CN117129366A - Method for testing high molecular weight protein extraction rate in protein extract - Google Patents
Method for testing high molecular weight protein extraction rate in protein extract Download PDFInfo
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- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 98
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 98
- 238000000034 method Methods 0.000 title claims abstract description 59
- 238000012360 testing method Methods 0.000 title claims abstract description 50
- 238000000751 protein extraction Methods 0.000 title claims abstract description 24
- 238000000502 dialysis Methods 0.000 claims abstract description 305
- 239000007788 liquid Substances 0.000 claims abstract description 42
- 238000000605 extraction Methods 0.000 claims abstract description 37
- 239000007864 aqueous solution Substances 0.000 claims abstract description 17
- 239000002736 nonionic surfactant Substances 0.000 claims abstract description 16
- 230000008569 process Effects 0.000 claims abstract description 11
- FASSJHMWVWKHAR-DKWTVANSSA-N (2r)-2-amino-3-sulfanylpropanoic acid;urea Chemical compound NC(N)=O.SC[C@H](N)C(O)=O FASSJHMWVWKHAR-DKWTVANSSA-N 0.000 claims abstract description 9
- 239000002131 composite material Substances 0.000 claims abstract description 8
- 238000004108 freeze drying Methods 0.000 claims abstract description 5
- 238000001556 precipitation Methods 0.000 claims abstract description 5
- 239000002994 raw material Substances 0.000 claims abstract description 5
- 239000004627 regenerated cellulose Substances 0.000 claims abstract description 5
- 239000000693 micelle Substances 0.000 claims abstract description 4
- 239000000843 powder Substances 0.000 claims abstract description 4
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 16
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 16
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 16
- 239000000243 solution Substances 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 230000000717 retained effect Effects 0.000 claims description 14
- 239000012530 fluid Substances 0.000 claims description 13
- 238000004090 dissolution Methods 0.000 claims description 11
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims description 10
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 7
- 239000004202 carbamide Substances 0.000 claims description 7
- 239000000385 dialysis solution Substances 0.000 claims description 7
- 239000004201 L-cysteine Substances 0.000 claims description 5
- 235000013878 L-cysteine Nutrition 0.000 claims description 5
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 4
- 229920000053 polysorbate 80 Polymers 0.000 claims description 4
- 230000014759 maintenance of location Effects 0.000 claims description 3
- 238000011049 filling Methods 0.000 description 7
- 239000008399 tap water Substances 0.000 description 6
- 235000020679 tap water Nutrition 0.000 description 6
- 239000003513 alkali Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000002791 soaking Methods 0.000 description 4
- 102000011782 Keratins Human genes 0.000 description 3
- 108010076876 Keratins Proteins 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000002699 waste material Substances 0.000 description 3
- 239000012620 biological material Substances 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000009471 action Effects 0.000 description 1
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- 230000009286 beneficial effect Effects 0.000 description 1
- 229920001222 biopolymer Polymers 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
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- 230000005484 gravity Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000002121 nanofiber Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 235000019633 pungent taste Nutrition 0.000 description 1
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N5/00—Analysing materials by weighing, e.g. weighing small particles separated from a gas or liquid
- G01N5/04—Analysing materials by weighing, e.g. weighing small particles separated from a gas or liquid by removing a component, e.g. by evaporation, and weighing the remainder
Abstract
The invention relates to the technical field of protein testing, and provides a method for testing the extraction rate of high molecular weight protein in protein extract, which comprises the following steps: firstly, placing the protein extract into a dialysis bag with a molecular weight cutoff of 8-12 kDa and made of regenerated cellulose for dialysis, and after the dialysis is finished, obtaining protein powder by adopting isoelectric point precipitation and freeze drying processes, and calculating the protein extraction rate; the protein extract is obtained by dissolving hair serving as a raw material in alkaline L-cysteine-urea composite liquid; when the protein extract is placed in a dialysis bag for dialysis for the first time, the adopted dialysis liquid is an aqueous solution containing a nonionic surfactant, and the concentration of the nonionic surfactant in the dialysis liquid is lower than the critical micelle concentration of the nonionic surfactant.
Description
Technical Field
The invention relates to the technical field of protein testing, in particular to a method for testing the extraction rate of high molecular weight protein in protein extract.
Background
Proteins are biopolymers composed of amino acids as basic units. The protein has a primary structure, a secondary structure, a tertiary structure and a quaternary structure, and the proteins with different structures have different functions, so that the method for extracting the high molecular weight protein from the waste hair has very important significance for high-value utilization of the waste hair in the field of biological materials. In the existing extraction method, the method for extracting the hair keratin by using L-cysteine can promote the protein to be converted into a beta-sheet structure from an alpha-helical structure part to a certain extent, the protein with more beta-sheet structures is more beneficial to improving the performance of biological materials prepared by the protein, and the method also has the characteristics of simple and mild extraction process, but because the method adopts high-concentration urea-alkali solution to promote the dissolution of the keratin, the obtained extract liquid needs to adopt a dialysis method to remove the protein with small molecular weight, so that the extraction effect of the method on the protein with high molecular weight can be determined.
In order to accurately measure the extraction rate of the high molecular weight protein in the extracting solution, the traditional dialysis method is to put the extracting solution into a dialysis bag with the interception molecular weight of 8-12 kDa and the material of regenerated cellulose, put the whole into water (dialyzate) for dialysis so as to minimize the content of urea, alkali, small molecular protein and the like, and then adopt isoelectric point precipitation and/or freeze drying to obtain the protein quality contained in the retaining solution in the dialysis bag, thereby calculating the extraction rate. In the dialysis process, small molecular proteins, urea, alkali and the like in the dialysis bag continuously enter the dialysis liquid, water in the dialysis liquid also continuously enters the dialysis bag, and when the content of the small molecular proteins in the reserved liquid in the dialysis bag is low enough, the dialysis can reach equilibrium, and then the dialysis is finished. During the test, it was found that, due to the large amount of water entering the dialysis bag, the volume of the liquid in the dialysis bag is often increased, and in order to avoid the expansion and rupture of the dialysis bag due to the increase of the volume of the liquid in the dialysis bag, the laboratory often adopts the following modes: during dialysis, reserving more than 50% of the volume in the dialysis bag; however, the probability of breaking the dialysis bag is still high, which results in the forced interruption and failure of the test process for the high molecular weight protein extraction rate, and further, repeated experiments are needed, but the dialysis bag cannot be ensured to be broken, which wastes manpower, material resources and time, increases the test cost, and prolongs the test period.
In view of this, the present invention has been proposed.
Disclosure of Invention
By analyzing the cause of the rupture of the dialysis bag, it is found that the monitoring of the dialysis time is not reasonable in the first dialysis, and the dialysis bag is ruptured when the dialysis time is not reached. Solves the defects of low test efficiency and high test cost caused by overlong dialysis time in the prior art,
specifically, the invention provides a method for testing the extraction rate of high molecular weight protein in protein extract, which comprises the following steps:
firstly, placing the protein extract into a dialysis bag with a molecular weight cutoff of 8-12 kDa and made of regenerated cellulose for dialysis, and after the dialysis is finished, obtaining protein powder by adopting isoelectric point precipitation and freeze drying processes, and calculating the protein extraction rate;
the protein extract is obtained by dissolving hair serving as a raw material in alkaline L-cysteine-urea composite liquid;
when the protein extract is placed in a dialysis bag for dialysis for the first time, the adopted dialysis liquid is an aqueous solution containing nonionic surfactant, and the concentration of the nonionic surfactant in the dialysis liquid is below the critical micelle concentration.
The calculation formula of the protein extraction rate in the invention is as follows: a=w1/w×100%; wherein a is the protein extraction rate, w is the mass of hair dissolution in the protein extract, and w1 is the mass of protein powder.
Generally, the new dialysis bag can be put into service by soaking it in hot water at 80 ℃ for 10 minutes before use. And the use times of the dialysis bag are 3-4 times. The dialysis bag of the present invention needs to reserve a certain volume after being filled with the liquid containing the high molecular weight protein, and the air in the dialysis bag is discharged before the dialysis bag is sealed by adopting the sealing clamp, and the dialysis bag of the present invention is completely filled with the liquid, when one end of the dialysis bag containing the liquid is lifted, the lifted end is observed to be completely filled with no air under the action of self gravity, and the whole dialysis bag is cylindrical.
By observing the traditional dialysis method, the pungent taste of the extracting solution is reduced along with the prolongation of the dialysis time, and is basically not easy to find in the second dialysis, and as the early-stage main micromolecular substances such as urea, alkali and the like enter the dialysis solution from the dialysis bag in dialysis, the early-stage dialysis rate is the fastest, the time for completely filling the dialysis bag in the early stage is shorter than 2 hours basically, and the dialysis bag is easier to break because the overspray is not detected in time, so that the testing process cannot be normally carried out. Wherein the concentration of the nonionic surfactant in the dialysate is critical, and if the concentration exceeds the critical micelle concentration, the subsequent dialysis and testing accuracy will be adversely affected.
According to the method for testing the high molecular weight protein extraction rate in the protein extract, the dialysis process is as follows:
dialysis 1 st: the protein extract is filled into a first batch of dialysis bags, the reserved volume in the first batch of dialysis bags is 50-70%, the dialysis is carried out at the temperature of 5-35 ℃ by taking the aqueous solution containing the nonionic surfactant as the dialysis liquid, the 1 st dialysis is finished until the dialysis bags are completely filled, and the next dialysis is carried out immediately;
performing n times of dialysis on the retention solution obtained by the 1 st time of dialysis until the dialysis is finished; in n times of dialysis, during each time of dialysis, part or all of the reserved liquid after n-1 times of dialysis is filled into n batches of dialysis bags, and the reserved volume in the n batches of dialysis bags is 50-70%; and in each dialysis, the dialysate is replaced by fresh water which is not used as the dialysate, and the n-1 th dialysis bag and the n-th dialysis bag filled with the reserved liquid are continuously dialyzed at the temperature of 5-35 ℃ for more than 8 hours; wherein n is more than or equal to 2;
when the dialysis is performed for more than 8 hours in the n-1 th dialysis, the dialysis bag is completely filled, and the n-th dialysis is performed;
if the dialysis is performed for 8 hours or more in the n-1 th dialysis, the dialysis bag is not filled, and the end of the dialysis is determined.
In general, in order to increase the dialysis efficiency, the retained fluid in the dialysis bag is dispensed into a new dialysis bag when the dialysis fluid is changed for the first time, and the volume is also reserved by 50% or more. In the test, the dialysis bag is used in the mode, so that the swelling and cracking of the dialysis bag can be effectively avoided. Because of the limitation of the mechanical properties of the dialysis bag, the length of the dialysis bag is generally not more than 40cm, and each batch of dialysis bags can comprise more than 1, when the volume of the reserved liquid to be split in the previous batch is too large, a plurality of dialysis bags are needed to be used for dialysis, and the batch of dialysis bags comprise a plurality of dialysis bags.
According to the method for testing the high molecular weight protein extraction rate in the protein extract, the volume ratio of the retention solution in the dialysis bag to the dialysis solution is 200 ml:40-60L during dialysis.
According to the method for testing the extraction rate of the high molecular weight protein in the protein extract, the nonionic surfactant is Tween 20 and/or Tween 80.
According to the method for testing the extraction rate of the high molecular weight protein in the protein extract, the nonionic surfactant is Tween 20.
In the test, although tween 20 and tween 80 can both effectively shorten the time of first dialysis and reduce the probability of test failure, tween 80 has a lower effect of improving the overall dialysis efficiency than tween 20. Further preferably, the dialysate used in the first dialysis is an aqueous solution of tween 20 at a concentration of 0.015 to 0.035mm to minimize its effect on the subsequent rate of dialysate dialysis.
According to the method for testing the high molecular weight protein extraction rate in the protein extract, the nth dialysis bag is placed in an aqueous solution containing a nonionic surfactant for soaking for 20-40 min and then used, wherein n is more than or equal to 2.
According to the method for testing the high molecular weight protein extraction rate in the protein extract, except for the first batch of dialysis bags, all the dialysis bags are soaked in hot water at the temperature of below 80 ℃ for 8-15 minutes, and then are soaked in aqueous solution of tween 20 with the concentration of below 0.04-0.055 mM for 20-40 minutes for use.
The dialysis bag is longer in use time, the dialysis effect is poorer, the dialysis bag is generally subjected to 3 times of dialysis to an equilibrium state, and needs to be replaced by a new dialysis bag, so that the extract with higher extraction rate needs a long time to reach the end of dialysis, the test cost is high, moreover, the dialysis rate gradually changes gradually at the later stage of dialysis, in order to shorten the dialysis time, the use of the dialysis bag is reduced, although the prior art suggests the use of flowing dialysate, heating the dialysate, centrifuging and the like, other equipment or a specific dialysis bag device is required to be introduced, or the heating process can lead to further degradation of the extract, which is unfavorable for the accuracy of the test, therefore, the invention deeply researches the relation of the movement process of protein in the dialysis process and the quality of the high molecular weight protein which is concerned by the test extraction rate, and discovers that before the dialysis bag is put into use, the dialysis bag is soaked in the aqueous solution of 20 in addition to the conventional cleaning mode, the end of dialysis is facilitated in a shorter time, and the influence on the accuracy of the extraction rate is small.
According to the method for testing the high molecular weight protein extraction rate in the protein extract, the dissolution rate of human hair in the protein extract is more than or equal to 30%, and the protein extraction rate is more than or equal to 25%.
According to the method for testing the high molecular weight protein extraction rate in the protein extract, the dissolution rate of human hair in the protein extract is more than or equal to 70%.
The method for extracting the protein in the hair by using the L-cysteine has the advantages that the extraction rate is generally within 60%, the higher the extraction rate is, the higher the dissolution rate is, the higher the amount of small molecular weight protein in the protein extract with high extraction rate is, and the corresponding dialysis time is obviously increased.
According to the method for testing the high molecular weight protein extraction rate in the protein extract, the protein extract is obtained by dissolving human hair as a raw material in an L-cysteine-urea composite liquid with the pH value of more than or equal to 8;
in the L-cysteine-urea composite liquid, the content of L-cysteine is 2-20 g/L, and the content of urea is more than 1M; the urea content is preferably 8M;
the mass volume ratio of the human hair to the L-cysteine-urea composite liquid is 1 g:15-25 ml.
According to the method for testing the high molecular weight protein extraction rate in the protein extract, provided by the invention, the specific surfactant is used in a specific method in the dialysis process, so that the time for completely filling the dialysis bag in the first dialysis can be shortened, the dialysis efficiency is improved, and the condition of test failure caused by human negligence is avoided.
Furthermore, by carrying out specific pretreatment on the dialysis bag, the dialysis balance can be achieved by using fewer dialysis bags and shorter dialysis time, so that the test efficiency and test cost of the high molecular weight protein extraction rate in the protein extract are greatly improved.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or equipment used were conventional products available for purchase by regular vendors without the manufacturer's attention. The dialysis bag adopted by the invention has the specification as follows: the molecular weight cut-off is 8k, the material is regenerated cellulose, the flattening width is 30-45 mm, and the length of the dialysis bag is 35-40 cm when the dialysis bag is used each time. According to the method disclosed by the invention, protein extract A and protein extract B are obtained according to the method of paper fluorescent keratin nanofiber preparation and application performance research thereof, wherein during extraction, the urea content is 8M, the L-cysteine content is 20g/L and 10g/L respectively, and the corresponding dissolution rate and the extraction rate tested according to the traditional method are as follows:
extract a: the dissolution rate is 85% and the extraction rate is 48%.
Extract B: the dissolution rate is 43 percent, and the extraction rate is 34 percent.
Example 1
A method for testing the extraction rate of high molecular weight protein in protein extract comprises the following steps:
(1) Dialysis 1 st: 200ml of extracting solution A is filled into a first batch of dialysis bags, two ends of the dialysis bags are sealed by dialysis bag clamps, the reserved volume in the first batch of dialysis bags is 70%, aqueous solution containing Tween 20 is taken as dialysis solution, dialysis is carried out at 25 ℃, the volume ratio of the extracting solution to the dialysis solution is 200ml:50L during dialysis, the concentration of Tween 20 in the dialysis solution is 0.02mM, and before the dialysis bags are used, the dialysis bags are placed into 80 ℃ hot water for 10 minutes for use. The discovery is as follows: at 1.5h of dialysis, the dialysis bag was completely filled and was immediately subjected to dialysis at 2 nd time.
(2) Dialysis 2 nd: taking out the dialysis bags in the step (1), and filling 50% of the retained fluid into a second batch of dialysis bags, wherein the reserved volume in the second batch of dialysis bags is 50%. Wherein, before the second batch of dialysis bags is used, the dialysis bags are soaked in hot water at 80 ℃ for 10 minutes, and then are placed in an aqueous solution of Tween 20 with the concentration of 0.05mM, and soaked for 30 minutes at 25 ℃.
Simultaneously, the equal volume of the dialysate is replaced by fresh tap water which is not used as the dialysate, the first and second batches of dialysis bags filled with the reserved liquid are continuously dialyzed for 8 hours at normal temperature, the dialysis bags are found to be completely filled, and the 3 rd dialysis is immediately carried out.
(3) Dialysis 3 rd: and (3) taking out the dialysis bags in the step (2), and filling 50% of the retained fluid into a third batch of dialysis bags, wherein the reserved volume in the third batch of dialysis bags is 50%. Wherein, before the third batch of dialysis bags is used, the dialysis bags are soaked in hot water at 80 ℃ for 10 minutes, and then the dialysis bags are soaked in aqueous solution of Tween 20 with the concentration of 0.05mM for 30 minutes at normal temperature.
And simultaneously, replacing the equal volume of the dialysis liquid with fresh tap water which is not used as the dialysis liquid, continuing to dialyze the first, second and third batches of dialysis bags filled with the reserved liquid at normal temperature for 8 hours, and immediately dialyzing the first, second and third batches of dialysis bags for 4 th time after the dialysis bags are completely filled.
(4) Dialysis 4 th time: taking out the dialysis bags in the step (3), and filling all the retained fluid in the first batch of dialysis bags, 50% of the retained fluid in the second batch of dialysis bags and the third batch of dialysis bags into a fourth batch of dialysis bags, wherein the reserved volume in the fourth batch of dialysis bags is 50%. Before the fourth lot of dialysis bags are used, the dialysis bags are soaked in hot water at 80 ℃ for 10 minutes, and then the dialysis bags are placed in an aqueous solution of Tween 20 with the concentration of 0.05mM, and soaked for 30 minutes at normal temperature.
Simultaneously, the equal volume of the dialysate is replaced by fresh tap water which is not used as the dialysate, the second, the third and the fourth dialysis bags filled with the reserved liquid are continuously dialyzed for 8 hours at normal temperature, and the 5 th dialysis is immediately carried out after the dialysis bags are found to be completely filled.
(5) Dialysis 5 th time: and (3) taking out the dialysis bags in the step (4), and filling 50% of all the retained fluid in the second lot of dialysis bags, the third lot of dialysis bags and the fourth lot of dialysis bags into a fifth lot of dialysis bags, wherein the reserved volume in the fifth lot of dialysis bags is 50%. Before the fifth lot of dialysis bags are used, the dialysis bags are soaked in hot water at 80 ℃ for 10 minutes, and then the dialysis bags are soaked in aqueous solution of Tween 20 with the concentration of 0.05mM for 30 minutes at normal temperature.
Meanwhile, the equal volume of the dialysate is replaced by fresh tap water which is not used as the dialysate, the third, fourth and fifth batches of dialysis bags filled with the reserved liquid are continuously dialyzed at normal temperature, the volume of the reserved liquid in the dialysis bags is slowly increased, and the dialysis bags are not fully filled when the dialysis is performed for 8 hours, so that the end of the dialysis is considered.
(6) Isoelectric point precipitation.
(7) And (5) freeze-drying and weighing, wherein the extraction rate of the high-molecular-weight protein in the extracting solution A is 48.3 percent according to a protein extraction rate calculation formula.
Example 2
The procedure for testing the extraction yield of high molecular weight proteins in the protein extract was essentially the same as in example 1, except that:
the dialysate in step (1) is replaced by water in equal volume.
The result shows that:
at dialysis 1, when dialysis was carried out for 2.3 hours, the dialysis bag was found to be broken, the operation was repeated, and the dialysis 1 was continuously monitored, and when dialysis was carried out for 2 hours, the dialysis bag was found to be completely filled, and the dialysis 2 nd was carried out immediately.
The dialysis was carried out up to the 5 th time, and at the 5 th dialysis, the volume of the retained liquid in the dialysis bag was slightly larger than in example 1, but it was not completely filled, and the high molecular weight protein extraction rate in the extract A was calculated to be 48.2% in step (7).
Example 3
A method for testing the extraction rate of high molecular weight protein in a protein extract is basically the same as in example 1, except that:
all the dialysis bags except the first batch of dialysis bags are put into use by soaking the dialysis bags in hot water at 80 ℃ for 10 minutes before use.
The result shows that:
although the dialysis bag was completely filled at 1.5h of dialysis, the dialysis bag was completely filled at 5h to 8h of dialysis. The following 6 th and 7 th dialysis were continued:
dialysis 6 th: the dialysis bag for the 5 th dialysis was removed, and the whole of the retained fluid in the third lot of dialysis bags, the fourth lot of dialysis bags and 50% of the retained fluid in the fifth lot of dialysis bags were filled into the sixth lot of dialysis bags, the reserved volume in the sixth lot of dialysis bags being 50%. Wherein, before the sixth batch of dialysis bags is used, the dialysis bags are only put into use after being soaked in hot water at 80 ℃ for 10 minutes. Simultaneously, the equal volume of the dialysate is replaced by fresh tap water which is not used as the dialysate, the fourth, fifth and sixth dialysis bags filled with the reserved liquid are continuously dialyzed for 8 hours at normal temperature, and the 7 th dialysis is immediately carried out after the dialysis bags are found to be completely filled.
Dialysis 7 th time: the dialysis bag for the 6 th dialysis was removed, and the entire retained fluid in the fourth dialysis bag, the fifth dialysis bag, and 50% of the retained fluid in the sixth dialysis bag were filled into the seventh dialysis bag, the reserved volume in the seventh dialysis bag being 50%. Wherein, before the seventh lot of dialysis bags are used, the dialysis bags are only put into use after being soaked in hot water at 80 ℃ for 10 minutes. Meanwhile, the equal volume of the dialysate is replaced by fresh tap water which is not used as the dialysate, the fifth, sixth and seventh dialysis bags filled with the reserved liquid are continuously dialyzed at normal temperature, the volume of the reserved liquid in the dialysis bags is slowly increased, and the dialysis bags are not fully filled when the dialysis is performed for 8 hours, so that the end of the dialysis is considered.
And further subjected to the operations of step (6) and step (7) in example 1, it was found that the high molecular weight protein extraction rate in the extract A was 48%.
This means that pretreatment of the dialysis bag with an aqueous solution of tween 20 at a concentration of 0.05mM plays a significant role in improving the dialysis efficiency, and since the larger the volume of the retained fluid in the dialysis bag, the more the dialysis bag is used, and accordingly, the method also greatly saves the use of the dialysis bag, significantly reduces the test cost, and at the same time, the method of the present invention does not affect the test result of the extraction rate.
Example 4
A method for testing the extraction rate of high molecular weight protein in a protein extract is basically the same as in example 1, except that:
the equivalent volume of extract A of step (1) of example 1 was replaced with extract B.
It was found that the 1 st dialysis was completely filled in the dialysis bag at 1.1h, the 2 nd dialysis was completely filled in the dialysis bag, the 3 rd dialysis was slow in the volume increase of the remaining solution in the dialysis bag, and the dialysis was completed as the dialysis was completed without filling in the dialysis bag at 8 h.
And further subjected to the operations of step (6) and step (7) in example 1, it was found that the high molecular weight protein extraction rate in this extract B was 34.2%.
Example 5
A method for testing the extraction rate of high molecular weight protein in a protein extract is basically the same as in example 4, except that:
all the other dialysis bags except the first batch of dialysis bags are put into use by soaking the dialysis bags in hot water at 80 ℃ for 10 minutes before use.
It was found that the dialysis bag was completely filled during dialysis for 1.1h, and the dialysis bag was completely filled during dialysis for 2 nd, and the volume of the retained fluid in the dialysis bag was slightly slower during dialysis for 3 rd than in example 4, and the end of dialysis was determined.
And further subjected to the operations of step (6) and step (7) in example 1, it was found that the high molecular weight protein extraction rate in this extract B was 34.1%.
From the results of examples 4 and 5 described above, it can be seen that the extent to which the proposed method improves the dialysis efficiency of the extract decreases significantly as the content of small molecular proteins in the extract decreases, and thus the method of the present invention is more suitable for testing the extraction rate of high molecular weight proteins in the extract with a very high dissolution rate.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A method for testing the extraction rate of high molecular weight protein in protein extract is characterized by comprising the following steps:
firstly, placing the protein extract into a dialysis bag with a molecular weight cutoff of 8-12 kDa and made of regenerated cellulose for dialysis, and after the dialysis is finished, obtaining protein powder by adopting isoelectric point precipitation and freeze drying processes, and calculating the protein extraction rate;
the protein extract is obtained by dissolving hair serving as a raw material in alkaline L-cysteine-urea composite liquid;
when the protein extract is placed in a dialysis bag for dialysis for the first time, the adopted dialysis liquid is an aqueous solution containing nonionic surfactant, and the concentration of the nonionic surfactant in the dialysis liquid is below the critical micelle concentration.
2. The method for testing the extraction rate of high molecular weight protein in a protein extract according to claim 1, wherein the dialysis process is as follows:
dialysis 1 st: the protein extract is filled into a first batch of dialysis bags, the reserved volume in the first batch of dialysis bags is 50-70%, the dialysis is carried out at the temperature of 5-35 ℃ by taking the aqueous solution containing the nonionic surfactant as the dialysis liquid, and the 1 st dialysis is finished when the dialysis bags are completely filled;
performing n times of dialysis on the retention solution obtained by the 1 st time of dialysis until the dialysis is finished; in n times of dialysis, during each time of dialysis, part or all of the reserved liquid after n-1 times of dialysis is filled into n batches of dialysis bags, so that the reserved volume in the n batches of dialysis bags is 50-70%; and in each dialysis, the dialysate is replaced by water which is not used as the dialysate, and the n-1 th dialysis bag and the n-th dialysis bag filled with the reserved liquid are continuously dialyzed at the temperature of 5-35 ℃ for more than 8 hours; wherein n is more than or equal to 2;
when the dialysis is performed for more than 8 hours in the n-1 th dialysis, the dialysis bag is completely filled, and the n-th dialysis is performed;
if the dialysis is performed for 8 hours or more in the n-1 th dialysis, the dialysis bag is not filled, and the end of the dialysis is determined.
3. The method for testing the extraction rate of high molecular weight protein in protein extract according to claim 2, wherein the volume ratio of the retained fluid in the dialysis bag to the dialysis fluid is 200 ml:40-60L during dialysis.
4. The method for testing the extraction rate of high molecular weight protein in a protein extract according to claim 1, wherein the nonionic surfactant is tween 20 and/or tween 80.
5. The method for measuring the extraction rate of high molecular weight protein in a protein extract according to claim 4, wherein the nonionic surfactant is Tween 20.
6. The method for testing the extraction rate of high molecular weight proteins in a protein extract according to claim 2, wherein the nth dialysis bag is used after being immersed in an aqueous solution containing a nonionic surfactant for 20-40 min, wherein n is not less than 2.
7. The method according to claim 6, wherein all the other dialysis bags except the first dialysis bag are immersed in an aqueous solution of tween 20 having a concentration of 0.04 to 0.055mm for 20 to 40 minutes.
8. The method for testing the extraction rate of high molecular weight protein in a protein extract according to any one of claims 1 to 7, wherein the dissolution rate of human hair in the protein extract is not less than 30% and the protein extraction rate is not less than 25%.
9. The method for testing the extraction rate of high molecular weight protein in a protein extract according to claim 8, wherein the dissolution rate of human hair in the protein extract is not less than 70%.
10. The method for testing the extraction rate of high molecular weight protein in protein extract according to claim 8, wherein the protein extract is obtained by dissolving human hair as a raw material in an L-cysteine-urea complex liquid with a pH value of not less than 8;
in the L-cysteine-urea composite liquid, the content of L-cysteine is 2-20 g/L, and the content of urea is more than 1M;
the mass volume ratio of the human hair to the L-cysteine-urea composite liquid is 1 g:15-25 ml.
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