CN103555698A - Extracting process of complex chymosin - Google Patents
Extracting process of complex chymosin Download PDFInfo
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- CN103555698A CN103555698A CN201310552506.9A CN201310552506A CN103555698A CN 103555698 A CN103555698 A CN 103555698A CN 201310552506 A CN201310552506 A CN 201310552506A CN 103555698 A CN103555698 A CN 103555698A
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- rennh
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6478—Aspartic endopeptidases (3.4.23)
- C12N9/6483—Chymosin (3.4.23.4), i.e. rennin
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/23—Aspartic endopeptidases (3.4.23)
- C12Y304/23004—Chymosin (3.4.23.4), i.e. rennin
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract
The invention relates to an extracting method of complete chymosin, belonging to the technical field of biological pharmacy. The extracting process of the complex chymosin comprises a step of stimulating and activating abomasums powder of a lamb by using acid liquor; in the extracting process, each step and each parameter are matched with each other and co-acted, so that the extracting process is not only short in reaction time, gentle in reaction condition, not applicable to much organic solvent and environment-friendly, but also high in yield. The obtained complex chymosin has high in-vitro milk clotting activity and high protein activity, so that a new thought is developed for researching the extracting process of the complex chymosin.
Description
Technical field
The present invention relates to a kind of extraction process of biological medicament, relate in particular to a kind of extraction process of Compound rennh, belong to biological pharmacy technical field.
Background technology
According to " 12 ", the status of " 13 " plan-pharmaceutical industries in national economy, constantly rise, biological medicine has been listed in national strategy new industry key areas.In addition, along with science constantly develops, biochemical drug is from organism, and composition belongs to biomacromolecule more, easily absorbed by the body, and participates in the metabolism of human body directly, can regulate, supplement, recover and maintain the normal function of human body.Biochemical drug good effect, side effect is little, targeting is strong, and instant effect is more and more paid attention to by people.
Disease of stomach is the organic or functional disease that betides stomach.Common are clinically chronic gastritis, peptide ulceration, gastroptosis and cancer of the stomach etc.The cause of disease is very complicated, comprises physicochemical irritation, infection, toxin, heredity, mental element, dysplasia, operation impact etc.Cardinal symptom has epigastric pain, the heart that burns, feel sick, vomiting, belch, sour regurgitation, poor appetite, spitting blood, have blood in stool, lump etc.Disease of stomach is having a strong impact on the healthy of people.
For complying with medicine company development trend and solving the problem that disease of stomach perplexs human health for a long time, develop biological medicine for stomach dynamic technology, giving full play to biochemical drug is the magnificent mission that pharmacy corporation is born to the result for the treatment of of human body.
Summary of the invention
Technical problem to be solved by this invention is the extraction process that the defect that overcomes prior art provides a kind of Compound rennh, and this extraction process has that the time is short, reaction conditions is gentle, free from environmental pollution, the external curdled milk vigor of gained Compound rennh advantages of higher.
Technical problem of the present invention is realized by following technical scheme.
An extraction process for Compound rennh, it comprises the step of using the acid solution thorn broken end of activating signal activation lamb sheep fourth stomach.
The extraction process of above-mentioned Compound rennh, the hydrochloric acid that described acid solution is 5%-10% or glacial acetic acid solution.
The extraction process of above-mentioned Compound rennh, the broken last diameter of described lamb sheep fourth stomach is 140mm-220mm.
The extraction process of above-mentioned Compound rennh, it comprises the steps:
(1) former material preparation: get lamb sheep fourth stomach 300g, put the broken end that is twisted into 140mm-220mm diameter in mincer, standby;
(2) thorn activating signal activation: the broken end of step (1) gained is put into beaker and added purified water 120-400ml, then put in water-bath and heat, be warmed up to 35-47 ℃, after stirring, the hydrochloric acid with 5%-10% or glacial acetic acid solution regulate pH value to 3.5-4.5, constantly stir and activate 3 to 4 hours, let cool, 80 order filter-cloth filterings, collect filtrate, standby;
(3) collecting precipitation: the acetone or alcohol to adding 2.5-4.5 times of filtrate weight in step (2) gained filtrate, stir 20 minutes, standing 20 minutes, the supernatant liquor collecting precipitation thing that inclines, standby;
(4) target product: step (3) gained throw out is put in beaker and added 750ml acetone or alcohol, stir 10 minutes, the acetone or alcohol that inclines, by this method repetitive scrubbing 4-5 time, then puts washes in vacuum drying oven 40 ℃ and be dried, and obtains Compound rennh.
The extraction process of preferred above-mentioned Compound rennh, it comprises the steps:
(1) former material preparation: get lamb sheep fourth stomach 300g, put the broken end that is twisted into 180mm diameter in mincer, standby;
(2) thorn activating signal activation: the broken end of step (1) gained is put into beaker and added purified water 350ml, then put in water-bath and heat, be warmed up to 41 ℃, after stirring, the hydrochloric acid with 8% or glacial acetic acid solution regulate pH value to 4, constantly stir and activate 3.5 hours, let cool, 80 order filter-cloth filterings, collect filtrate, standby;
(3) collecting precipitation: the acetone or alcohol to adding 3.5 times of filtrate weight in step (2) gained filtrate, stir 20 minutes, standing 20 minutes, the supernatant liquor collecting precipitation thing that inclines, standby;
(4) target product: step (3) gained throw out is put in beaker and is added 750ml acetone or alcohol, stir 10 minutes, the acetone or alcohol that inclines, by this method repetitive scrubbing 5 times, then washes is put in vacuum drying oven 40 ℃ dry, obtain Compound rennh.
The extraction process of a kind of Compound rennh of the present invention, between each step, each parameter, cooperatively interact, acting in conjunction, make this extraction process have that not only the reaction times is short, reaction conditions is gentle, inapplicable a large amount of organic solvents are free from environmental pollution, and yield is up to 25.6%, the external curdled milk vigor of gained Compound rennh is up to 950 curdled milk units, and protein vigor is up to 900 units, for the Study on extraction of Compound rennh has been opened up new thinking.
Embodiment
Below in conjunction with embodiment, the present invention is described in further details.
The extraction of embodiment 1 Compound rennh
Step is as follows:
(1) former material preparation: get lamb sheep fourth stomach 300g, put the broken end that is twisted into 140mm diameter in mincer, standby;
(2) thorn activating signal activation: the broken end of step (1) gained is put into beaker and added purified water 200ml, then put in water-bath and heat, be warmed up to 35 ℃, after stirring, the hydrochloric acid with 5% or glacial acetic acid solution regulate pH value to 3.5, constantly stir and activate 3 hours, let cool, 80 order filter-cloth filterings, collect filtrate, standby;
(3) collecting precipitation: the acetone to adding 2.5 times of filtrate weight in step (2) gained filtrate, stir 20 minutes, standing 20 minutes, the supernatant liquor collecting precipitation thing that inclines, standby;
(4) target product: step (3) gained throw out is put in beaker and is added 750ml acetone, stir 10 minutes, the acetone that inclines, by this method repetitive scrubbing 4 times, then washes is put in vacuum drying oven 40 ℃ dry, obtain 64 grams of Compound rennhs, yield 21.3%.
The extraction of embodiment 2 Compound rennhs
Step is as follows:
(1) former material preparation: get lamb sheep fourth stomach 300g, put the broken end that is twisted into 220mm diameter in mincer, standby;
(2) thorn activating signal activation: the broken end of step (1) gained is put into beaker and added purified water 500ml, then put in water-bath and heat, be warmed up to 47 ℃, after stirring, glacial acetic acid solution with 10% regulates pH value to 4.5, constantly stirs and activates 4 hours, lets cool, 80 order filter-cloth filterings, collect filtrate, standby;
(3) collecting precipitation: the ethanol to adding 4.5 times of filtrate weight in step (2) gained filtrate, stir 20 minutes, standing 20 minutes, the supernatant liquor collecting precipitation thing that inclines, standby;
(4) target product: step (3) gained throw out is put in beaker and is added 750ml ethanol, stir 10 minutes, the ethanol that inclines, by this method repetitive scrubbing 5 times, then washes is put in vacuum drying oven 40 ℃ dry, obtain 75 grams of Compound rennhs, yield 25.3%.
The extraction of embodiment 3 Compound rennhs
Step is as follows:
(1) former material preparation: get lamb sheep fourth stomach 300g, put the broken end that is twisted into 180mm diameter in mincer, standby;
(2) thorn activating signal activation: the broken end of step (1) gained is put into beaker and added purified water 350ml, then put in water-bath and heat, be warmed up to 41 ℃, after stirring, hydrochloric acid soln with 8% regulates pH value to 4, constantly stirs and activates 3.5 hours, lets cool, 80 order filter-cloth filterings, collect filtrate, standby;
(3) collecting precipitation: the acetone to adding 3.5 times of filtrate weight in step (2) gained filtrate, stir 20 minutes, standing 20 minutes, the supernatant liquor collecting precipitation thing that inclines, standby;
(4) target product: step (3) gained throw out is put in beaker and is added 750ml acetone, stir 10 minutes, the acetone that inclines, by this method repetitive scrubbing 5 times, then washes is put in vacuum drying oven 40 ℃ dry, obtain 77 grams of Compound rennhs, yield 25.6%.
The extraction of embodiment 4 Compound rennhs
Step is as follows:
(1) former material preparation: get lamb sheep fourth stomach 300g, put the broken end that is twisted into 180mm diameter in mincer, standby;
(2) thorn activating signal activation: the broken end of step (1) gained is put into beaker and added purified water 350ml, then put in water-bath and heat, be warmed up to 41 ℃, after stirring, glacial acetic acid solution with 8% regulates pH value to 4, constantly stirs and activates 3.5 hours, lets cool, 80 order filter-cloth filterings, collect filtrate, standby;
(3) collecting precipitation: the ethanol to adding 3.5 times of filtrate weight in step (2) gained filtrate, stir 20 minutes, standing 20 minutes, the supernatant liquor collecting precipitation thing that inclines, standby;
(4) target product: step (3) gained throw out is put in beaker and is added 750ml ethanol, stir 10 minutes, the ethanol that inclines, by this method repetitive scrubbing 5 times, then washes is put in vacuum drying oven 40 ℃ dry, obtain 78 grams of Compound rennhs, yield 26.3%.
Embodiment 5 gets the Compound rennh that embodiment 1 to 3 extracts and carries out titration
[rennin]
1. whole milk powder 12g is got in the preparation of substrate solution, adds calcium chloride solution and (gets sodium-acetate 2.05g, calcium chloride 1.11g, add water and make to be dissolved into 1000ml, regulate pH value to 6.3 ± 0.1) appropriate, grind evenly, put in 100ml measuring bottle, add calcium chloride solution to scale, shake up.This liquid should face with newly joining.
2. rennet standard product are got in the preparation of standard solution, accurately weighed, add phosphate buffered saline buffer (PH6.3) and (get SODIUM PHOSPHATE, MONOBASIC 7.8g, Sodium phosphate dibasic 2.7g, add water and make to be dissolved into 1000ml, regulate pH value to 6.3 ± 0.05) dissolve and be diluted to the solution that contains 0.4～0.6 unit of activity in every 1ml.This liquid should remain on below 20 ℃, and uses in latter 2 hours of preparation.
3. the Compound rennh that the Preparation Example 1 to 4 of need testing solution is extracted proceeds as follows respectively; get the about 0.1g of this product; accurately weighed; put in mortar; add phosphate buffered saline buffer (pH6.3) a small amount of, grinds evenly, puts in 100ml measuring bottle; add phosphate buffered saline buffer (pH6.3) to scale, shake up.This liquid should remain on below 20 ℃, faces with newly joining.
4. assay method precision measures substrate solution 10ml, put in test tube, in 30 ℃ ± 0.5 ℃ water bath with thermostatic control, be incubated 10 minutes, precision adds standard solution 1ml, shake up and timing immediately, with glass stick, constantly dip solution and flow down along tube wall, make into even thin layer, when visible particle appears in thin layer, show curdled milk, accurate recording after standard solution adds to the time that occurs curdled milk phenomenon.Replication three times, the relative standard deviation of three measured values must not be greater than 5%.Get the mean value of three times as curdled milk time of standard solution.Take the prepared need testing solution 1ml of Compound rennh of embodiment 1 to 4 extraction, operation, is calculated as follows in accordance with the law.
The vigor of S-------standard substance in formula, Ru/mg;
The average curdled milk time of Ts-------standard solution, second;
The average curdled milk time of T--------need testing solution, second;
In the every 1ml of Ws--------standard solution, contain the amount of rennet standard product, g;
W-------trial-product sampling amount, g;
N--------trial-product extension rate.
Note: curdled milk time circulation ratio in the time of 180～240 seconds is best.Otherwise test liquid concentration should be adjusted.
[proteolytic enzyme]
1. the preparation precision of reference product solution takes that through 105 ℃, to be dried to constant weight tyrosine appropriate, adds hydrochloric acid soln (get 1mol/L hydrochloric acid soln 65ml, add water to 1000ml) and makes in every 1ml the solution containing 0.4mg.
2. the about 0.2g of this product is got in the preparation of need testing solution, accurately weighed, with above-mentioned hydrochloric acid soln, grinds and makes to dissolve, and puts in 100ml measuring bottle, adds above-mentioned hydrochloric acid soln to scale, shakes up.
3. assay method is measured according to the method under stomach egg enzyme activity determination item (Chinese Pharmacopoeia 2010 version two 530 pages).
[result] is in Table 1.
Tiring of the Compound rennh that table 1 embodiment 1 to 4 extracts
| ? | Embodiment 1 | Embodiment 2 | Embodiment 3 | Embodiment 3 |
| External curdled milk vigor | 870 curdled milk units | 910 curdled milk units | 950 curdled milk units | 940 curdled milk units |
| Proteinase activity | 830 units | 870 units | 900 units | 900 units |
Above-described embodiment is only explanation technical conceive of the present invention and advantage; the present invention also can have other variation; as well known to the skilled person; above-described embodiment only plays the exemplary role in foregoing invention protection domain; for those of ordinary skills; in the protection domain limiting in the present invention, also have a lot of conventional distortion and other embodiment, these distortion and embodiment are by within the protection domain awaiting the reply in the present invention.
Claims (5)
1. an extraction process for Compound rennh, it comprises the step of using the acid solution thorn broken end of activating signal activation lamb sheep fourth stomach.
2. the extraction process of Compound rennh according to claim 1, is characterized in that, the hydrochloric acid that described acid solution is 5%-10% or glacial acetic acid solution.
3. the extraction process of Compound rennh according to claim 2, is characterized in that, the broken last diameter of described lamb sheep fourth stomach is 140mm-220mm.
4. the extraction process of Compound rennh according to claim 3, is characterized in that, it comprises the steps:
(1) former material preparation: get lamb sheep fourth stomach 300g, put the broken end that is twisted into 140mm-220mm diameter in mincer, standby;
(2) thorn activating signal activation: the broken end of step (1) gained is put into beaker and added purified water 200-500ml, then put in water-bath and heat, be warmed up to 35-47 ℃, after stirring, the hydrochloric acid with 5%-10% or glacial acetic acid solution regulate pH value to 3.5-4.5, constantly stir and activate 3 to 4 hours, let cool, 80 order filter-cloth filterings, collect filtrate, standby;
(3) collecting precipitation: the acetone or alcohol to adding 2.5-4.5 times of filtrate weight in step (2) gained filtrate, stir 20 minutes, standing 20 minutes, the supernatant liquor collecting precipitation thing that inclines, standby;
(4) target product: step (3) gained throw out is put in beaker and added 750ml acetone or alcohol, stir 10 minutes, the acetone or alcohol that inclines, by this method repetitive scrubbing 4-5 time, then puts washes in vacuum drying oven 40 ℃ and be dried, and obtains Compound rennh.
5. the extraction process of Compound rennh according to claim 4, is characterized in that, it comprises the steps:
(1) former material preparation: get lamb sheep fourth stomach 300g, put the broken end that is twisted into 180mm diameter in mincer, standby;
(2) thorn activating signal activation: the broken end of step (1) gained is put into beaker and added purified water 350ml, then put in water-bath and heat, be warmed up to 41 ℃, after stirring, the hydrochloric acid with 8% or glacial acetic acid solution regulate pH value to 4, constantly stir and activate 3.5 hours, let cool, 80 order filter-cloth filterings, collect filtrate, standby;
(3) collecting precipitation: the acetone or alcohol to adding 3.5 times of filtrate weight in step (2) gained filtrate, stir 20 minutes, standing 20 minutes, the supernatant liquor collecting precipitation thing that inclines, standby;
(4) target product: step (3) gained throw out is put in beaker and is added 750ml acetone or alcohol, stir 10 minutes, the acetone or alcohol that inclines, by this method repetitive scrubbing 5 times, then washes is put in vacuum drying oven 40 ℃ dry, obtain Compound rennh.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310552506.9A CN103555698B (en) | 2013-11-11 | 2013-11-11 | Extracting process of complex chymosin |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310552506.9A CN103555698B (en) | 2013-11-11 | 2013-11-11 | Extracting process of complex chymosin |
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| Publication Number | Publication Date |
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| CN103555698A true CN103555698A (en) | 2014-02-05 |
| CN103555698B CN103555698B (en) | 2015-06-10 |
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| CN201310552506.9A Active CN103555698B (en) | 2013-11-11 | 2013-11-11 | Extracting process of complex chymosin |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1924012A (en) * | 2006-09-01 | 2007-03-07 | 上海阿敏生物技术有限公司 | Preparation method for rennin |
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2013
- 2013-11-11 CN CN201310552506.9A patent/CN103555698B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1924012A (en) * | 2006-09-01 | 2007-03-07 | 上海阿敏生物技术有限公司 | Preparation method for rennin |
Non-Patent Citations (2)
| Title |
|---|
| FOLTMANN B: "A review on prorennin and rennin", 《COMPT REND TRAV LAB CARLSBERG》 * |
| 赵胜娟等: "羔羊皱胃酶提取工艺研究", 《河南农业科学》 * |
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