0.04 weight part, ammonium nitrate 0.02 weight part, sal epsom 0.07 weight part; The substratum of 60 parts by volume under 102 ℃ of conditions, was sterilized 38 minutes, cooling after the sterilization, and the access slant strains so that the slant strains volume accounts for 7% of substratum, at 24 ℃, is fermented under the rotating speed 220r/min condition, cultivates 3 days, as liquid fermentation and culture liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, hydrochloric benazepril 0.05 weight part of the every parts by volume of the solution of making, after millipore filtration sterile filtration as substrate; Enzyme reaction: utilizing liquid fermentation and culture liquid as the enzyme source, substrate is added in the liquid fermentation and culture liquid, contain substrate 0.00008 weight part to every parts by volume fermentation culture, is 29 ℃ in invert point, and transformation time is 9 hours, namely gets composition.
19. the preparation method of benazepril hydrochloride composition as claimed in claim 16 is characterized in that the method is:
Slant strains is cultivated: the mould CICC 2436 of selected songs carries out spawn culture, and agar is added the worts of 4 degree, makes pH and be 7.5 substratum, so that every parts by volume substratum contains agar 0.04 weight part; Sterilize under 108 ℃ of conditions, cooling after the sterilization makes inclined-plane and inoculation, cultivates 4 days under 37 ℃ of conditions, as slant strains; Liquid fermenting: with glucose, extractum carnis, peptone, ammonium nitrate, sal epsom are added to the water that to make pH be 7.5 substratum, so that every parts by volume substratum contains glucose 0.08 weight part, extractum carnis 0.05 weight part, peptone 0.03 weight part, ammonium nitrate 0.04 weight part, sal epsom 0.02 weight part; The substratum of 70 parts by volume under 117 ℃ of conditions, was sterilized 24 minutes, cooling after the sterilization, and the access slant strains so that the slant strains volume accounts for 7% of substratum, at 36 ℃, is fermented under the rotating speed 105r/min condition, cultivates 3 days, as liquid fermentation and culture liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, hydrochloric benazepril 0.1 weight part of the every parts by volume of the solution of making, after millipore filtration sterile filtration as substrate; Enzyme reaction: utilizing liquid fermentation and culture liquid as the enzyme source, substrate is added in the liquid fermentation and culture liquid, contain substrate 0.00004 weight part to every parts by volume fermentation culture, is 34 ℃ in invert point, and transformation time is 43 hours, namely gets composition.
20. the preparation method of benazepril hydrochloride composition as claimed in claim 16 is characterized in that the method is:
Slant strains is cultivated: the mould CICC 2436 of selected songs carries out spawn culture, and agar is added the worts of 4 degree, makes pH and be 7 substratum, so that every parts by volume substratum contains agar 0.04 weight part; Sterilize under 116 ℃ of conditions, cooling after the sterilization makes inclined-plane and inoculation, cultivates 3 days under 34 ℃ of conditions, as slant strains; Liquid fermenting: with glucose, extractum carnis, peptone, ammonium nitrate, sal epsom is added to the water that to make pH be 8 substratum, so that every parts by volume substratum contains glucose 0.05 weight part, and extractum carnis 0.01 weight part, peptone 0.06 weight part, ammonium nitrate 0.04 weight part, sal epsom 0.02 weight part; The substratum of 100 parts by volume under 108 ℃ of conditions, was sterilized 21 minutes, cooling after the sterilization, the access slant strains is so that the slant strains volume accounts for 10% of substratum, at 30 ℃, ferment under the rotating speed 105r/min condition, cultivated 5 days, as liquid fermentation and culture liquid; The preparation of substrate: with aqueous solution dissolved salt Benazepril Hydrochloride, hydrochloric benazepril 0.1 weight part of the every parts by volume of the solution of making, after millipore filtration sterile filtration as substrate; Enzyme reaction: utilizing liquid fermentation and culture liquid as the enzyme source, substrate is added in the liquid fermentation and culture liquid, contain substrate 0.00004 weight part to every parts by volume fermentation culture, is 40 ℃ in invert point, and transformation time is 25 hours, namely gets composition.
21. such as the application of the arbitrary described benazepril hydrochloride composition of claim 1-5,11-15 in the preparation Altace Ramipril.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1517331A (en) * | 2003-01-13 | 2004-08-04 | 上海天伟生物制药有限公司 | Purification method of pravastiatin sodium |
| CN1886125A (en) * | 2003-11-24 | 2006-12-27 | 特瓦药厂私人有限公司 | Method of purifying pravastatin |
| CN1903825A (en) * | 2005-07-25 | 2007-01-31 | 丘比株式会社 | Method for manufacturing pravastatin sodium |
| CN101012474A (en) * | 2007-02-05 | 2007-08-08 | 大连理工大学 | Method for preparing Chinese yam saponin by microorganism transformation process |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1517331A (en) * | 2003-01-13 | 2004-08-04 | 上海天伟生物制药有限公司 | Purification method of pravastiatin sodium |
| CN1886125A (en) * | 2003-11-24 | 2006-12-27 | 特瓦药厂私人有限公司 | Method of purifying pravastatin |
| CN1903825A (en) * | 2005-07-25 | 2007-01-31 | 丘比株式会社 | Method for manufacturing pravastatin sodium |
| CN101012474A (en) * | 2007-02-05 | 2007-08-08 | 大连理工大学 | Method for preparing Chinese yam saponin by microorganism transformation process |
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