CN110988249B - Method for measuring concentration of water-soluble peanut protein in solution containing reducing monosaccharide - Google Patents
Method for measuring concentration of water-soluble peanut protein in solution containing reducing monosaccharide Download PDFInfo
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Abstract
The invention discloses a method for measuring the concentration of water-soluble peanut protein in a solution containing reducing monosaccharide, which comprises the following steps: (1) pretreating a sample by adopting acetonitrile; (2) determining the concentration of various reducing monosaccharides in the sample obtained by pretreatment by ion chromatography; (3) after color development, determining the molar absorption coefficient of various reducing monosaccharides at 562nm and the total absorbance value of a sample to be detected at the wavelength by using an ultraviolet-visible spectrum; (4) and based on the parameter values determined in the steps, taking bovine serum albumin as a reference standard substance of the peanut protein, deducting the contribution of reducing monosaccharide to the total absorbance value at the wavelength of 562nm, and calculating to obtain the concentration of the water-soluble peanut protein in the sample obtained by pretreatment. The method eliminates the interference of different reducing monosaccharides in the sample on the determination of the concentration of the water-soluble peanut protein, and the experimental result shows that the slightly high rate of the concentration of the water-soluble peanut protein caused by the reducing monosaccharides reaches 22-50%.
Description
Technical Field
The invention belongs to the technical field of chemical detection, and particularly relates to a method for determining the concentration of water-soluble peanut protein in a solution containing reducing monosaccharide.
Background
Peanut is one of six important raw materials for producing edible oil in China. At present, the annual output of peanuts in China is the first in the world and accounts for more than 40% of the total output of peanuts in the world. Apart from being used for eating, export and crop seeds, about 50-60% of peanuts are used for preparing edible oil every year. The remainder of the peanuts after being pressed to produce oil is called peanut meal, and the annual output of the peanut meal reaches over 300 ten thousand tons. Except carbohydrate and a small amount of fat substances, the content of protein in the peanut meal is up to more than 50 percent. After effective extraction, purification and refining, the peanut protein can be directly used as a natural additive of starch and meat products, so that the nutritional value of the food is improved; after being hydrolyzed by protease, separated, purified and refined, the compound enzyme can generate a plurality of active polypeptide substances besides 8 essential amino acids needed by human body, and has the obvious effect of enhancing the immunity of human body.
The water-soluble protein is the protein of peanut protein which is most easily extracted and utilized. In order to increase the extraction rate of water-soluble peanut protein, biological enzymes (such as hemicellulase, cellulase, lipase and the like) are generally adopted to hydrolyze glycan and oligosaccharide in peanut meal, so that the accessibility of the peanut meal is increased. However, the components of the extracting solution generated by the enzyme-assisted extraction technology are complex, and besides the lack of the water-soluble protein purification and refining technology, the existing protein concentration determination method has the defects of complicated steps or low accuracy, so that the values of the self enzymatic hydrolysis, separation and purification and refining of the water-soluble peanut protein are difficult to accurately evaluate, most of the peanut meal or the extract thereof is directly used as a feed additive for fish and poultry, the high-valued conversion and utilization of the water-soluble peanut protein in China are obviously influenced, and the serious waste of resources is caused.
In the early stage, researchers mainly adopt a Kjeldahl method to measure the concentration of water-soluble protein in a peanut meal extracting solution (Li Xiao just, Zhang Yongdan. peanut meal enzymolysis new process research [ J ] food industry science and technology, 2004,25(2): 102-. However, the method involves complicated operation steps such as concentrated sulfuric acid digestion of a sample, distillation of ammonia, hydrochloric acid titration and the like, and the analysis titration has large artificial operation errors, which affects the accuracy of the determination. Furthermore, although some researchers used BCA reagents to measure serum (Chilobrachys zurabrachys, Zhuwenya, Penduling, etc.. BCA method for preliminary evaluation of trace proteins [ J ] J. Clin. clinical examination, 1992(3): 134-.
Although the published chinese patent invention (a method for determining the protein content in a protein sample rich in reducing sugars, application No. 201710213757.2) uses a 3, 5-dinitrosalicylic acid (DNS) method to eliminate interference of reducing sugars with the determination of the protein content in a tobacco leaf extract, an apple pulp extract and a diabetes sample. On the one hand, previous studies (Differential diagnosis of the dinitrosalicylic acid (DNS) reagent reagents mono-and di-saccharoide donors, Biomass and Bioenergy,2011,35,4748-4750) showed that monosaccharides (such as glucose) and oligosaccharides (such as cellobiose) at the same concentration produced different absorbance values after color reaction with DNS solvent, i.e., different molar absorbances of monosaccharides and oligosaccharides at specific wavelengths. The plant extract (for example, Chinese patent application No. 201710076652.7, a tobacco leaf polysaccharide extract, an extraction method and an application thereof, and a method for simultaneously extracting mulberry leaf protein and mulberry leaf polysaccharide, application No. 201811481746.3) basically contains monosaccharide and oligosaccharide, and the accuracy of total soluble sugar measured by a DNS method is poor. On the other hand, the invention discovers that different monosaccharide (such as glucose and xylose) with the same concentration react with the BCA reagent after color development, and the generated absorbance values are different, namely the molar absorption coefficients of the different monosaccharide at specific wavelength are different (see figures 2, 3 and 4); meanwhile, cellobiose does not substantially undergo a color reaction with the BCA reagent. Therefore, according to the method of the disclosed invention patent (application No. 201710213757.2), first, the concentration of reducing sugar in a sample measured by the DNS method is not accurate by itself because of the difference in molar absorption coefficients at specific wavelengths after the chromogenic reaction of glucose and cellobiose with DNS; secondly, because the difference of the molar absorption coefficients of glucose, xylose and the BCA reagent at a specific wavelength after color reaction is larger, the molar absorption coefficients of reducing sugar (including different monosaccharides) at the specific wavelength are determined by taking the glucose as a standard substance, and the accuracy of the interference of the reducing sugar on the total absorbance value of the sample is further inaccurate by calculating the concentration of the reducing sugar according to the DNS method. In general, the disclosed invention patent (application No. 201710213757.2) has the following significant disadvantages: (1) the concentration of reducing sugar measured by a DNS method is inaccurate; (2) after chromogenic reaction with BCA reagent, the large difference in molar absorption coefficients at specific wavelengths for different monosaccharides (such as glucose and xylose) was ignored.
Disclosure of Invention
In order to solve the defects of the prior art, the invention mainly aims to provide a method for measuring the concentration of water-soluble peanut protein in a solution containing reducing monosaccharide.
The invention is realized by the following technical scheme:
a method for determining the concentration of water-soluble peanut protein in a solution containing reducing monosaccharides, comprising the steps of:
(1) taking a solution sample containing reducing monosaccharide, diluting the solution sample with acetonitrile or acetone, adding the acetonitrile or acetone into the solution sample, wherein the volume of the acetonitrile or acetone is 2-4 times of that of the solution sample, centrifuging the solution sample after protein in the solution sample is precipitated, and taking supernate after centrifugation for subsequent ion chromatography determination;
(2) ion chromatography standard curves for various sugars:
respectively dissolving arabinose, galactose, glucose, xylose and mannose standard substances into ultrapure water, and diluting into a series of concentration gradients, wherein the concentration range of sugar is 0.1-2 mg/L;
determining the peak emergence time of various sugars by adopting ion chromatography, measuring the peak areas of the sugars, and establishing a standard curve of arabinose, galactose, glucose, xylose and mannose according to the configured sugar concentration;
the detector, chromatographic column and electrode used in the ion chromatography are respectively a pulse ampere electrochemical detector, a carbohydrate analysis column and a gold electrode, the leacheate consists of ultrapure water and 200-250 mmol/L NaOH solution, and various sugars in the filtrate are separated and quantitatively determined by adopting a gradient elution mode;
(3) ion chromatography determination of the concentration of various monosaccharides in the supernatant:
diluting the supernatant with deionized water, filtering the supernatant, and determining peak areas of various monosaccharides in the filtrate according to the ion chromatography in the step (2);
(4) based on the standard curve established in the step (2), calculating the concentration of various reducing monosaccharides in the solution sample according to the peak areas of various sugars, the deionized water dilution times and the acetonitrile dilution times in the chromatogram in the step (3);
(5) ultraviolet-visible spectroscopy determination of absorbance values of standard sugar solutions:
mixing an A reagent and a B reagent in a commercial BCA kit according to the volume ratio of 45: 1-50: 1 to obtain a BCA reagent serving as a color developing agent of protein and reducing monosaccharide;
mixing 0.1-0.15 mL of acetic acid-sodium acetate buffer solution with the concentration of 40-50 mmol/L and the pH value of 5.0-6.0 and 3-4.5 mL of BCA reagent, placing the mixture in a condition of 30-37 ℃ for reaction for 30-45 min, pouring the mixture into a quartz cuvette with the width of 1cm after reaction, performing background scanning of ultraviolet-visible spectrum after standing, and recording the absorbance value at the wavelength of 562 nm;
(6) respectively dissolving arabinose, galactose, glucose, xylose and mannose standard substances into an acetic acid-sodium acetate buffer solution with the concentration of 40-50 mmol/L and the pH of 5.0-6.0, wherein the concentration range of sugar is 0.1-8 g/L;
scanning a sample of an ultraviolet-visible spectrum according to the method in the step (5), and recording the absorbance values of various reducing monosaccharide solutions at the wavelength of 562 nm;
establishing a linear standard curve between the concentration of the standard solution and its absorbance value at 562nm wavelength according to lambert beer's law:
wherein the content of the first and second substances,
is the absorbance value of a sugar at 562 nm;
the molar absorptivity of a certain sugar at 562nm, L/g; cSugarConcentration of a formulated sugar, g/L;
molar absorptivity of a sugar at 562nm based on the measured absorbance value and the configured sugar concentration
Calculating according to equation 1;
(7) ultraviolet-visible spectrometry determination of peanut protein concentration in solution samples:
another part of peanut protein solution sample containing reducing monosaccharide is taken, diluted by 5-20 times by acetic acid-sodium acetate buffer solution with the concentration of 40-50 mmol/L and the pH of 5.0-6.0, and the total absorbance value of water-soluble peanut protein and reducing monosaccharide in the 562nm position in the solution sample is determined according to the methods in the steps (5) and (6)
；
At a certain wavelength, the principle of superposition of contributions of different substances to absorbance is known:
wherein the content of the first and second substances,
is the total absorbance value of the solution sample at 562 nm;
and
the absorbance values of arabinose, galactose, glucose, xylose, mannose and water-soluble peanut protein at 562nm respectively;
according to the concentrations of the various reducing monosaccharides in the solution sample determined in step (4) and equation 1, the net absorbance value of water-soluble peanut protein in the solution sample at 562nm can be calculated as follows:
wherein the content of the first and second substances,
and
molar absorption coefficients of arabinose, galactose, glucose, xylose and mannose at 562nm, respectively; cAra、CGal、CXyl、CGluAnd CManThe concentrations of arabinose, galactose, glucose, xylose and mannose in the solution sample measured in the step (4) respectively;
(8) concentration of water-soluble peanut protein in solution sample:
dissolving a certain mass of bovine serum albumin in an acetic acid-sodium acetate buffer solution with the concentration of 40-50 mmol/L and the pH of 5.0-6.0, diluting the bovine serum albumin by a certain multiple through the same acetic acid-sodium acetate buffer solution, measuring the absorbance values of the bovine serum albumin solutions with different concentrations at 562nm by adopting the methods in the steps (5) and (6), establishing a standard curve according to the Lambert beer law, and calculating the molar absorption coefficient
；
Mole absorptivity based on bovine serum albumin without deduction of monosaccharide interference
The concentration of water-soluble peanut protein in the solution sample is calculated using the formula:
to avoid interference from monosaccharides, the concentration of water-soluble peanut protein in the solution sample can be calculated according to the following formula, combining equation 3 and the molar absorption coefficient of bovine serum albumin:
wherein, CPPThe concentration of the water-soluble peanut protein in the solution sample is g/L;
is the molar absorption coefficient of bovine serum albumin at 562nm, L/g.
In general, Bovine Serum Albumin (BSA) is often used as a standard for the determination of various protein concentrations.
Preferably, the process conditions of the centrifugation in the step (1) are as follows: the centrifugal rotating speed and the time range are 10000-12000 r/min and 15-20 min respectively.
Preferably, the supernatant is diluted by deionized water in the step (3), and the dilution range is 1000-3000 times.
Preferably, the filtration in the step (3) is performed by using an aqueous microporous filter membrane with a pore size of 0.22-0.45 μm.
Preferably, in the above steps (2) and (3), the gradient elution conditions are: 0-18min 1mmol/L NaOH solution, 18.1-23min 50mmol/L NaOH solution, 23.1-30min 50-250mmol/L NaOH solution, 30.1-55min 250mmol/L NaOH solution, 55.1-70min 1mmol/L NaOH solution;
the temperature of the column box is 25-30 ℃; the temperature of the detector is 30-35 ℃; the flow rate of the leaching solution is 0.4-0.5 mL/min; the injection volume is 20-25 mu L.
Preferably, the solution containing the reducing monosaccharide is defatted peanut powder extract; the degreased peanut powder is peanut meal obtained by dry hot pressing or dry cold pressing peanut oil, and the peanut meal is ground and then screened by a 60-80-mesh screen.
Preferably, the preparation of the defatted peanut flour extract comprises the following steps:
(a) adding a certain volume of acetic acid-sodium acetate buffer solution and a certain mass of hemicellulase into a container for dissolving;
(b) adding a certain mass of defatted peanut powder into a container, and placing the container in an environment with the temperature of 45-55 ℃ for protein extraction; after extraction, carrying out centrifugal separation, and taking supernatant as crude extraction liquid of the defatted peanut powder;
(c) heating the crude extract in the step (b) at 85 ℃ for 15min, then centrifuging to remove the hemicellulase, cellulase and cellobiase of the denatured precipitate, and taking the supernatant as the extract of the defatted peanut powder.
Preferably, in the step (a), the concentration and the pH value of the acetic acid-sodium acetate buffer solution are respectively 40-50 mmol/L and 4.5-6.0; the mass ratio of the defatted peanut powder to the acetic acid-sodium acetate buffer solution in absolute dry mass ranges from 1:8 to 1: 12.
Preferably, in the step (a), the dosage of the hemicellulase is 5-30 mg of protein per gram of oven-dried defatted peanut powder.
Preferably, in the step (b), after the extraction is finished, the solid-liquid mixture is subjected to centrifugal separation, and the centrifugal rotation speed and the time range are 5000-8000 rpm and 10-15 min respectively.
Preferably, the specific steps of heating the crude extract in step (c) at 85 ℃ for 15min are:
and (c) pouring the crude extraction liquid in the step (b) into a round-bottom flask, wherein the upper part of the round-bottom flask is provided with a snake-shaped condensation pipe, tap water is introduced into the snake-shaped condensation pipe from top to bottom for cooling, and the round-bottom flask is placed in an oil bath and heated for 15min at 85 ℃.
Compared with the prior art, the invention has the following beneficial effects:
the invention adopts a method of hemicellulase assisted hydrolysis to improve the dissolution rate of water-soluble peanut protein in the defatted peanut powder.
Measuring the molar absorption coefficient of the monosaccharide standard substance at a specific wavelength, measuring the concentrations of various monosaccharides in the extracting solution by ion chromatography, further calculating the contribution of the monosaccharides to the absorbance value at the specific wavelength according to the known molar absorption coefficient and concentration, eliminating the interference of the monosaccharides in the extracting solution to protein measurement, and accurately measuring the concentration of water-soluble peanut protein in the defatted peanut powder extracting solution by using a BCA reagent.
Due to the existence of reducing monosaccharide, the influence on the concentration of water-soluble peanut protein in the extract liquid sample of the defatted peanut powder is very obvious, and the higher rate of the concentration reaches 22.2-49.7 percent.
Drawings
FIG. 1 is a UV-Vis spectrum of Bovine Serum Albumin (BSA) at different concentrations after reaction with BCA reagent;
FIG. 2 is a UV-Vis spectrum of glucose at different concentrations after reaction with BCA reagent;
FIG. 3 is a UV-Vis spectrum of xylose after reaction with BCA reagent at different concentrations;
FIG. 4 is a graph showing the correlation between the concentration of various substances and their absorbance values after reaction with the BCA reagent.
Detailed Description
Examples are given below to specifically describe the present invention, and raw materials used in examples and comparative examples are commercially available.
Commercial BCA kit: available from Thermo Scientific corporation of America, including PierceTMBCA Protein Assay Reagent A and Reagent B.
Examples 1 to 5 and comparative examples 1 to 5
A method for measuring the concentration of water-soluble peanut protein in a solution containing reducing monosaccharide takes a defatted peanut powder extracting solution as a test sample, and comprises the following steps:
(1) preparing an extracting solution of defatted peanut powder:
firstly, adding a certain volume of acetic acid-sodium acetate buffer solution and a certain mass of hemicellulase into a triangular flask, and fully shaking to completely dissolve the three enzymes;
then, adding certain mass of degreased peanut powder into the triangular flask, and tightly plugging the triangular flask by using a rubber plug;
finally, the triangular flask with the closed bottle mouth is placed in an air bath shaker for protein extraction.
And after extraction is finished, carrying out centrifugal separation on the solid-liquid mixture, and taking the supernatant as a crude extraction liquid of the degreased peanut powder.
Wherein the defatted peanut powder is peanut meal obtained by dry pressing peanut oil, and the peanut meal is ground and then screened by a 80-mesh screen.
Wherein the concentration and pH value range of the acetic acid-sodium acetate buffer solution are respectively 40-50 mmol/L and 4.5-6.0;
the mass ratio of the defatted peanut powder to the acetic acid-sodium acetate buffer solution (solid-to-liquid ratio for short, g: ml) in absolute dry mass ranges from 1:8 to 1: 12.
Wherein the dosage of the hemicellulase, the cellulase and the cellobiase is respectively 5-30 mg of protein per gram of oven-dried defatted peanut powder.
Wherein the operation temperature, the operation time and the rotation speed of the air bath shaking table are respectively 45-55 ℃, 12-48 h and 150-250 r/min.
Wherein the rotating speed and the time of the centrifugal separation of the solid-liquid mixture obtained by extraction are respectively 5000-8000 revolutions/min and 10-15 min.
(2) Pretreatment of the extracting solution:
pouring the crude extraction liquid in the step (1) into a round-bottom flask, matching the upper part of the round-bottom flask with a snake-shaped condenser pipe, connecting the snake-shaped condenser pipe with tap water from top to bottom for cooling, placing the round-bottom flask into an oil bath, heating at 85 ℃ for 15min, centrifuging to remove denatured and precipitated hemicellulase, wherein the centrifugation rotating speed and the centrifugation time range are 10000-12000 r/min and 15-20 min respectively, and taking the supernatant as an extraction liquid of the degreased peanut powder.
Dividing the extracting solution into two equal parts (extracting solution E1 and extracting solution E2) according to volume, and respectively filling into glass vials;
diluting the extract by 5-20 times with acetic acid-sodium acetate buffer solution (40-50 mmol/L, pH 5.0.0-6.0), and directly using the extract E1 for ultraviolet-visible spectrum determination;
diluting the extracting solution E2 with acetonitrile, adding acetonitrile with the volume 2-4 times of that of the extracting solution E2, precipitating water-soluble peanut protein extracted from the degreased peanut powder, further centrifuging the diluted solution, wherein the centrifugation speed and the time range are 10000-12000 r/min and 15-20 min respectively, centrifuging, and taking supernatant (E2-1 solution) to be placed in another clean glass vial with a cover for the subsequent ion chromatography determination.
(3) Ion chromatography determination of sugar concentration in E2-1 solution:
further diluting the E2-1 solution by deionized water with the dilution multiple range of 1000-3000 times, then filtering the diluted solution by a water-based microporous filter membrane with the aperture of 0.45 mu m, and further determining the concentration of sugar in the filtrate by using ion chromatography;
the detector, chromatographic column and electrode used in ion chromatography are pulse ampere electrochemical detector, carbohydrate analysis column and gold electrode, the leacheate is composed of ultrapure water and 250mmol/L NaOH solution, and the multiple sugars in the E2-1 solution are separated and quantitatively determined by adopting a gradient elution mode.
Wherein, the gradient elution conditions are as follows: 0-18min 1mmol/L NaOH solution, 18.1-23min 50mmol/L NaOH solution, 23.1-30min 50-250mmol/L NaOH solution, 30.1-55min 250mmol/L NaOH solution, 55.1-70min 1mmol/L NaOH solution.
Wherein the temperature of the column box is 25-30 ℃; the temperature of the detector is 30-35 ℃; the flow rate of the leaching solution is 0.4-0.5 mL/min; the injection volume is 20-25 mu L.
In addition, only arabinose, galactose, glucose, xylose, mannose, xylose.
According to the linear relation between the chromatographic peak area and the sugar concentration, various sugar standards are dissolved in ultrapure water and diluted into a series of concentration gradients (0.1-2 mg/L), and standard curves of arabinose, galactose, glucose, xylose, mannose, xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose are established;
then, based on the established standard curve, the concentrations of various sugars in the extract E2 can be calculated according to the peak areas of various sugars in the chromatogram, the dilution times of deionized water and the dilution times of acetonitrile.
(4) Ultraviolet-visible spectroscopy determination of absorbance values of standard sugar solutions:
first, a commercial BCA kit (purchased from Thermo Scientific, USA, containing Pierce) was preparedTMBCA protein assay Reagent and Reagent B) in a volume ratio of 50:1 (mL: mL), and the mixed Reagent (called BCA Reagent) is used as a color developing agent of protein and sugar;
taking 0.1mL of acetic acid-sodium acetate buffer solution (40-50 mmol/L, pH 5.0.0-6.0) and 3mL of BCA reagent in a glass sample bottle, placing the sample bottle in a water bath kettle at 37 ℃ for reaction for 30min, pouring the reaction solution into a quartz cuvette with the width of 1cm after the reaction, and performing background scanning of ultraviolet-visible spectrum after standing for 1 min;
then, dissolving the standard substances of various sugars in an acetic acid-sodium acetate buffer solution (40-50 mmol/L, pH 5.0.0-6.0) respectively, wherein the concentration range of the sugars is 0.1-8 g/L, taking 0.1mL of sugar solution and 3mL of BCA reagent in a glass sample bottle, carrying out color development reaction and sample scanning of ultraviolet-visible spectrum by adopting the same method, and recording the absorbance value at the wavelength of 562 nm.
Establishing a linear standard curve between the concentration of a particular standard sugar solution and its absorbance value at a wavelength of 562nm according to lambert beer's law:
wherein the content of the first and second substances,
is the absorbance value of a sugar at 562 nm;
molar absorptivity (L/g) of a certain sugar at 562 nm; cSugarIs the concentration (g/L) of a certain standard sugar formulated.
Molar absorptivity at 562nm for a particular saccharide based on the measured absorbance values and configured saccharide concentrations
Then the calculation can be made according to equation 1
In addition, only the arabino-sugars, galactose, glucose, xylose and mannose had absorption at 562nm as seen by uv-vis spectroscopy, and no xylo-oligosaccharides had absorption.
(5) Ultraviolet-visible spectrometry of peanut protein concentration in extract E1:
the total absorbance value at 562nm of the protein and monosaccharide in extract E1 was measured according to the method of step (4). At a specific wavelength (e.g. 562nm), there is a superposition principle of the contributions of different substances to absorbance:
wherein the content of the first and second substances,
is the sum of the absorbance values of the mixture at 562 nm;
and
arabinose, galactose, glucose, xylose, mannose and absorbance values of the extracted peanut proteins at 562nm, respectively.
From the concentrations of the various sugars in extract E2 determined in step (3) and equation 1, the net absorbance value at 562nm for peanut proteins in extract E1 can be calculated as follows:
wherein the content of the first and second substances,
and
molar absorption coefficients (L/g) at 562nm for arabinose, galactose, glucose, xylose and mannose, respectively; cAra、CGal、CXyl、CGluAnd CManThe concentrations (g/L) of arabinose, galactose, glucose, xylose and mannose in the extract E2 were measured by ion chromatography, respectively.
In general, Bovine Serum Albumin (BSA) is often used as a standard for the determination of various protein concentrations.
Dissolving a certain mass of BSA in an acetic acid-sodium acetate buffer solution (40-50 mmol/L, pH 5.0.0-6.0), diluting by a certain multiple with the same acetic acid-sodium acetate buffer solution, measuring the absorbance values of BSA solutions with different concentrations at 562nm by adopting the method in the step (4), establishing a standard curve according to the Lambert beer law, and calculating the molar absorption coefficient (A)
，L/g)。
In the case where monosaccharide interference is not subtracted, the concentration of peanut proteins in extract E1 is generally calculated based on the molar absorption coefficient of BSA using the following formula:
to avoid interference from monosaccharides, combining equation 3 and the molar absorption coefficient of BSA, the concentration of peanut proteins in extract E1 can be calculated according to the following formula:
wherein, CPPFor extracting defatted peanut powderThe concentration (g/L) of water-soluble peanut protein in the liquid;
is the molar absorption coefficient (L/g) of bovine serum albumin at 562 nm.
Ultraviolet-visible spectra after the reaction of Bovine Serum Albumin (BSA), glucose, xylose and BCA reagent with different concentrations are shown in figures 1-3. The correlation between the concentration of different substances after reaction with the BCA reagent and their absorbance values is shown in FIG. 4.
The measurement conditions of the examples and comparative examples are shown in table 1:
TABLE 1 measurement of peanut protein concentration in different extracts before and after deduction of monosaccharide interference
Note: in all comparative examples and examples, the buffers used were acetic acid-sodium acetate buffers, the concentrations of which were 50mmol/L except for the pH; the centrifugation speed and the centrifugation time adopted in each group of experiments are the same. When monosaccharide interference is not deducted, calculating the peanut protein concentration in the extracting solution according to equation 4; when monosaccharide interference is subtracted, the peanut protein concentration in the extract is calculated according to equation 5.
As can be seen from Table 1, the existence of the reducing monosaccharide has a very significant effect on the concentration of the water-soluble peanut protein in the defatted peanut powder extract sample, and the concentration is slightly higher than 22-50%.
Claims (7)
1. A method for determining the concentration of water-soluble peanut protein in a solution containing reducing monosaccharides, comprising the steps of:
(1) taking a solution sample containing reducing monosaccharide, diluting the solution sample with acetonitrile or acetone, adding the acetonitrile or acetone into the solution sample, wherein the volume of the acetonitrile or acetone is 2-4 times of that of the solution sample, centrifuging the solution sample after protein in the solution sample is precipitated, and taking supernate after centrifugation for subsequent ion chromatography determination;
(2) ion chromatography standard curves for various sugars:
respectively dissolving arabinose, galactose, glucose, xylose and mannose standard substances into ultrapure water, and diluting into a series of concentration gradients, wherein the concentration range of sugar is 0.1-2 mg/L;
determining the peak emergence time of various sugars by adopting ion chromatography, measuring the peak areas of the sugars, and establishing a standard curve of arabinose, galactose, glucose, xylose and mannose according to the configured sugar concentration;
the detector, chromatographic column and electrode used in the ion chromatography are respectively a pulse ampere electrochemical detector, a carbohydrate analysis column and a gold electrode, the leacheate consists of ultrapure water and 200-250 mmol/L NaOH solution, and various sugars in the filtrate are separated and quantitatively determined by adopting a gradient elution mode;
(3) ion chromatography determination of the concentration of various monosaccharides in the supernatant:
diluting the supernatant with deionized water, filtering the supernatant, and determining peak areas of various monosaccharides in the filtrate according to the ion chromatography in the step (2);
(4) based on the standard curve established in the step (2), calculating the concentration of various reducing monosaccharides in the solution sample according to the peak areas of various sugars, the deionized water dilution times and the acetonitrile dilution times in the chromatogram in the step (3);
(5) ultraviolet-visible spectroscopy determination of absorbance values of standard sugar solutions:
mixing an A reagent and a B reagent in a commercial BCA kit according to the volume ratio of 45: 1-50: 1 to obtain a BCA reagent serving as a color developing agent of protein and reducing monosaccharide;
mixing 0.1-0.15 mL of acetic acid-sodium acetate buffer solution with the concentration of 40-50 mmol/L and the pH value of 5.0-6.0 and 3-4.5 mL of BCA reagent, placing the mixture in a condition of 30-37 ℃ for reaction for 30-45 min, pouring the mixture into a quartz cuvette with the width of 1cm after reaction, performing background scanning of ultraviolet-visible spectrum after standing, and recording the absorbance value at the wavelength of 562 nm;
(6) respectively dissolving arabinose, galactose, glucose, xylose and mannose standard substances into an acetic acid-sodium acetate buffer solution with the concentration of 40-50 mmol/L and the pH of 5.0-6.0, wherein the concentration range of sugar is 0.1-8 g/L;
scanning a sample of an ultraviolet-visible spectrum according to the method in the step (5), and recording the absorbance values of various reducing monosaccharide solutions at the wavelength of 562 nm;
establishing a linear standard curve between the concentration of the standard solution and its absorbance value at 562nm wavelength according to lambert beer's law:
wherein the content of the first and second substances,
is the absorbance value of a sugar at 562 nm;
the molar absorptivity of a certain sugar at 562nm, L/g; cSugarConcentration of a formulated sugar, g/L;
molar absorptivity of a sugar at 562nm based on the measured absorbance value and the configured sugar concentration
Calculating according to equation 1;
(7) ultraviolet-visible spectrometry determination of peanut protein concentration in solution samples:
another part of peanut protein solution sample containing reducing monosaccharide is taken, diluted by 5-20 times by acetic acid-sodium acetate buffer solution with the concentration of 40-50 mmol/L and the pH of 5.0-6.0, and the total absorbance value of water-soluble peanut protein and reducing monosaccharide in the 562nm position in the solution sample is determined according to the methods in the steps (5) and (6)
At a certain wavelength, the principle of superposition of contributions of different substances to absorbance is known:
wherein the content of the first and second substances,
is the total absorbance value of the solution sample at 562 nm;
and
the absorbance values of arabinose, galactose, glucose, xylose, mannose and water-soluble peanut protein at 562nm respectively;
according to the concentrations of the various reducing monosaccharides in the solution sample determined in step (4) and equation 1, the net absorbance value at 562nm of the water-soluble peanut proteins in the solution sample is calculated as follows:
wherein the content of the first and second substances,
and
molar absorption coefficients of arabinose, galactose, glucose, xylose and mannose at 562nm, respectively; cAra、CGal、CXyl、CGluAnd CManRespectively measuring the content of the Ara in the solution samples in the step (4)Concentrations of primary sugars, galactose, glucose, xylose, and mannose;
(8) concentration of water-soluble peanut protein in solution sample:
dissolving a certain mass of bovine serum albumin in an acetic acid-sodium acetate buffer solution with the concentration of 40-50 mmol/L and the pH of 5.0-6.0, diluting the bovine serum albumin by a certain multiple through the same acetic acid-sodium acetate buffer solution, measuring the absorbance values of the bovine serum albumin solutions with different concentrations at 562nm by adopting the methods in the steps (5) and (6), establishing a standard curve according to the Lambert beer law, and calculating the molar absorption coefficient
Mole absorptivity based on bovine serum albumin without deduction of monosaccharide interference
The concentration of water-soluble peanut protein in the solution sample was calculated using the following formula:
to avoid interference from monosaccharides, the concentration of water-soluble peanut protein in the solution sample is calculated according to the following formula, combining equation 3 and the molar absorption coefficient of bovine serum albumin:
wherein, CPPThe concentration of the water-soluble peanut protein in the solution sample is g/L;
the molar absorption coefficient of bovine serum albumin at 562nm, L/g;
the solution containing the reducing monosaccharide is a defatted peanut powder extracting solution; the preparation method of the degreased peanut powder extracting solution comprises the following steps:
(a) adding a certain volume of acetic acid-sodium acetate buffer solution and a certain mass of hemicellulase into a container for dissolving;
(b) adding a certain mass of defatted peanut powder into a container, and placing the container in an environment with the temperature of 45-55 ℃ for protein extraction; after extraction, carrying out centrifugal separation, and taking supernatant as crude extraction liquid of the defatted peanut powder;
(c) heating the crude extract in the step (b) at 85 ℃ for 15min, then centrifuging to remove the hemicellulase of the denatured precipitate, and taking the supernatant as a defatted peanut powder extract;
the solution for extracting the water-soluble peanut protein in the peanut meal is an acetic acid-sodium acetate buffer solution, and the concentration and the pH value range of the acetic acid-sodium acetate buffer solution are respectively 40-50 mmol/L and 4.5-6.0.
2. The method for determining the concentration of water-soluble peanut protein in a solution containing a reducing monosaccharide as claimed in claim 1, wherein the centrifugation in the step (1) is carried out under the following conditions: the centrifugal rotating speed and the time range are 10000-12000 r/min and 15-20 min respectively.
3. The method for determining the concentration of water-soluble peanut protein in a solution containing reducing monosaccharide according to claim 1, wherein the supernatant in the step (3) is diluted by deionized water in a range of 1000 to 3000 times.
4. The method for determining the concentration of water-soluble peanut protein in a solution containing a reducing monosaccharide as claimed in claim 1, wherein the filtration in the step (3) is performed by using an aqueous microfiltration membrane having a pore size of 0.22 to 0.45 μm.
5. The method for determining the concentration of water-soluble peanut protein in the solution containing the reducing monosaccharide according to claim 1, wherein the defatted peanut powder is peanut meal obtained by dry hot pressing or dry cold pressing peanut oil, and the peanut meal is ground and then screened by a 60-80-mesh screen.
6. The method for determining the concentration of water-soluble peanut proteins in a solution containing reducing monosaccharides according to claim 5, wherein the ratio of the mass of defatted peanut powder to the volume of acetic acid-sodium acetate buffer solution in absolute dry mass is in the range of 1:8 to 1: 12.
7. The method for determining the concentration of water-soluble peanut protein in a solution containing monosaccharide reducibility according to claim 5, wherein the enzyme for auxiliary extraction of the water-soluble peanut protein in the peanut meal is hemicellulase, and the dosage of the hemicellulase is 5-30 mg of protein per gram of oven-dried defatted peanut powder.
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| CN106841079A (en) * | 2017-04-01 | 2017-06-13 | 重庆理工大学 | A kind of method for determining protein content in the protein sample rich in reduced sugar |
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