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 by 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 polypeptide 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 liquor 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 common 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 glycine in contrast.
Blood polypeptide of the present invention can be made this area regular dosage form, as 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.
The embodiment of form is described in further detail foregoing of the present invention again by the following examples.But this should be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following example.All technology that realizes based on foregoing of the present invention all belong to scope of the present invention.
Beneficial effect by following shaker test proof blood polypeptide of the present invention and preparation method thereof.
The 1 screening cell wall breaking test of test example
With 200ml fresh pig blood by 1: 1,1: 2,1: 3,1: 4,1: 5,1: 6 (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, the gained supernatant liquor adds in isopyknic 95% ethanol, 6000g * 10min is centrifugal, and the precipitation 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 repeats 3 times, with three average out to statisticses, the results are shown in 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 |
By experimental result as can be known, adding almost unanimity of 0.01% sodium citrate aqueous solution gained protein, aminoacids content by 3~6 times of volumes, is to save 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, it is centrifugal that room temperature leaves standstill after 1 hour 10000g * 10min, the gained supernatant liquor adds in isopyknic 95% ethanol, 6000g * 10min is centrifugal, the precipitation 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 determine the add-on effect relationship of pancreatin.
The temperature of keeping 50 ℃ of solution in the reactor is half an 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 agitator continuously stirring, begin enzyme digestion reaction, adjust the quantity of steam that feeds the reactor interlayer during this time and make temperature maintenance at 37 ℃, enzyme digestion reaction finishes after adding pancreatin 6h.Test repeats 3 times, with three average out to statisticses, the results are shown in 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 |
By experimental result as can be known, pancreatin enzyme amount is during with 1g/1L~1g/0.5L, and along with the increase of enzyme amount, total protein content/aminoacids content is almost constant, for 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, the gained supernatant liquor adds in isopyknic 95% ethanol, 6000g * 10min is centrifugal, the precipitation 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 repeats 3 times, with three average out to statisticses, the results are shown in 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 |
By experimental result as can be known, hydrolysis time 3 hours to 9 hours, total protein content, aminoacids content are almost constant, consider for saving the production time, 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, it is centrifugal that room temperature leaves standstill after 1 hour 10000g * 10min, the gained supernatant liquor adds in isopyknic 95% ethanol, 6000g * 10min is centrifugal, the precipitation 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 determine best drying temperature.
Test repeats 3 times, with three average out to statisticses, the results are shown in Table 4.
Table 4 spray drying experiment
Temperature |
120℃ |
150℃ |
180℃ |
200℃ |
220℃ |
250℃ |
300℃ |
Water content/% |
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 |
By experimental result as can be known, 220~300 ℃ of water content of drying temperature are approaching, consider for saving the production time, and 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 by 1: 3, the gained supernatant liquor adds in isopyknic 95% ethanol, 6000g * 10min is centrifugal, and the precipitation 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 inlet temperature, 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 (%), the results are shown in Table 5.
Table 5 blood polypeptide wettability test of the present invention
From the result, it is very weak to extract the powder water absorbability, need not add auxiliary material and solve its water absorbability.
The 6 blood polypeptide study on the stability tests of the present invention of test example
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 results are shown in 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 liquor 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 as follows:
1, claims sample: 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: add 6mol/L hydrochloric acid 10ml~15ml (deciding on blood polypeptide protein content of the present invention) in the hydrolysis pipe, 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, the hydrolysis pipe that will seal at inflated with nitrogen state lower sealing or tighten the screws lid is placed in the thermostatic drying chamber of 1 ℃ in 110 ℃ of soil, 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 filtrate 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 with the damping fluid dissolving of 1ml pH2.2, is used for Instrument measuring.
3, measure: accurately draw O.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---the amino acid whose content of blood polypeptide 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 hypoxia tolerance effect (experiment of normal pressure hypoxia tolerance) of test example 7 blood polypeptides of the present invention
Experimental animal: 36 of KM kind mouse, body weight 20 ± 2g.
Test is the blood polypeptide that test example 6 is adopted with blood polypeptide.
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 dosage group 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.Illustrate that blood polypeptide of the present invention has the hypoxia tolerance effect.The results are shown in Table 7.
Table 7 blood polypeptide is to the influence of mouse hypoxia tolerance
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 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.
By above every test-results as can be seen, blood polypeptide of the present invention does not have obvious toxic-side effects, and raw material sources are abundant, the method of extracting and be prepared into corresponding healthcare products, food there is no 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.