CN112858561A - Hyperbolic method for determining casein content in formula milk powder - Google Patents
Hyperbolic method for determining casein content in formula milk powder Download PDFInfo
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Abstract
A hyperbolic curve method for determining casein content in formula milk powder is provided. In the prior art, the content ratio of casein to whey protein is obtained by measuring each spectral band of casein and whey protein separated according to the molecular weight sequence by using a densitometer, and the accuracy and precision of the measurement result are poor. The method comprises the following steps: hydrolyzing a detected sample at 110 ℃ for 22 hours by 6mol/L hydrochloric acid, diluting the hydrolyzed solution to 50mL volume, filtering, simultaneously deriving the sample filtrate and the standard solution by using a derivative, separating the derivative by using a high performance liquid chromatograph or an ultra high performance liquid chromatograph, detecting by using an ultraviolet detector, determining the content of valine, alanine and proline, determining that a curve relation of a quadratic function exists between the content of casein in the sample and the content ratio of two amino acids contained in the sample, and preparing two quantitative curve equations by using the values of casein with different contents and the ratio of two amino acids contained in the valine, alanine and proline. The invention is a hyperbolic method for determining casein content in infant formula.
Description
Technical Field
The invention relates to the technical field of infant milk powder formulas, in particular to a hyperbolic method for determining the content of casein in formula milk powder. Background
At present, GB/T5413.2-1997 determination of infant formula and milk powder whey protein is used for determining the ratio of casein to whey protein in infant formula, after SDS-polyaniline gel electrophoresis (SDS-PAGE, Laemmli method) is carried out on a sample, each band of casein and whey protein separated according to the sequence of molecular weight is determined by a densitometer in the prior art, the content ratio of casein to whey protein is obtained, and the technology has poor operability, accuracy of determination results and precision. The method overcomes the technical defects of determination of casein and whey protein in the infant formula milk powder, has strong operability, and greatly improves the accuracy and precision of the method.
Disclosure of Invention
The invention aims to provide a hyperbolic method for measuring casein content in formula milk powder.
The above purpose is realized by the following technical scheme:
a hyperbolic method for determining the casein content of a milk formula powder, the method comprising the steps of: firstly, hydrolyzing a measured sample at 110 ℃ for 22 hours by 6mol/L hydrochloric acid, fixing the volume of a hydrolysis solution to 50mL, filtering, simultaneously derivatizing a sample filtrate and a standard solution by using a derivatizing agent, separating the derivatizing agent by using a high performance liquid chromatograph or an ultra high performance liquid chromatograph, detecting by using an ultraviolet detector, determining the contents of valine, alanine and proline, making two quantitative curve equations by using the ratios of casein with different contents and two amino acids contained in the casein, respectively introducing the corresponding amino acid ratios measured in the measured sample into the corresponding curve equations, calculating two casein content values, and taking an average value as a determination result;
(1) preparation of the samples
A. Preparation of amino acid sample: weighing a proper amount of sample, wherein the protein content is 10.0-30.0 mg, placing the sample in a hydrolysis tube with a sealing gasket threaded cover, adding 6mL of warm water at 45-50 ℃ into the hydrolysis tube, slightly shaking to dissolve the sample, adding 6mL of concentrated hydrochloric acid, slightly shaking and uniformly mixing, filling nitrogen for 1min, screwing a screw cover, placing the hydrolysis tube in a constant-temperature drying box at 110 +/-1 ℃ to hydrolyze for 22 h, taking out and cooling to room temperature; transferring the hydrolysate with ultrapure water and fixing the volume to a 50mL volumetric flask, washing the residual liquid for three times, combining the washing liquids, fixing the volume, uniformly mixing, filtering with filter paper, sucking 1 mL of filtrate, putting the filtrate into a finger-type flask, concentrating the filtrate on a nitrogen blowing instrument at 60 +/-2 ℃ until the filtrate is dry or distilling the filtrate under reduced pressure at the temperature lower than 50 ℃ until the filtrate is dry, and dissolving the residue with 1 mL of ultrapure water to obtain a sample solution;
B. preparation of free amino acid sample: accurately weighing about 2.5 g of the uniformly mixed sample, accurately weighing 0.0001 g of the uniformly mixed sample, putting the sample into a 100 mL conical flask, adding about 25 mL of warm water at 45-50 ℃ for dissolution, cooling to room temperature, adjusting the pH value of the sample solution to 1.7 +/-0.1 by using a hydrochloric acid solution, standing for about 1min, adjusting the pH value of the sample solution to 4.5 +/-0.1 by using a sodium hydroxide solution, carrying out ultrasonic oscillation for about 10min, transferring by using water, fixing the volume to 50mL volumetric flasks for uniform mixing, filtering by using a filter paper, and filtering by using a 0.22 mu m filter membrane to obtain a filtrate, namely the sample solution;
(2) making curves
A. Amino acid standard curve preparation: and injecting the standard working solution derivative solution into an ultra-high performance liquid chromatograph or a high school liquid chromatograph to obtain the peak area of the amino acid standard working solution, drawing a standard curve by taking the peak area of the standard working solution as a vertical coordinate and the concentration mu mol/mL of the standard working solution as a horizontal coordinate, wherein the curve is used for calculating the concentration and the content of each amino acid, and the content is expressed by the gram of amino acid contained in each 100 grams of protein.
And preparing a hyperbola for quantitative calculation of the sample: taking prepared different casein contents as ordinate and the reciprocal of valine/alanine, valine/proline or the ratio thereof obtained after the determination by the above machine as abscissa, respectively making two quantitative calculation curves, namely hyperbolic curves, to obtain two curve equations, and requiring that the square value of the correlation coefficient is more than or equal to 0.9980;
(3) determination of sample solutions
Injecting the sample solution to be detected into an ultra-high performance liquid chromatograph or a high performance liquid chromatograph to obtain peak area, obtaining the concentration mu mol/mL of the required amino acid in the sample solution to be detected according to an amino acid standard curve, and calculating the content and the required amino acid ratio;
(4) determination of Total protein
Determining total protein in the sample, decomposing protein under catalytic heating condition, combining generated ammonia with sulfuric acid to generate ammonium sulfate, absorbing ammonia free by boric acid during alkalization distillation, titrating with sulfuric acid or hydrochloric acid, and calculating according to the consumed volume of acid multiplied by conversion coefficient to obtain protein content;
(5) calculation of amino acids
The content of each amino acid in the milk protein in the sample is calculated according to the following formula:
wherein:
x is the content of amino acids in the sample, in grams per hundred grams of protein;
x1 — total amino acid content in samples, in grams per hundred grams of protein;
x2 — content of free amino acids in the sample, in grams per hundred grams of protein;
c1-obtaining the concentration of the total amino acid determination solution of the sample from the standard curve, wherein the unit is micromole per milliliter mu mol/mL;
c2-concentration of free amino acid assay in micromoles per milliliter μmol/mL from standard curve;
m 1-weighing the mass of the hydrolyzed amino acid sample in grams;
m 2-weight of sample to be measured for free amino acid, in grams;
MAA-the molar mass of each amino acid in grams per mole g/mol;
v1 — volume of hydrolyzed amino acid sample was determined in ml;
v2 — volume of free amino acid sample determined in ml;
n 1-determination of dilution factor of hydrolyzed amino acid sample;
n 2-determination of dilution factor of free amino acid sample;
p-whey protein content in grams per hundred grams;
pz-total protein content in grams per hundred grams;
PF-milk casein content in grams per hundred grams;
(6) casein content calculation
Respectively substituting corresponding amino acid content ratios of the tested samples obtained by experiments into respective hyperbolic equations to obtain two casein contents, taking an average value as a measurement result, keeping the average value to one bit behind a decimal point, and subtracting the casein content from 100 to obtain the whey protein content if the whey protein needs to be calculated;
(7) precision degree
The absolute difference between the two casein contents measured in the hyperbola should not exceed 5% of the arithmetic mean.
Has the advantages that:
1. the invention relates to a hyperbolic method for measuring casein content in formula milk powder, which specifically adopts the hyperbolic method to calculate the casein content in a measured sample, prepares a group of quantitative samples, selects three amino acids, simultaneously makes two quantitative curves, has high (hyperbolic) measuring efficiency and can achieve the effect of twice with half the effort.
The method provided by the invention fills the technical defects of determination of casein and whey protein in the infant formula milk powder, has strong operability, and greatly improves the accuracy and precision of the method.
The abscissa of each group of curves is composed of different amino acid ratios, is equivalent to a natural internal standard and is superior to an internal standard substance added later, and the measured matrix backgrounds of the two amino acids are completely consistent, so that matrix interference is eliminated, and the defect that the casein content in the formula milk powder is inaccurate due to the fact that the internal standard substance added later is dissociated in a sample and is not completely consistent with the matrix of a component to be measured in other traditional methods is solved.
The invention adopts a hyperbolic curve method to simultaneously obtain two curves by processing the same sample once to obtain two measurement data, interference factors are mutually counteracted, and the precision of the measurement data is obviously improved.
The method of the invention adopts natural internal standard substance, eliminates more measurement errors and effectively improves the accuracy of the measurement result.
Description of the drawings:
FIG. 1 is an ultra-high performance liquid chromatogram of an amino acid according to the present invention.
FIG. 2 is a high performance liquid chromatogram of an amino acid according to the present invention.
FIG. 3 is a schematic hyperbolic diagram of the quantitative determination of casein in accordance with the present invention.
FIG. 4 is a second schematic diagram of the casein quantitative determination of the present invention.
The specific implementation mode is as follows:
example 1:
a hyperbolic method for determining the casein content of a milk formula powder, the method comprising the steps of: firstly, hydrolyzing a measured sample at 110 ℃ for 22 hours by 6mol/L hydrochloric acid, fixing the volume of a hydrolysis solution to 50mL, filtering, simultaneously derivatizing a sample filtrate and a standard solution by using a derivatizing agent, separating the derivatizing agent by using a high performance liquid chromatograph or an ultra high performance liquid chromatograph, detecting by using an ultraviolet detector, determining the contents of valine, alanine and proline, making two quantitative curve equations by using the ratios of casein with different contents and two amino acids contained in the casein, respectively introducing the corresponding amino acid ratios measured in the measured sample into the corresponding curve equations, calculating two casein content values, and taking an average value as a determination result;
(1) preparation of the samples
A. Preparation of amino acid sample: weighing a proper amount of sample, wherein the protein content is 10.0-30.0 mg, placing the sample in a hydrolysis tube with a sealing gasket threaded cover, adding 6mL of warm water at 45-50 ℃ into the hydrolysis tube, slightly shaking to dissolve the sample, adding 6mL of concentrated hydrochloric acid, slightly shaking and uniformly mixing, filling nitrogen for 1min, screwing a screw cover, placing the hydrolysis tube in a constant-temperature drying box at 110 +/-1 ℃ to hydrolyze for 22 h, taking out and cooling to room temperature; transferring the hydrolysate with ultrapure water and fixing the volume to a 50mL volumetric flask, washing the residual liquid for three times, combining the washing liquids, fixing the volume, uniformly mixing, filtering with filter paper, sucking 1 mL of filtrate, putting the filtrate into a finger-type flask, concentrating the filtrate on a nitrogen blowing instrument at 60 +/-2 ℃ until the filtrate is dry or distilling the filtrate under reduced pressure at the temperature lower than 50 ℃ until the filtrate is dry, and dissolving the residue with 1 mL of ultrapure water to obtain a sample solution;
B. preparation of free amino acid sample: accurately weighing about 2.5 g of the uniformly mixed sample, accurately weighing 0.0001 g of the uniformly mixed sample, putting the sample into a 100 mL conical flask, adding about 25 mL of warm water at 45-50 ℃ for dissolution, cooling to room temperature, adjusting the pH value of the sample solution to 1.7 +/-0.1 by using a hydrochloric acid solution, standing for about 1min, adjusting the pH value of the sample solution to 4.5 +/-0.1 by using a sodium hydroxide solution, carrying out ultrasonic oscillation for about 10min, transferring by using water, fixing the volume to 50mL volumetric flasks for uniform mixing, filtering by using a filter paper, and filtering by using a 0.22 mu m filter membrane to obtain a filtrate, namely the sample solution;
reference chromatographic conditions:
reference conditions of ultra-high performance liquid chromatography:
a chromatographic column: a UPLC BEH C18 column, 2.1X 100 mm, 1.7 μm or other column of similar properties; flow rate: 0.6 mL/min; sample introduction volume: 1 mu L; an ultraviolet detector: the wavelength is 248 nm; column temperature: 60 ℃; temperature of the sample chamber: 10 ℃;
reference to the derivatization conditions: respectively taking 10 mu L of each sample solution and mixed standard working solution, adding 70 mu L of each sample buffer solution, and uniformly mixing by oscillation. Adding 20 μ L of derivatization agent while oscillating, and standing in an incubator at 55 + -1 deg.C for 10min after oscillating to obtain standard working derivatization solution and sample solution to be tested;
table 1: reference condition for gradient elution of ternary pump
High performance liquid chromatography reference conditions:
a chromatographic column: a C18 column, 4.6X 250mm, 5 μm or other column of similar properties; flow rate: 1.0 mL/min; sample introduction volume: 10 muL; an ultraviolet detector: the wavelength is 254 nm; column temperature: 35 ℃;
reference to the derivatization conditions: respectively taking 200 mu L of each sample solution and mixed standard working solution, placing the sample solution and the mixed standard working solution into a 1.5mL centrifuge tube, respectively adding 100 mu L of triethylamine acetonitrile solution, uniformly mixing, then adding 100 mu L of phenyl isothiocyanate acetonitrile solution, uniformly mixing by oscillation, and reacting for 1 h. Then adding 400 mu L of n-hexane, shaking, standing for 10min, sucking the subnatant, filtering with a 0.45 mu m needle filter, taking 200 mu L of the filtrate, adding 800 mu L of purified water for dilution, and mixing uniformly to obtain a standard working derivative solution and a sample solution to be tested;
table 2: gradient elution reference conditions for high performance liquid chromatography
(2) Preparation of Standard Curve
A. Amino acid standard curve preparation: and injecting the standard working solution derivative solution into an ultra-high performance liquid chromatograph or a high school liquid chromatograph to obtain the peak area of the amino acid standard working solution, drawing a standard curve by taking the peak area of the standard working solution as a vertical coordinate and the concentration mu mol/mL of the standard working solution as a horizontal coordinate, wherein the curve is used for calculating the concentration and the content of each amino acid, and the content is expressed by the gram of amino acid contained in each 100 grams of protein.
And preparing a hyperbola for quantitative calculation of the sample: taking prepared different casein contents as ordinate and the reciprocal of valine/alanine, valine/proline or the ratio thereof obtained after the determination by the above machine as abscissa, respectively making two quantitative calculation curves, namely hyperbolic curves, to obtain two curve equations, and requiring that the square value of the correlation coefficient is more than or equal to 0.9980;
(3) determination of sample solutions
Injecting the sample solution to be detected into an ultra-high performance liquid chromatograph or a high performance liquid chromatograph to obtain peak area, obtaining the concentration mu mol/mL of the required amino acid in the sample solution to be detected according to an amino acid standard curve, and calculating the content and the required amino acid ratio;
(4) determination of Total protein
Determining total protein in the sample, decomposing protein under catalytic heating condition, combining generated ammonia with sulfuric acid to generate ammonium sulfate, absorbing ammonia free by boric acid during alkalization distillation, titrating with sulfuric acid or hydrochloric acid, and calculating according to the consumed volume of acid multiplied by conversion coefficient to obtain protein content;
(5) calculation of amino acids
The content of each amino acid in the milk protein in the sample is calculated according to the following formula:
wherein:
x-the content of amino acids in a sample in grams per hundred grams of protein (g/100 g protein);
X1-Total amino acid content in sample in grams per hundred grams protein (g/100 g protein);
x2-free amino acid content in samples in grams per hundred grams of protein (g/100 g protein);
c1-obtaining the concentration of the total amino acid determination solution of the sample from the standard curve, wherein the unit is micromole per milliliter mu mol/mL;
c2-concentration of free amino acid assay in micromoles per milliliter μmol/mL from standard curve;
m 1-weighing the mass of the hydrolyzed amino acid sample in grams;
m 2-weight of sample to be measured for free amino acid, in grams;
MAA-the molar mass of each amino acid in grams per mole g/mol;
v1 — volume of hydrolyzed amino acid sample was determined in ml;
v2 — volume of free amino acid sample determined in ml;
n 1-determination of dilution factor of hydrolyzed amino acid sample;
n 2-determination of dilution factor of free amino acid sample;
p-whey protein content in grams per hundred grams (g/100 g);
Pz-Total protein content in grams per hundred grams (g/100 g);
PF-milk casein content in grams per hundred grams (g/100 g);
(6) casein content calculation
Respectively substituting corresponding amino acid content ratios of the tested samples obtained by experiments into respective hyperbolic equations to obtain two casein contents, taking an average value as a measurement result, keeping the average value to one bit behind a decimal point, and subtracting the casein content from 100 to obtain the whey protein content if the whey protein needs to be calculated;
(7) precision degree
The absolute difference between the two casein contents measured hyperbolically must not exceed 5% of the arithmetic mean;
(8) others
The standard has the following quantitative limit for each amino acid:
the quantitative limit of alanine is 0.025 mu mol/mL;
the quantitative limit of proline is 0.025 mu mol/mL;
the limit of valine quantification is 0.025 mu mol/mL;
example 2:
a hyperbolic method for determining the casein content of a formula milk powder according to example 1, which is suitable for the determination of the casein content of formula milk powder prepared using cow's milk (from holstein cow or san cow or yak cow's milk), goat's milk (from saanen goat or guan goat or sheep) as the main raw material;
(1) reagents and materials: unless otherwise stated, all reagents used in the method are analytically pure, and water is primary water specified in GB/T6682;
A. reagent
Ammonium formate: the top grade is pure; concentrated hydrochloric acid: the concentration is more than or equal to 36 percent, and the product is of high-grade purity; sodium hydroxide; formic acid: carrying out chromatographic purification; acetonitrile: carrying out chromatographic purification; anhydrous sodium acetate; glacial acetic acid: the top grade is pure; triethylamine: the top grade is pure; n-hexane: the top grade is pure; boric acid; sodium borate; sulfuric acid standard solution: purchasing certified materials or calibrating after self preparation; n-hydroxysuccinimide-active heterocyclic carbamates (AQCs) or other derivatizing agents of similar function, such as ninhydrin or phenylisothiocyanate (guaranteed purity);
B. reagent preparation
Hydrochloric acid solution (6.0 mol/L) mixing hydrochloric acid with water according to the ratio of 1: 1;
hydrochloric acid solution (1.0 mol/L): fixing the volume of 90mL of hydrochloric acid to 1000 mL;
hydrochloric acid solution (0.1 mol/L): 9mL of hydrochloric acid is added to reach the constant volume of 1000 mL;
sodium hydroxide solution (1.0 mol/L): dissolving 40g of sodium hydroxide to a constant volume of 1000 mL;
sodium hydroxide solution (0.1 mol/L): dissolving 4g of sodium hydroxide and fixing the volume to 1000 mL;
sample buffer solution (sodium borate-boric acid buffer): 5% sodium tetraborate +95% water, pH adjusted = 8.8;
triethylamine acetonitrile solution: 2.8 mL of triethylamine is added into 17.2 mL of acetonitrile and mixed evenly;
phenyl isothiocyanate acetonitrile solution: accurately sucking 100 mu L of phenyl isothiocyanate, adding 8mL of acetonitrile, and uniformly mixing;
reference derivatizing agent: accurately weighing 10 mg of N-hydroxysuccinimide-active heterocyclic carbamate (AQC) and dissolving the AQC in 1 mL of acetonitrile, and preparing other derivatization agents such as phenyl isothiocyanate and ninhydrin ketone according to the corresponding derivatization requirements;
ultra-high performance liquid chromatography mobile phase: mobile phase A: accurately weighing 3.153 g of ammonium formate, diluting to 1000mL with water, adjusting pH to =5.00 with formic acid, wherein the solution is 50 mmol/L, and filtering with 0.22 μm filter membrane; mobile phase B: acetonitrile; mobile phase C: ultrapure water;
high performance liquid chromatography mobile phase: mobile phase A: weighing 15.2g of anhydrous sodium acetate, adding 1850 mL of water for dissolving, adjusting the pH to be =6.50 by using acetic acid, adding 140mL of acetonitrile, uniformly mixing, and filtering by using a 0.45-micron filter membrane; mobile phase B: adding 800mL of acetonitrile into 200mL of water, and uniformly mixing;
C. standard or reference substance
Alanine (abbreviated Ala): the molecular weight is 89.09, and the purity is more than or equal to 99 percent;
proline (Pro for short): molecular weight 115.13, purity greater than or equal to 99%;
valine (Val for short): the molecular weight is 117.15, and the purity is more than or equal to 99 percent;
whey powder (purchased with witness reference or prepared and rated by oneself): whey powder obtained by purifying and drying whey protein separated by an acid method is subjected to protein measurement by GB 5009.5-2016 (determination of protein in national food safety standard) to determine the content of the whey protein;
casein powder (purchased certified reference substance or prepared and rated by oneself): casein powder obtained by purifying and drying casein separated by an acid method is subjected to protein measurement by GB 5009.5-2016 (national food safety standard) protein measurement to determine the content of the casein;
whole or defatted bovine (sheep) milk powder (purchased with witness reference or prepared and rated by oneself): is derived from cow (sheep) milk, and the protein content is determined by protein determination with GB 5009.5-2016 (determination of protein in food safety national standard food). Casein accounts for 76% of goat milk proteins; 80% of milk proteins are casein; casein accounts for 80% of sheep milk protein;
full-fat or defatted yak milk powder (purchased with evidence reference substance or prepared and assigned by oneself): is derived from yak milk, and is subjected to protein measurement by GB 5009.5-2016 (measurement of protein in national food safety standard) to determine the protein content. Casein accounts for 80% of yak milk protein;
D. standard or reference substance
Amino acid standard stock solution: respectively and accurately weighing 0.0446 g of amino acid standard substance alanine, 0.0576 g of proline and 0.0586g of valine (accurate to 0.0001 g), dissolving with 0.1mol/L hydrochloric acid, and making into 50mL volumetric flasks, wherein each standard stock solution is 10.0 mu mol/mL;
amino acid mixed standard working solution: sequentially sucking 0.5 mL, 1.0 mL, 5.0 mL, 10.0 mL and 20.0 mL of the standard stock solutions into a 100 mL volumetric flask, diluting with ultrapure water, fixing the volume to a scale, and preparing mixed standard working solutions with the concentrations of 0.05, 0.10, 0.50, 1.00 and 2.00 (mu mol/mL);
preparation of a milk casein quantitative curve sample: preparing a casein quantitative curve sample from the same source as the tested sample according to the tested sample ingredient table and the milk raw material source, such as a milk source sample, and preparing a milk casein powder-milk whey protein powder combination; preparing a sample from goat milk, namely preparing a goat cheese protein powder-goat whey protein powder combination; preparing a whole or defatted goat milk powder-bovine whey protein powder combination by mixing a goat milk and cow milk source sample; mixing yak milk and cow milk to obtain a mixture of whole milk or defatted yak milk powder and whey protein powder. Preparing a group of quantitative curve samples with different milk casein contents corresponding to each type of sample, preparing a reasonable range of milk casein in the total protein content of the tested sample as the center, not easily dispersing, and preparing at least five quantitative curve samples with different proportions, such as: for the cow milk source sample, based on the limit requirement that the whey protein/total protein in the infant formula milk powder is more than or equal to 60%, when preparing a cow milk casein powder-cow whey protein powder combined quantitative curve sample, the casein/total protein is between 30% and 50%, and 5 samples are prepared in total.
The instrument and equipment that this application adopted:
an ultra-high performance liquid chromatograph or a high performance liquid chromatograph with an ultraviolet detector;
balance: the induction is 0.1 mg;
a pH meter: the precision is 0.01;
a vortex mixer;
a nitrogen blowing instrument;
a constant temperature drying oven;
a reduced pressure distillation device;
an ultrasonic oscillator;
a hydrolysis pipe: the volume of the pressure-resistant threaded cover glass tube is 10-30 mL.
Claims (1)
1. A hyperbolic method for measuring the casein content in formula milk powder is characterized in that: the method comprises the following steps: firstly, hydrolyzing a measured sample at 110 ℃ for 22 hours by 6mol/L hydrochloric acid, fixing the volume of a hydrolysis solution to 50mL, filtering, simultaneously derivatizing a sample filtrate and a standard solution by using a derivatizing agent, separating the derivatizing agent by using a high performance liquid chromatograph or an ultra high performance liquid chromatograph, detecting by using an ultraviolet detector, determining the contents of valine, alanine and proline, making two quantitative curve equations by using the ratios of casein with different contents and two amino acids contained in the casein, respectively introducing the corresponding amino acid ratios measured in the measured sample into the corresponding curve equations, calculating two casein content values, and taking an average value as a determination result;
(1) preparation of the samples
A. Preparation of amino acid sample: weighing a proper amount of sample, wherein the protein content is 10.0-30.0 mg, placing the sample in a hydrolysis tube with a sealing gasket threaded cover, adding 6mL of warm water at 45-50 ℃ into the hydrolysis tube, slightly shaking to dissolve the sample, adding 6mL of concentrated hydrochloric acid, slightly shaking and uniformly mixing, filling nitrogen for 1min, screwing a screw cover, placing the hydrolysis tube in a constant-temperature drying box at 110 +/-1 ℃ to hydrolyze for 22 h, taking out and cooling to room temperature;
transferring the hydrolysate with ultrapure water and fixing the volume to a 50mL volumetric flask, washing the residual liquid for three times, combining the washing liquids, fixing the volume, uniformly mixing, filtering with filter paper, sucking 1 mL of filtrate, putting the filtrate into a finger-type flask, concentrating the filtrate on a nitrogen blowing instrument at 60 +/-2 ℃ until the filtrate is dry or distilling the filtrate under reduced pressure at the temperature lower than 50 ℃ until the filtrate is dry, and dissolving the residue with 1 mL of ultrapure water to obtain a sample solution;
B. preparation of free amino acid sample: accurately weighing about 2.5 g of the uniformly mixed sample, accurately weighing 0.0001 g of the uniformly mixed sample, placing the sample into a 100 mL conical flask, adding about 25 mL of warm water of 45-50 ℃ for dissolution, cooling to room temperature, adjusting the pH value of the sample solution to 1.7 +/-0.1 by using a hydrochloric acid solution, placing for about 1min, adjusting the pH value of the sample solution to 4.5 +/-0.1 by using a sodium hydroxide solution, carrying out ultrasonic oscillation for about 10min, transferring by using water, fixing the volume to 50mL of a volumetric flask for uniform mixing, filtering by using a filter paper, and filtering by using a 0.22 mu m filter membrane to obtain a filtrate, namely the sample solution;
(2) making curves
A. Amino acid standard curve preparation: injecting the standard working solution derivative solution into an ultra-high performance liquid chromatograph or a high school liquid chromatograph to obtain the peak area of the standard amino acid working solution, drawing a standard curve by taking the peak area of the standard working solution as a vertical coordinate and the concentration [ mu ] mol/mL of the standard working solution as a horizontal coordinate, wherein the curve is used for calculating the concentration and the content of each amino acid, and the content is expressed by the gram of amino acid contained in each 100 grams of protein;
B. hyperbolic preparation of sample quantitative calculation: taking prepared different casein contents as ordinate and the reciprocal of valine/alanine, valine/proline or the ratio thereof obtained after the determination by the above machine as abscissa, respectively making two quantitative calculation curves, namely hyperbolic curves, to obtain two curve equations, and requiring that the square value of the correlation coefficient is more than or equal to 0.9980;
(3) determination of sample solutions
Injecting the sample solution to be detected into an ultra-high performance liquid chromatograph or a high performance liquid chromatograph to obtain peak area, obtaining the concentration mu mol/mL of the required amino acid in the sample solution to be detected according to an amino acid standard curve, and calculating the content and the required amino acid ratio;
(4) determination of Total protein
Determining total protein in the sample, decomposing protein under catalytic heating condition, combining generated ammonia with sulfuric acid to generate ammonium sulfate, absorbing ammonia free by boric acid during alkalization distillation, titrating with sulfuric acid or hydrochloric acid, and calculating according to the consumed volume of acid multiplied by conversion coefficient to obtain protein content;
(5) calculation of amino acids
The content of each amino acid in the milk protein in the sample is calculated according to the following formula:
wherein:
x is the content of amino acids in the sample, in grams per hundred grams of protein;
x1 — total amino acid content in samples, in grams per hundred grams of protein;
x2 — content of free amino acids in the sample, in grams per hundred grams of protein;
c1-obtaining the concentration of the total amino acid determination solution of the sample from the standard curve, wherein the unit is micromole per milliliter mu mol/mL;
c2-concentration of free amino acid assay in micromoles per milliliter μmol/mL from standard curve;
m 1-weighing the mass of the hydrolyzed amino acid sample in grams;
m 2-weight of sample to be measured for free amino acid, in grams;
MAA-the molar mass of each amino acid in grams per mole g/mol;
v1 — volume of hydrolyzed amino acid sample was determined in ml;
v2 — volume of free amino acid sample determined in ml;
n 1-determination of dilution factor of hydrolyzed amino acid sample;
n 2-determination of dilution factor of free amino acid sample;
p-whey protein content in grams per hundred grams;
pz-total protein content in grams per hundred grams;
PF-milk casein content in grams per hundred grams;
(6) casein content calculation
Respectively substituting the amino acid content ratios corresponding to the tested samples obtained by the experiment into respective hyperbolic equations to obtain two casein contents, taking the average value as the measurement result, keeping the average value to one bit behind a decimal point, and subtracting the casein content from 100 to obtain the whey protein content if the whey protein needs to be calculated;
(7) precision degree
The absolute difference between the two casein contents measured in the hyperbola should not exceed 5% of the arithmetic mean.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113777067A (en) * | 2021-09-13 | 2021-12-10 | 上海药明生物技术有限公司 | Method for measuring molar extinction coefficient of protein |
| WO2022143936A1 (en) * | 2021-08-06 | 2022-07-07 | 北京三元食品股份有限公司 | Method for measuring content of whey protein and content of casein in powdered milk and/or ratio of two |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022143936A1 (en) * | 2021-08-06 | 2022-07-07 | 北京三元食品股份有限公司 | Method for measuring content of whey protein and content of casein in powdered milk and/or ratio of two |
| JP2023509124A (en) * | 2021-08-06 | 2023-03-07 | ベイジン サンユアン フーズ カンパニー リミテッド | Method for detecting whey protein, casein content and/or ratio of both in powdered milk |
| JP7305894B2 (en) | 2021-08-06 | 2023-07-10 | ベイジン サンユアン フーズ カンパニー リミテッド | Method for detecting whey protein, casein content and/or ratio of both in powdered milk |
| CN113777067A (en) * | 2021-09-13 | 2021-12-10 | 上海药明生物技术有限公司 | Method for measuring molar extinction coefficient of protein |
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