CN104988129A - Extraction method of efficient active enzymes of fig latex - Google Patents
Extraction method of efficient active enzymes of fig latex Download PDFInfo
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- CN104988129A CN104988129A CN201510483905.3A CN201510483905A CN104988129A CN 104988129 A CN104988129 A CN 104988129A CN 201510483905 A CN201510483905 A CN 201510483905A CN 104988129 A CN104988129 A CN 104988129A
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- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
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- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/22—Cysteine endopeptidases (3.4.22)
- C12Y304/22003—Ficain (3.4.22.3)
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Abstract
The invention discloses an extraction method of efficient active enzymes of fig latex. The method comprises the steps that acetone precipitation, gel chromatography separation, anion chromatography and vacuum freeze-drying are sequentially conducted on the fig latex which is pretreated at the temperature of 4 DEG C to obtain efficient active enzyme powder. Due to the fact that pretreatment and heat exchange are conducted and the extraction process is conducted under a low-temperature condition, the influence of conditions such as long-term high temperature on reducing enzyme activity can be effectively reduced; by means of milling action of a colloid mill, fig enzymes are dissolved in material liquid as many as possible, and the subsequent extraction is facilitated. By means of continuous separation through a disc-type centrifuge and a tubular bowl centrifuge, clear fig protease material liquid is obtained. The vacuum freezing and drying technology is adopted, due to the fact that when water in materials becomes ice, the water directly sublimates to be discharged, it is guaranteed that drying is conducted on the enzymes in the low-temperature condition, and the loss of enzyme mass and reducing of the enzyme activity in the drying process are reduced.
Description
Technical field
The present invention relates to field of food.Be specifically related to a kind of high-efficiency activated enzyme extraction method of Fructus Fici milk.
Background technology
Fructus Fici (Ficus carica Linn.) is a kind of flowering plant, is under the jurisdiction of Moraceae Ficus, primary growth in some torrid zone and the place in temperate zone, genus subtropics defoliation small arbor.Fructus Fici there will be a known 800 kinds at present, and the overwhelming majority is all evergreen kind, and that is only longer than place, temperate zone is only fallen leaves kind.Fruit is bulbous, and afterbody has an aperture, and pollen is propagated by wasp.Because of the pharmaceutical use that it is good, more and more welcomed by the people.
Ficin (Papain) is extensively present in kind Fructus Fici stem leaf and fruit, is the highest with the content of immature fruit milk, can (precipitator method) and biotechnological means prepare and obtain to chemically.Ficin platymiscium proteolytic enzyme, be called for short ficoin, because of its Heat stability is good, there is very strong capacity of decomposition to animal/vegetable protein, fat, acid amides etc., industry and the fields such as Medicines and Health Product, food, cosmetics, chemical industry process hides can be widely used in.
In recent years, a lot of report has been had to the extraction of ficin and application, but what use in the market is mostly the thick enzyme of ficin, because the extraction process of thick enzyme is simple, production cost is low, general employing warm air drying, obtained ficin enzyme activity is only at about 10000u/g; Meanwhile, due to other refining ficin complex manufacturing, production cost is high, is unfavorable for that the industrialization of refining ficin is produced, thus also limit the application at the higher-end businesses such as Medicines and Health Product and cosmetics of ficin.
Summary of the invention
Solve the problems referred to above, the present invention proposes the high-efficiency activated enzyme extraction method of Fructus Fici milk.
In order to realize foregoing invention object, the present invention adopts following technical scheme:
The present invention adopts following technical scheme:
A kind of high-efficiency activated enzyme extraction method of Fructus Fici milk, by pretreated Fructus Fici milk, obtains high-efficiency activated enzyme powder successively at the temperature that can keep enzymic activity after acetone precipitation, gel chromatography separation, anion chromatographic chromatography, vacuum-freeze-dry.
Find in practical study, the sedimentation effect of ethanol to Fructus Fici milk organized enzyme is poor, cannot meet the requirement of high efficiency extraction.Acetone precipitation protein requirements necessarily carries out at low temperatures, and comparatively lower protein precipitation is very fast for temperature.The effect of acetone and water, can destroy the hydration shell of protein, and protein is separated because of dissolubility difference in certain density organic solvent.Compared with ethanol, very fast, the successful of protein precipitation under same low temperature, albumen precipitation is more thorough.
The described temperature of enzymic activity that can keep is 4 DEG C.
The present invention adopts anionite-exchange resin SefinoseFastFlow to be separated hemolytic toxin albumen with gel filtration resin SephadexG-75, have the advantages that flow velocity is fast, resolving power is high, productive rate is high, and separating step is few, only just can obtain purer proteolytic enzyme through two step column chromatography for separation, effectively shorten disengaging time, decrease the loss of activity of proteolytic enzyme.
Further, described pretreatment condition is: collect fresh latex from Fructus Fici branch incision, latex is centrifugal 30-40min under 10000-12000rpm, gets supernatant liquor and is stored in 4 ~ 6 DEG C, to obtain final product.
Further, the concrete steps of described acetone precipitation are: precipitated in 20 – 100% (v/v) acetone solns by latex, centrifugal, go drying precipitate, are dissolved in the Tris-hydrochloride buffer of 10-12mM, pH8.0-8.5.Multiple enzyme reaction be protected and be participated in the PKa value of Tris-hydrochloride buffer, by the concentration of damping fluid and the impact of use temperature, can.The PKa value of existing PBS solution can change along with the dilution of damping fluid, and suppresses the activity of most enzyme, comprises kinases, Starch phosphorylase, desaturase etc.
Further, the condition that described gel chromatography is separated is: the enzyme liquid after acetone precipitation is adopted 25-30mM, pH3-9 through gel chromatographic columns SephadexG-75, the Tris-hydrochloride buffer of Triton X-100 (w/v) containing 5/1000ths carries out separation and purification with 20-25mL/h flow velocity, detect protein peak, totally 2 protein peaks, through Enzyme assay, collect first protein liquid.Triton X-100 is a kind of nonionogenic tenside, good lubricant, can play the effect being separated enzyme component simultaneously.But concentration should reduce as far as possible, avoid lytic enzyme albumen.Therefore, the content of the preferred Triton X-100 of the present invention is 0.5% (w/v) of Tris-hydrochloride buffer.
Further, described gel chromatographic columns Sephadex G-75 Sephadex G-50, Sephadex G-100, SephadexG-150 type replaces.
Further, the concrete steps of described anion chromatographic chromatography are that the elutriant warp that gel chromatography is collected contains Sefinose FastFlow anion-exchange column, adopt elutriant to carry out purifies and separates with 50-60mL/h flow velocity, collect elutriant and concentrate with the super filter tube of 3 ~ 5K at low temperatures.
Further, the concrete steps of described vacuum-freeze-dry are: the proteolytic enzyme concentrated solution of anion chromatographic chromatography is put into vacuum freeze drier or Ultralow Temperature Freezer (freezer) quick-frozen to temperature is-30 ~-25 DEG C; Start to vacuumize, continue to maintain refrigeration 2 ~ 2.5 hours; Refrigeration is become heating, when slowly heating makes feed temperature rise to 30 ~ 35 DEG C, obtains ficin drying solid.
Beneficial effect of the present invention:
(1) by pre-treatment, heat exchange keep leaching process to carry out under cryogenic, the impact that the conditions such as long term high temperature reduce enzyme activity can effectively be reduced;
(2) by the milling action of colloidal mill, ficoin is as far as possible molten in feed liquid, be beneficial to the extraction of postorder.
(3) be continuously separated by disk centrifugal separator and tubular-bowl centrifuge the ficin feed liquid obtaining clarifying.
(4) adopt Vacuum Freezing & Drying Technology, directly distil when becoming ice by the water in material and be discharged, ensure that enzyme carries out under cryogenic in drying process, reduce the loss of enzyme quality in drying course and the reduction of enzyme activity.
(5) high, high (the enzymic activity 25700U/mg of vigor of ficin purity that extracts of present method, final yield 9.25%), Heat stability is good, the indexs such as outward appearance, physics and chemistry, microbial limit all meet or exceed the domestic and international relevant food drug standard, the product of different size can be produced by modulation, industry and the fields such as Medicines and Health Product, food, cosmetics, chemical industry process hides can be widely used in.
(6) the method technique is simple, production cost is low, and output is high, can suitability for industrialized production.
(7) coordinated between each step in the present invention, mutually promote, achieve ficin purity, vigor and extraction yield synchronously to improve, from the characteristic of ficin, select corresponding extraction process, and in conjunction with the feature of back extraction process, subsequent extracted method selected and adjusts, achieving the maximization of extraction efficiency.
Embodiment
Mode by the following examples further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, conventionally selects with condition.
Embodiment 1
Collect fresh latex from Fructus Fici branch incision, latex centrifugal 30 minutes through 10000rpm, remove natural gum and other insoluble compositions, residue clarified liq is natural emulsion, is stored in 4 DEG C of purifying for enzyme.After this step is all carried out at 4 DEG C, prevents degraded and the depolymerization of enzyme.
Protein content detects and adopts Coomassie Brilliant Blue to measure, and does typical curve with bovine serum albumin.
Enzyme assay adopts tyrosine to do typical curve, take casein as substrate, forint phenol reagent color reaction.
1, acetone precipitation
Latex is first through acetone precipitation.Utilize the enzyme in 20 – 40% (v/v) acetone soln precipitation latexes respectively, then 10000rpm centrifugal treating.By enzyme vacuum-drying, removing acetone, is dissolved in the Tris-hydrochloride buffer (pH8.0) of 10mM.Measure protein concentration.Enzyme yield 85.4% (with centrifugal rear clarification latax albumen above for benchmark).Now 15200U/mg.
2, chromatographic separation
Enzyme liquid utilizes Sephadex G-75 (2cm*90) post to filter, and sloughs foreign protein and inorganic salt.Equilibrating buffer A (the Tris-hydrochloride buffer of 25mM, pH7.6.0, the Triton X-100 containing 5/1000ths).Sample crosses post, speed 25mL/h through using same buffer A, and Simultaneously test protein content (absorbancy at 595nm place), albumen yield is 25%.Detect protein peak, totally 2 protein peaks, through Enzyme assay, collect first protein liquid.Now enzyme work is at 17650U/mg.
3, anion chromatographic chromatography
Collect the protein eluate being associated with activeconstituents, upper Sefinose Fast Flow anion-exchange chromatography post continues to be separated, and utilizes buffer B (the Tris-hydrochloride buffer of 25mM, pH7.6), rushes post, flow velocity 50mL/h.Enzymic activity 25700U/mg, final yield 9.25%.
Quaternary amine anion column purifying sloughs metallic cation, is further purified enzyme.
4, vacuum-freeze-dry, obtains enzyme powder.
Embodiment 2
Concrete extraction step is with embodiment 1, and its difference is, adopts Sephadex G-50 post to filter in step 2.Enzymic activity 25600U/mg, final yield 9.15%.
Embodiment 3
Concrete extraction step is with embodiment 1, and its difference is, adopts Sephadex G-100 post to filter in step 2.The enzymic activity 25650U/mg of the ficin of final extraction, final yield 9.21%.
Embodiment 4
Concrete extraction step is with embodiment 1, and its difference is, adopts Sephadex G-150 post to filter in step 2.The enzymic activity 25760U/mg of the ficin of final extraction, final yield 9.22%.
Embodiment 5
Concrete extraction step is with embodiment 1, and its difference is, adopts Sephadex G-150 post to filter in step 2.The enzymic activity 25770U/mg of the ficin of final extraction, final yield 9.26%.
Embodiment 6
Concrete extraction step is with embodiment 1, its difference is, fresh latex is collected from Fructus Fici branch incision, latex centrifugal 30 minutes through 10000rpm, remove natural gum and other insoluble compositions, residue clarified liq is natural emulsion, regulates latex pH value to be 6.6, is stored in 5 DEG C of purifying for enzyme.After this step is all carried out at 5 DEG C, prevents degraded and the depolymerization of enzyme.The ficin of final extraction enzymic activity 25870U/mg, final yield 9.29%.
Although above-mentioned, the specific embodiment of the present invention is described; but not limiting the scope of the invention; one of ordinary skill in the art should be understood that; on the basis of technical scheme of the present invention, those skilled in the art do not need to pay various amendment or distortion that creative work can make still within protection scope of the present invention.
Claims (7)
1. the high-efficiency activated enzyme extraction method of Fructus Fici milk, it is characterized in that, by pretreated Fructus Fici milk, at the temperature that can keep enzymic activity, after acetone precipitation, gel chromatography separation, anion chromatographic chromatography, vacuum-freeze-dry, obtain high-efficiency activated enzyme powder successively.
2. the method for claim 1, is characterized in that, described pretreatment condition is: collect fresh latex from Fructus Fici branch incision, latex is centrifugal 30 ~ 35min under 10000-12000rpm, gets supernatant liquor and is stored in 4 ~ 6 DEG C, to obtain final product.
3. the method for claim 1, it is characterized in that, the concrete steps of described acetone precipitation are: precipitated in 20 – 100% (v/v) acetone solns by latex, centrifugal, go drying precipitate, are dissolved in the Tris-hydrochloride buffer of 10-15mM, pH8.0-8.5.
4. the method for claim 1, it is characterized in that, the condition that described gel chromatography is separated is: the enzyme liquid after acetone precipitation is adopted 25-30mM, pH3-9 through gel chromatographic columns Sephadex G-75, the Tris-hydrochloride buffer of Triton X-100 (w/v) containing 5/1000ths carries out separation and purification with 20-25mL/h flow velocity, collect the elutriant of first protein peak.
5. method as claimed in claim 4, is characterized in that, described gel chromatographic columns Sephadex G-75 SephadexG-50, Sephadex G-100, Sephadex G-150 type replaces.
6. the method for claim 1, it is characterized in that, the concrete steps of described anion chromatographic chromatography are that the elutriant warp that gel chromatography is collected contains Sefinose Fast Flow anion-exchange column, adopt elutriant to carry out purifies and separates with 50-60mL/h flow velocity, collect elutriant and concentrate with the super filter tube of 3 ~ 5K at low temperatures.
7. the method for claim 1, is characterized in that, the concrete steps of described vacuum-freeze-dry are: the proteolytic enzyme concentrated solution of anion chromatographic chromatography is put into vacuum freeze drier or Ultralow Temperature Freezer quick-frozen to temperature is-30 ~-25 DEG C; Start to vacuumize, continue to maintain refrigeration 2 ~ 2.5 hours; Refrigeration is become heating, when slowly heating makes feed temperature rise to 30 ~ 35 DEG C, obtains ficin drying solid.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107048341A (en) * | 2017-01-16 | 2017-08-18 | 星康桥医药科技（南京）有限公司 | A kind of application technology of enzyme and the medicine edible animal glue for which improving digestibility |
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| CN100439495C (en) * | 2006-12-22 | 2008-12-03 | 王晓平 | Preparation process of ficin |
| CN104768535A (en) * | 2012-11-05 | 2015-07-08 | 株式会社Bmi韩国 | Stabilizer for hyaluronidase and liquid formulation comprising hyaluronidase |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100439495C (en) * | 2006-12-22 | 2008-12-03 | 王晓平 | Preparation process of ficin |
| CN104768535A (en) * | 2012-11-05 | 2015-07-08 | 株式会社Bmi韩国 | Stabilizer for hyaluronidase and liquid formulation comprising hyaluronidase |
Non-Patent Citations (2)
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| 郭冬青: "无花果蛋白酶的研究进展", 《北方园艺》 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107048341A (en) * | 2017-01-16 | 2017-08-18 | 星康桥医药科技（南京）有限公司 | A kind of application technology of enzyme and the medicine edible animal glue for which improving digestibility |
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