Method for determination of natural amino acids in food products protein composition

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.

2 tbl


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

Known various methods for determining the composition of amino acids in proteins. The most commonly used method is the classic high-performance liquid chromatography (HPLC), which allows the separation and identification of most of the amino acids in the mixture, but the lack of specificity of the method does not allow full differentiation of all 20 natural amino acids, in particular not there is a complete separation and proper identification from serine valine, phenylalanine from Proline and some other pairs of amino acids with similar chromatographic mobility in the conditions of HPLC [Neklyudov A.D., A. Ivankin. Collagen: synthesis, properties and application: monograph. - M.: GOU VPO at MSFU, 2007].

There is also known a method of increasing the accuracy of determining the sequence of amino acid residues of a biopolymer according to mass spectrometric analysis using computational system [Patent RU No. 2408011 C2]. The method is accurate, but is a complex technical implementation and requires dorogostojascego mass spectrometric detection.

The known method of identification 11-16 amino acids by capillary electrophoresis [Patent RU No. 2346931 C1], however this method does not allow to identify the full range of natural amino acids.

Closest to the claimed method is the separation of basic amino acids, which is that amino acids shared by two-dimensional thin-layer chromatography and sequential use of two new chromatographic systems solvents: isopropyl alcohol - acetone - 24-25% solution of ammonia - 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 detection of amino acids using ninhydrin reagent and optionally reagents Ehrlich or Nessler [Application RU # 94028253 A1, CL G01N 30/02]. The method is time consuming and does not allow for quick identification in automatic mode.

The technical objective of the claimed invention is to accelerate the process of separating amino acids from food and increase detection accuracy by reducing losses and use of sensitive material.

The problem is solved in the method comprising the acidic hydrolysis of the sample filter and the chromatographic separation of the hydrolysate followed automatically the second identification and quantification of amino acids on the automated analyzer.

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

The inventive method of determination carried out by the following method.

Analysis of the amino acids being performed on the amino acid analyzer, which is injected hydrolyzate food product. For the separation of amino acids using a buffer system components A-F, the composition of the buffer solutions is listed in table 1. pH buffer solutions pre-installed with the use of orthophosphoric acid and sodium hydroxide, the solution before introduction into the analyzer is filtered through a Teflon filter with a pore size of 0.22 μm.

Table 1
The composition of the buffer solutions
pH3,33,6 4,511,011,0
Sodium acetate, g8,28,28,28,20
Methanol ml750000
Formic acid, ml3,03,02,01,20
Acetic acid, ml15,020,01,55,00
Boric acid, g0002,00
Ethylenediaminetetraacetate disodium salt, g 0000,50
Sodium hydroxide, g0006,0to 12.0
Caprylic acid, ál100100100100100
Deionized water for HPLC, lto 1to 1to 1to 1to 1

The principle of determination of amino acids is as follows. 20 ál of the test sample is placed in a plastic vessel, which is mounted in rotating tripod automatic temperature-controlled injector. Sakol samples is carried out in accordance with the program after washing and regeneration of the chromatography column having a temperature of 47°C and filled with ion exchanger is a cation exchange resin with carboxylic ionic groups in the Na+-form. The flow of eluent is carried out by capillary high-pressure hoses with an inner diameter of 0.2 mm Separation AMI is ocelot is carried out automatically in accordance with a given program (table 2). After separation column, the solution comes in a temperature-controlled reactor for color reaction. The feed to the reactor ninhydrin solution containing 15 g/l of ninhydrin, 0.55 g/l hydrogentartrate, 1% methylcellosolve, 10% methanol in 0.1 M acetate buffer pH of 4.8, if the reactor temperature 125°C to implement a color reaction analyzed and last amino acids with ninhydrin. Next colored solution with a complex of amino acids with ninhydrin is pumped in the UV detector. Registration of amino acids that are related in complex with ninhydrin, carried out automatically at the same time at 570 nm, Proline assayed at 440 nm, as this is the maximum absorbance data of the designated amino acids.

Operation of the analyzer is performed according to the program, providing for a simultaneous step change of the type of buffer and temperature of the chromatographic column at each stage. Standard analysis time from the moment you enter the sample until the completion of the last peak, which corresponds to arginine, 50 minutes at a feed rate of the eluent 0.2 ml/min

Table 2
Program analysis of amino acids (P in column (50 ATM, flow 0.2 ml/min)
Time minto 12.05,57,59,03,011,09,08,00,18,04,06,10,4
The input sampleX
Ninhydrin bufferXXX XXXXX
Buffer AXX
Buffer BX
Buffer CXX
Buffer DXXX
The column temperature, °C4747484849525660607070 5555

The method used allows to determine with accuracy of ±(5-10)% availability of all natural amino acids with a minimum level of their content in the solution (0,500±0,006) µmol/ml Minimum interval reliable determination signal of amino acids constituting a >200 mV, to get a concentration of >0.3 ág/ml was taken for analysis of protein in the sample.

Example. 0.1 g of the sample (food or protein of unknown composition containing the protein component of more than 20%) treated with 6 M HCl solution (1 ml at a temperature of 120°C for 24 h in an atmosphere of Ar, the resulting hydrolysate is mixed with 5 ml of buffer with a pH of 2.2, contains: sodium citrate of 9.8 g of concentrated HCl to 8.3 ml, 1 ml thiodiglycol and Caprylic acid 50 ál 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 get the chromatogram, the content of amino acids (X) in μm/ml (or mg/ml, %, conventional machine units mm or the height or the peak areas in accordance with the preset automatic calibration processing of chromatograms) carried out automatically by the formula: X=S1/S2·C, where S1- the peak area defined by amino acids on the aminogram are determined; S2the peak area of the same amino acid standard mixture; C is the concentration of the situation amino acids in the standard mixture, μm/ml as a calibration using standard solution containing 2.5 μmol/ml of ASP, THR, GLU, PRO, GLY, ALA, CYS, VAL, MET, ILEY, LEY, TYR, PHE, HIS, LYS, TRP, and ARG. The results of determination calculated to the second decimal place and rounded to the first decimal place after the decimal point.

The method of determining the composition and content of amino acids in proteins food products that include acid hydrolysis of the sample, filtration, chromatographic separation and subsequent identification and quantification of amino acids, characterized in that the chromatographic separation and subsequent identification and quantification of amino acids in a concentration of 0.5 mmol/ml solution mixture initially carried out in the system buffer solutions of variable composition with increasing normality of from 0.1 to 0.3 on a chromatographic column with cation exchange resin with carboxylic ionic groups in the Na+form in the interval of rising temperatures ranging from 47 to 70°C, then in the buffer with a normality of 0.1 in the interval decreasing temperature 70-55°C with simultaneous detection of separated at the output of amino acids in the form of a colored complex with ninhydrin at two wavelengths 440 and 570 nm in an automatic mode.


Same patents:

FIELD: food industry.

SUBSTANCE: one performs weighing of a sample of marmalade or sweet jelly body. One places the sample into a measuring flask. Distilled water is added. The mixture is stirred until the sample dissolution; the solution is diluted to volume with distilled water and centrifuged. Then the transparent solution is relocated into a vessel for study of the microelements complex composition by way of capillary electrophoresis using a buffer solution consisting of 5-25 mmol/l of benzimidazole, 2-7 mmol/l of tartaric acid, 1.5-2.5 mmol/l of 18-Crown-6 at pH equal to 5.1-6.2. Detection is performed in a diode matrix detector, the thermostate temperature equal to 19-24°C and voltage at the capillary ends equal to 10-25 kV. Calculation of the potassium and calcium peaks height is performed at wave length amounting to 254 nm. Then the apple puree weight fraction is determined from the formula: M=1,25h2h1m100% (1) where M - weight fraction of apple puree in the product, %, m - weight of the product sample batch, g; h1 - sum total of the potassium and calcium peaks heights in the electrophoregramme of the microelements standard solution with the weight fraction of each microelement equal to 2 mg/l, transmission units; h2 - sum total of the potassium and calcium peaks heights in the electrophoregramme of the sample solution, transmission units; 1.25 being the coefficient accounting for concentration of microelements in the standard solution, the sample dilution and sum total of microelement (potassium and calcium) in apple puree equal to 0.264%.

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1 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: disclosed is an express, safe and cheap method of determining mycotoxins in animal and vegetable products. Determination is carried out from 2 g of a sample, the QuEChERS purified extract is divided into three portions of 2 ml each and 300 mcl of chloroform is used as a dispersant in dispersion liquid-liquid micro-extraction. The obtained extracts are collected in micro-vials. The solvent is evaporated. The residue in the first and third micro-vials is dissolved in 50 mcl acetonitrile and the residue in the second micro-vial is dissolved in 50 mcl hexane. Aflatoxins (B1, B2, G1, G2), zearalenone and ochratoxin A are determined in the first micro-vial by HPLC with a fluorimetric detector. Trichothecene mycotoxins (deoxynivalenol, nivalenol, HT-2, T-2, diacetoxyscirpenol, 13-, 15-acetyl deoxynivalenol), penicidin, ochratoxin A and zearalenone are determined in the second micro-vial by gas-liquid chromatography with an electron capture detector. Penicidin and zearalenone are determined in the third micro-vial by HPLC with a donor-matrix detector. The duration of determining mycotoxins is 1.5-2 hours when operating with three chromatographs simultaneously. Sample preparation requires 10.1 ml aceonitrile, 0.9 ml chloroform and 0.05 ml hexane.

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1 tbl, 1 ex

FIELD: ecology.

SUBSTANCE: method includes extraction of all available mature firebugs from territories, generation of an image of each adult firebug, arrangement of landmarks on the image of each adult firebug, as it is provided in the figure, calculation of the centroid size for each adult firebug and calculation of the average size of the centroid for extracted firebugs. The average size of the centroid value making at least 1.73*10-2 m, and the values of content of each of heavy metals that are below the values of limit permissible concentrations, are accepted as parameters of chemical safety of food for a human body.

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1 dwg, 2 tbl, 2 ex

FIELD: food industry.

SUBSTANCE: for determination of amidated pectin weight fraction in marmalade one prepares gauging solutions. For this purpose amidated pectin in an amount of 0.25 g, 0.50 g, 0.75g, 1.25 g (respectively) is placed into flasks. Then one adds distilled water in an amount of 10 cm3 in each flask. One heats the flasks in a water bath until complete dissolution of amidated pectin. Then the solution mass is diluted to 25 g with distilled water. The gauging solutions are alternatively placed into the frustrated total internal reflection pan of an IR spectrophotometer. The spectre is registered; the solution absorption is measured with frequencies equal to 1644, 1640 and 1637 cm-1. One calculates the absorption peak height A for the wave number equal to 1640 cm-1 (optic density units) from the formula A=A0A1+A22                                                                          (1)         (1), where A0, A1, A2 - the solution absorption with the frequency equal to 1640 cm-1, 1644 cm-1, 1637 cm-1, optic density units, respectively. One draws a gauging graph. Then marmalade in an amount of 12.5 g is weighed. One adds distilled water in an amount of 10 cm3 in the flask. One performs heating in a water bath. The produced solution is diluted to 25 g with distilled water. One performs placement into the pan, registration of the spectre and measurement of the absorption peak height for the wave number equal to 1640 cm-1. From the gauging graph, one identifies the weight fraction of amidated pectin. Amidated pectin weight fraction (M) is calculated according to the formula: M=0.44·T·2 (2), where 0.44 - coefficient accounting for influence of organic acids contained in the product; T - amidated pectin weight fraction in the solution under study, %; 2 - coefficient accounting for the sample dilution.

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1 dwg, 1 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry, particularly a method for specific collection of DNA molecules (DNA aptamers) with high affinity for a recombinant protein target. Said method involves synthesis of a single polypeptide chain of a recombinant protein containing a fragment of glutathione S-transferase, a protein target, a peptide sequence split by the B. Anthracis lethal factor, a peptide which is biotinylated in vitro under the action of an E.coli biotin-ligase enzyme, binding the obtained recombinant polypeptide with an oligonucleotide library and immobilising the protein on paramagnetic particles bearing glutathione, washing the paramagnetic particles with the immobilised polypeptide from unbound oligonucleotides in a liquid stream, splitting the protein target with the bound DNA aptamers from the surface of paramagnetic particles with the B. anthracis lethal factor, separating and amplifying the DNA sequence with affinity to the recombinant protein target in a polymerase chain reaction and obtaining a set of single-chain DNA aptamers that are specific to the protein target.

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4 dwg, 4 ex

FIELD: food industry.

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3 cl, 3 dwg

FIELD: measurement equipment.

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4 cl, 3 ex, 1 dwg

FIELD: tobacco industry.

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1 tbl, 1 ex

FIELD: food industry.

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3 ex, 1 tbl, 3 dwg

FIELD: food industry.

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17 cl, 3 dwg, 10 ex

FIELD: metallurgy.

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13 cl, 3 dwg

FIELD: machine building.

SUBSTANCE: aspirator-dust sampler consists of a casing, a diaphragm pump with electric drive, a system to stabilise the volume speed of air pumping, systems to measure the volume of the pumped air, a sampling tube and a filter holder with a filter. The diaphragm pump is made as two chambers being set towards each other, rigidly interconnected and driven by an eccentric mechanism. The eccentric mechanism is installed on the electric motor axis so that the front diaphragm position of one chamber corresponds to the opposite diaphragm position of the other chamber. Suction of air into one chamber is accompanied by the discharge of air from the other chamber. Suction valves are set on the movable diaphragms, and the discharge valves - on the stationary chambers' casing. Both chambers are the walls of the air-tight pump casing connected to a suction branch pipe in which a rarefaction sensor is built-in. The two-chamber pump is placed in another external air-tight casing where air from the chambers is discharged to and one wall of which is replaced by a rubber diaphragm serving as a damper together with the inner casing space. An air mass flow metre is built-in in the other wall. The rarefaction sensor and the flow metre are connected to a motor mode control unit and to the unit for the data on air flow, volume of pumped air, weight of dust on the filter and dust concentration.

EFFECT: improved accuracy of sampling and measuring the pumped air volume, keeping and measuring the constant volume speed of air pumping through the filter with dust residues, increased reliability of aspirator performance both at dust sampling and in the course of operation, simplified valve design, simplified measuring procedure for pumped air volume reduced to standard conditions.

3 cl, 1 dwg

FIELD: testing equipment.

SUBSTANCE: prismatic sample has a prism shape, longitudinal and transverse planes of symmetry, two side ledges, arranged longitudinally, at the ends of the prism - support surfaces, and in its central part - surface of loading with a transverse test load. The prismatic sample is additionally equipped with inclined support surfaces arranged on side longitudinal ledges of the prism and characterised by angles of inclination to the longitudinal plane of the prism symmetry 5…20°.

EFFECT: simplification and reduction of cost of prismatic sample testing process with concentrators of mechanical stresses in complex stressed condition, provision of necessary accuracy of modelling of a type of stressed-deformed condition of structure material in focus of its damage.

2 cl, 4 dwg

FIELD: medicine.

SUBSTANCE: invention refers to a medical sampling container, particularly to a modified multifunctional sampling container. The container comprises a body (1), a lid (2) on a body (1) opening, a fixed rotating rod (4) mounted on the lid (2) and freely rotating about the lid (2), and a sampling spoon (5) placed at the bottom of the fixed rotating rod (4). The container comprises a mesh filter (3) placed inside the body (1) perpendicular to the lid (2), and a separator connected to the bottom of the mesh filter (3). The above mesh filter (3) and the separator divide the container body (1) on a pump-down chamber (11) and a pump-off chamber (12).

EFFECT: preventing laboratory contamination and contagion, as well as providing odour-control treatment in the laboratory.

7 cl, 3 dwg

FIELD: process engineering.

SUBSTANCE: set of invention relates to entrapment of biological particles suspended in fluid for preparation of biological specimens for cytological analysis. It relates also to preparation of biological specimens with the help of this device, to platform and system for multianalysis. This device comprises pipe with first and second ends. Note here that pipe first end is closed by filter membrane surface glued to pipe wall cross-section. It includes the piston composed by rod engaged with thrust element. Note also that said rod can slide in axis parallel with pipe wall. Besides, it comprises the unit of hydrophilic absorbent arranged in said pipe between filter membrane inner surface and piston thrust element. Proposed process comprises placing aforesaid device in vessel with fluid wherein suspended are biological particles, retaining said device in said vessel for time sufficient for entrapment of a portion of biological particles on filter membrane surface. Then, this device is removed from said vessel to collect trapped biological particles from membrane filter.

EFFECT: production of high-quality cytological preparations using simpler device.

18 cl, 11 dwg

FIELD: tobacco industry.

SUBSTANCE: invention relates to the field of elaboration of biocatalysts intended for usage as part of biological gas purification filters and may be used during conductance of laboratory experiments with samples of biocatalysts removing volatile components of natural tobacco raw material from air as well as for creation of selective conditions in the process of isolation and study of microorganisms constituting the biologically active components of the sais type catalysts. For the method implementation tobacco raw material is moistened with water, the moistened tobacco raw material mass is placed into the extractor and heated to a temperature exceeding that of water boiling with subsequent pumping of purified air through the extractor to produce the model gas-and-air mixture.

EFFECT: production of a highly efficient biocatalyst for deodorisation of gas-and-air emissions.

5 cl

FIELD: chemistry.

SUBSTANCE: method of determining the coefficient of heterogeneity of a mixture of hard-to-separate granular materials involves determining the number of samples, the minimum allowable weight of a sample, collecting samples of the mixture and components thereof. The samples are distributed in a uniform layer on a smooth surface and photographed. Pixel-by-pixel analysis of images of miscible components is performed to obtain histograms of distribution of pixels of the image on shades of gray with respect to the total number thereof, followed by determination of the threshold shade. Concentration values of the key component in samples of the mixture are then determined as a ratio of the number of pixels corresponding thereto to the total number of pixels of the image of the sample and the coefficient of heterogeneity of the mixture is then calculated. When calculating the value of the threshold shade, coordinates of the centroids of areas of the histograms of distribution of pixels of the components of the mixture are found and the value corresponding to the abscissa of the middle of the section between the centroids of the areas of the histograms is assigned the threshold shade.

EFFECT: simple and accurate method of determining the coefficient of heterogeneity of a mixture of hard-to-separate components with minimum time consumption.

1 dwg

FIELD: mining.

SUBSTANCE: invention refers primarily to mine-mill industry in part of operating procedure and equipment development and may be used during pulp sampling in quality control system for products preparation for analysis. Pulp flow sampling method includes installation of slot-type sampling device to sampled pulp pipeline using mating flanges, pulp flow supply to reforming chamber of slot-type sampling device and taking primary sample using slot-type shutoff device. After pulp flow delivery to reforming chamber, pulp flow is reformed to horizontal average flow directed to expansion chamber. Pulp average flow is used for continuous taking of primary sample supplied through inlet chamber and sample outlet branch to feeding funnel of sample reducing module. Reduced sample taking is performed using cutoff-bucket, reduced sample from cutoff-bucket is directed to flow reducer, in which after mechanical mixing of flow on homogenisation disk flow is reformed to vertical down flow through homogenisation chamber, and then - to annular flow directed to reducing chamber walls outgoing from reducing disk, and controlled continuous taking of final reduced sample is performed from pulp annular flow.

EFFECT: obtaining final reduced accumulated sample with minimum error.

3 cl, 3 dwg

FIELD: physics.

SUBSTANCE: system and method for ground material characterisation in a grinding system use an irradiation section through which at least a part of the ground material stream is fed and with irradiation means for irradiating the particles in the part of the stream with electromagnetic radiation; and a detection section for passage, having a detection means for detecting electromagnetic radiation emitted from the particles of the part of the ground material stream fed through the irradiation section The detection means comprises an imaging system and a colour image sensor for imaging the particles thereon using the electromagnetic radiation emitted by the particles. The colour image sensor comprises image elements for spectrally selective detection of the electromagnetic radiation imaged on the sensor image elements. The detection section comprises a luminous means or is made and arranged to detect particles of the ground material using a combination of transmitted and incident light.

EFFECT: high rate and accuracy of detecting properties of a stream of a grinding product.

26 cl, 3 dwg

FIELD: veterinary.

SUBSTANCE: method for detecting bird's dysbiosis is characterised by the fact that it provides for use of a detection device of an electronic nose type based on a block of 8 piezosensors with basis oscillation frequency of 10-15 MHz, the electrodes of which are modified with coatings sensitive to nitrogen-containing organic compounds, aliphatic acids, compound ethers, aromatic and aliphatic amines, amino acids, ammonium, sulphur-containing compounds, and water. At investigation of birds by means of the detection device of the electronic nose type for dysbiosis of their bowels, first, total content of gases-markers of the disease is determined; for that purpose, average sample of the bird's manure with the weight of 5.00 g is placed into a sterile glass vessel with a soft membrane, thermally regulated at the temperature of 25°C during 20 minutes, extracted with an individual syringe (5 cm3) of an equilibrium gas phase and introduced to a detection cell; maximum signals of the block of piezosensors are recorded by means of the programme. After that, a compressor is switched on for 1-2 minutes to clean the detection cell and the piezosensors. Then, a provisional index of dysbiosis is calculated as a ratio of maximum signals of sensors with films of polyvinyl pyrrolidone (PVP) to polydiethyleneglycol succinate (PDEGS), which reflect the ratio of content of free water to amines in equilibrium gas phase above the sample if the dysbiosis index is less than 1.10±0.05; a conclusion is made on availability of dysbiosis state of birds; with that, maximum signals of other sensors in the block, which are related to content of other gases-markers such as compound ethers, aliphatic acids and amino acids, sulphur-containing compounds, shall be lower in equilibrium gas phase than signals of water-detecting sensors, with a polyvinyl pyrrolidone coating, and amines, with a polydiethyleneglycol succinate coating; with that, content of individual classes of compounds ("щ") is calculated by a standardisation method.

EFFECT: improving accuracy and reliability, diagnostics informativity as per a provisional dysbiosis index - the ratio of content of free water and amines; detection of different classes of organic substances and equilibrium gas phase above manure samples is simplified.

3 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to application of peptide, which has sequence originating from amino acid sequence of protein SNAP-25, for treatment of pain and/or inflammation.

EFFECT: obtaining novel composition.

9 cl, 1 dwg, 1 tbl, 2 ex