CN110836936A - Method for measuring protein hydrolysis degree - Google Patents

Method for measuring protein hydrolysis degree Download PDF

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CN110836936A
CN110836936A CN201911178827.0A CN201911178827A CN110836936A CN 110836936 A CN110836936 A CN 110836936A CN 201911178827 A CN201911178827 A CN 201911178827A CN 110836936 A CN110836936 A CN 110836936A
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polypeptide
protein
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amino acid
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王静
杨桂兰
耿凤
郭文逸
王欣
郭学平
刘爱华
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Huaxi Biotechnology Co Ltd
Bloomage Biotech Co Ltd
Shandong Bloomage Hyinc Biopharm Co Ltd
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Shandong Bloomage Hyinc Biopharm Co Ltd
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Abstract

The invention belongs to the technical field of product inspection, and relates to a method for measuring the hydrolysis degree of protein, which comprises the following steps: measuring the molecular weight distribution of the polypeptide by using a high-efficiency gel filtration chromatography method for the protein hydrolysate, calculating the mass percentage content of the polypeptide with different molecular weight ranges, and further calculating to obtain the content of the polypeptide, the content of free amino acid and the degree of proteolysis; and the content of free amino acid, the content of polypeptide and the degree of protein hydrolysis in a series of protein standard solutions with different degrees of hydrolysis can be measured by an accurate method, and a standard curve is drawn to quantify more accurately. The method realizes the simultaneous determination of the polypeptide content, the free amino acid content and the hydrolysis degree in the sample by using the high-efficiency gel filtration chromatography, and is simpler and quicker than the traditional determination method. The method has high correlation and accuracy between the result and the measurement result of the traditional method, and provides a quick and reliable method for monitoring the process of industrially producing the oligopeptide.

Description

Method for measuring protein hydrolysis degree
Technical Field
The invention belongs to the technical field of product inspection, and relates to a method for measuring the hydrolysis degree of protein.
Background
The oligopeptide is polypeptide consisting of 2-10 amino acids, has higher nutritive value and health care function, and is the most economic component in a polypeptide product. In the processes of protein enzymolysis and polypeptide preparation, in order to improve the oligopeptide content, the enzymolysis degree of protein needs to be controlled, and the content of free amino acids and macromolecular peptides needs to be reduced as much as possible. Therefore, in order to control the enzymatic hydrolysis reaction process, the polypeptide product is usually characterized by taking the polypeptide content, the free amino acid content, the hydrolysis degree and the molecular weight distribution of the polypeptide as indexes.
The polypeptide content refers to the content of peptide containing 2-50 amino acids, usually trichloroacetic acid is used to precipitate macromolecular protein in the hydrolysate, then the amino acid content is determined by Kjeldahl nitrogen determination method, and the polypeptide content is obtained by subtracting the free amino acid content. The content determination of free amino acid comprises high performance liquid chromatography, ion exchange chromatography, gas chromatography and capillary electrophoresis, wherein the pre-column derivatization high performance liquid chromatography has the advantages of short analysis time, high sensitivity and the like, and is the most widely applied determination method at present. The degree of hydrolysis is the ratio of peptide bonds of protein molecules to total peptide bonds, and commonly used determination methods include ninhydrin colorimetry, formaldehyde titration, pH-stat method and the like. The molecular weight distribution of the polypeptide is mainly determined by high-efficiency gel filtration chromatography, has the characteristics of high efficiency, high speed and high sensitivity, and is a method for rapidly and slightly determining the molecular weight distribution of the polypeptide, such as the method for determining the molecular weight distribution of the bovine bone polypeptide by high-efficiency gel chromatography in patent CN 106309372A.
In the production process, in order to control the enzymolysis degree, various indexes of the polypeptide product need to be measured respectively, which not only increases the production difficulty, but also wastes time and cannot be measured continuously. Therefore, it is necessary to develop a method for simultaneously detecting the degree of proteolysis, the content of polypeptide and free amino acid, so as to be suitable for rapid real-time detection in industrial production.
Disclosure of Invention
Aiming at the problem that a method for simultaneously detecting the protein hydrolysis degree and the content of polypeptide and free amino acid is lacked at present, the invention provides the method for measuring the protein hydrolysis degree, which does not need special equipment, is simple and has high reliability.
In order to achieve the purpose, the invention adopts the following technical scheme.
A method for measuring the degree of hydrolysis of a protein, comprising the steps of:
(1) diluting the hydrolyzed protein solution into protein hydrolysate with proper concentration by using buffer solution;
(2) measuring the molecular weight distribution of the polypeptide with high performance gel filtration chromatography, and calculating the mass percentage content X of the polypeptide with different molecular weight rangesaWherein a is molecular weight;
(3) and (3) result and calculation:
(a) the polypeptide content in the sample is calculated according to the following formula:
polypeptide content (%) = Σ Xa，
Wherein a = 200Da-10000 Da;
(b) the content of free amino acids in the sample was calculated according to the following formula:
free amino acid content (%) = Xa，
Wherein a <200 Da;
(c) the degree of proteolysis, calculated according to the following formula:
degree of proteolysis (%) = Σ (128X)a/a’），
Wherein 128 is the average molecular weight of the amino acid,
a' is the average molecular weight of polypeptides of different molecular weight ranges.
In the step (1), the concentration of the protein hydrolysate is 20-200mg protein/mL.
In the step (1), the pH value of the buffer solution is 6.5-8.0; the concentration of the buffer solution is 20-50mmol/L (mM). Preferably, the buffer is selected from the group consisting of phosphate buffered saline (PBS buffer), phosphate buffered saline (PB buffer), Britton-Robinson's Universal buffer, Clark-Lubs buffer, Tris-HCl buffer.
In the step (2), the detection conditions of the high performance gel filtration chromatography can be obtained by adopting technical means known in the field; preferably, the chromatographic conditions of the high performance gel filtration chromatography are as follows:
a chromatographic column: superdex peptide 10/300 GL;
mobile phase: 0.10-0.20M NaCl, 20-50mM pH7.2 phosphate buffer;
flow rate: 0.2-1.0 mL/min;
detection wavelength: 214 nm;
pressure: not more than 1.8 Mpa;
sample introduction volume: 20-100 muL.
Preferably, the method for determining the degree of proteolysis further comprises the steps of preparing a standard curve and calculating the accurate polypeptide content, free amino acid content and degree of proteolysis content:
(i) respectively measuring the content of free amino acid, the content of polypeptide and the degree of protein hydrolysis of a series of protein standard solutions with different degrees of hydrolysis according to the methods of the steps (1) to (3);
(ii) (ii) determining the exact content of free amino acids and polypeptides and the exact degree of proteolysis from a series of protein standard solutions of different degrees of hydrolysis in (i);
(iii) taking the content of free amino acid in the step (i) as a vertical coordinate, and taking the accurate content of the free amino acid as a horizontal coordinate to make a free amino acid content standard curve;
taking the polypeptide content in (i) as a vertical coordinate, and taking the accurate polypeptide content as a horizontal coordinate to make a polypeptide content standard curve;
(ii) taking the degree of proteolysis in the step (i) as an ordinate, and taking the accurate degree of proteolysis as an abscissa to make a standard curve of the degree of proteolysis;
(iv) and (5) respectively substituting the free amino acid content, the polypeptide content and the protein hydrolysis degree of the protein hydrolysate obtained by determination into the standard curve in the step (iii) to calculate and obtain the accurate free amino acid content, polypeptide content and protein hydrolysis degree.
In steps (ii) and (iii), the free amino acid content can be determined by paper chromatography or pre-column derivatization high performance liquid chromatography;
the polypeptide content determination method can use a biuret method, an o-phthalaldehyde method, an ultraviolet absorption method or a trichloroacetic acid method;
the degree of proteolysis may be determined by pH-state, trinitrobenzene sulfonic acid, formaldehyde titration or ninhydrin colorimetry.
The invention has the following advantages:
the method realizes the simultaneous determination of the polypeptide content, the free amino acid content and the hydrolysis degree in the sample by using the high-efficiency gel filtration chromatography, and is simpler and quicker than the traditional determination method. The method has high correlation, namely high accuracy, with the measurement result of the traditional method, and provides a quick and reliable method for monitoring the process of industrially producing oligopeptides.
Drawings
FIG. 1 is a molecular weight standard curve for a polypeptide;
FIG. 2 is a standard curve of the free amino acid content of BSA protein hydrolysate;
FIG. 3 is a standard curve of the content of free amino acids in rice protein hydrolysate;
FIG. 4 is a standard curve for the polypeptide content of BSA protein hydrolysate;
FIG. 5 is a standard curve of polypeptide content in rice protein hydrolysate;
FIG. 6 is-NH2A base content standard curve;
FIG. 7 is a standard curve of the degree of proteolysis of BSA proteolysis solutions;
FIG. 8 is a standard curve of the degree of proteolysis of rice protein hydrolysate.
Detailed Description
The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited to the following examples.
EXAMPLE 1 preparation of protein hydrolysate
Taking a certain amount of Bovine Serum Albumin (BSA) and rice protein, preparing into a protein solution with the concentration of 10mg/mL in a 20mM phosphate buffer solution with the pH of 8.0 at 50 ℃, adding trypsin with the concentration of 1% of the protein mass, performing enzymolysis for 1h, 2h, 3h, 4h, 5h and 6h, respectively taking 10mL samples, immediately placing the samples in a water bath with the temperature of 100 ℃ for 10min for enzyme deactivation after taking out the samples, and obtaining proteolytic samples with different degrees of hydrolysis.
Example 2 determination of the degree of proteolysis
The molecular weight distribution of the polypeptide was determined by high performance gel filtration chromatography on the proteolyzed sample of example 1, and the degree of proteolysis was calculated:
(1) polypeptide molecular weight standard curve: standard molecular weight polypeptides: cytochrome C (Mw 12500), aprotinin (Mw 6512), oxidized glutathione (Mw 615), reduced glutathione (Mw 310), glycine (Mw 6512) and aprotinin (Mw 75) are prepared into 10mg/mL solutions respectively in 20mM phosphate buffer solution with pH 8.0;
(2) chromatographic conditions are as follows: a chromatographic column: superdex peptide 10/300 GL; mobile phase: 0.15M NaCl, 50mM pH7.2 phosphate buffer; detection wavelength: 214 nm; flow rate: 0.5 ml/min; pressure: not more than 1.8 Mpa; elution volume: 30mL (60 min); sample introduction volume: 50 muL; column temperature: room temperature;
(3) and (3) detection: detecting the protein hydrolysate with different degrees of hydrolysis and the polypeptide solution with standard molecular weight in the example 1 according to the chromatographic conditions in the step (2);
(4) the method for calculating the molecular weight distribution of the sample comprises the following steps: the molecular weight distribution range of the sample can be obtained by substituting chromatographic data of the sample into a polypeptide molecular weight standard curve by using active G2182AA GPC data processing software (A.02.02). The relative percentages of peptides in different molecular weight ranges were calculated by peak area normalization according to the following formula:
Figure 3994DEST_PATH_IMAGE001
in the formula:
Xathe mass fraction of the peptide segment in the sample based on a certain relative molecular mass
A-peak area of peptide segment of certain relative molecular mass;
Ageneral assembly-sum of peak areas of the peptide fragments of respective relative molecular masses.
(5) Calculating the polypeptide content, the free amino acid content and the protein hydrolysis degree in the protein hydrolysate:
(a) the polypeptide content in the sample is calculated according to the following formula:
polypeptide content (%) = Σ Xa
Wherein a = 200Da-10000 Da;
(b) the content of free amino acids in the sample was calculated according to the following formula:
free amino acid content (%) = Xa
Wherein a <200 Da;
(c) the degree of proteolysis, calculated according to the following formula:
degree of proteolysis (%) = Σ (128X)a/a’）
Wherein 128 is the average molecular weight of the amino acid,
a' is the average molecular weight of polypeptides of different molecular weight ranges.
(6) Results
The retention times and retention volumes of the polypeptides with different standard molecular weights are shown in Table 1, and a polypeptide molecular weight standard curve lgM is preparedwT, as shown in FIG. 1, the fitting equation is lgMw= 6.3053- 0.0894t，R2=0.9713；
TABLE 1 Retention time and Retention volume for Standard molecular weight Polypeptides
Figure 124397DEST_PATH_IMAGE002
。
Substituting the chromatographic data of the sample into the equation according to the formula in the step (4) to calculate, and obtaining the molecular weight distribution of the polypeptide in the enzymolysis liquid obtained by different enzymolysis time of the sample BSA and the rice protein, as shown in tables 2 and 3:
TABLE 2 molecular weight distribution of polypeptides in BSA enzymatic solutions at different enzymatic hydrolysis times
Figure 169713DEST_PATH_IMAGE003
TABLE 3 polypeptide molecular weight distribution of rice protein enzymatic hydrolysate at different enzymatic hydrolysis times
Figure 269650DEST_PATH_IMAGE004
。
Calculating the polypeptide content (%) of the enzymatic hydrolysate obtained by the different enzymatic hydrolysis times of BSA and rice protein according to the formula of steps (5) - (a) and the data in tables 2 and 3, and the results are shown in Table 4:
TABLE 4 polypeptide content of BSA and rice protein hydrolysate
Figure 349601DEST_PATH_IMAGE005
。
Calculating the content (%) of free amino acids in the enzymatic hydrolysate obtained by the different enzymatic hydrolysis times of BSA and rice protein according to the formulas of steps (5) - (b) and the data in tables 2 and 3, and the results are shown in Table 5:
TABLE 5 free amino acid content of BSA and rice protein hydrolysate
Figure 172064DEST_PATH_IMAGE006
。
Calculating the proteolysis degree (%) of the enzymatic hydrolysate obtained by different enzymatic hydrolysis time of BSA and rice protein according to the formulas of steps (5) - (c) and the data of tables 2 and 3, wherein the values of a' are 12000, 8000, 4500, 2100, 700, 128 respectively, which represent the average molecular weight of polypeptides of >10000Da, 10000-:
TABLE 6 degree of proteolysis of BSA and rice protein hydrolysates
Figure 642359DEST_PATH_IMAGE007
。
(7) Method for determining content of free amino acid by using precolumn derivatization high performance liquid chromatography
Norleucine internal standard solution: about 10mg of norleucine was weighed and dissolved in 10mL of 0.1mol/L hydrochloric acid solution.
Mixed amino acid standard solution (table 7): 1.0mL of amino acid standard solution is accurately measured and placed in a 5mL volumetric flask, 0.1mol/L hydrochloric acid solution is added to the volumetric flask to be constant volume to a scale, and the solution is diluted by 5 times.
Accurately measuring 200 mu L of each of the amino acid standard solution and the hydrolyzed protein solution obtained in the example 1, and respectively placing the amino acid standard solution and the hydrolyzed protein solution in a 1.5mL centrifuge tube; accurately adding 20 mu L of norleucine internal standard solution into each centrifuge tube; adding 100 mu L of triethylamine acetonitrile solution and 100 mu L of phenyl isothiocyanate acetonitrile solution into each centrifuge tube respectively, mixing uniformly, and standing for 1 hour at room temperature; then adding 400 mu L of normal hexane into each centrifugal tube, shaking and standing for 10 min; taking the lower layer solution (PTC-AA), and filtering with 0.45 μm needle filter; 200 μ L of the filtrate was taken, diluted with 800 μ L of water, shaken well and injected with 10 μ L of the sample.
TABLE 7 amino acid standard solution concentrations
Figure 106839DEST_PATH_IMAGE008
Chromatographic conditions are as follows: venusil AA amino acid analytical column 4.6X 250nm, 5 μm; column temperature: 40 ℃; detection wavelength: 254 nm; mobile phase A: weighing 15.2g of anhydrous sodium acetate, adding 1850mL of water, dissolving, adjusting the pH to 6.5 with glacial acetic acid, adding 140mL of acetonitrile, mixing uniformly, and filtering with a 0.45-micron filter membrane; mobile phase B: 80% acetonitrile in water; the mobile phase gradient was as follows:
Figure DEST_PATH_IMAGE010AA
。
the amino acid concentration in the sample was calculated according to the following formula:
certain amino acid concentration (μ g/mL) = f1/f2×C
In the formula:
f1-peak area of certain amino acid/peak area of internal standard in sample solution;
f2mixing the peak areas of the amino acids in the amino acid standard solution/the peak area of the internal standard;
c-concentration of certain amino acids controls (. mu.g/mL).
Total amino acid content (%) = concentration of various amino acids and × V × 10-6×100/W
In the formula:
v is the volume (mL) of the sample solution;
w is the mass (g) of the sample.
TABLE 8 determination of free amino acid content by pre-column derivatization HPLC
Figure 713401DEST_PATH_IMAGE011
As can be seen from the data in Table 8 in combination with the data in Table 5, the measurement results of gel filtration chromatography are slightly higher than those of pre-column derivatization HPLC because some small molecular impurities exist in the part with molecular weight less than 200Da, and the gel filtration chromatography can only distinguish different substances according to the molecular weight and cannot distinguish impurities and amino acids.
The free amino acid contents in table 5 and table 8 were plotted on the ordinate and the abscissa, respectively, to obtain standard curves, the BSA protein hydrolysate sample was as shown in fig. 2, and the rice protein hydrolysate sample was as shown in fig. 3, which were fitted with y = -0.12352+ 1.22402x and y = 1.6029+ 0.93429x, respectively, and the correlation coefficients (R) thereof were 0.9778 and 0.9910, respectively. It can be seen that the correlation between the two measurements is very good, indicating that gel filtration chromatography can be used to more accurately characterize the amino acid content.
(8) Determination of polypeptide content by trichloroacetic acid precipitation method
Taking 10mL of sample, adding 10mL of 10% trichloroacetic acid solution, mixing uniformly, standing for 30min, centrifuging at 12000rpm for 5min, taking supernatant, and determining the content of the polypeptide by using a Kjeldahl method. Calculating the content of free amino acid determined in step (7) and according to the following formula:
Figure 910027DEST_PATH_IMAGE012
in the formula:
content (%) of X-polypeptide;
v-volume of HCl standard solution consumed by the sample (mL);
V0-volume of blank consumed HCl standard solution (mL);
concentration of N-HCl standard solution (mol/L);
0.014-1 mol/1 mL of LHCl standard solution corresponds to the gram of nitrogen;
m-mass of sample (g);
conversion factor of F-nitrogen (5.95 for rice protein, 6.25 for BSA).
TABLE 9 determination of polypeptide content by trichloroacetic acid precipitation
Figure 664356DEST_PATH_IMAGE013
As can be seen from the data in Table 9 and the data in Table 4, the polypeptide contents of the BSA sample and the rice protein sample measured by the two methods showed increasing trends as the enzymolysis time increased, and the results of the two methods were similar.
The polypeptide content in table 4 is plotted as ordinate and the polypeptide content in table 9 is plotted as abscissa, the BSA protein hydrolysate sample is shown in fig. 4, the rice protein hydrolysate sample is shown in fig. 5, the fitting curves are y = -6.96975+ 1.14191x and y = 24.92128+ 0.70646x, and the correlation coefficients are 0.9889 and 0.9813, respectively. As can be seen from the figure, the polypeptide contents of the BSA sample and the rice protein sample obtained by the two measuring methods have better correlation, so that the polypeptide content can be measured and calculated by using a gel filtration chromatography, and then the result is corrected by using a standard curve to obtain an accurate polypeptide content result.
(9) Ninhydrin colorimetric method for determining degree of proteolysis
Drawing a standard curve: 0.1000g of dried glycine is dissolved and then the volume is determined to be 100mL, 2.00mL of dried glycine is taken out and the volume is determined to be 100mL, and 20 mu g/mL of solution is obtained. The solution is taken out and diluted into solution with the content of 2-20 mu g/mL respectively for drawing a standard curve. Adding 1.00mL of color developing agent into 2.00mL of diluent for determination in a test tube, uniformly mixing, heating in a boiling water bath for 15min, and simultaneously performing a blank test; then cooling with cold water, adding 5.00mL of 40% ethanol solution, mixing, standing for 15min, using a 1cm cuvette, zeroing with a blank tube, and measuring A value at 570 nm.
NH in hydrolyzed protein solution2Determination of the base: 0.50mL of the hydrolyzed protein solution obtained in example 1 was taken and the volume was adjusted to 50mL, 0.4mL of the diluted solution was put in a test tube, 1.60mL of distilled water and 1.00mL of a color developing agent were added, and after mixing, the mixture was heated in a boiling water bath for 15min and a blank test was conducted. The same standard curve was followed. calculating-NH in the enzymolysis liquid by using a standard curve2Content of groups (. mu. mol/mL), degree of proteolysis (DH) was calculated according to the following formula:
DH（%）=
Figure DEST_PATH_IMAGE014
in the formula:
n-nitrogen content, mg/mL;
h-number of peptide bonds cleaved, mmol/g.
The standard curve is shown in fig. 6, the fitted curve is c = 0.00101+ 0.54535a, the correlation coefficient is 0.9998, and the calculated degree of proteolysis is shown in table 10:
TABLE 10 Ninhydrin colorimetric determination of degree of proteolysis
Figure DEST_PATH_IMAGE015
As can be seen from the data in table 10 and table 6, the change tendency of the proteolysis degree of the BSA sample and the rice protein sample measured by the gel filtration chromatography and the ninhydrin colorimetry were the same and the results were similar.
Standard curves were prepared with the degrees of proteolysis in table 6 as ordinate and the degrees of proteolysis in table 10 as abscissa, respectively, the fitting curves for the BSA protein hydrolysate sample as shown in fig. 7 and the rice protein hydrolysate sample as shown in fig. 8 were y =2.72769+ 1.07549x and y = -3.68374+ 1.04719x, respectively, and the correlation coefficients (R) were 0.97778 and 0.9798, respectively. It can be seen that there is a better correlation between the two assays. Therefore, the measurement result of the gel filtration chromatography can be corrected by using the standard curve to obtain an accurate result of the degree of proteolysis.
EXAMPLE 3 determination of the degree of proteolysis
Taking a certain amount of rice protein in 20mM phosphate buffer solution with pH8.0 at 50 ℃ to prepare protein solution with the concentration of 10mg/mL, adding trypsin with the concentration of 1% of the protein by mass, performing enzymolysis for 6h, taking 10mL of sample, immediately placing the sample in 100 ℃ water bath for 10min for enzyme deactivation treatment after taking out the sample, and obtaining a protein hydrolysis sample.
The molecular weight distribution of the polypeptide was determined from the proteolytic sample by high performance gel filtration chromatography as described in example 2, and the free amino acid content, polypeptide content and degree of proteolysis were calculated and corrected using the standard curve in example 2, respectively, to give a free amino acid content of 14.32%, a polypeptide content of 77.62% and a degree of proteolysis of 23.11%.

Claims (7)

1. A method for measuring the degree of hydrolysis of a protein, comprising the steps of:
(1) diluting the hydrolyzed protein solution into protein hydrolysate with proper concentration by using buffer solution;
(2) measuring the molecular weight distribution of the polypeptide with high performance gel filtration chromatography, and calculating the mass percentage content X of the polypeptide with different molecular weight rangesaWherein a is the relative molecular weight;
(3) and (3) result and calculation:
(a) the polypeptide content in the sample is calculated according to the following formula:
polypeptide content (%) = Σ Xa，
Wherein, a = 200Da-10000 Da;
(b) the content of free amino acids in the sample was calculated according to the following formula:
free amino acid content (%)= Xa，
Wherein a <200 Da;
(c) the degree of proteolysis, calculated according to the following formula:
degree of proteolysis (%) = Σ (128X)a/a’），
Wherein 128 is the average molecular weight of amino acids,
a' is the average molecular weight of polypeptides of different molecular weight ranges.
2. The assay method according to claim 1, wherein in the step (1), the concentration of the protein hydrolysate is 20 to 200mg protein/mL.
3. The assay method according to claim 1, wherein in step (1), the buffer has a pH of 6.5 to 8.0; the concentration of the buffer solution is 20-50 mmol/L.
4. The assay of claim 3, wherein the buffer is selected from the group consisting of phosphate buffer, Britton-Robinson wide buffer, Clark-Lubs buffer, Tris-HCl buffer.
5. The method according to claim 1, wherein the chromatographic conditions of the high performance gel filtration chromatography in step (2) are as follows:
a chromatographic column: superdex peptide 10/300 GL;
mobile phase: 0.10-0.20M NaCl, 20-50mM pH7.2 phosphate buffer;
flow rate: 0.2-1.0 mL/min;
detection wavelength: 214 nm;
pressure: not more than 1.8 Mpa;
sample introduction volume: 20-100 muL.
6. The assay of claim 1, further comprising the steps of preparing a standard curve and calculating the exact polypeptide content, free amino acid content and degree of proteolysis:
(i) respectively measuring the content of free amino acid, the content of polypeptide and the degree of protein hydrolysis of a series of protein standard solutions with different degrees of hydrolysis according to the methods of the steps (1) to (3);
(ii) (ii) determining the exact content of free amino acids and polypeptides and the exact degree of proteolysis from a series of protein standard solutions of different degrees of hydrolysis in (i);
(iii) taking the content of free amino acid in the step (i) as a vertical coordinate, and taking the accurate content of the free amino acid determined by the pre-column derivatization high performance liquid chromatography as a horizontal coordinate to prepare a free amino acid content standard curve;
taking the polypeptide content in the step (i) as a vertical coordinate, and taking the accurate polypeptide content of a trichloroacetic acid precipitation method as a horizontal coordinate to make a polypeptide content standard curve;
taking the protein hydrolysis degree in the step (i) as an ordinate, and taking the accurate protein hydrolysis degree measured by a ninhydrin colorimetric method as an abscissa to make a protein hydrolysis degree standard curve;
(iv) and (5) respectively substituting the free amino acid content, the polypeptide content and the protein hydrolysis degree of the protein hydrolysate obtained by determination into the standard curve in the step (iii) to calculate and obtain the accurate free amino acid content, polypeptide content and protein hydrolysis degree.
7. The assay of claim 6, wherein in steps (ii) and (iii), the free amino acid content is determined by a method selected from the group consisting of paper chromatography or pre-column derivatization high performance liquid chromatography;
the method for measuring the content of the polypeptide is selected from a biuret method, an o-phthalaldehyde method, an ultraviolet absorption method or a trichloroacetic acid method;
the degree of proteolysis is selected from the group consisting of pH-state, trinitrobenzene sulfonic acid, formaldehyde titration, and ninhydrin colorimetry.
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Application publication date: 20200225