CN103122363A - Preparation method of donkey-hide gelatin oligopeptide - Google Patents
Preparation method of donkey-hide gelatin oligopeptide Download PDFInfo
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Abstract
The invention discloses a preparation method of donkey-hide gelatin oligopeptide, and the preparation method comprises the following steps of: dissolving a pure donkey-hide gelatin stock solution into water; heating and stirring; regulating the pH value; adding Alcalase 2.4L FG hydrolyzing prolease for hydrolysis reaction; and increasing temperature, deactivating enzyme, cooling, filtering and spray-drying to obtain the donkey-hide gelatin oligopeptide. The preparation method disclosed by the invention has the advantages that the average molecular weight of hydrolyzed donkey-hide gelatin is less than 3000, and the donkey-hide gelatin effectively exerts physiological characteristics by being easier to absorb by a human body through the hydrolyzed donkey-hide gelatin stock solution.
Description
Technical field
The present invention relates to a kind of preparation method of Effective Component of Chinese Medicine, be specifically related to a kind of preparation method of donkey-hide gelatin oligopeptide, belong to medical technical field.
Background technology
Donkey-hide gelatin (Colla Corii Asini) is the hematopoietic in Chinese medicine, its instructions of taking is molten, or make the pulpous state ointment of different ingredients, all adopt oral administration, but in donkey-hide gelatin, collagen protein accounts for 80%, absorb than indigestibility, active low to weakness of the spleen and the stomach patient's digestive tube endoenzyme particularly, its digestion ability is poor, and oral donkey-hide gelatin is digested and assimilated not exclusively, do not reach due drug effect, also increase simultaneously the stomach and intestine burden.
Found in recent years, after donkey-hide gelatin is water-soluble, through certain reaction, the molecular weight of Colla Corii Asini collagen is reduced greatly, when the collagen molecules amount of donkey-hide gelatin lower than 3000 the time, can be absorbed fully by human body, and its pharmacodynamic experiment confirmation, this material have stronger qi and blood, improve immunity of organisms, the anti-ageing effect of waiting for a long time.
In the donkey-hide gelatin preparation process, for the ease of final moulding, generally can add some auxiliary materials, the net result of doing like this in donkey-hide gelatin is that this donkey-hide gelatin is larger in viscosity after molten, and not dissolving appears in the part activeconstituents, thereby makes the drug effect loss of donkey-hide gelatin.Simultaneously, because can exist grease such as sesame oil etc. in donkey-hide gelatin liquid, make in the donkey-hide gelatin product after decomposition and have impurity, the donkey-hide gelatin product purity is low.The present invention overcomes above-mentioned shortcoming, adopts the donkey-hide gelatin raw material that does not add any auxiliary material to add a kind of proteolytic enzyme to be decomposed into the method for donkey-hide gelatin oligopeptide under certain condition.
Summary of the invention
The object of the invention is to provide a kind of preparation method of donkey-hide gelatin oligopeptide.
The present invention utilizes the pure donkey-hide gelatin stoste of Alcalase2.4L FG protolysate enzymic hydrolysis to prepare the Colla Corii Asini collagen of molecular-weight average≤3000Da.
Preparation method of the present invention comprises the steps:
After being dissolved in pure donkey-hide gelatin stoste in 1-3 times of weight water, homo(io)thermism between 30 ℃-80 ℃, heated and stirred; Regulate pH to 6-9; Add Alcalase2.4L FG hydrolysising protease, the usage quantity of enzyme is the 0.1-0.4% of pure donkey-hide gelatin stock solution quality, hydrolysis reaction 3-5 hour; The Alcalase2.4LFG hydrolysising protease that adds again 0.1-0.4%, hydrolysis 3-5 is termination reaction as a child; Be warming up to 80-100 ℃ and went out enzyme 3-8 minute, cooling, paper pulp filtering, spraying drying, and get final product.
Preparation method of the present invention is preferably as follows step:
After being dissolved in pure donkey-hide gelatin stoste in 2 times of weight, homo(io)thermism between 50 ℃-60 ℃, heated and stirred; Regulate pH to 6.5-8.5; Add Alcalase2.4L FG hydrolysising protease, the usage quantity of enzyme is 0.25% of pure donkey-hide gelatin stock solution quality, hydrolysis reaction 4 hours; Add 0.25% Alcalase2.4L FG hydrolysising protease, hydrolysis 4 is termination reaction as a child again; Be warming up to 90 ℃ of enzymes 5 minutes of going out, cooling, paper pulp filtering, spraying drying, and get final product.
Preparation method of the present invention is preferably as follows step:
After being dissolved in pure donkey-hide gelatin stoste in 1.5 times of weight, homo(io)thermism between 60 ℃, heated and stirred; Regulate pH to 8; Add Alcalase2.4L FG hydrolysising protease, the usage quantity of enzyme is 0.2% of pure donkey-hide gelatin stock solution quality, hydrolysis reaction 3.5 hours; Add 0.35% Alcalase2.4L FG hydrolysising protease, hydrolysis 4.5 is termination reaction as a child again; Be warming up to 85 ℃ of enzymes 4 minutes of going out, cooling, paper pulp filtering, spraying drying, and get final product.
Preparation method of the present invention is preferably as follows step:
After being dissolved in pure donkey-hide gelatin stoste in 2.5 times of weight water, homo(io)thermism between 55 ℃, heated and stirred; Regulate pH to 7; Add Alcalase2.4L FG hydrolysising protease, the usage quantity of enzyme is 0.35% of pure donkey-hide gelatin stock solution quality, hydrolysis reaction 4.5 hours; Add 0.15% Alcalase2.4L FG hydrolysising protease, hydrolysis 3.5 is termination reaction as a child again; Be warming up to 95 ℃ of enzymes 6 minutes of going out, cooling, paper pulp filtering, spraying drying, and get final product.
The donkey-hide gelatin oligopeptide of the present invention's preparation can add conventional auxiliary material, according to common process, make tablet, capsule, powder, soft capsule, dripping pill, honeyed bolus, pill, granule, soft extract with bee honey agent, sustained release preparation, quick releasing formulation, controlled release preparation, oral liquid.
Pure donkey-hide gelatin stoste of the present invention can be to extract after the dissolving of donkey-hide gelatin piece, also can directly buy from producer the donkey-hide gelatin stoste that not yet becomes piece.
Donkey-hide gelatin molecular-weight average after this inventive method hydrolysis is below 3000.Preferred donkey-hide gelatin stoste is effectively brought into play physiological property thereby the donkey-hide gelatin stoste after hydrolysis more easily is absorbed by the body it.
The below's experiment and embodiment are used for further illustrating but being not limited to the present invention.
One, test design
1. enzyme reagent is selected
Sumizyme MP is to make through bacterium Protoplast Mutation method the 2709 withered grass bar microorganisms that educate to reach the refining a kind of proteolytic ferment that forms by submerged fermentation, extraction, belong to the crisp outer high alkaline proteases of a kind of Serine, it can generate polypeptide or amino acid by protein hydrolysate molecule peptide chain, it is a kind of restriction endonuclease, catalytic site is Serine, molecular weight is about 27300, has the ability of stronger decomposing protein.What Sumizyme MP was selected is the product A lcalase2.4L FG hydrolysising protease of Novi's letter.
2. initial reactant determines
Commercially available donkey-hide gelatin piece hydrolysis reaction liquid not only fishy smell on mouthfeel, oleaginous taste is extremely heavy, and because contained grease in product is too much, has also brought trouble for later preparation work.The donkey-hide gelatin stoste that employing does not add the donkey hide of any additive to boil need not to remove the floating oil of enzymolysis liquid level after enzymolysis, can directly filter; The hydrolyzed solution that contains on the other hand lipid should not be preserved at ambient temperature, and the hydrolyzed solution that makes of the donkey-hide gelatin stoste of oil-containing can not deposited about 10 days at ambient temperature, is more conducive to preserve; The enzymolysis rate of recovery of oil-containing donkey-hide gelatin is generally less than 60%, and the donkey-hide gelatin stoste of oil-containing is not hydrolyzed the rate of recovery greater than 75%, and the clarity of hydrolysate has also obtained very large improvement.
3.pH the selection of value and temperature
According to the Sumizyme MP working conditions, have higher hydrolysis ability or hydrolytic activity in order to make Sumizyme MP when reacting, use temperature and pH value will meet the service requirements of Sumizyme MP.In method of the present invention, the Sumizyme MP pH value scope of application 6～9 transfers to 7.0 with the pH value that is hydrolyzed initial reaction liquid, and is close with the suitable pH value of Sumizyme MP.Hydrolysis temperature is take 60 ℃ of the optimal temperatures of Sumizyme MP as hydrolysis temperature.
4. hydrolysis end-point detection method
Can find a lot of detection methods about the hydrolysis terminal point from document, but these methods respectively there are its relative merits.OPA detection method that this has tested final choice, more simple and easy to do in operation with respect to other method although this method also has its limitation, also can relatively simplify the work on quality control procedure from now on.
4.1.OPA method is surveyed protein degree
4.1.1 degree of hydrolysis definition
Degree of hydrolysis (Degree of Hydrolysis, DH) is the per-cent of the peptide bond number that is hydrolyzed.
The peptide bond number of DH=hydrolysis/total peptide bond number * 100%=h/h
tot* 100%
(h
totThe type that depends on raw material.H is Serine several function that rubs in the least.H=(serine NH
2-β)/α mmol/ gram protein.Most food proteins, amino acid whose molecular-weight average 125g/mol, the peptide bond sum is 8, namely every kg albumen approximately contains 8 peptide bonds that rub.）
4.1.2 experimental principle
Peptide bond of the every hydrolysis of proteolytic enzyme will discharge a free amine group.Free amine group and OPA reagent react generate yellowish complex compound, can survey its absorbancy with spectrophotometer in 340 nanometers.
4.1.3 laboratory apparatus and equipment
Ultraviolet spectrophotometer (Shimadzu UV-1700), analytical balance (METTLE TOLEDO AT400),
Magnetic stirring apparatus, vibrator, liquid-transfering gun, volumetric flask (100ml, 200ml), test tube (10ml)
4.1.4 experiment reagent
Ten hydration sodium tetraborate decahydrate (analytical pure, the Shantou Xilong Chemical Factory), sodium lauryl sulphate (SDS) (ancient cooking vessel state biotech development center, Beijing, the sigma packing, L5750), o-phthalaldehyde(OPA) (OPA) (chemical pure, Beijing chemical reagents corporation), dithiothreitol (DTT) (DTT) (Merck233156, purity 〉=99%), dehydrated alcohol (analytical pure, Beijing chemical reagents corporation), Serine (ancient cooking vessel state biotech development center, Beijing, Merck.7769)
4.1.5OPA the preparation of reagent
1) sodium tetraborate that accurately takes 7.620g adds the deionized water of 150ml left and right in beaker.
2) sodium lauryl sulphate (SDS) that accurately takes 200mg adds the deionized water of 10ml left and right in small beaker.
3) small beaker is risen with the tinfoil paper paper bag, accurately takes 160mg o-phthalaldehyde(OPA) 97%(OPA) in small beaker, add the dehydrated alcohol of 4ml.
4) accurately take 176mgDTT(99%) in small beaker, add the deionized water of 10ml left and right.
5) add rotor in the above-mentioned beaker that four kinds of reagent are housed, rim of a cup is sealed with sealed membrane, and the beaker that OPA is housed also will cover rim of a cup with masking foil.Then four beakers being transferred to magnetic stirring apparatus stirs and makes its dissolving.
6) prepare the volumetric flask of a 200ml, it wrapped with masking foil, with the agent transfer of above-mentioned four kinds of dissolve completes in volumetric flask.Transfer sequence is: sodium tetraborate, SDS, OPA, DTT.
7) use the deionized water rinsing beaker, after pouring into again washing fluid in volumetric flask.
8) use the deionized water constant volume to 200ml.
(annotate: this reagent is prepared when used the same day photaesthesia again.）
4.1.6 the preparation of Serine standard model
Take the Serine standard substance of 10mg in the volumetric flask of 100ml, be settled to 100ml(0.9516 with deionized water to rub in the least/liter).
Standardized solution can store for three weeks under 5 degrees centigrade.
4.1.7 the preparation of testing sample solution
Take the donkey-hide gelatin hydrolyzation sample of about 0.6g-0.7g in the volumetric flask of 100ml, be settled to 100ml (0.25-2.5 rub in the least amino nitrogen/liter) with deionized water.
4.1.8 experimental procedure
1) open the preheating of uv-spectrophotometric instrument, arranging and detecting wavelength is 340nm, makes zero and makes reference with deionized water.
2) experimental sequence:
1: standard model, detecting number of times is 2 times;
2: blank, detecting number of times is 4 times;
3: testing sample, each testing sample is surveyed 2 times;
4: standard model, detecting number of times is 2 times.
3) mensuration of standard model: get a test tube that contains 3ml OPA reagent, add 400 microlitre Serine standard models to it, the beginning timing, the concussion mixing is after accurately reacting 2 minutes, in 340nm place's survey absorbancy.(get the mean value of four standard sample determinations during calculating.）
4) blank determination: get a test tube that contains 3ml OPA reagent, add 400 microlitre deionized waters to it, the beginning timing, the concussion mixing is after accurately reacting 2 minutes, in 340nm place's survey absorbancy.(get the mean value of four blank determinations during calculating.）
5) mensuration of testing sample: get a test tube that contains 3ml OPA reagent, add 400 microlitre testing sample diluents to it, the beginning timing, the concussion mixing is after accurately reacting 2 minutes, in 340nm place's survey absorbancy.(testing sample measures twice, averages during calculating.）
6) remarks: because of the absorbancy temporal evolution, so guarantee that accurately reaction was measured after 2 minutes.Reference value: standard OD is about 0.77-0.82, and blank OD is about 0.065-0.075.
4.1.9 result is calculated
1) mensuration of h:
In formula:
Serine-NH
2: serine NH rubs in the least
2/ g albumen
X: example weight
P: the protein content in sample
0.1: sample volume changes into L
H is calculated as follows: h=(Serine NH
2-β)/α meqv/g protein
α, β value are tabled look-up.
2) calculate DH:
DH=h/h
tot*100%
h
totNeed table look-up.
5. molecular weight detection
Adopt the Maldi-tof method to detect.MALDI-TOF is a kind of soft ionization technology, does not produce or produce less fragmention.It can directly apply to the analysis of mixture, also can be used to whether contain in test sample the molecular weight of impurity and impurity.Molecular weight is also primary parameter during biomacromolecule such as polypeptide, protein etc. are identified, is also one of significant data of gene engineering product declaration.The accuracy of MALDI-TOF is up to 0.1%-0.01%, far away higher than present conventional SDS electrophoresis and the efficient gel chromatographic technique of using.
6. donkey-hide gelatin protolysate is hydrolyzed scheme
Donkey-hide gelatin protolysate hydrolysis scheme, namely take 10% donkey-hide gelatin stoste as substrate, the water-bath that is placed in 60 ℃ stirs, and is incubated adjust pH to 7.0 after a hour, adds 0.5% Alcalase2.4L FG hydrolysising protease, is hydrolyzed after 8 hours the high temperature enzyme that goes out.
7. product forms is selected
In tentative test, once attempted the spray of donkey-hide gelatin hydrolyzed solution is done, but because the particle of spray powder is very thin, very easily produced electrostatic adhesion, need to debug preparation technology whole if make solid preparation.By contrast, use the product after donkey-hide gelatin stoste is hydrolyzed instead, fishy smell is little, and clarity is fine, and has changed the proterties that freezes under donkey-hide gelatin low temperature, and is if select the formulation oral liquid, relatively simple.
Two, scale-up
1, scale-up equipment
30L fermentor tank water-bath, saccharometer (0-32%), pH meter, centrifuge tube;
All the other experiment materials see Table one.
Table one amplification test material and key instrument equipment
2, scale-up test reaction conditions
The amplification test reaction conditions sees Table two.
Table two amplification test reaction conditions
Remarks:
★Add 15ml in the time of the 0th hour, add 15ml after the 4th hour finishes
3, scale-up hydrolysis process flow process
Get a certain amount of donkey-hide gelatin stoste and be placed in fermentor tank, add the dilution of twice water; Start fermentor tank at sequence of threads, steam heating, manually temperature control, regulate rotating speed of agitator; After solution temperature rises to 55 ℃ in fermentor tank, add a certain amount of edible alkali to regulate the pH value to 25 ℃ of 7.0(room temperatures) left and right; Add the Alcalase2.4L FG hydrolysising protease of donkey-hide gelatin stock solution quality 5 ‰, timing begins; Each hour sampling, 95 ℃ of enzymes that go out were cooled to room temperature after 5 minutes, measured degree Beaume, pH value and viscosity; Heat after reaction in 8 hours finishes cooling after 5 minutes to 95 ℃ of enzymes that go out, hydrolyzed solution is packed into preserve in container; The sample of taking-up hourly takes to make of the OPA method surveys protein degree, and the sampling sample detection protein molecular weight of the 8th hour detects.
4 scale-up test-results
4.1 the scale-up of donkey-hide gelatin hydrolysis for the first time
4.1.1 (concrete outcome sees Table three to experimental phenomena.）
1) solid substance that can observe from the fermentor tank form in glue along with the carrying out that reacts slowly becomes in small, broken bits.
2) sampling in 1-8 hour places to spend the night all has precipitation to produce afterwards, and upper strata liquid is muddy, and the sample of other first hour has obvious settling to produce.
3) temperature control shows 55 ℃, but actual temperature is 57 ℃, temperature control and actual error arranged.
4) the hydrolysis after product has a little bitter taste.
Table three is amplification test degree Beaume, viscosity, pH, DH result for the first time
Annotate: 0# is that hydrolysis reaction begins the front sample of getting.
4.1.2 molecular weight determination result:
Molecular-weight average: 2212
4.2 the scale-up of donkey-hide gelatin hydrolysis for the second time research
4.2.1 experimental phenomena
When 1) reaction was carried out about 1 hour, solid particle had become very in small, broken bits from the glue of form observation fermentor tank.
Observe the sample of front 5 hours when 2) reaction has been carried out 5 and a half hours, wherein:
Enzyme-added front sample: centrifuge tube bottom and tube wall have a large amount of settlings, and upper strata liquid is muddy.
The 1st hour sample: see a small amount of precipitation bottom centrifuge tube, have small part adherent, upper strata liquid is muddy.
The 2nd hour sample: centrifuge tube bottom and tube wall precipitation are less than the 1st hour sample, and upper strata liquid is muddy.
The 3rd hour sample: precipitate seldom, upper strata liquid is muddy.
4th, 5 hours samples: also do not observe precipitation this moment, solution is muddy.
Observe the 1st ~ 7 hour institute's sample thief when 3) reaction proceeds to 7 hours 40 minutes, wherein:
The 1st hour sample: the part settling is arranged at the bottom of centrifuge tube, and upper strata liquid is muddy, the 5th and a half hours viewed amount of unnecessary reaction, but obviously be less than hydrolysis reaction sample before.
The 2nd hour sample: at the bottom of centrifuge tube, precipitation is less than the 1st hour sample, and upper strata liquid is muddy.
The 3rd hour sample: at the bottom of centrifuge tube, precipitation is less than the 2nd hour sample, and upper strata liquid is muddy.
The 4th hour sample: a little precipitation is arranged at the bottom of centrifuge tube, be less than the 3rd hour sample, upper strata liquid is muddy.
5th, 6 hours samples: the minute quantity precipitation is arranged at the bottom of centrifuge tube, and upper strata liquid is muddy.
The 7th hour sample: there is no precipitation.
4) after sample spent the night, throw out was all arranged at centrifuge tube bottom, and upper strata liquid is muddy.
5) the hydrolysis after product has a little bitter taste.
Concrete outcome sees Table four.
Table four is test-results degree Beaume, viscosity, pH, DH result for the second time
Annotate: 0# is that hydrolysis reaction begins the front sample of getting.
4.3 the scale-up of donkey-hide gelatin hydrolysis for the third time research trial result
4.3.1 experimental phenomena
1) solid substance that can observe from the fermentor tank form in glue along with the carrying out that reacts slowly becomes in small, broken bits.
When going out enzyme after 2) sample takes out, can observe the more large stretch of flocks of Guan Zhongyou, solution is comparatively limpid.
3) after sample goes out and is cooled to room temperature after enzyme and places for some time, solution by light brown add gradually be deep to brown.
4) after sample spent the night, precipitation in small, broken bits was arranged at centrifuge tube bottom, and along with the increase in reaction times, precipitation capacity reduces gradually, and upper strata liquid is comparatively limpid.
Concrete experimental result sees Table five
Table five is amplification test degree Beaume, viscosity, pH, DH result for the third time
Annotate: 0# is that hydrolysis reaction begins the front sample of getting.
4.3.2 molecular weight detection result
Molecular-weight average: 2193
4.4 the 4th donkey-hide gelatin hydrolysis scale-up research trial result
4.4.1 experimental phenomena
1) solid substance that can observe from the fermentor tank form in glue along with the carrying out that reacts slowly becomes in small, broken bits.
When going out enzyme after 2) sample takes out, can observe the more large stretch of flocks of Guan Zhongyou, solution is comparatively limpid.
3) after sample goes out and is cooled to room temperature after enzyme and places for some time, solution by light brown add gradually be deep to brown.
4) after sample spent the night, precipitation in small, broken bits was arranged at centrifuge tube bottom, and along with the increase in reaction times, precipitation capacity reduces gradually, and upper strata liquid is comparatively limpid.
The 4th amplification test degree Beaume of table six, viscosity, pH, DH result
| No. | Time | Brix | Viscosity (s) Φ 0.6 | pH | DH%(OPA) | DH(pH-state) |
| 0# | 10:05 | / | / | 6.99 | 3.02 | / |
| 1# | 11:05 | 12.1 | 144 | 6.58 | 11.54 | 0.535 |
| 2# | 12:05 | 12.0 | 137 | 6.54 | 13.98 | 0.779 |
| 3# | 13:05 | 12.1 | 131 | 6.47 | 15.13 | 0.934 |
| 4# | 14:05 | 12.0 | 127 | 6.48 | 16.24 | 0.938 |
| 5# | 15:05 | 12.0 | 136 | 6.50 | 16.21 | 1.010 |
| 6# | 16:05 | 12.1 | 135 | 6.51 | 17.72 | 1.021 |
| 7# | 17:05 | 12.2 | 126 | 6.43 | 17.43 | 1.125 |
| 8# | 18:05 | 12.2 | 123 | 6.41 | 17.70 | 1.127 |
Annotate: 0# is that hydrolysis reaction begins the front sample of getting.
4.5 the 5th donkey-hide gelatin hydrolysis scale-up research trial result
4.5.1 experimental phenomena
1) solid substance that can observe from the fermentor tank form in glue along with the carrying out that reacts slowly becomes in small, broken bits.
When going out enzyme after 2) sample takes out, can observe the more large stretch of flocks of Guan Zhongyou, solution is comparatively limpid.
3) after sample goes out and is cooled to room temperature after enzyme and places for some time, solution by light brown add gradually be deep to brown.
4) after sample spent the night, precipitation in small, broken bits was arranged at centrifuge tube bottom, and along with the increase in reaction times, precipitation capacity reduces gradually, and upper strata liquid is comparatively limpid.
The 6th amplification test degree Beaume of table seven, viscosity, pH, DH result
Annotate: 0# is the enzyme-added rear sample of getting in 2 minutes (enzyme has gone out).
5. technology stability is investigated experiment
5.1 materials variance
In five scale-up tests, we have adopted donkey-hide gelatin stoste as experimental raw, and in hydrolysising experiment and other related experiment, glue is compared, glue exists difference and is accompanied by hydrolysis degree and the difference of hydrolyzed solution physico-chemical property as can be seen from the results.Therefore, need donkey-hide gelatin stoste is carried out quality control before production.
5.2 temperature control and pH span of control in hydrolysis reaction
The suitableeest working temperature of donkey-hide gelatin hydrolysis reaction food grade Alcalase2.4L FG used hydrolysising protease is 50 ℃ ~ 60 ℃, and the suitableeest working pH value is 6.0 ~ 9.5.Therefore, in hydrolysis reaction, temperature and pH value all being controlled at the suitableeest scope gets final product.
5.3 alr mode and rotating speed of agitator
Scale-up technique more for the first time and for the second time is in the situation that the identical alr mode that changes of other conditions can find out that from twice final degree of hydrolysis result of scale-up the mass transfer effect that uses stirring rake to stir is better than pneumatic blending.The rotating speed of agitator aspect, because mass transfer is to be determined by the linear velocity of stirring rake, therefore after testing scale-up, rotating speed of agitator should reduce, change 150rpm into by before 300rpm when the 4th scale-up after, hydrolysis effect is still stable, not only guarantees quality product but also little rotating speed that can be energy-conservation therefore adopt.
5.4 enzyme add mode
Adopted two kinds to add enzyme method in the scale-up test: disposable enzyme-added and in batches enzyme-added.Enzyme-added benefit is to replenish when prolongation along with the time reduces enzymic activity the enzyme into high activity again in batches, can make hydrolysis reaction remain on continuously higher speed of response.As can be seen from the results, when 8 hours finished reaction, degree of hydrolysis obviously improved, and the quality of hydrolysate is also promoting thereupon, the pH of the 5th scale-up, viscosity variation are also relatively stably simultaneously, and this also provides strong assurance for the on-line Control in production process.Therefore determine that enzyme addition is in batches enzyme-added.
5.5 degree of hydrolysis and molecular weight index
Guaranteeing under the stay-in-grade prerequisite of donkey-hide gelatine raw feed liquid, can guarantee that the molecular-weight average of donkey-hide gelatin Collagen Hydrolysate is below 3000 when degree of hydrolysis reaches 17.5% when above.
6, donkey-hide gelatin stoste quality control experiment
In the tentative test of carrying out, at first the donkey-hide gelatin raw material adopts donkey-hide gelatin liquid, now the aqueous solution is made in its pulverizing during experiment, product after hydrolysis not only fishy smell on mouthfeel, oleaginous taste is extremely heavy, and because contained grease in product is too much, also brought trouble for later preparation work, considered in addition Cost Problems, the cost of donkey-hide gelatin is relatively high.The donkey-hide gelatin stoste that has adopted afterwards the donkey hide that do not add any additive to boil, and carried out again hydrolysising experiment under identical hydrolysising condition, find that on taste, fishy smell is light a lot, and because contained grease is few, the clarity of product has also obtained very large improvement, therefore determines to adopt donkey-hide gelatin stoste.
At the trial, the variation tendency of some testing datas demonstrates the difference of different batches donkey-hide gelatin stoste, so we are to the protein content of different batches donkey-hide gelatin stoste, water content, ash content, solid content, initially hydrolysis and outward appearance are measured and estimated.
6.1 different batches donkey-hide gelatine raw liquid eggs white matter assay
Different batches donkey-hide gelatin stoste measurement result sees Table eight:
Table eight different batches donkey-hide gelatin stoste measurement result
Protein content has certain influence to final molecular-weight average and degree of hydrolysis, therefore will guarantee the stable product quality, needs protein content is proposed respective standard; Can estimate the content of non-proteinaceous matter from water content and protein content, non-proteic substance might affect hydrolysis process and result; The ash content of different batches donkey-hide gelatin stoste is all less than 1%, but therefore first glue sees that from the angle of ash content the quality outline of second batch glue is better than first quality greater than second batch; The different solid content of hydrolyzed solution may be to be determined by protein content in donkey-hide gelatin stoste and non-proteinaceous matter content, has embodied the concentration of hydrolyzed solution; The measuring method of initial degree of hydrolysis is the OPA method, found out that from the data of scale-up test identical technical process and stable zymin are not all very large on the impact of degree of hydrolysis, the hydrolysis degree of two batches of donkey-hide gelatin is certain, and therefore initial degree of hydrolysis is controlled and can be predicted within the specific limits final degree of hydrolysis with the process of controlled hydrolysis reaction.
6.2 freezing power measures
Freeze power, refer to again the ability of congealing, it is the important indicator that checks under the gelatin item.Because donkey-hide gelatin and gelatin have similarity, therefore can monitor the power of freezing of donkey-hide gelatin with reference to the inspection of gelatin freezing concentration in national standard.
Correlative study shows, the molecular weight distribution of gelatin has greatly affected the physicochemical property of gelatin, and especially gelatin freezes power and viscosity.The molecular weight of gelatin is larger, and the viscosity of gelatin and the power of freezing are also higher.Freeze the larger donkey-hide gelatin quality of power better, yet this may cause the reduction of degree of hydrolysis, thereby affect the hydrolysis degree of protein, cause the molecular-weight average of final hydrolysate higher.Melting in the glue process before hydrolysis reaction begins taked heat tracing for some time the method for (30 ~ 60 minutes), donkey-hide gelatin freeze power and viscosity all can reduce greatly, carry out smoothly to guarantee hydrolysis reaction.
6.3 protein molecular weight distributes
The final molecular-weight average of protein molecular weight affects hydrolysis that donkey-hide gelatin stoste is initial, namely initial molecular weight is distributed in larger molecular weight ranges, so through after the hydrolysis of 8 hours, no matter is its degree of hydrolysis or molecular-weight average, all can be less than normal.But what of restriction enzyme site also might have influence on net result.Therefore the distribution range of suitably controlling protein molecular weight may produce a desired effect at the hydrolysis terminal point.
6.4 enzymatic determination
Proteolytic enzyme is a kind of protein of less stable, often produces irreversible active decline because of impacts such as temperature, pH, ionic strengths.In process of production, for the activity that enzyme was kept relative stability in the long as far as possible time, need to control in real time temperature, the pH value is regulated.
In hydrolytic process, food grade Alcalase2.4L FG hydrolysising protease used is the High Efficient Bacteria proteolytic enzyme of developing specially for the hydrolysis range protein, and it is a kind of endo-protease.Its suitableeest working temperature is 50 ℃ ~ 60 ℃, and the suitableeest working pH value is 6.0 ~ 9.5.Aborning, temperature and pH value can be controlled in the above scope, to guarantee Enzymic stability.
Three, the experimental study of donkey-hide gelatin Collagen Hydrolysate to the rat nutritional anemia
1. experiment purpose
By low iron forage feed rat method nutritional anemia experimental model, observe the donkey-hide gelatin Collagen Hydrolysate to the impact of rat model index of correlation, estimate given the test agent to improving the effect of animal nutritional anaemia.
2. experiment material
2.1 reagent
Protoporphyrin, sigma company produces.
The cyanmethemoglobin reference liquid, Shanghai Yi Hua medical science and technology company limited produces, lot number: 20128005.
Hemoglobinometry reagent, anticoagulant heparin agent, 5% diatomite normal saline suspension, ethyl acetate and acetic acid (4:1) suspension, protoporphyrin reference liquid.
2.2 laboratory animal and raising situation
(1) laboratory animal
The SD Weanling rat, the SPF level, male, available from Department Of Medicine, Peking University Laboratory Animal Science section.License licensed licenser licence numbering: SCXK(capital) 2006-0008.
(2) feed
Low iron feed formulation:
Composition: glucose (crystal water) 49.38%, without VITAMIN casein 20.00%, take off navel yellow corn powder 15.00%, gelatin 5.00%, Semen Maydis oil 5.00%, SODIUM PHOSPHATE, MONOBASIC 2.00%, calcium carbonate 2.00, Repone K 0.50%, mixed trace elements
1.0.27%, choline chloride 60 0.15, mixed vitamin
2.0.10%,
DL-methionine(Met) 0.10%
1. mixed trace elements (g%): MgSO4H
2O73.816g, ZnSO
47H
2O19.657g, MnSO
4H
2O5.733g, CuSO
45H
2O0.7315g, KIO
30.0625g.
2. mixed vitamin (g%): vitamin A (500,000 IU/g) 1.00g, Vitamin D3 500,000 I.U/GM (200,000 IU/g) 0.75g, α-Vitamin E-acetate (gelatin that contains 25%VE) 12.50g, vitamin K 0.04g, vitamin 0.30g, riboflavin 0.30g, vitamins B
60.30g, general junket calcium 0.60g, nicotinic acid 3.00g, folic acid 0.10g, vitamins B
12(contain 0.1%B
12Gelatin) 2.00g, sucrose 79.11g.
(3) raising condition
Experimental animal feeding in SPF level animal housing, fluorescent lamp lighting, the 12h light and shade cycle, freely drink water (deionized water), diet (low iron feed), 22 ℃ ± 2 ℃ of temperature ,-10 times/h blows 6 times.
2.3 laboratory apparatus
UV2300 ultraviolet/visible spectrophotometer, U.S. scientific instrument company limited of Shanghai section.
Shimadzu RF5301 fluorophotometer, Japan.
The BCD-257SL of Haier refrigerating refrigerator, Qingdao HaiEr Co., Ltd.
Sartorious BT124S precise electronic balance, Sai Duolisi scientific instrument (Beijing) company limiteds.
IKAMS3 digital oscillator: Germany.
3. experimental technique
3.1 dosage setting
(1) donkey-hide gelatin
In the Pharmacopoeia of the People's Republic of China 2005 editions, regulation donkey-hide gelatin quantity is 3g-9g, and it is 9g/ people/per daily dose that dosage is selected in this experiment.6 times of rat bioequivalence dosage behaviour clinical application amount are set in this experiment, and people's standard body weight is drafted and is 60kg, so the equivalent dosage of rat is 9g crude drug ÷ 60kg * 6=0.9g crude drug/kg, this behaviour clinical equivalent dosage.
(2) donkey-hide gelatin Hy drolyzed Skin Powder
After donkey-hide gelatin is hydrolyzed by technique, the ratio of gained protein powder is about the 1.25:1(quality).The theoretical consumption of people by the above-mentioned quantity conversion of donkey-hide gelatin donkey-hide gelatin Collagen Hydrolysate is 9g/1.25=7.2g/ people/day, calculate by above-mentioned rat bioequivalence dosage coefficient and people's standard body weight, the theoretical equivalent dosage of the rat of donkey-hide gelatin Collagen Hydrolysate is 7.2g crude drug ÷ 60kg * 6=0.72g crude drug/kg, and this is theoretical people's clinical equivalent dosage; Establish two dosage according to 1/2 and 1/4 multiple respectively downwards, totally three dosage 0.72g/kg, 0.36g/kg, 0.18g/kg are equivalent to respectively 1 times, 1/2 times, 1/4 times of the theoretical clinical application amount of people.
3.3 experimentation on animals and grouping
Weanling rat adaptability was raised after 2 days, raised and low iron feed and deionized water, adopted Rotating Stainless Steel Cage and food tank.From the 3rd every weekly selection part rat of week beginning, survey oxyphorase (Hb) content from the tail venous blood sampling, until most animals Hb content when following lower than 100g/L, is measured the Hb of whole rats.Choose the rat of Hb＜100g/kg as laboratory animal, be divided at random 5 groups according to Anemia in Rats Hb level: dosage group and donkey-hide gelatin Collagen Hydrolysate low dose group in low iron control group, donkey-hide gelatin group, donkey-hide gelatin Collagen Hydrolysate high dose group, donkey-hide gelatin Collagen Hydrolysate.The donkey-hide gelatin group gives donkey-hide gelatin 0.9g crude drug/kg; The high, medium and low dosage group of donkey-hide gelatin Collagen Hydrolysate gives respectively donkey-hide gelatin Collagen Hydrolysate 0.72g/kg, 0.36g/kg, 0.18g/kg; Low iron control group gives the equal-volume deionized water.Route of administration is per os pipe gastric infusion, and the gastric infusion volume is 5ml/kg body weight rat.
Be subjected to continuously test product after 4 weeks, the eye socket venous plexus is got whole blood, respectively the level of free protoporphyrin in Measuring hemoglobin content and red corpuscle.
4. statistical method
Experimental data is with " mean+SD
" expression.Data are first carried out homogeneity test of variance.Variance selects One-Way ANOVA method to carry out comparing between each group together; Heterogeneity of variance select t ' check organize between relatively.
5. experimental result
(1) impact of donkey-hide gelatin Collagen Hydrolysate on rat hemoglobin sees Table nine
The impact of table nine donkey-hide gelatin Collagen Hydrolysate on rat hemoglobin
| Grouping | Dosage | N | Oxyphorase (g/L) |
| Low iron control group | -- | 12 | 90.22±7.23 |
| The donkey-hide gelatin group | 0.9g/kg | 12 | 118.34±10.64 ** |
| Donkey-hide gelatin Collagen Hydrolysate high dose group | 0.72g/kg | 12 | 150.37±10.43 ***△△ |
| Dosage group in the donkey-hide gelatin Collagen Hydrolysate | 0.36g/kg | 12 | 137.56±8.56 ***△ |
| Donkey-hide gelatin Collagen Hydrolysate low dose group | 0.18g/kg | 12 | 120.58±7.52 ** |
Annotate: compare with low iron control group,
*P＜0.01,
* *P＜0.001; Compare with the donkey-hide gelatin group,
△P＜0.05,
△ △P＜0.01.
Result of study shows, oral administration is subjected to 4 weeks after test product, and donkey-hide gelatin group and donkey-hide gelatin Collagen Hydrolysate three each dosage groups are remarkable elevation model rat hemoglobin content all, relatively there is significant difference (to distinguish P＜0.01 with low iron control group, 0.001,0.001,0.01).Compare with the donkey-hide gelatin group, the donkey-hide gelatin Collagen Hydrolysate is high, middle dosage group content of hemoglobin significantly raises (P＜0.01,0.05 respectively); Donkey-hide gelatin Collagen Hydrolysate low dose group and donkey-hide gelatin group comparison there was no significant difference (P〉0.05).
(2) impact of donkey-hide gelatin Collagen Hydrolysate on the free protoporphyrin of Rat Erythrocytes
The results are shown in Table ten.
The impact of table ten donkey-hide gelatin Collagen Hydrolysate on the free protoporphyrin of Rat Erythrocytes
| Grouping | Dosage | N | Free protoporphyrin (μ g/L whole blood) |
| Low iron control group | -- | 12 | 1525±135 |
| The donkey-hide gelatin group | 0.9g/kg | 12 | 1329±122 ** |
| Donkey-hide gelatin Collagen Hydrolysate high dose group | 0.72g/kg | 12 | 1093±142 ***△△ |
| Dosage group in the donkey-hide gelatin Collagen Hydrolysate | 0.36g/kg | 12 | 1275±137 ***△ |
| Donkey-hide gelatin Collagen Hydrolysate low dose group | 0.18g/kg | 12 | 1357±193 * |
Annotate: compare with low iron control group,
*P＜0.05,
*P＜0.01,
* *P＜0.001; Compare with the donkey-hide gelatin group,
△P＜0.05,
△ △P＜0.01.
Result of study shows, oral administration is subjected to 4 weeks after test product, and donkey-hide gelatin group and donkey-hide gelatin Collagen Hydrolysate three each dosage groups all can significantly reduce free protoporphyrin level in the rat model red corpuscle, relatively there is significant difference (to distinguish P＜0.01 with low iron control group, 0.001,0.001,0.05).Compare with the donkey-hide gelatin group, the donkey-hide gelatin Collagen Hydrolysate is high, the interior free protoporphyrin level of middle dosage group red corpuscle significantly reduces (P＜0.01,0.05 respectively); Donkey-hide gelatin Collagen Hydrolysate low dose group and donkey-hide gelatin group comparison there was no significant difference (P〉0.05).
6. experiment conclusion
(1) adopt low iron forage feed to prepare rat hypoferric anemia model, donkey-hide gelatin Collagen Hydrolysate 0.72g/kg, 0.36g/kg, three dosage groups of 0.18g/kg are after the per os gavage gave for 4 weeks, all elevation model rat hemoglobin content and reduce free protoporphyrin level in red corpuscle in various degree, have the effect that improves preferably the iron deficiency nutritional anemia.
(2) compare with the donkey-hide gelatin group, donkey-hide gelatin Collagen Hydrolysate 0.72g/kg, 0.36g/kg group free protoporphyrin level in elevation model rat hemoglobin content and reduction red corpuscle has some superiority, and significant difference is both relatively arranged.The effect of donkey-hide gelatin Collagen Hydrolysate 0.18g/kg group is close with the donkey-hide gelatin group, both compares there was no significant difference.
Following embodiment all can realize the effect of above-mentioned experimental example.
Embodiment one: the preparation of tablet
After being dissolved in pure donkey-hide gelatin stoste in 2 times of weight, homo(io)thermism between 50 ℃-60 ℃, heated and stirred; Regulate pH to 6.5-8.5; Add Alcalase2.4L FG hydrolysising protease, the usage quantity of enzyme is 0.25% of pure donkey-hide gelatin stock solution quality, hydrolysis reaction 4 hours; Add 0.25% Alcalase2.4L FG hydrolysising protease, hydrolysis 4 is termination reaction as a child again; Be warming up to 90 ℃ of enzymes 5 minutes of going out, cooling, paper pulp filtering, spraying drying adds conventional auxiliary material, according to common process, makes tablet.
Embodiment two: the preparation of capsule
After being dissolved in pure donkey-hide gelatin stoste in 1.5 times of weight, homo(io)thermism between 60 ℃, heated and stirred; Regulate pH to 8; Add Alcalase2.4L FG hydrolysising protease, the usage quantity of enzyme is 0.2% of pure donkey-hide gelatin stock solution quality, hydrolysis reaction 3.5 hours; Add 0.35% Alcalase2.4L FG hydrolysising protease, hydrolysis 4.5 is termination reaction as a child again; Be warming up to 85 ℃ of enzymes 4 minutes of going out, cooling, paper pulp filtering, spraying drying, and get final product, add conventional auxiliary material, according to common process, make capsule.
Embodiment three: the preparation of powder
After being dissolved in pure donkey-hide gelatin stoste in 2.5 times of weight water, homo(io)thermism between 55 ℃, heated and stirred; Regulate pH to 7; Add Alcalase2.4L FG hydrolysising protease, the usage quantity of enzyme is 0.35% of pure donkey-hide gelatin stock solution quality, hydrolysis reaction 4.5 hours; Add 0.15% Alcalase2.4L FG hydrolysising protease, hydrolysis 3.5 is termination reaction as a child again; Be warming up to 95 ℃ of enzymes 6 minutes of going out, cooling, paper pulp filtering, spraying drying, and get final product, add conventional auxiliary material, according to common process, make powder.
Embodiment four: the preparation of granule
After being dissolved in pure donkey-hide gelatin stoste in 1.8 times of weight, homo(io)thermism between 52 ℃, heated and stirred; Regulate pH to 7; Add Alcalase2.4L FG hydrolysising protease, the usage quantity of enzyme is 0.25% of pure donkey-hide gelatin stock solution quality, hydrolysis reaction 4 hours; Add 0.25% Alcalase2.4L FG hydrolysising protease, hydrolysis 4 is termination reaction as a child again; Be warming up to 90 ℃ of enzymes 5 minutes of going out, cooling, paper pulp filtering, spraying drying adds conventional auxiliary material, according to common process, makes tablet.
Embodiment five: the preparation of oral liquid
After being dissolved in pure donkey-hide gelatin stoste in 2.2 times of weight water, homo(io)thermism between 58 ℃, heated and stirred; Regulate pH to 8; Add Alcalase2.4L FG hydrolysising protease, the usage quantity of enzyme is 0.35% of pure donkey-hide gelatin stock solution quality, hydrolysis reaction 4.5 hours; Add 0.15% Alcalase2.4L FG hydrolysising protease, hydrolysis 3.5 is termination reaction as a child again; Be warming up to 95 ℃ of enzymes 6 minutes of going out, cooling, paper pulp filtering, spraying drying, and get final product, add conventional auxiliary material, according to common process, make oral liquid.
Claims (6)
1. the preparation method of a donkey-hide gelatin oligopeptide, after it is characterized in that the method comprises the steps: to be dissolved in pure donkey-hide gelatin stoste in 1-3 times of weight water, homo(io)thermism between 30 ℃-80 ℃, heated and stirred; Regulate pH to 6-9; Add Alcalase2.4L FG hydrolysising protease, the usage quantity of enzyme is the 0.1-0.4% of pure donkey-hide gelatin stock solution quality, hydrolysis reaction 3-5 hour; The Alcalase2.4L FG hydrolysising protease that adds again 0.1-0.4%, hydrolysis 3-5 is termination reaction as a child; Be warming up to 80-100 ℃ and went out enzyme 3-8 minute, cooling, paper pulp filtering, spraying drying, and get final product.
2. as the preparation method of claim 1 described a kind of donkey-hide gelatin oligopeptide, after it is characterized in that the method comprises the steps: to be dissolved in pure donkey-hide gelatin stoste in 2 times of weight, homo(io)thermism between 50 ℃-60 ℃, heated and stirred; Regulate pH to 6.5-8.5; Add Alcalase2.4L FG hydrolysising protease, the usage quantity of enzyme is 0.25% of pure donkey-hide gelatin stock solution quality, hydrolysis reaction 4 hours; Add 0.25% Alcalase2.4L FG hydrolysising protease, hydrolysis 4 is termination reaction as a child again; Be warming up to 90 ℃ of enzymes 5 minutes of going out, cooling, paper pulp filtering, spraying drying, and get final product.
3. as the preparation method of claim 1 described a kind of donkey-hide gelatin oligopeptide, after it is characterized in that the method comprises the steps: to be dissolved in pure donkey-hide gelatin stoste in 1.5 times of weight, homo(io)thermism between 60 ℃, heated and stirred; Regulate pH to 8; Add Alcalase2.4L FG hydrolysising protease, the usage quantity of enzyme is 0.2% of pure donkey-hide gelatin stock solution quality, hydrolysis reaction 3.5 hours; Add 0.35% Alcalase2.4L FG hydrolysising protease, hydrolysis 4.5 is termination reaction as a child again; Be warming up to 85 ℃ of enzymes 4 minutes of going out, cooling, paper pulp filtering, spraying drying, and get final product.
4. as the preparation method of claim 1 described a kind of donkey-hide gelatin oligopeptide, after it is characterized in that the method comprises the steps: to be dissolved in pure donkey-hide gelatin stoste in 2.5 times of weight water, homo(io)thermism between 55 ℃, heated and stirred; Regulate pH to 7; Add Alcalase2.4L FG hydrolysising protease, the usage quantity of enzyme is 0.35% of pure donkey-hide gelatin stock solution quality, hydrolysis reaction 4.5 hours; Add 0.15% Alcalase2.4L FG hydrolysising protease, hydrolysis 3.5 is termination reaction as a child again; Be warming up to 95 ℃ of enzymes 6 minutes of going out, cooling, paper pulp filtering, spraying drying, and get final product.
5. as the preparation method of the arbitrary described donkey-hide gelatin oligopeptide of claim 1-4, it is characterized in that adding conventional auxiliary material, according to common process, make tablet, capsule, powder, soft capsule, dripping pill, honeyed bolus, pill, granule, soft extract with bee honey agent, sustained release preparation, quick releasing formulation, controlled release preparation, oral liquid.
6. as the preparation method of the arbitrary described donkey-hide gelatin oligopeptide of claim 1-4, it is characterized in that with the donkey-hide gelatin molecular-weight average after the method hydrolysis below 3000.
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| CN107019215A (en) * | 2017-05-03 | 2017-08-08 | 山东东阿东盛阿胶产品科技开发有限公司 | A kind of gelatin powder production technology |
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| CN107008053A (en) * | 2017-05-03 | 2017-08-04 | 山东东阿东盛阿胶产品科技开发有限公司 | A kind of donkey-hide gelatin stoste filtering technique |
| CN107019215A (en) * | 2017-05-03 | 2017-08-08 | 山东东阿东盛阿胶产品科技开发有限公司 | A kind of gelatin powder production technology |
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