Embodiment
The preparation method of blood polypeptide of the present invention comprises the separating animal's blood protein, enzymolysis, drying and other steps.
Animal blood of the present invention derives from mammals such as the pig, ox, sheep of raising, and birds such as chicken, duck, and these animals must be the Jiankang animals through quarantine.Animal blood refers to that the scale meat producing plant adopts the collected clean animal blood that can be used for further consumption processing that meets the food sanitation requirement of certain means in slaughtering process.Pancreatin is trypsinase again, is a kind of proteolytic ferment that is extracted in ox or the pig pancreas, is endopeptidase, and it can cut off the carboxyl side in Methionin in the polypeptied chain and the arginine residues, plays the effect of digestive ferment.Because it is pancreatin comes from animal pancreatic, therefore with strong points in the degraded of animal proteinum.
The method of above-mentioned separating animal's blood protein is: the volume proportion by 1 part of animal blood, 3~6 parts of cell pyrolysis liquids mixes, and leaves standstill; Get and leave standstill the back centrifugal, the gained supernatant adds in isopyknic 95% ethanol, and centrifugal gained is animal blood albumen.According to shaker test, consider that from cost and effect aspect the volume proportion of preferred animal blood and cell pyrolysis liquid is 1 part of animal blood, 3 parts of cell pyrolysis liquids; Particularly, cell pyrolysis liquid is 0.01%~0.03% sodium citrate aqueous solution.
Be dissolved in the water of 10 times of volumes at animal blood albumen in the above-mentioned enzymolysis process, add pancreatin 0.6~2g in every liter of lysate, in 35~40 ℃ of reactions 1~9 hour.According to shaker test, consider to add preferred every liter of lysate pancreatin 1~2g from cost and effect aspect, in 35~40 ℃ of reactions 3~9 hours.Preferred embodiment was to add pancreatin 1g in every liter of lysate, in 37 ℃ of reactions 3 hours.
When dry, control blood polypeptide moisture content is lower than 5% and gets final product.The spray-dired mode of general employing, method is simple for this, is the domestic method that drying contains the liquid of albumen, polypeptide, aminoacid component.The condition that adopts during spraying drying is not higher than 220 ℃, the about 5ml/min of sample introduction speed.
Through method for preparing and blood polypeptide contain following composition: total nitrogen 10.7~14.8%, total free aminoacids (in N) 0.37~0.58%, iron 0.05~0.07%.Blood polypeptide quality index of the present invention is a quality control standard with animal blood enzymolysis amino acid, adopts ninhydrin to measure, with glycocoll as contrast.
Blood polypeptide of the present invention can be processed this area regular dosage form, like powder, tablet, capsule, sustained-release preparation or the like.
Obviously, according to foregoing of the present invention,,, can also make modification, replacement or the change of other various ways not breaking away under the above-mentioned basic fundamental thought of the present invention prerequisite according to the ordinary skill knowledge and the customary means of this area.
Below, foregoing of the present invention is remake further detailed description through the embodiment of embodiment form.But should this be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following instance.All technology that realizes based on foregoing of the present invention all belong to scope of the present invention.
Beneficial effect through following shaker test proof blood polypeptide of the present invention and preparation method thereof.
Test Example 1 screening cell wall breaking test
(v: v) add 0.01% sodium citrate aqueous solution, slowly stirred 5 minutes, it is centrifugal that room temperature leaves standstill after 1 hour 10000g * 10min by 1: 1,1: 2,1: 3,1: 4,1: 5,1: 6 with 200ml fresh pig blood; The gained supernatant adds in isopyknic 95% ethanol; 6000g * 10min is centrifugal, and the deposition that obtains is redissolved in the distilled water of 10 times of volumes, and every liter of lysate adds self-control pancreatin 1 gram; In 37 ℃ of reactions 1 hour, measure its protein and amino acid equal size.Test repetition 3 times, with three average out to statisticses, the result sees table 1.
The volume proportion of table 1 screening cell pyrolysis liquid and animal blood
|
1∶1 |
1∶2 |
1∶3 |
1∶4 |
1∶5 |
1∶6 |
Total nitrogen (in N)/% |
1.36 |
5.59 |
12.8 |
13.1 |
13.1 |
13.2 |
Total free aminoacids (in N)/% |
0.05 |
0.19 |
0.35 |
0.36 |
0.36 |
0.39 |
Iron/% |
0.03 |
0.04 |
0.05 |
0.05 |
0.05 |
0.05 |
Can be known by experimental result, add almost unanimity of 0.01% sodium citrate aqueous solution gained protein, aminoacids content by 3~6 times of volumes, be to practice thrift cost, and 0.01% sodium citrate aqueous solution add-on is good with 3 times.
The test of Test Example 2 pancreatin enzyme dose-effects
With 200ml fresh pig blood by 1: 3 (v: v) add 0.01% sodium citrate aqueous solution; Slowly stirred 5 minutes; Room temperature leaves standstill after 1 hour that 10000g * 10min is centrifugal, and the gained supernatant adds in isopyknic 95% ethanol, and 6000g * 10min is centrifugal; The deposition that obtains is redissolved in the distilled water of 10 times of volumes, add a certain amount of pancreatin.Pancreatin enzyme amount is set is: 1g/500ml, 1g/700ml, 1g/1L, 1g/1.3L, 1g/1.5L carry out the test of enzyme amount, to confirm the add-on effect relationship of pancreatin.
The temperature of keeping 50 ℃ of solution in the reactor is half a hour at least; Feed tap water by interlayer then and reduce in the pot solution temperature to 37 ℃; The pancreatin that adds requirement; Under the whisking appliance continuously stirring, begin enzyme digestion reaction, during the adjustment quantity of steam that feeds the reactor interlayer make temperature maintenance at 37 ℃, enzyme digestion reaction finishes after adding pancreatin 6h.Test repetition 3 times, with three average out to statisticses, the result sees table 2.
The test of table 2 pancreatin enzyme dose-effect
The enzyme amount |
1g/500ml |
1g/700ml |
1g/1000ml |
1g/1300ml |
1g/1500ml |
Total nitrogen (in N)/% |
12.8 |
12.8 |
12.7 |
13.6 |
14.1 |
Total free aminoacids (in N)/% |
0.35 |
0.35 |
0.35 |
0.17 |
0.06 |
Iron/% |
0.05 |
0.05 |
0.05 |
0.05 |
0.05 |
Can be known that by experimental result pancreatin enzyme amount is during with 1g/1L~1g/0.5L, along with the increase of enzyme amount, total protein content/aminoacids content is almost constant, from the consideration of production cost, is good with the enzyme amount with 1g/1L.
The test of Test Example 3 hydrolysis processs
With 200ml fresh pig blood by 1: 3 (v: v) add 0.01% sodium citrate aqueous solution; Slowly stirred 5 minutes, it is centrifugal that room temperature leaves standstill after 1 hour 10000g * 10min, and the gained supernatant adds in isopyknic 95% ethanol; 6000g * 10min is centrifugal; The deposition that obtains is redissolved in the distilled water of 10 times of volumes, press 1g/1L and add pancreatin, investigate hydrolysis time.The design hydrolysis time is respectively and came observing time to extracting the influence of component content in 1,2,3,4,5,6,7,8,9 hour.Test repetition 3 times, with three average out to statisticses, the result sees table 3.
The test of table 3 hydrolysis process
Time |
1h |
2h |
3h |
4h |
5h |
6h |
7h |
8h |
9h |
Total nitrogen (in N)/% |
14.2 |
13.7 |
12.9 |
12.8 |
12.7 |
12.7 |
12.8 |
127 |
12.7 |
Total free aminoacids (in N)/% |
0.06 |
0.21 |
0.35 |
0.35 |
0.35 |
0.36 |
0.36 |
0.37 |
0.37 |
Iron/% |
0.05 |
0.05 |
0.05 |
0.05 |
0.05 |
0.05 |
0.05 |
0.05 |
0.05 |
Can know by experimental result, hydrolysis time 3 hours to 9 hours, total protein content, aminoacids content are almost constant, consider from practicing thrift the PT, be good with 3 hours.
Test Example 4 spray drying experiment
With fresh pig blood by 1: 3 (v: v) add 0.01% sodium citrate aqueous solution; Slowly stirred 5 minutes; Room temperature leaves standstill after 1 hour that 10000g * 10min is centrifugal, and the gained supernatant adds in isopyknic 95% ethanol, and 6000g * 10min is centrifugal; The deposition that obtains is redissolved in the distilled water of 10 times of volumes, press 1g/1L and add pancreatin reaction 3 hours.With the hydrolyzed solution spraying drying.Setting drying temperature is 120 ℃, 150 ℃, 180 ℃, 200 ℃, 220 ℃, 250 ℃ and 300 ℃, to confirm best drying temperature.
Test repetition 3 times, with three average out to statisticses, the result sees table 4.
Table 4 spray drying experiment
Temperature |
120℃ |
150℃ |
180℃ |
200℃ |
220℃ |
250℃ |
300℃ |
Water cut/% |
11.63 |
9.56 |
8.40 |
6.89 |
4.98 |
4.76 |
4.76 |
Solvability |
0.5% clarification |
0.5% clarification |
0.5% clarification |
0.5% clarification |
0.5% clarification |
0.5% clarification |
0.5% clarification |
Can be known that by experimental result 220~300 ℃ of water cut of drying temperature are approaching, consider from practicing thrift the PT, be good with 220 ℃.
Test Example 5 blood polypeptide wettability tests
Adopt following method to prepare blood polypeptide, then its water absorbability is measured.Adopt the closed blood-collecting method to gather fresh pig blood at normal temperatures.(v: v) add 0.01% sodium citrate aqueous solution, slowly stirred 5 minutes, it is centrifugal that room temperature leaves standstill after 1 hour 10000g * 10min, and the gained supernatant adds in isopyknic 95% ethanol by 1: 3; 6000g * 10min is centrifugal, and the deposition that obtains is redissolved in the distilled water of 10 times of volumes, and every liter of lysate adds self-control pancreatin 1 gram; In 37 ℃ of reactions 3 hours, spraying drying, 220 ℃ of EATs; 60 ℃ of air outlet temperatures, sample rate 5mL/min, drying process keeps stirring.Get the blood polypeptide powder.
It is an amount of accurately to take by weighing above-mentioned blood polypeptide powder, puts in the worn-out mouthful flat weighing bottle, under 25 ℃ of 75% relative humidity condition, weighs at interval by certain hour, calculates moisture uptake and rate of moisture absorption (%), and the result sees table 5.
Table 5 blood polypeptide wettability test of the present invention
See from the result, extract the powder water absorbability very a little less than, need not add auxiliary material and solve its water absorbability.
Test Example 6 blood polypeptide study on the stability tests of the present invention
Temperature, humidity investigation blood polypeptide stability of the present invention are set, and the time was respectively 0,3,6,9,12,16,18,24 month, and full the detection decided, and the result sees table 6.
Test is to get with following prepared with blood polypeptide:
The method of A, separating animal's blood protein is: the volume proportion by 1 part of animal blood, 3~6 parts of cell pyrolysis liquids mixes, and leaves standstill; Get and leave standstill the back centrifugal, the gained supernatant adds in isopyknic 95% ethanol, and centrifugal gained is animal blood albumen;
B, enzymolysis: animal blood albumen is dissolved in the water of 10 times of volumes, adds pancreatin 0.6~2g in every liter of lysate, in 35~40 ℃ of reactions 1~10 hour;
C, drying: be dried to the blood polypeptide moisture content and be lower than 5%, promptly get blood polypeptide of the present invention.
Concrete steps are following:
1, claims appearance: accurately take by weighing the blood polypeptide of a certain amount of good uniformity, be accurate to 0.0001g, the blood polypeptide of the present invention that weighs up is put in the hydrolysis pipe.
2, hydrolysis: in the hydrolysis pipe, add 6mol/L hydrochloric acid 10ml~15ml (look blood polypeptide protein contnt of the present invention and decide), add 3-4 of new distillatory phenol, again the hydrolysis pipe is put into refrigerant, freezing 3min-5min receives on the extraction pipe of vacuum pump again.Vacuumize (near 0Pa), charge into high pure nitrogen then; Vacuum nitrogen filling gas again behind the triplicate, in the hydrolysis pipe that inflated with nitrogen state lower sealing or tighten the screws lid will seal is placed on 110 ℃ ± 1 ℃ thermostatic drying chamber, behind the hydrolysis 22h, takes out cooling.Open the hydrolysis pipe, after hydrolyzed solution is filtered, repeatedly wash the hydrolysis pipe, hydrolyzed solution is all transferred to 50ml with deionized water.Use the deionized water constant volume in the volumetric flask.Draw filtrating 1ml in the 5ml volumetric flask, 40 ℃~50 ℃ dryings, residue is with 1ml~2ml water dissolution, and is dry again, carries out twice repeatedly with vacuum drier.Last evaporate to dryness, the damping fluid dissolving with 1ml pH2.2 supplies Instrument measuring to use.
3, measure: accurately draw 0.200ml kilnitamin standard; Sodium citrate buffer solution with pH2.2 is diluted to 5ml; This standard diluent concentration is 5.00nmol/50 μ l; As the amino acid standard of last machine mensuration usefulness, measure the aminoacids content of liquid with the external standard method present composition with automatic analyzer for amino acids.
4, the result calculates
Be calculated as follows:
In the formula:
X---blood polypeptide content of amino acids of the present invention, unit is gram every hectogram (g/100);
C---blood polypeptide of the present invention is measured aminoacids content in the liquid, and unit is per 50 microlitres of nmole (nmol/50 μ l);
F---blood polypeptide extension rate of the present invention;
V---blood polypeptide constant volume of the present invention after the hydrolysis, unit is milliliter (ml);
M---amino acid molecular amount;
M---blood polypeptide quality of the present invention, unit is gram (g);
1/50---be converted to the aminoacids content that every milliliter of blood polypeptide of the present invention is measured, unit is every liter of micromole (μ mol/L);
10
9---blood polypeptide content of the present invention is converted to the coefficient of gram (g) by nanogram (ng).
Table 6 detected result
Test item |
0 month |
March |
June |
September |
December |
16 months |
18 months |
24 months |
Total nitrogen content (in N)/% |
12.8 |
12.8 |
12.8 |
12.8 |
12.8 |
12.8 |
12.7 |
12.7 |
Moisture/% |
4.97 |
4.97 |
4.98 |
4.96 |
5.01 |
4.99 |
4.98 |
5.01 |
Muriate (in NaCl)/% |
1.09 |
1.10 |
1.09 |
1.09 |
1.08 |
1.07 |
1.11 |
1.12 |
Iron/% |
0.05 |
0.05 |
0.05 |
0.05 |
0.05 |
0.05 |
0.05 |
0.05 |
pH(1%,25℃) |
4.66 |
4.65 |
4.63 |
4.64 |
4.66 |
4.65 |
4.63 |
4.62 |
Solvability |
0.5% clarification |
0.5% clarification |
0.5% clarification |
0.5% clarification |
0.5% clarification |
0.5% clarification |
0.5% clarification |
0.5% clarification |
Total free aminoacids (in N)/% |
0.35 |
0.35 |
0.36 |
0.35 |
0.34 |
0.35 |
0.35 |
0.34 |
Evidence: blood polypeptide of the present invention has good stability, and stores after 24 months, and product composition is stable, and protein, aminoacids content are stablized.
The anoxia effect of Test Example 7 blood polypeptides of the present invention (experiment of normal pressure anoxia)
Experimental animal: 36 of KM kind mouse, body weight 20 ± 2g.
The blood polypeptide that test uses blood polypeptide to be adopted as Test Example 6.
With mouse be divided into high dose group (dosage: 0.4g blood polypeptide/10g), low dose group (dosage: 0.2g blood polypeptide/10g) and model group (water: 0.4ml/10g), every group each 12, male and female half and half, every day oral administration, 30d continuously.60min after the last administration places airtight 250ml port grinding bottle with mouse.The record mouse is because of the dead time of anoxic.
Test-results: show that through one-way analysis of variance the equal nonsignificance of difference between each dose groups mouse body weight and the control group does not see that blood polypeptide of the present invention is influential to the mouse body weight.Each organizes the difference significance (P<0.05) of hypoxia endurance time average.Through the F check, high dose group is compared difference with control group have utmost point significance (P<0.01), and low dose group has been compared significant difference (P<0.05) with control group.Explain that blood polypeptide of the present invention has the anoxia effect.The result sees table 7.
Table 7 blood polypeptide is to the influence of mouse anoxia
Annotate: compare with the normal control group:
*P<0.01,
*P<0.05.
Test Example 8 toxicity are observed
(1) do not cause death to mouse filling maximum administration concentration of food and dosage, can not measure the medium lethal dose(LD&-{50}) of blood polypeptide of the present invention.
(2) carried out rat teratogenic test, Salmonella reversion test, mouse bone marrow cells micronucleus test and chromosomal aberration test with blood polypeptide of the present invention, proved that blood polypeptide of the present invention does not have teratogenesis and mutagenesis.
Each item test-results by above can be found out; Blood polypeptide of the present invention does not have obvious toxic-side effects, and raw material sources are abundant, and the method for extracting and be prepared into corresponding healthcare products, food does not have particular requirement and restriction; Has the comprehensive pharmacological action of ideal aspect the raising body's hypoxia tolerance; Have the prospect that well is applied to healthcare products, food, can bring into play satisfied assisting therapy, health care and rehabilitative action in the anoxia of treatment due to a variety of causes.