CN104878063A - Crocodilian serum protein with antineoplastic function and preparation method thereof - Google Patents
Crocodilian serum protein with antineoplastic function and preparation method thereof Download PDFInfo
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Abstract
The invention relates to the field of the processing of functional foods, and particularly provides crocodilian serum protein with an antineoplastic function and a preparation method thereof. The preparation method of the crocodilian serum protein comprises the following steps of ultrasonically treating a crocodilian serum solution, adding flavorzyme into the crocodilian serum solution to carry out enzymolysis for obtaining enzymatic hydrolysate, and freeze-drying the enzymatic hydrolysate to obtain the crocodilian serum protein with the antineoplastic function. According to the crocodilian serum protein with the antineoplastic function and the preparation method thereof, the crocodilian serum protein is treated by utilizing an ultrasonic technique; the antineoplastic function of the serum protein can be improved obviously; the growth of a tumor cell is effectively controlled.
Description
Technical field
The present invention relates to functional foodstuff manufacture field, specifically, relate to a kind of crocodile blood white protein and the preparation method with anti-tumor function.
Background technology
Current pig, bovine blood be existing certain application in foodstuffs industry, mainly by blood after degraded, decolouring, dry, disintegrating process, make and be rich in iron food.Utilize blood to prepare the aspect products such as protolysate, hemochrome, superoxide-dismutase, immunoglobulin (Ig) and also have certain research.Crocodile is a kind of ancient Reptilia survived the cretaceous period.From functional performance and the exploitation aspect of current related animal blood, the utilization for livestock and poultry blood reaches its maturity.And for Reptilia crocodile, people are mainly limited to the introduction in the Compendium of Material Medica of Chinese medicine treasured book LI Shi-Zhen to its understanding, pertinent literature has the partial function characteristic of report crocodile blood, as anti-microbial activity and oxidation-resistance, but the concrete research in crocodile blood anti-tumor function is also little, more have no the report for the albuminised production of crocodile blood, research and correlation function.
Summary of the invention
In order to solve problems of the prior art, the object of this invention is to provide a kind of crocodile blood white protein and the preparation method with anti-tumor function.
In order to realize the object of the invention, first the present invention provides a kind of crocodile blood white protein with anti-tumor function, the albuminised preparation method of described crocodile blood is: by crocodile serum solution through ultrasonication, adds flavor protease and carries out enzymolysis and obtain enzymolysis solution, after lyophilize and get final product.
Further, described ultrasonication condition is: 80 ~ 130KHz process 10 ~ 20 minutes.
Further, described enzymatic hydrolysis condition is: enzyme digestion reaction 5 ~ 20 minutes at 40 ~ 50 DEG C.
Further, the preparation method of described crocodile serum solution comprises the steps:
(1) gather crocodile blood, add the sodium citrate solution of total blood volume 0.8 ~ 1.2%, be cooled to 15 ~ 20 DEG C, centrifuging and taking supernatant; Lyophilize, obtains crocodile serum powder;
(2) mixed with the ratio of water in 8 ~ 12% (w/w) by crocodile serum powder, 20 ~ 35 DEG C are stirred 10 ~ 20 minutes, make it fully dissolve, obtain crocodile serum solution.
Wherein, the addition of described flavor protease is 0.2 ~ 0.5% of crocodile serum grain weight amount.
Wherein, the concentration of described sodium citrate solution is 25% (w/w).
Further, described centrifugal condition is: under 8000 ~ 10000g condition centrifugal 5 ~ 10 minutes.
Further, after enzymolysis terminates, in 20 minutes, be cooled to room temperature, obtain crocodile serum enzymolysis solution; In 60 minutes, start lyophilize, obtain the crocodile blood white protein with anti-tumor function.
Present invention also offers a kind of albuminised preparation method of crocodile blood with anti-tumor function, described method be by crocodile serum solution through ultrasonication, add flavor protease and carry out enzymolysis and obtain enzymolysis solution, after lyophilize and get final product.
Further, described method comprises the steps:
(1) gather crocodile blood, the sodium citrate solution adding total blood volume 0.8 ~ 1.2%, as antithrombotics, is cooled to 15 ~ 20 DEG C, within centrifugal 5 ~ 10 minutes under 8000 ~ 10000g condition, gets supernatant; Lyophilize, obtains crocodile serum powder;
(2) mixed with the ratio of water in 8 ~ 12% (w/w) by crocodile serum powder, 20 ~ 35 DEG C are stirred 10 ~ 20 minutes, make it fully dissolve, obtain crocodile serum solution; Through ultrasonic wave 80 ~ 130KHz process 10 ~ 20 minutes; Change the weave construction of albumen;
(3) in the crocodile serum solution through ultrasonication, add flavor protease according to the weight percent ratio of crocodile serum grain weight amount 0.2 ~ 0.5%, under 40 ~ 50 DEG C of conditions, carry out enzyme digestion reaction 5 ~ 20 minutes; In 20 minutes, be cooled to room temperature after enzymolysis terminates, obtain crocodile serum enzymolysis solution;
(4) by the crocodile serum enzymolysis solution of step (3) gained, started to freeze and lyophilize in 60 minutes, obtain the crocodile blood white protein with anti-tumor function.
Beneficial effect of the present invention is:
The present invention utilizes ultrasonic technology process crocodile blood white protein, can significantly improve the anti-tumor function of serum protein, and effectively control the growth of tumour cell.
The product safety of the present invention's exploitation, adopt food grade flavor protease, through in a mild condition, through appropriate enzymolysis, do not add any additive, the crocodile blood white protein powder of exploitation has good local flavor.
The crocodile blood white protein of the present invention's exploitation, can be widely used as the raw material of medicine, functional food.
Crocodile blood white protein preparation method provided by the invention is simple, more easily realizes industrialization and produces.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
Embodiment 1 one kinds has the albuminised preparation method of crocodile blood of anti-tumor function
(1) 50 gram of crocodile blood is collected in container, add the sodium citrate solution (sodium citrate solution concentration is 25%) of 0.4 gram in a reservoir as antithrombotics, be cooled to 15 DEG C, under 10000g condition centrifugal 5 minutes, collect crocodile blood supernatant liquor; Supernatant liquor, by lyophilize, obtains 3.5 grams, crocodile serum powder.
(2) get 3.5 grams, crocodile serum powder, mix in the ratio of 12% crocodile serum powder (w/w) with water, 30 DEG C are stirred 10 minutes, make it fully dissolve, preparation crocodile serum solution.Then in ultrasonic generator, process 10 minutes through ultrasonic wave (frequency is 130kH), change the weave construction of albumen.
(3) according to crocodile serum grain weight amount 0.5% weight percent ratio in the crocodile serum powder liquid through ultrasonication, add flavor protease 0.0175 gram, under 45 DEG C of conditions, carry out enzyme digestion reaction 5 minutes; In 20 minutes, be cooled to room temperature after enzymolysis terminates, obtain crocodile serum enzymolysis solution;
(4) by the crocodile serum enzymolysis solution of step (3) gained, started to freeze and lyophilize in 60 minutes, by lyophilize, obtain 3.3 grams, the crocodile blood white protein powder with better local flavor and anti-tumor function.
Embodiment 2 one kinds has the albuminised preparation method of crocodile blood of anti-tumor function
(1) 100 gram of crocodile blood is collected in container, add the sodium citrate solution (sodium citrate solution concentration is 25%) of 1.2 grams in a reservoir as antithrombotics, be cooled to 20 DEG C, under 8000g condition centrifugal 10 minutes, collect crocodile blood supernatant liquor; Supernatant liquor, by lyophilize, obtains 7.2 grams, crocodile serum powder.
(2) get crocodile serum powder, mix in the ratio of 8% crocodile serum powder (w/w) with water, 20 DEG C are stirred 10 ~ 20 minutes, make it fully dissolve, preparation crocodile serum solution.Then in ultrasonic generator, process 20 minutes through ultrasonic wave (frequency is 90kH), change the weave construction of albumen.
(3) in the crocodile serum powder liquid through ultrasonication, add flavor protease 0.0144 gram according to the weight percent ratio of crocodile serum grain weight amount 0.2%, under 50 DEG C of conditions, carry out enzyme digestion reaction 20 minutes; In 20 minutes, be cooled to room temperature after enzymolysis terminates, obtain crocodile serum enzymolysis solution;
(4) by the crocodile serum enzymolysis solution of step (3) gained, start in 60 minutes to freeze and lyophilize, by lyophilize, obtain 6.8 grams, the crocodile blood white protein powder with better local flavor and anti-tumor function.
Embodiment 3 one kinds has the albuminised preparation method of crocodile blood of anti-tumor function
(1) 10 gram of crocodile blood is collected in container, add the sodium citrate solution (sodium citrate solution concentration is 25%) of 0.1 gram in a reservoir as antithrombotics, be cooled to 15 DEG C, under 10000g condition centrifugal 10 minutes, collect crocodile blood supernatant liquor; Supernatant liquor, by lyophilize, obtains 0.7 gram, crocodile serum powder.
(2) get crocodile serum powder, mix in the ratio of 10% crocodile serum powder (w/w) with water, 25 DEG C are stirred 20 minutes, make it fully dissolve, preparation crocodile serum solution.Then in ultrasonic generator, process 15 minutes through ultrasonic wave (frequency is 130kH), change the weave construction of albumen.
(3) in the crocodile serum powder liquid through ultrasonication, add flavor protease 0.00035 gram according to the weight percent ratio of crocodile serum grain weight amount 0.5%, under 45 DEG C of conditions, carry out enzyme digestion reaction 15 minutes; In 30 minutes, be cooled to room temperature after enzymolysis terminates, obtain crocodile serum enzymolysis solution;
(4) by the crocodile serum enzymolysis solution of step (3) gained, started to freeze and lyophilize in 60 minutes, by lyophilize, obtain 0.69 gram, the crocodile blood white protein powder with better local flavor and anti-tumor function.
Embodiment 4 antitumor crocodile blood white protein is to the inhibiting mensuration of human hepatoma cell HepG 2 proliferation
(1) cell cultures, go down to posterity, freezing and thawing
Cell cultures: by HepG2 cell cultures containing in 10% foetal calf serum and 1% dual anti-DMEM substratum, be placed in 37 DEG C of 5%CO
2cO2gas incubator in cultivate.
Passage: when cell is at 25cm
2when fusion degree in culturing bottle reaches 80 ~ 90%, can carry out going down to posterity or inoculating.First remove original substratum, add the aseptic PBS rinse twice of 3mL, after removing PBS, add the trypsin digestion and cell that 0.5mL contains EDTA.Examine under a microscope the contraction situation of cell, treat that intercellular space increases, add perfect medium after cell rounding and carry out termination digestion, and blow and beat cell uniformly, until all cell is blown and beaten and is scattered in uniformly in substratum.Finally go down to posterity for experiment in the ratio of 1:2.
Cell cryopreservation: get the cell dissociation being in logarithmic phase and be collected in the centrifuge tube of 15mL, centrifugal 5 minutes of 1000g, removes original substratum, and the perfect medium adding 900 μ L is resuspended, transfers them in the cryopreservation tube containing 100 μ L DMSO, then mixes.After finishing mark.Cryopreservation tube is placed in program temperature reduction box in-60 DEG C of Ultralow Temperature Freezer gradient coolings.Cell cryopreservation tube is taken out and is placed in liquid nitrogen container after 24h and preserve for a long time.
Cell recovery: take out frozen cell from liquid nitrogen container, the water being placed in 37 DEG C makes it dissolve rapidly in the quick shake of pot, then be transferred in the culturing bottle containing 4mL perfect medium and cultivate, when cell covers with 80 ~ 90%, carry out Secondary Culture, treat after twice goes down to posterity, can be used for experiment.
(2) mtt assay
Cell fishplate bar: collect the HepG2 cell being in logarithmic phase growth and make single cell suspension, with 1 × 10
4individual cell/mL density inoculating cell is in 96 orifice plates, and every hole 150 μ L, in 37 DEG C of 5%CO
2cO2gas incubator in cultivate.
Dosing: when Growth of Cells area reaches 50% ~ 60%, careful absorption abandoning supernatant, every hole adds containing different concns (125 μ g/ml ~ 5000 μ g/ml, totally 8 concentration) each 150 μ L of DMEM perfect medium of sample (see table 1), each concentration establishes 6 parallel holes, not add the substratum of medicine for negative control, in the incubator of 37 DEG C of 5% carbonic acid gas, cultivate 72h.
MTT process: after cell cultures terminates, discards normal incubation medium and pastille substratum, adds MTT (final concentration 0.5mg/mL) the 150 μ L diluted with complete DMEM, the culture plate adding MTT solution is put into incubator and continues to cultivate 4h.
Colour developing: MTT process discards the substratum containing MTT after cultivating, draws clean by the substratum in hole.Then every hole adds formazan in 150 μ L DMSO dissolved cells.
Measure absorbance (OD value): fully shaken by culture plate before measuring, after DMSO abundant dissolved cell Zhong formazan, use microplate reader OD
490measure.With cellular control unit vigor for 100%, medicine to each cell inhibitory rate calculation formula is: inhibiting rate %=(OD contrasts-OD test)/OD contrasts × 100; Measurement result is in table 1.
The antitumor crocodile blood white protein of table 1 is to the experimental result of the suppression of human liver cancer cell HepG2
The mensuration of embodiment 5 crocodile blood white protein induction human liver cancer cell HepG2 apoptosis:
Dosing: when the Growth of Cells area in 6 orifice plates reaches 50%-60%, careful absorption also abandoning supernatant, it is 300 μ g/mL that every hole adds sample concentration, 1000 μ g/mL, the each 2mL of DMEM perfect medium of 5000 μ g/mL, each concentration establishes 3 parallel holes, with not
The substratum adding medicine is negative control, in the incubator of 37 DEG C of 5% carbonic acid gas, cultivate 72h.
Measure: after collected by trypsinisation cell, in room temperature 2000rpm centrifugal 6 minutes, collecting cell, with precooling 1 × PBS (4 DEG C), re-suspended cell is once, centrifugal 6 minutes of 2000rpm, washed cell, adds 1 × Binding Buffer suspension cell of 300 μ L, after adding the Annexin V-FITC mixing of 5 μ L, lucifuge, incubated at room 15 minutes, upper machine adds the PI dyeing of 5 μ L for first 5 minutes again, uses flow cytomery.Measurement result is in table 2.
Table 2 antitumor crocodile blood white protein induction human hepatoma HepG2 cell apoptosis rate
As can be seen from Table 2, antitumor crocodile blood white protein prepared by the present invention has obvious restraining effect to tumour cell HepG2, and its action effect is significantly higher than the crocodile blood white protein of crocodile blood white protein and ultrasonication.The restraining effect of crocodile blood white protein to tumour cell HepG2 of ultrasonication is better than crocodile blood white protein.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. have a crocodile blood white protein for anti-tumor function, it is characterized in that, the albuminised preparation method of described crocodile blood is: by crocodile serum solution through ultrasonication, adds flavor protease and carries out enzymolysis and obtain enzymolysis solution, after lyophilize and get final product.
2. crocodile blood white protein according to claim 1, is characterized in that, described ultrasonication condition is: 80 ~ 130KHz process 10 ~ 20 minutes.
3. crocodile blood white protein according to claim 1 and 2, is characterized in that, described enzymatic hydrolysis condition is: enzyme digestion reaction 5 ~ 20 minutes at 40 ~ 50 DEG C.
4. crocodile blood white protein according to claim 3, is characterized in that, the preparation method of described crocodile serum solution comprises the steps:
(1) gather crocodile blood, add the sodium citrate solution of total blood volume 0.8 ~ 1.2%, be cooled to 15 ~ 20 DEG C, centrifuging and taking supernatant; Lyophilize, obtains crocodile serum powder;
(2) by crocodile serum powder and water in 8 ~ 12% ratio mix, 20 ~ 35 DEG C are stirred 10 ~ 20 minutes, make it fully dissolve, obtain crocodile serum solution.
5. crocodile blood white protein according to claim 4, is characterized in that, the addition of described flavor protease is 0.2 ~ 0.5% of crocodile serum grain weight amount.
6. the crocodile blood white protein according to claim 4 or 5, is characterized in that, the concentration of described sodium citrate solution is 25%.
7. crocodile blood white protein according to claim 6, is characterized in that, described centrifugal condition is: under 8000 ~ 10000g condition centrifugal 5 ~ 10 minutes.
8. the crocodile blood white protein according to claim 1 ~ 7 any one, is characterized in that, after enzymolysis terminates, in 20 minutes, is cooled to room temperature, obtains crocodile serum enzymolysis solution; In 60 minutes, start lyophilize, obtain the crocodile blood white protein with anti-tumor function.
9. there is the albuminised preparation method of crocodile blood of anti-tumor function, it is characterized in that, described method be by crocodile serum solution through ultrasonication, add flavor protease and carry out enzymolysis and obtain enzymolysis solution, after lyophilize and get final product.
10. method according to claim 9, is characterized in that, described method comprises the steps:
(1) gather crocodile blood, add the sodium citrate solution of total blood volume 0.8 ~ 1.2%, be cooled to 15 ~ 20 DEG C, within centrifugal 5 ~ 10 minutes under 8000 ~ 10000g condition, get supernatant; Lyophilize, obtains crocodile serum powder;
(2) mixed with the ratio of water in 8 ~ 12% (w/w) by crocodile serum powder, 20 ~ 35 DEG C are stirred 10 ~ 20 minutes, make it fully dissolve, obtain crocodile serum solution; Through ultrasonic wave 80 ~ 130KHz process 10 ~ 20 minutes;
(3) in the crocodile serum solution through ultrasonication, add flavor protease according to the weight percent ratio of crocodile serum grain weight amount 0.2 ~ 0.5%, under 40 ~ 50 DEG C of conditions, carry out enzyme digestion reaction 5 ~ 20 minutes; In 20 minutes, be cooled to room temperature after enzymolysis terminates, obtain crocodile serum enzymolysis solution;
(4) by the crocodile serum enzymolysis solution of step (3) gained, started to freeze and lyophilize in 60 minutes, obtain the crocodile blood white protein with anti-tumor function.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105168255A (en) * | 2015-09-08 | 2015-12-23 | 浙江海洋学院 | Zymolytic tuna blood oral liquid for antithrombotic and thrombolytic therapies and preparation method thereof |
| CN105249124A (en) * | 2015-09-08 | 2016-01-20 | 浙江海洋学院 | Tuna blood anti-fatigue electuary and preparation method therefor |
| CN106136197A (en) * | 2016-07-05 | 2016-11-23 | 成都大学 | A kind of method utilizing animal blood to make Natural meat flavor |
| CN106174396A (en) * | 2016-07-05 | 2016-12-07 | 成都大学 | A kind of method utilizing animal blood to make seasoning bottom material of chafing dish |
| CN108743910A (en) * | 2018-06-20 | 2018-11-06 | 百生康（北京）健康产业科技有限公司 | A kind of preparation method of crocodile blood peptide freeze-dried powder |
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| WO2003007874A2 (en) * | 2001-07-19 | 2003-01-30 | Natural Cure Ltd. | Anti-tumor activity from reptile serum |
| CN103393191A (en) * | 2013-07-09 | 2013-11-20 | 广州中医药大学 | Crocodile meat oral liquid and preparation method thereof |
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2015
- 2015-06-10 CN CN201510313743.9A patent/CN104878063B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003007874A2 (en) * | 2001-07-19 | 2003-01-30 | Natural Cure Ltd. | Anti-tumor activity from reptile serum |
| CN103393191A (en) * | 2013-07-09 | 2013-11-20 | 广州中医药大学 | Crocodile meat oral liquid and preparation method thereof |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105168255A (en) * | 2015-09-08 | 2015-12-23 | 浙江海洋学院 | Zymolytic tuna blood oral liquid for antithrombotic and thrombolytic therapies and preparation method thereof |
| CN105249124A (en) * | 2015-09-08 | 2016-01-20 | 浙江海洋学院 | Tuna blood anti-fatigue electuary and preparation method therefor |
| CN106136197A (en) * | 2016-07-05 | 2016-11-23 | 成都大学 | A kind of method utilizing animal blood to make Natural meat flavor |
| CN106174396A (en) * | 2016-07-05 | 2016-12-07 | 成都大学 | A kind of method utilizing animal blood to make seasoning bottom material of chafing dish |
| CN108743910A (en) * | 2018-06-20 | 2018-11-06 | 百生康（北京）健康产业科技有限公司 | A kind of preparation method of crocodile blood peptide freeze-dried powder |
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