RU2473699C2 - Quantitative food proteins content determination method - Google Patents

Abstract

FIELD: food industry.

SUBSTANCE: quantitative food proteins content determination method involves the following operations, sequentially performed: mixing test samples of the substrate and a fermentative substance coupled with a stabilising solution, incubation of the produced mixture at a temperature of 37°C, centrifugation of the fermentative-and-substrate complex and determination of quantitative food proteins content by way of calculation. The fermentative substance is represented by pancreatic juice preliminarily diluted with a stabilising solution down to 50% concentration, the ratio of the solution to the substrate test sample weight equal to 1:10. The prepared mixture incubation is performed during 5-15 minutes. Before quantitative food proteins content determination by way of calculation, the pure liquid fraction volume produced as a result of centrifugation is diluted with the stabilising solution at a ratio of 1:100-200. The food protein quantity is determined as equal to percentage expenditure of protease enzymes in comparison with a reference sample of pancreatic juice solution.

EFFECT: enhanced accuracy of determination of the quantitative food proteins content in food products.

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Description

The claimed invention relates to the field of biochemistry and biotechnology and can be used in biochemical studies to determine the quantitative content of not only food proteins, but also food carbohydrates and fats in products of plant and animal origin.

A known method for determining the quantitative content of food proteins, including sequentially mixing experimental samples of the substrate and the enzymatic substance in combination with a stabilizing solution, incubating the resulting mixture at a temperature of 37 ° C and determining the quantitative content of food proteins by calculation (copyright certificate SU No. 1247750, IPC G01N 33 / 48).

Since in this known method the quantitative content of food proteins is determined by the amount of total residual protein in the experimental sample of the substrate in comparison with the control sample in the form of a experimental sample of the substrate, which is not incubated in the enzyme solution, this leads to an increased duration and multi-stage process of the study, the need the use of more complex technological equipment and reagents and does not provide sufficient accuracy in determining the quantitative content of food fired proteins.

A known method for determining the quantitative content of food proteins, including sequentially mixing experimental samples of the substrate and the enzyme substance in combination with a stabilizing solution, incubating the resulting mixture at 37 ° C, centrifuging the enzyme-substrate complex formed after incubation to obtain a pure liquid fraction and determining the quantitative content food proteins by calculation (patent RU No. 2022021, IPC C12Q 1/00, 1/37).

This method of determining the quantitative content of food proteins, which is the closest in combination of features to the claimed method, also does not allow to provide the necessary accuracy in determining the quantitative content of food proteins and requires increased time and multi-stage research process and more complex technological equipment and expensive reagents, since the definition quantitative content of food proteins is produced by the amount of total protein content, respectively, in about torture and control samples. In addition, this method does not have the ability to determine the quantitative content of dietary carbohydrates and fats, since only proteolytic enzymes (pepsins, papains) that are not able to determine the content of dietary fats and carbohydrates are used as an enzymatic preparation.

The technical result of the proposed method for determining the quantitative content of food proteins is to increase the accuracy of determining the quantitative content of food proteins.

The specified technical result is achieved by the fact that in the method for determining the quantitative content of food proteins, which includes sequentially mixing experimental samples of the substrate and the enzymatic substance in combination with a stabilizing solution, incubating the resulting mixture at a temperature of 37 ° C, centrifuging the enzymatic-substrate complex formed after incubation to obtain pure liquid fraction and quantification of food proteins by calculation, mixing about torture samples are produced with an enzymatic substance in the form of pancreatic juice, previously diluted with a stabilizing solution up to 50% concentration at its ratio with the mass of the experimental sample of the substrate 1:10, incubation of the prepared mixture is carried out for 5-15 minutes, before determining the quantitative content of food proteins calculated by centrifugation, the volume of the pure liquid fraction is diluted with a stabilizing solution in a ratio of 1: 100-200, while the quantitative content of food O protein is defined as equal percentage flow protease enzymes in comparison with a control sample of pancreatic juice solution.

In addition, at the stage of determining the quantitative content of food proteins by calculation, if necessary, additionally determine the quantitative content of dietary carbohydrates and fats, while the content of dietary carbohydrates is determined as equal to the percentage consumption of amylase enzymes in comparison with the control sample of a solution of pancreatic juice, and the content of dietary fat is determined as equal to the percentage consumption of lipase enzymes in comparison with a control sample of a solution of pancreatic juice.

The use in the proposed method for determining the quantitative content of food proteins as an enzymatic substance is a solution of pancreatic juice, which is a natural enzymatic substance containing a full range of active enzymes, including proteolytic enzymes, as well as creating conditions for a biochemical reaction as close as possible to digestion in a living organism, and determining the quantitative content of dietary proteins by the percentage consumption of protease enzymes in comparison with con a trial breakdown of a solution of pancreatic juice can improve the accuracy of determining the quantitative content of food proteins while eliminating the multi-stage process of the study and the need for complex equipment and expensive reagents.

Comparative analysis with the prototype shows that the proposed method for determining the quantitative content of food proteins is characterized in that the mixing of the test samples is carried out with an enzymatic substance in the form of pancreatic juice, previously diluted with a stabilizing solution up to 50% concentration when its ratio with the mass of the test sample substrate is 1:10, incubation of the prepared mixture is carried out for 5-15 minutes, before determining the quantitative content of food proteins by calculation, obtained the volume of the pure liquid fraction resulting from centrifugation is diluted with a stabilizing solution in a ratio of 1: 100-200, while the quantitative content of food proteins is determined as equal to the percentage consumption of protease enzymes in comparison with a control sample of a pancreatic juice solution. In addition, at the stage of determining the quantitative content of food proteins by calculation, if necessary, an additional assessment is made of the quantitative content of dietary carbohydrates and fats, while the content of dietary carbohydrates is determined as equal to the percentage consumption of amylase enzymes in comparison with a control sample of a solution of pancreatic juice, and the content of dietary fat is as equal to the percentage consumption of lipase enzymes in comparison with a control sample of a solution of pancreatic juice. This difference from the prototype gives reason to argue that the proposed method meets the patentability criterion of the invention of "novelty." Comparison of the proposed method for determining the quantitative content of food proteins not only with the prototype, but also with other similar technical solutions in this area did not allow us to identify signs similar to the distinctive features, which allows us to conclude that the claimed method meets the patentability condition of the invention “inventive step”.

The proposed method for determining the quantitative content of food proteins is as follows.

In the optimal mode established experimentally, the method is as follows. A portion of a test substrate sample (100 mg), pre-dried to an air-dry substance and crushed, for example, grain of a crop, is mixed with pancreatic juice, previously diluted with a stabilizing solution, in particular Ringer's solution, to a 50% concentration at a concentration of at its ratio with the mass of the experimental sample of the substrate 1:10, i.e. 1 ml of a solution of pancreatic juice is mixed with 100 mg of a test sample of the substrate. The mixture thoroughly ground to a homogeneous mass is poured into three centrifuge tubes, the first of which is used to determine the quantitative content of food proteins, the second and third tubes can be used if necessary to determine the quantitative content of food carbohydrates and fats. In a separate fourth test tube, 1 ml of a solution of pancreatic juice of 50% concentration is poured without the addition of a substrate intended as a control sample. Incubation of the prepared mixture in the first three test tubes and a control sample of a solution of pancreatic juice in the fourth test tube is carried out in an thermostat at a temperature of 37 ° C for 10 minutes using a stopwatch. The temperature of 37 ° C is stable and corresponds to the normal temperature of the internal organs of animals. Formed in the first three centrifuge tubes during the biochemical reaction that occurred during the incubation period, the enzyme-substrate complex is centrifuged at 3000g for 2 minutes. The volumes of the pure liquid fraction obtained as a result of centrifugation from each centrifuge tube are poured into three separate tubes and then diluted with Ringer's stabilizing solution in a ratio of 1: 100. In this state and at this ratio, which meets the condition of the optimal ratio of substrate to enzymes, the contents in the first three tubes are ready to determine the quantitative content of dietary proteins, carbohydrates and fats. The fourth test tube, containing 1 ml of a solution of pancreatic juice of 50% concentration without a substrate and having passed the incubation stage, is required as a control sample for use at the stage of determining the quantitative content of food proteins, carbohydrates and fats. The determination and calculation of the quantitative content of dietary proteins is carried out by the percentage consumption of protease enzymes in comparison with a control sample of a solution of pancreatic juice and on the basis of the well-known method of splitting casein with photometric control (Photometric determination of the activity of proteolytic enzymes in the pancreas, juice to reduce the concentration of casein. C.ZH .Batoev Sat Scientific Works Buryat. Agricultural Institute. 1971 No. 25, pp. 122-126). The determination and calculation of the quantitative content of dietary carbohydrates is carried out according to the percentage consumption of amylase enzymes in comparison with a control sample of a solution of pancreatic juice and on the basis of a known method for assessing amylase activity, i.e. on the hydrolysis of starch (Comparative evaluation of saccharification and dextracting methods in determining the activity of amylase in the blood of healthy and patients with acute pancreatitis. V. M. Merina-Gluzkinai. Lab. delo, 1965, No. 3, p. 143). The determination and calculation of the quantitative content of edible fats is carried out by the percentage consumption of lipase enzymes in comparison with a control sample of a solution of pancreatic juice and based on the well-known method of hydrolysis of sunflower oil (Determination of lipase activity of pancreatic juice by hydrolysis of sunflower oil. C.Zh. Batoev, G.Ts. Tsybekmitova, Diseases of farm animals in Transbaikalia and the Far East and measures to combat them (Blagoveshchensk, 1985, pp. 70-73).

Having determined by calculation the percentage consumption of protease enzymes, amylase enzymes, and lipase enzymes and being guided by the well-known scientific position that the molecules of these enzymes interact exclusively only with food protein, carbohydrate, and fat molecules and strictly in a 1: 1 ratio, we can state that in the analyzed substrate, the quantitative content of food proteins is equal to the percentage consumption of protease enzymes, the quantitative content of food carbohydrates is equal to the percentage consumption of amylase enzymes, quantities The content of dietary fat is equal to the percentage consumption of lipase enzymes. In particular, it can be seen from Table 2 that, in the optimal mode, 33% of the consumption of protease enzymes is 33% of the content of dietary proteins, 54% of the consumption of amylase enzymes is 54% of the content of dietary carbohydrates and 27% of the consumption of lipase enzymes is 27% of the content of edible fats, and from Table 4 shows that meat and bone meal contains proteins 43.8%, fats 7.99%, carbohydrates 0.78%.

For the practical implementation of the proposed method for determining the quantitative content of food proteins, it is proposed to use pancreatic juice as an enzymatic substance obtained in stationary conditions from constant donors, preferably chickens or other poultry. The pancreatic homogenate of animals can be used as an equivalent enzyme composition substitute for pancreatic juice.

In the experimental mode, studies were carried out to determine the optimal concentration of a solution of pancreatic juice, the optimal ratio of the solution of pancreatic juice to the mass of the experimental sample of the substrate, the time period of incubation, and the ratio of the pure liquid fraction to Ringer's solution. As shown by the experimental data shown in Table 1, the optimal concentration of a solution of pancreatic juice required for mixing with the substrate of the prototype and the incubation should be 50%. The experimental data shown in Table 1 also give reason to conclude that the optimal ratio of pancreatic juice of 50% concentration to the mass of the substrate of the experimental sample should be recognized as 1:10. The experimental data shown in Table 2 give reason to conclude that the permissible incubation time range necessary for optimal biochemical interaction of protease, amylase, and lipase enzymes with test substrates is within 5-15 minutes. The experimental data shown in Table 3 give reason to conclude that the optimal ratio of pure liquid fraction to Ringer's solution is 1: 100-200.

Thus, as shown by experimental data, the inventive method for determining the quantitative content of food proteins, based on the use of pancreatic juice as an enzymatic substance and the determination of the quantitative content of food proteins, carbohydrates and fats by the percentage of the corresponding enzymes for the biochemical relationship solely with food proteins, carbohydrates, fats can improve the accuracy of determining the quantitative content of these components in p search products while reducing multistage and eliminating the need for sophisticated process equipment and expensive reagents. In addition, the proposed method provides the ability to determine the quantitative content of not only dietary proteins, but also dietary carbohydrates and fats.

Table 1 Comparative data on the biochemical interaction of different concentrations and volumes of pancreatic juice with 0.1 g of the substrate of the experimental sample (peas) The concentration of pancreatic juice,% one hundred fifty Pancreatic volume. juice (ml) 1.0 2.0 3.0 1.0 2.0 3.0 Protease enzyme activity (mg / ml / min) control sample 435 ± 4.5 440 ± 4.5 440 ± 5.6 223 ± 5.6 270 ± 3.37 280 ± 2.25 Protease enzyme activity (mg / ml / min) prototype 155 ± 2.25 260 ± 3.3 370 ± 3,5 152 ± 4.5 290 ± 2.25 287 ± 3.37 The difference between the activity of enzymes in the control and experimental samples,% 35.6 59.1 84.1 68.2 107.4 102.5

table 2 The influence of incubation time on the process of biochemical interaction of pancreatic juice enzymes with the substrate of the test samples with a ratio of 1.0 ml of pancreatic juice solution to 100 mg of the test sample substrate (peas) Incubation time, min 5 10 fifteen Samples The control
ny Experience
ny The consumption of enzymes,% The control
ny Experience
ny Consumption of fragments,% The control
ny Experience
ny Consumption of fragments,% Protease activity
(mg / ml / min) 150 ± 10.6 200 ± 20.6 210 ± 18.6 140 ± 10.1 33 140 ± 10.1 100 ± 8.3 29th The amount of protein,% - 33 29th Amylase Activity (mg / ml / min) 2280 ± 125.6 1680 ± 112.3 26.3 2280 ± 150.2 1320 ± 20.8 54 960 ± 86.1 1320 ± 95.7 - The amount of carbohydrates,% 26.3 - 54 - - Lipase activity (mg / ml / min) 18 ± 0.6 18 ± 0.5 - 18 ± 0.6 17.5 ± 0.2 2.7 18 ± 0.6 18 ± 0.5 - The amount of fat,% - 2.7 -

Table 3 The effect of different ratios of pure liquid fraction to Ringer's solution on enzymatic activity free of the enzyme-substrate complex The ratio of pure liquid fraction to Ringer's solution Lipase activity, μmol / ml / min Amylase activity, mg / ml / min The activity of proteases, mg / ml / min The control Experience The amount of fat,% The control Experience The amount of carbohydrates,% The control Experience The amount of protein,% 1:50 5.0 ± 0.2 5.0 ± 0.1 - 2400 ± 145.1 1600 ± 95.7 33.3 270 ± 17.1 220 ± 12.8 18.5 1: 100 15.0 ± 0.3 14.5 ± 0.1 3.4 2160 ± 123.9 960 ± 79.3 55.6 400 ± 12.7 290 ± 18.9 27.5 1: 200 25.0 ± 0.3 23.0 ± 0.2 8.0 720 ± 52.8 400 ± 10.6 44,4 720 ± 14.5 500 ± 32.3 30.5

Claims (1)

A method for determining the quantitative content of food proteins, including sequentially mixing test samples of the substrate and the enzyme substance in combination with a stabilizing solution, incubating the resulting mixture at 37 ° C, centrifuging the enzyme-substrate complex formed after incubation to obtain a pure liquid fraction and determining the quantitative content of food proteins by calculation, characterized in that the mixing of the experimental samples is carried out with the farm native substance in the form of pancreatic juice, previously diluted with a stabilizing solution to a 50% concentration when its ratio with the mass of the experimental sample of the substrate 1:10, incubation of the prepared mixture is carried out for 5-15 minutes, before determining the quantitative content of food proteins by calculation, obtained as a result of centrifugation, the volume of the pure liquid fraction is diluted with a stabilizing solution in the ratio 1: 100-200, while the amount of food proteins is determined as equal to the percentage course of protease enzymes in comparison with a control sample of pancreatic juice solution.