CN102108349A - Refining method of papain - Google Patents
Refining method of papain Download PDFInfo
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- CN102108349A CN102108349A CN2009102007502A CN200910200750A CN102108349A CN 102108349 A CN102108349 A CN 102108349A CN 2009102007502 A CN2009102007502 A CN 2009102007502A CN 200910200750 A CN200910200750 A CN 200910200750A CN 102108349 A CN102108349 A CN 102108349A
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Abstract
The invention provides a refining method of papain. The method comprises the following steps: 1) milling; 2) centrifugation; 3) ultra-filtration; and 4) drying. The papain obtained by adopting the refining method has high purity, high vitality (the highest vitality achieves 3 million u/g) and good heat stability, and indexes of appearance, physical and chemical indexes, microbial limit and the like achieve or exceed related food and medicine standards at home and abroad; furthermore, the method has simple process, low production cost and high yield, and can realize industrial production.
Description
Technical field
The present invention relates to the refining of enzyme, specifically, the present invention relates to a kind of process for purification of papoid.
Background technology
Papoid (Papain) is called for short papain, is called Fructus Chaenomelis ferment again, is the milk that utilizes in the immature papaya fruit, adopts modern biotechnology to refine the pure-natural biological enzyme preparation that forms.It is to dredge base (SH) endopeptidase has the activity of proteolytic enzyme and esterase, and specificity is more widely arranged a kind of containing, animal/vegetable protein, polypeptide, ester, acid amides etc. there is stronger hydrolysis ability, simultaneously, also have complex functionality, can synthesize proteinoid to proteolysate.Its applied industry field is more extensive, comprising: fields such as medicine industry, foodstuffs industry, cosmetics industry, fodder industry, leather industry, textile industry.Because papoid relates to the extensive of field, people are more and more deep to its understanding, and its using value and field are also increasing.Domestic existing a part of enterprise begins to produce papoid now, but most of product still belongs to rough-wrought product, and Application Areas is limited.
Papoid mainly is present in the milk of prematurity papaya fruit, it can decompose protein commonly used such as gelatin, casein, gluten, elastin, sphaeroprotein and parapeptone effectively, be widely used in industry such as tenderization, clarify beer, gelatin manufacturing, degumming silkworm cocoons, leather depilation, purposes is wider aspect health care, so but do not send out the choosing area because of the papaya growing area mainly is distributed in economy. the proteolytic enzyme production technique is backward relatively, and the mode that a lot of areas still adopt the sun to be exposed to the sun is produced papoid.By the unstable product quality that this method is produced, most products do not reach specification of quality, therefore, how to improve production technique, improving the quality of products becomes the problem that current urgent need solves.
Extraction, the method for purification of papoid are a lot, and its separating and extracting method mainly contains: affinity chromatography partition method, ultra-filtration membrane partition method, precipitation separation, extraction separation method and flocculence etc.Disclose a kind of preparation method of papoid as CN1908164, comprised centrifugal, micro-filtration, three grades of ultrafiltration; CN1944640 also discloses a kind of physical extracting method of papoid, it may further comprise the steps: pawpaw slurry, freezing, pre-treatment, coarse filtration, smartly filter, the two-stage ultrafiltration, adopt ultra-filtration technique purifying papoid, the operational condition gentleness, be difficult for causing that biochemical substances destroys, but, in ultra-filtration process, can produce the concentration polarization phenomenon in the accumulation of film surface because papoid belongs to macromolecular substance, ultrafiltration rate is descended gradually, and have problems such as organic solvent residue; Publication number is the preparation method that the patent application of CN 1597946A discloses a kind of papoid, it adopts salting-out process to carry out thick enzyme and the preparation of smart enzyme, smart enzyme activity 2,000,000 units/gram, the papoid activity that adopts salting-out process to extract is lower, differ far away with medicinal requirements, and owing to used a large amount of salt, make ash content increase in the enzyme, reduced every gram papoid activity simultaneously, utilize the organic solvent extraction papoid, the program complexity, extraction yield is lower, has problems such as organic solvent residue; Flocculence extraction specificity is not strong, and the purity of papain that makes is not high, and foreign matter content is higher.Though the papoid purity that the affinity chromatography partition method is extracted is higher, complicated operation is difficult for industrial amplification production, and the pretreatment liquid that is used for affinity chromatography need be the enzyme liquid through preliminary purification.
Processes such as the said extracted method need be passed through various fractionation precipitations mostly, saltoutd, crystallization, washing, chromatography, these method extraction cost height yield poorly, and operate more loaded down with trivial details, and the papaya fruit after tapping milk fails to be fully utilized, thereby causes waste.
Summary of the invention
At the problem that prior art exists, the present inventor has done improvement on production technique, changed traditional complete processing, has designed a kind of process for purification of papoid, and this method technology is simple, production cost is low, output is high.
The object of the present invention is to provide the process for purification of described a kind of papoid.
Process for purification according to a kind of papoid provided by the invention said method comprising the steps of:
1) mills: the pawpaw slurry is poured in the colloidal mill, fully ground, obtain the papain feed liquid;
2) centrifugal: as, to obtain clarifying papain cooking by constantly separating continuously;
3) ultrafiltration: remove small molecular weight impurity and water, concentrate feed liquid, volume ratio is 4～6: 1;
4) drying: vacuumize ,-30 ℃ freezing 3 hours, slowly be heated to 30 ℃ again, the papoid drying solid.
According to process for purification of the present invention, in described centrifugation step, adopt disk centrifugal separator or tubular-bowl centrifuge.
According to process for purification of the present invention, centrifugation rate is 3000-5000rpm.
According to process for purification of the present invention, before described ultrafiltration step, carry out micro-filtration.
According to process for purification of the present invention, in described ultrafiltration step, adopting molecular weight cut-off is the hyperfiltration membrane assembly of 8000-16000.
The present invention has carried out breakthrough improvement to the production technique of papoid; not only effectively reduce production cost; and promoted the quality of product significantly; at aspects such as the activity of enzyme, purity, solvability, effective constituent resist technologies outstanding performance is arranged; compare with the existing papoid product in market, have following characteristics:
1, the milling action by colloidal mill is dissolved into papain in the feed liquid as far as possible, is beneficial to subsequent extracted.
2, can remove microorganism and small molecular weight impurity by hyperfiltration membrane assembly, guarantee quality product; Concentrating the material liquid volume ratio is 4～6: 1, increases material proportion, reduces drying cost.
3, adopt vacuum lyophilization, directly distil when becoming ice and drained, guarantee that enzyme carries out in drying process under cold condition, reduce the loss of the enzyme quality in the drying course and the reduction of enzyme quality by the water in the material.
4, purity of papain height, vigor height (the highest vigor reaches 3,000,000 u/g), the Heat stability is good that adopts process for purification of the present invention to obtain, indexs such as outward appearance, physics and chemistry, microbial limit all meet or exceed the domestic and international relevant food drug standard, and this method technology is simple, production cost is low, output height, but suitability for industrialized production.
Embodiment
Below for a more detailed description with embodiment to the present invention.These embodiment only are the descriptions to best mode for carrying out the invention, scope of the present invention are not had any restriction.
Embodiment 1
The pawpaw slurry is poured in the colloidal mill, fully ground, obtain the papain feed liquid; The papain feed liquid reduces temperature to 12 ℃ rapidly by plate-type heat exchanger, and keeps feed liquid in this temperature range in extraction separation process after this by the cold water recycle system; The papain feed liquid is constantly separated continuously by disk centrifugal separator, and centrifugation rate is 3000rpm, obtains clarifying papain clarification feed liquid; Papain clarification feed liquid is that 10000 hyperfiltration membrane assembly is removed small molecular weight impurity and water by molecular weight cut-off, concentrates feed liquid, and volume ratio is 5: 1; The papoid concentrated solution is put into vacuum freeze drier or Ultralow Temperature Freezer (freezer) quick-frozen to temperature is-30 ℃, begin to vacuumize, continue to keep refrigeration 3 hours, and again refrigeration was become heating, get the papoid drying solid when slowly heating makes feed temperature rise to 30 ℃.
Just can be packaged into the finished product of different size behind the pulverizing of papoid drying solid process, interpolation auxiliary material, the mixing.
Embodiment 2
The pawpaw slurry is poured in the colloidal mill, fully ground, obtain the papain feed liquid; The papain feed liquid reduces temperature to 12 ℃ rapidly by plate-type heat exchanger, and keeps feed liquid in this temperature range in extraction separation process after this by the cold water recycle system; The papain feed liquid is constantly separated continuously by tubular-bowl centrifuge, centrifugation rate is 4500rpm, obtain clarifying papain clarification feed liquid: papain clarification feed liquid is removed microorganism and macromolecule impurity by micro-filtration membrane module, be that 15000 hyperfiltration membrane assembly is removed small molecular weight impurity and water by molecular weight cut-off again, concentrate feed liquid, volume ratio is 4.5: 1; The papoid concentrated solution is put into vacuum freeze drier or Ultralow Temperature Freezer (freezer) quick-frozen to temperature is-30 ℃, begin to vacuumize, continue to keep refrigeration 3 hours, refrigeration is become heating, get the papoid drying solid when slowly heating makes feed temperature rise to 30 ℃.
Just can be packaged into the finished product of different size behind the pulverizing of papoid drying solid process, interpolation auxiliary material, the mixing.
Embodiment 3
The pawpaw slurry is poured in the colloidal mill, fully ground, obtain the papain feed liquid; The papain feed liquid reduces temperature to 12 ℃ rapidly by plate-type heat exchanger, and keeps feed liquid in this temperature range in extraction separation process after this by the cold water recycle system; The papain feed liquid is constantly separated continuously by tubular-bowl centrifuge, and centrifugation rate is 5000rpm, obtains clarifying papain clarification feed liquid; Papain clarification feed liquid is that 13000 hyperfiltration membrane assembly is removed small molecular weight impurity and water by molecular weight cut-off, concentrates feed liquid, and volume ratio is 6: 1; The papoid concentrated solution is put into vacuum freeze drier or Ultralow Temperature Freezer (freezer) quick-frozen to temperature is-30 ℃, begin to vacuumize, continue to keep refrigeration 3 hours, and again refrigeration was become heating, get the papoid drying solid when slowly heating makes feed temperature rise to 30 ℃.Just can be packaged into the finished product of different size behind the pulverizing of papoid drying solid process, interpolation auxiliary material, the mixing.
The test example
1, the mensuration of papoid enzyme activity
In every test tube, add 250 caseins (1.0mg/ml), 37 ℃ of insulation 10min, add the papoid that an amount of embodiment 1 obtains in each test tube, 37 ℃ accurately are incubated 10min, add 5ml Xylene Brilliant Cyanine G solution (100mg Xylene Brilliant Cyanine G afterwards, 50ml 95% ethanol, 100ml 85% phosphoric acid, be settled to 1000ml), mixing, room temperature is placed 20min, surveys the photoabsorption of 595nm, the reference protein concentration standard curve is found corresponding proteins concentration, and calculating enzyme activity mean value is 2,900,000 u/g.An enzyme activity unit is defined as 37 ℃, the required enzyme amount of hydrolysis 1mg casein in the 1min.
2, measure purity of papain
Ion exchange chromatography among the FPLC can calculate the purity of papoid.The purifying papoid of 25ul (25G albumen) is 20ml glycine-sodium hydroxide buffer zone buffering of 10.6 at PH, papoid and other albumen behind the wash-out, use from 0 to 0.5 meter (the about 24ml of the total amount) flow velocity liquid chromatography of linear gradient chlorination sodium of programming to be 1.0ml/min subsequently.Record chromatogram fritter A280 and gradient are formed and elution amount, and calculating proteolytic enzyme purity is 99.69%.
The present invention has carried out breakthrough improvement to the production technique of papoid; not only effectively reduce production cost; and promoted the quality of product significantly; activity at enzyme; purity; solvability; there is outstanding performance aspects such as effective constituent resist technology; the purity of papain height that adopts process for purification of the present invention to obtain; vigor height (the highest vigor reaches 3,000,000 u/g); Heat stability is good; outward appearance; physics and chemistry; indexs such as microbial limit all meet or exceed the domestic and international relevant food drug standard; and this method technology is simple; production cost is low; output height, but suitability for industrialized production.
Claims (5)
1. the process for purification of a papoid is characterized in that, described process for purification may further comprise the steps:
1) mills: the pawpaw slurry is poured in the colloidal mill, fully ground, obtain the papain feed liquid;
2) centrifugal: as, to obtain clarifying papain cooking by constantly separating continuously;
3) ultrafiltration: remove small molecular weight impurity and water, concentrate feed liquid, volume ratio is 4～6: 1;
4) drying: vacuumize ,-30 ℃ freezing 3 hours, slowly be heated to 30 ℃ again, the papoid drying solid.
2. process for purification according to claim 1 is characterized in that, in described centrifugation step, adopts disk centrifugal separator or tubular-bowl centrifuge.
3. process for purification according to claim 1 and 2 is characterized in that, centrifugation rate is 3000-5000rpm.
4. process for purification according to claim 1 is characterized in that, carries out micro-filtration before described ultrafiltration step.
5. process for purification according to claim 1 is characterized in that, in described ultrafiltration step, adopting molecular weight cut-off is the hyperfiltration membrane assembly of 8000-16000.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009102007502A CN102108349A (en) | 2009-12-25 | 2009-12-25 | Refining method of papain |
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| CN2009102007502A CN102108349A (en) | 2009-12-25 | 2009-12-25 | Refining method of papain |
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| CN102108349A true CN102108349A (en) | 2011-06-29 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104293750A (en) * | 2014-10-15 | 2015-01-21 | 重庆骄王天然产物股份有限公司 | Method for efficiently extracting sterile papain |
| CN106554952A (en) * | 2016-11-13 | 2017-04-05 | 孔龙 | A kind of extracting method of papain |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1944640A (en) * | 2006-08-03 | 2007-04-11 | 谢丹青 | Physical extracting method for papain |
| CN101570748A (en) * | 2008-05-04 | 2009-11-04 | 徐闻县美仑生物制品有限公司 | Method for refining papain |
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2009
- 2009-12-25 CN CN2009102007502A patent/CN102108349A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1944640A (en) * | 2006-08-03 | 2007-04-11 | 谢丹青 | Physical extracting method for papain |
| CN101570748A (en) * | 2008-05-04 | 2009-11-04 | 徐闻县美仑生物制品有限公司 | Method for refining papain |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104293750A (en) * | 2014-10-15 | 2015-01-21 | 重庆骄王天然产物股份有限公司 | Method for efficiently extracting sterile papain |
| CN106554952A (en) * | 2016-11-13 | 2017-04-05 | 孔龙 | A kind of extracting method of papain |
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Application publication date: 20110629 |