CN1169950C - Process for preparing calf rennase - Google Patents
Process for preparing calf rennase Download PDFInfo
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- CN1169950C CN1169950C CNB021152608A CN02115260A CN1169950C CN 1169950 C CN1169950 C CN 1169950C CN B021152608 A CNB021152608 A CN B021152608A CN 02115260 A CN02115260 A CN 02115260A CN 1169950 C CN1169950 C CN 1169950C
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- chymosin
- supernatant liquor
- elutriant
- calf
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Abstract
The present invention relates to the technical field of chymosin preparation. A rennet stomach of a calf is used as material and is induced at low temperature, and the chymosin is extracted by water solution of NaCl and ethanol. Coarse chymosin liquid is gelated and purified through an ion exchange column and a molecular sieve so as to obtain partial purified chymosin. The optimum temperature of the chymosin reaction is 50 DEG C, the optimum pH value is 5.8, the chymosin activity is stable within the pH value range of 3.0 to 6.0, Ca2+ is an activating factor of the chymosin, and the chymosin activity is gradually enhanced when the total solid content of milk is more than 9%. The present invention has the advantages of easy source of raw material, low cost, time saving, economy, high chymosin content and high recovery activity. The investment of technological preparation equipment is little, and the technology is simple and practical. The present invention provides a new path for development and utilization of chymosin in China and market development of novel milk products.
Description
Technical field
The present invention relates to the preparation of rennin, relate in particular to the preparation method of calf rennase.
Background technology
Rennin is present in animal, plant and the microbe, and its effect is that cow's milk is solidified.The report of external relevant rennin is a lot of, and the rennin commodity of various ways such as liquid, glue, pulvis and tablet are arranged.But China is used milk-coagulating enzyme preparation at present, mainly comes from import, and the production that is applied to the rennin in the dairy products still is a blank.
Rennin can be divided into animal rennet, plant rennin and microbial rennet by its source.Animal rennet is the class of enzymes of using during cheese is produced the earliest, no matter at the rennin that all is better than other source aspect local flavor, quality and the output.The rennin proteolysis of microorganism and plant origin is relatively poor, can become the smaller polypeptides fragment to the K-casein hydrolysis, and easily produces bitter taste in ripening process, has limited their application aborning.
The rennin of animal-origin mainly is present in the Pepsin of calf and lamb.Nineteen sixty, 8,000,000 calves have been slaughtered for extracting rennin by the U.S..China is vast in territory, not deficient calf and lamb resource.Be used to make serum, pharmacy or meat calf stomach of butchering, carry out utilization of waste material as byproduct, the calf that can collect application at present has ten thousand of 1.8-2.5, and is still increasing.
The extraction of traditional calf rennase is that the calf abomasum after butchering is cleaned, and places the shady and cool ventilation place, and air-dry drying is used pulverizer powder powdered then, and powder is the thick enzyme that extracts.The extracting method of tradition calf rennase not only extraction time long, and enzyme is easy to inactivation in air drying process, activity recovery is not high, enzyme content is low.
Summary of the invention
Purpose of the present invention just is to seek out the preparation method that a kind of simple and easy to do, timing-saving and economic, enzyme content and vigor reclaim high calf rennase.
The present invention cleans back milk powder and CaCl with fresh or refrigerated calf abomasum
2Smear in the surface, after inducing under the cold condition, reject collagen, fat, chopping is weighed, and adds the extracting solution of 4 ℃ of precoolings, and extracting solution contains the aqueous solution of 5%NaCl and 10% dehydrated alcohol, and the volume of extracting solution is 3-5 with material weight in wet base ratio: 1; Homogenate then, stirring and leaching 30-240min gets supernatant liquor after centrifugal, and residue adds the extraction with aqueous solution liquid that contains 5%NaCl and 10% dehydrated alcohol of 4 ℃ of precoolings again, and the volume of extracting solution and material weight in wet base are than being 1-3: 1; Homogenate then, stirring and leaching 180-240min gets supernatant liquor after centrifugal; The supernatant liquor that merges twice gained, adding the volume number in supernatant liquor is the HCl of the 1mol/L of supernatant liquor 5%, and the precipitation foreign protein is centrifugal, gets supernatant liquor, and adding the weight number in supernatant liquor is the NaCl of supernatant liquor 5%, dissolves zymoprotein; After adjusting the pH value and be subacidity with the NaOH of 1mol/L, be the liquid crude enzyme.
With liquid crude enzyme DEAE ion-exchange chromatography, 0.05mol/L-0.4mol/L, pH5.4-6.4 citric acid-sodium citrate damping fluid carries out the pH linear gradient elution, the flow velocity of elutriant is 1.25ml/min, collector is collected elutriant, every pipe dress 5ml, being eluted to elutriant does not have absorption under the 280nm wavelength, 6 protein peaks appear in elutriant under the 280nm wavelength, and the 12-16 pipe of collection is peak III for peak I, 21-25 pipe for peak II, 30-34 pipe.Experimental results show that: wherein peak I, peak II, peak III and enzymic activity peak overlapping, remaining is the protein liquid that does not have enzymic activity, and the enzymic activity of peak I is the highest.Get peak I enzyme liquid, through dialysis, polyoxyethylene glycol be concentrated to original volume 1/3 after, use Sephadex chromatography column chromatography again, 0.05mol/L-0.4mol/L, pH5.4-6.4 citric acid-sodium citrate damping fluid carries out the pH linear gradient elution, the flow velocity of elutriant is 5ml/min, collector is collected elutriant, every pipe dress 5ml, be eluted to elutriant and under the 280nm wavelength, do not have absorption, 3 protein peaks appear in elutriant under the 280nm wavelength, wherein peak III and enzymic activity peak overlapping, the protein liquid of collected 19-26 pipe is the rennin of purifying, remaining is the protein liquid with enzymic activity.The rennin of purifying, it is a 171.43U/mg albumen than vigor, protein concentration is 0.007mg/ml.Its main zymologic property is as follows: the suitableeest temperature of the reaction of rennin is 55 ℃, and when temperature was lower than 25 ℃, curdled milk was very slow, and since 30 ℃, along with the rising of temperature, vigor also increases thereupon, but when being higher than 55 ℃, vigor descends rapidly; Rennin is handled 90min at 40 ℃, and enzyme activity is very stable, but along with the rising of temperature, the vigor of enzyme changes, and handles 90min at 55 ℃, and 36.7% vigor forfeiture is arranged; The optimum pH of milk coagulating enzyme action is 5.8, the pH value below 5.8 and more than, the vigor of enzyme all descends to some extent, is stable between pH3.0-6.0; The raw dairy dry matter content did not solidify in the 30min less than 8% o'clock, and since 9%, with the increase of dry-matter, its vigor also increases; Ca
2+Concentration is in the 0.025-0.125% scope, and with the increase of add-on, the vigor of rennin also increases, Ca
2+Concentration is more than 0.125%, and enzyme activity increases slowly.
Advantage of the present invention:
One, easy extraction, timing-saving and economic, enzyme content and vigor reclaim high.
Two, raw material sources are easy, inexpensive, adapt to the comprehensive development and utilization of product.
Three, the explained hereafter facility investment is few, and is simple for process, for the development and use and the novel dairy product market development of China's rennin provides new approach.
Specific embodiment
With 2 fresh or refrigerated calf abomasum cleaning back 20g milk powder and 2gCaCl
2Smear in the surface, induced under 4 ℃ cold condition 2-3 days, chopping, impurity elimination take by weighing 100g, add the extracting solution 400ml of 4 ℃ of precoolings, extracting solution is the aqueous solution that contains 5%NaCl and 10% dehydrated alcohol, homogenate, stir and extract 45min, the centrifugal 10min of 3000r/min gets supernatant liquor; Residue adds the extracting solution 200ml of 4 ℃ of precoolings again, and extracting solution is the aqueous solution that contains 5%NaCl and 10% dehydrated alcohol, and homogenate is stirred and extracted 240min, and the centrifugal 10min of 3000r/min gets supernatant liquor; The supernatant liquor that merges twice gained adds 30ml 1mol/L HCl, stirs, and the precipitation foreign protein, the centrifugal 10min of 3000r/min gets supernatant liquor; And then the NaCl of adding 60g, stirring and dissolving, adjusting the pH value with 1mol/L NaOH is 6.0, gets the about 600ml of liquid crude enzyme.
Get liquid crude enzyme 8ml DEAE23 ion-exchange chromatography, 0.05mol/L, pH5.4 citric acid-sodium citrate damping fluid carries out the pH linear gradient elution, the flow velocity of elutriant is 1.25ml/min, collector is collected elutriant, every pipe dress 5ml, being eluted to elutriant does not have absorption under the 280nm wavelength, 6 protein peaks appear in elutriant under the 280nm wavelength, and the 12-16 pipe of collection is peak III for peak I, 21-25 pipe for peak II, 30-34 pipe.Get peak I enzyme liquid 25ml, through dialysis, after polyethylene glycol 6000 is concentrated to 8ml, use Sephadex G-75 chromatography column chromatography again, 0.05mol/L, pH5.6 citric acid-citric acid are received damping fluid and are carried out the pH linear gradient elution, the flow velocity of elutriant is 5ml/min, collector is received elutriant, every pipe dress 5ml, and being eluted to elutriant does not have absorption under the 280nm wavelength, collected 19-26 pipe enzyme liquid 40ml, be the rennin of purifying, it is a 171.43U/mg albumen than vigor, and protein concentration is 0.007mg/ml.
Claims (1)
1, the preparation method of calf rennase: it is characterized in that: fresh or refrigerated calf abomasum are cleaned back 20g blockmilk and 2g solid CaCl
2Smear in the surface, after inducing under 4 ℃ of cold condition, reject collagen and fat, chopping, the extracting solution that adds 4 ℃ of precoolings, extracting solution is to contain 5% the NaCl and the aqueous solution of 10% dehydrated alcohol, extracting solution is 3-5 with material weight in wet base ratio: 1, homogenate, stirring and leaching 30-240min, centrifugal, get supernatant liquor, repeat 1-3 time, merge the supernatant liquor of gained several times, adding the volume number in supernatant liquor is the 1mol/L HCl precipitation foreign protein of supernatant liquor 5%, centrifugal, get supernatant liquor, the NaCl that adds the weight number and be supernatant liquor 5% in supernatant liquor dissolves zymoprotein, after being pH6 with 1mol/LNaOH adjustment pH value again, be the liquid crude enzyme, with crude enzyme liquid DEAE-23 ion-exchange chromatography, 0.05mol/L-0.4mol/L, pH5.4-6.4 citric acid-sodium citrate buffer solution carries out the pH linear gradient elution, and being eluted to elutriant does not have absorption under the 280nm wavelength, and the flow velocity of elutriant is 1.25ml/min, collector is collected elutriant, get the first protein peak enzyme liquid of collection, through dialysis, polyoxyethylene glycol be concentrated to original volume 1/3 after, use Sephadex G-75 column chromatography again, 0.05mol/L-0.4mol/L, pH5.4-6.4 citric acid-sodium citrate buffer solution carries out pH linear gradient elution to elutriant does not have absorption under the 280nm wavelength, the flow velocity of elutriant is 5ml/min, and collector is collected elutriant, and the 3rd protein peak that gets collection is the rennin of purifying.
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CNB021152608A CN1169950C (en) | 2002-05-23 | 2002-05-23 | Process for preparing calf rennase |
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CNB021152608A CN1169950C (en) | 2002-05-23 | 2002-05-23 | Process for preparing calf rennase |
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CN1379090A CN1379090A (en) | 2002-11-13 |
CN1169950C true CN1169950C (en) | 2004-10-06 |
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CNB021152608A Expired - Fee Related CN1169950C (en) | 2002-05-23 | 2002-05-23 | Process for preparing calf rennase |
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Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN100354416C (en) * | 2005-11-30 | 2007-12-12 | 马忠仁 | Chymosin powder preparation method |
CN105349513A (en) * | 2015-12-03 | 2016-02-24 | 光明乳业股份有限公司 | Chymosin storage method and chymosin liquid capable of being stored at normal temperature |
CN105274079A (en) * | 2015-12-03 | 2016-01-27 | 光明乳业股份有限公司 | Highly-concentrated chymosin and preparation method thereof |
CN110724681A (en) * | 2019-09-29 | 2020-01-24 | 山东职业学院 | Protective agent and method for improving heat stability of chymosin |
CN112410323B (en) * | 2020-11-19 | 2024-02-06 | 新疆生化药业有限公司 | Preparation method of lamb abomasum chymosin standard |
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2002
- 2002-05-23 CN CNB021152608A patent/CN1169950C/en not_active Expired - Fee Related
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