RU2517628C1 - Method for determination of natural amino acids in food products protein composition - Google Patents

Abstract

FIELD: food industry.

SUBSTANCE: method envisages the sample acid hydrolysis, the hydrolysate filtration and chromatographic separation with subsequent automatic identification and quantitative evaluation of amino acids content using an automatic analyser. The invention allows to determine amino acids in the food product proteins composition with amino acids content equal to nearly 0.1-3.5 g/100 g of the product (1.5-17 g/100 g of protein) with application of sequential elution of amino acids with a buffer solutions mixture and simultaneous detection of the components at two wave lengths being 440 and 570 nm.

EFFECT: acceleration of the process of amino acids isolation from the food product and determination accuracy enhancement due to losses decrease and highly sensitive material application.

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Description

The invention relates to the field of analysis of the biological value of food and medical purposes, in particular animal raw materials and products based on it, and can be used in medicine, food and perfume industry, as well as agriculture.

Various methods are known for determining the composition of amino acids in proteins. The most commonly used method is classical high performance liquid chromatography (HPLC), which makes it possible to separate and identify most amino acids in a mixture, however, the lack of specificity of the method does not allow for the complete differentiation of all 20 natural amino acids, in particular, there is no complete separation and corresponding identification of serine from valine, phenylalanine from proline and some other pairs of amino acids having close chromatographic mobility under the conditions of HPLC [Nek Lyudov A.D., Ivankin A.N. Collagen: production, properties and application: monograph. - M .: GOU VPO MGUL, 2007].

There is also a method of increasing the accuracy of determining the sequence of amino acid residues of biopolymers according to mass spectrometric analysis using a computer system [Patent RU No. 2408011 C2]. The method is accurate, but it is complicated in technical implementation and requires the use of expensive mass spectrometric detection.

A known method for identifying 11-16 amino acids by capillary electrophoresis [Patent RU No. 2346931 C1], however, this method does not allow to determine the entire spectrum of natural amino acids.

Closest to the claimed method is the separation of basic amino acids, which consists in the fact that the amino acids are separated by two-dimensional thin layer chromatography and the sequential use of two new chromatographic solvent systems: isopropyl alcohol - acetone - 24-25% ammonia solution - water in the ratio (19.0- 27.0) :( 20.0-26.0) :( 3.0-6.5) :( 6.0-10.0) (by volume) and chloroform - ethanol - glacial acetic acid - water in the ratio (23.0-27.0) :( 13.5-16.0) :( 1.3-3.5) (by volume). For the detection of amino acids using ninhydrin reagent and optionally reagents Ehrlich or Nessler [Application RU No. 94028253 A1, cl. G01N 30/02]. The method is time consuming and does not allow for quick identification in automatic mode.

The technical task of the claimed invention is to accelerate the process of separation of amino acids from a food product and increase the accuracy of determination by reducing losses and the use of highly sensitive material.

The problem is solved in a method involving acid hydrolysis of a sample, filtration and chromatographic separation of the hydrolyzate, followed by automatic identification and quantification of amino acid content on an automatic analyzer.

The technical result consists in the determination of amino acids in the protein composition of a food product with a content of about 0.1-3.5 g / 100 g of product (1.5-17 g / 100 g of protein) using sequential elution of amino acids with a mixture of buffer solutions and simultaneous detection components at two wavelengths of 440 and 570 nm.

The inventive method of determination is carried out according to the following method.

Amino acid analysis is performed on an amino acid analyzer into which a hydrolyzate of the food product is introduced. For the separation of amino acids, a buffer system of components A-F is used, the composition of the buffer solutions is shown in Table 1. The pH of the buffer solutions is pre-set using phosphoric acid and sodium hydroxide, the solutions are filtered before being introduced into the analyzer through a fluoroplastic filter with a pore size of 0.22 μm.

Table 1 The composition of the buffer solutions Name A B C D F pH 3.3 3.6 4,5 11.0 11.0 Normality 0.10 0.10 0.10 0.25 0.3 Sodium Acetate, g 8.2 8.2 8.2 8.2 0 Methanol, ml 75 0 0 0 0 Formic acid, ml 3.0 3.0 2.0 1,2 0 Acetic acid, ml 15.0 20,0 1,5 5,0 0 Boric acid, g 0 0 0 2.0 0 Ethylene diamine tetraacetate disodium salt, g 0 0 0 0.5 0 Sodium hydroxide, g 0 0 0 6.0 12.0 Caprylic acid, μl one hundred one hundred one hundred one hundred one hundred Deionized water for HPLC, l up to 1 up to 1 up to 1 up to 1 up to 1

The principle of determining amino acids is as follows. 20 μl of the analyzed sample is placed in a plastic vessel, which is installed in a rotating tripod of an automatic thermostatically controlled injector. Sample stabilization is carried out in accordance with the program after washing and regenerating the chromatographic column, which has a temperature of 47 ° C and is filled with an ion exchanger - cation exchanger with carboxyl ionogenic groups in the Na ⁺ form. The eluent is supplied via high pressure capillary hoses with an inner diameter of 0.2 mm. The separation of amino acids is carried out automatically in accordance with a given program (table 2). After the separation column, the solution enters the thermostatic reactor for a color reaction. Submission to the reactor of a ninhydrin solution containing: 15 g / l of ninhydrin, 0.55 g / l of hydridanthine hydrate, 1% methylcellosolve, 10% methanol in 0.1 M acetate buffer, pH 4.8, allows for color reaction at a temperature of 125 ° C the reaction of the analyzed and past amino acids with ninhydrin. Next, a colored solution with a complex of amino acids with ninhydrin is pumped into a UV detector. The registration of amino acids bound in complex with ninhydrin is carried out automatically simultaneously at 570 nm, proline is analyzed at 440 nm, because this is the maximum absorption for these determined amino acids.

The work of the analyzer is performed according to a program that provides for a simultaneous stepwise change in the type of buffer and the temperature of the chromatographic column at each stage. The standard analysis time from the moment of injection of the sample to the completion of the output of the last peak corresponding to arginine is 50 minutes at an eluent feed rate of 0.2 ml / min.

table 2 Amino Acid Analysis Program (P in column 50 atm, flow 0.2 ml / min) one 2 four 5 6 7 8 9 10 eleven 12 13 Time min 12.0 5.5 7.5 9.0 3.0 11.0 9.0 8.0 0.1 8.0 4.0 6.1 0.4 Sample Entry X Ninhydrin buffer X X X X X X X X Buffer A X X Buffer b X Buffer c X X Buffer D X X X Buffer f X X X X X Column temperature, ° C 47 47 48 48 49 52 56 60 60 70 70 55 55

The method used allows us to determine with accuracy ± (5-10)% the presence of all natural amino acids with a minimum level of their content in solution (0.500 ± 0.006) μmol / ml. The minimum interval for reliable determination of the amino acid signal of> 200 mV is obtained for a concentration of> 0.3 μg / ml taken for analysis of protein in the sample.

Example. 0.1 g of a sample (food product or protein of unknown composition with a protein component of more than 20%) is treated with 6 M HCl in an amount of 1 ml at 120 ° C for 24 hours in an Ar atmosphere, the resulting hydrolyzate is mixed with 5 ml of buffer with pH 2.2, containing: sodium citrate 9.8 g, concentrated HCl 8.3 ml, thiodiglycol 1 ml and caprylic acid 50 μl per liter, filtered through a membrane filter with pores of 0.45 μm and injected into the amino acid analyzer.

Upon completion of the automatic analysis, a chromatogram is obtained, the content of amino acid (X) in μM / ml (or in mg / ml,%, machine units or mm in height or peak area in accordance with a given automatic calibration of the calculation of chromatograms) is carried out automatically by the formula: X = S ₁ / S ₂ · C, where S ₁ is the peak area of the determined amino acid in the aminogram; S ₂ is the peak area of the same amino acid in the standard mixture; C is the concentration of amino acids in the standard mixture, μm / ml. A standard solution containing 2.5 μmol / ml ASP, THR, GLU, PRO, GLY, ALA, CYS, VAL, MET, ILEY, LEY, TYR, PHE, HIS, LYS, TRP and ARG is used as calibration. The results of the determination are calculated to the second decimal place and rounded to the first decimal place.

Claims (1)

A method for determining the composition and content of amino acids in food proteins, including acid hydrolysis of a sample, filtration, chromatographic separation followed by identification and quantification of amino acids, characterized in that chromatographic separation with subsequent identification and quantification of amino acids at a concentration of 0.5 μmol / ml the mixture solution is initially carried out in a system of buffer solutions of variable composition with increasing normality from 0.1 to 0.3 per chromatographic colo ke with ionogenic cation with carboxyl groups in Na ⁺ -form increasing temperatures ranging from 47 to 70 ° C, then in buffer with 0.1 normality in lowering the temperature range of 70-55 ° C with simultaneous detection of amino acids divided output as a color complex with ninhydrin at two wavelengths of 440 and 570 nm in automatic mode.