Quantitative food proteins content determination method

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.

3 tbl

 

The invention relates to the field of biochemistry and biotechnology and can be used in biochemical studies for the quantification of not only food proteins, but also of dietary carbohydrates and fats in foods of plant and animal origin.

There is a method of determining the quantitative content of dietary proteins, including consistently pursued mixing of sample substrate and enzymatic substances in connection with the stabilizing solution, incubating the formed mixture at a temperature of 37°C and determining the quantitative content of food proteins by calculation (inventor's certificate SU # 1247750, IPC G01N 33/48).

Since in this known method of determining the quantitative content of food proteins produced by the number of total residual protein in the prototype of the substrate in comparison with the control sample in the form of a prototype of the substrate that is not incubated in the enzyme solution, it leads to increased duration and multistage process research required the use of more sophisticated technological equipment and reagents, and does not provide sufficient accuracy in determining the quantitative content of food proteins.

There is a method of determining the quantitative soda is a place of food proteins includes consistently pursued mixing of sample substrate and enzymatic substances in connection with the stabilizing solution, incubating the formed mixture at a temperature of 37°C, centrifugation formed after incubation of enzyme-substrate complex to obtain pure liquid fraction and determining the quantitative content of food proteins by calculation (patent RU No. 2022021, IPC C12Q 1/00, 1/37).

This method of determining the quantitative content of dietary proteins, which is the closest to the set of features of the claimed method, cannot provide the necessary accuracy of the quantification of food proteins and requires increased time and multi-stepping on the research process and more sophisticated technological equipment and expensive reagents, since the determination of the quantitative content of dietary proteins produced by the quantity of content of total protein, respectively, in the experimental and control samples. In addition, this method has no way of determining the quantitative content of dietary carbohydrates and fats, as enzymatic drug use only proteolytic enzymes (pepsin, papain), which is not able to determine the content of edible fats and ug is Asadov.

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

This technical result is achieved in that in the method of determining the quantitative content of food proteins, including consistently pursued mixing of sample substrate and enzymatic substances in connection with the stabilizing solution, incubating the formed mixture at a temperature of 37°C, centrifugation formed after incubation of enzyme-substrate complex to obtain pure liquid fraction and determining the quantitative content of food proteins by calculation, mixing prototypes produced with an enzyme substance in the form of pancreatic juice, prediluted stabilizing solution of 50% concentration with the ratio of the mass of the prototype of the substrate 1:10, incubating the prepared mixture is conducted within 5-15 minutes, before carrying out the quantification of food proteins by calculation obtained by centrifugation net liquid fraction is diluted with a stabilizing solution in the ratio 1:100-200, while the quantitative content of food proteins localize the t as equal to the interest expense protease enzymes in comparison with the control sample solution pancreatic juice.

In addition, at the stage of determining the quantitative content of food proteins by calculation if necessary conduct a quantitative determination of the content of dietary carbohydrates and fats, while the content of dietary carbohydrates is defined as equal to the interest expense amylase enzymes in comparison with the control sample solution pancreatic juice, and the content of dietary fats is defined as equal to the interest expense of the enzyme lipase in comparison with the control sample solution pancreatic juice.

Using the proposed method of determining the quantitative content of food proteins as enzymatic substance solution pancreatic juice, representing natural enzymatic substance containing a full range of active enzymes, including proteolytic enzymes, as well as creating conditions biochemical reactions, as close as possible to the conditions of digestion in vivo, and determining the quantitative content of food proteins in interest expense protease enzymes in comparison with the control sample solution pancreatic juice improves the accuracy of the quantification of dietary protein while eliminating the multistage process of research and the need for applied what I sophisticated equipment and expensive reagents.

Comparative analysis of the prototype shows that the proposed method for the quantification of food proteins differs in that the mixing of prototypes produced with an enzyme substance in the form of pancreatic juice, prediluted stabilizing solution of 50% concentration with the ratio of the mass of the prototype of the substrate 1:10, incubating the prepared mixture is conducted within 5-15 minutes, before carrying out the quantification of food proteins by calculation obtained by centrifugation net liquid fraction is diluted with a stabilizing solution in the ratio 1:100-200, while the quantitative content of food proteins is defined as equal to the interest expense protease enzymes in comparison with the control sample solution pancreatic juice. In addition, at the stage of determining the quantitative content of food proteins by calculation if necessary assess the quantitative content of dietary carbohydrates and fats, while the content of dietary carbohydrates is defined as equal to the interest expense amylase enzymes in comparison with the control sample solution pancreatic juice, and the content of dietary fats is defined as equal to the interest expense of the enzyme lipase in the CPA is to control sample solution pancreatic juice. This difference from the prototype gives grounds to assert the consistency of the proposed method, the patentability criteria of "novelty". Comparison of the proposed method of determining the quantitative content of dietary proteins not only prototype, but with other similar technical solutions in this area failed to reveal any sign, similar distinctive signs, which allows to make a conclusion on the conformity of the proposed method to the condition of patentability "inventive step".

The proposed method for the quantification of food proteins is as follows.

Optimally established experimentally that the method is as follows. A portion of the prototype of the substrate (100 mg), previously dried to air-dry substance and crushed, such as grain crops, to the powder or meat and bone meal mixed with pancreatic juice, prediluted stabilizing solution, in particular ringer's solution, up to 50% concentration with the ratio of the mass of the prototype of the substrate 1:10, ie, 1 ml of pancreatic juice mixed with 100 mg of a test sample substrate. Received thoroughly ground to a homogeneous mass, the mixture was poured in Proc. of the centrifuge tubes the first of which is intended for the quantification of food proteins, second and third tubes can be used if necessary to determine the quantitative content of dietary carbohydrates and fats. In a separate fourth test tube pour 1 ml of pancreatic juice 50% concentration without addition of the substrate intended as a control sample. Incubation prepared mixture in the first three test tubes and the test sample solution pancreatic juice in the fourth tube are in thermostat at 37°C for 10 minutes on the 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 in the process of biochemical reactions that occurred during the period of incubation, enzyme-substrate complex is subjected to centifugation at 3000g for 2 minutes. Obtained by centrifugation volumes of pure liquid fraction from each centrifuge tubes merge in three separate test tubes and then diluted stabilizing ringer's solution at a ratio of 1:100. In this state, and the value that meets the condition of the optimum ratio of substrate to enzyme, the contents of the first three tubes is ready to determine the population of the quantitative content of food proteins carbohydrates and fats. The fourth tube containing 1 ml of pancreatic juice 50% concentration without substrate and last stage of incubation is required as a control sample for use at the stage of determining the quantitative content of dietary proteins, carbohydrates and fats. Defining and quantifying the content of dietary proteins provide interest expense protease enzymes in comparison with the control sample solution pancreatic juice and on the basis of known methods for the cleavage of casein by photometric monitoring (photometric determination of the activity of proteolytic enzymes in the pancreas, the juice to reduce the concentration of casein. Carbatol Sat. the scientific. proceedings of the Buryats. Kai. 1971 No. 25, p.122-126). Defining and quantifying the content of dietary carbohydrates provide interest expense amylase enzymes in comparison with the control sample solution pancreatic juice and on the basis of known methods for the estimation of amylase activity, i.e. the hydrolysis of starch (Comparative evaluation sharifinia and distrectious methods for determining the activity of amylase in the blood of healthy and patients with acute pancreatitis. Vmmain-Gluskin. Lab. matter, 1965, No. 3, str). Defining and quantifying the content of dietary fats Khujand who are in interest expense enzymes lipase in comparison with the control sample solution pancreatic juice and based on known methods of hydrolysis of sunflower oil (determination of the activity of pancreatic lipase juice hydrolysis of sunflower oil. Carbate, Gccireoa. Diseases of agricultural animals in Transbaikalia and the far East and measures of struggle against them. Blagoveshchensk, 1985, p.70-73).

After calculating the interest expense of the enzymes protease, amylase enzymes and enzymes lipase and guided known scientific position that the molecules of these enzymes interact only with molecules of food proteins, carbohydrates and fats and strictly in the ratio of 1:1, it can be stated that in the analyzed substrate quantitative content of food proteins is equal to the percentage consumption of protease enzymes, the quantitative content of dietary carbohydrates is equal to the percentage consumption of the enzymes amylase, the quantitative content of the edible fats equals interest expense of the enzyme lipase. In particular, Table 2 shows that in the optimal mode 33% of the consumption of protease enzymes is 33% content of food proteins, 54% of the expenditure of the enzymes amylase is equal to 54% of the content of dietary carbohydrates and 27% of the expenditure of the enzymes lipase equal to 27% of dietary fats, and Table 4 shows that in meat and bone meal contains protein 43,8%, fat 7,99%, carbohydrates 0,78%.

For the practical implementation of the proposed method of determining the quantitative content of dietary protein is suggested to use as an enzymatic prophetic is tion of pancreatic juice, get in stationary conditions from regular donors, preferably chickens or other poultry. As equivalent by the enzymatic composition of the substitute pancreatic juice can be used in the homogenate of the pancreas of animals.

Experimental studies were conducted to determine the optimal concentration of pancreatic juice, the optimal solution of pancreatic juice to the mass of the prototype of the substrate, the time period of incubation and the ratio of net liquid fraction to the ringer's solution. As shown by the experimental data shown in Table 1, the optimal solution concentration of pancreatic juice needed for mixing with the substrate of the prototype and conduct of incubation must be a value of 50%. Experimental data reported in Table 1 also provide a basis to conclude that the optimal ratio of pancreatic juice 50% concentration by weight of the substrate of the prototype should recognize 1:10. Experimental data shown in Table 2, provide a basis to conclude that a valid time range of incubation required for optimal biochemical interaction of the enzymes protease, amylase and lipase with substrates of prototypes, is in the limits of 5-15 minutes. Experimental data shown in Table 3, give reason to conclude that the optimal ratio of net liquid fraction to the ringer's solution is 1:100-200.

Thus, as shown by the experimental data, the inventive method of determining the quantitative content of dietary proteins based on the use of pancreatic juice as enzymatic substances and determining the quantitative content of dietary proteins, carbohydrates and fats in net interest expense related enzymes in biochemical relationship only with food proteins, carbohydrates, fats can improve the accuracy of determining the quantitative content of these components in food products while reducing multi-stepping and avoiding the use of complex technological equipment and expensive reagents. In addition, the proposed method provides the possibility to determine the quantitative content of not only food proteins and dietary carbohydrates and fats.

Table 1
Comparative data of biochemical interactions of different concentrations and volumes of pancreatic juice with 0.1 g of substrate experienced the th sample (pea)
The concentration of pancreatic juice, %10050
Volume pancreat. juice (ml)1.02.03.01.02.03.0
The activity of enzymes protease (mg/ml/min) control sample435±4.5440±4.5440±5,6223±5.6270±3.37280±2.25
The activity of enzymes protease (mg/ml/min) test sample155±2.25260±3.3370±3,5152±4.5290±2.25287±3.37
The difference between the activity of enzymes in control and experimental samples, %35.659,184,168.2107.4102.5

Table 2
The influence of the incubation time on the process of biochemical interaction of the enzymes of pancreatic juice with the substrate prototypes at a ratio of 1.0 ml of pancreatic juice to 100 mg of substrate prototype (pea)
The incubation time, min51015
SamplesControl
NYY
Experience
NYY
The consumption of enzymes, %Control
NYY
Experience
NYY
Consumption of Frontov, %Control
NYY
Experience
NYY
Consumption of Frontov, %
Protease activity,
(mg/ml/min)
150±10.6200±20.6210±18.6140±10.133140±10.1100±8.329
Protein, %-3329
The amylase activity (mg/ml/min)2280±was 125.61680±112.326.32280±150.21320±20.854960±86.11320±95.7-
The amount of carbohydrate, %26.3-54--
The lipase activity, (mg/ml/min)18±0.618±0.5-18±0.617.5±0.22.718±0.618±0.5-
Fat, % -2.7-

Table 3
The effect of different ratio of net liquid fraction to the ringer's solution at the free fermentable complex enzymatic activity
The ratio of net liquid fraction to the ringer's solutionThe lipase activity, µmol/ml/minThe amylase activity (mg/ml/minProtease activity (mg/ml/min
ControlExperienceFat, %ControlExperienceThe amount of carbohydrate, %ControlExperienceProtein, %
1:505,0±0,25,0±0,1-2400±RUB 145.133,3270±17,1220±12,818,5
1:10015,0±0,314,5±0,13,42160±123,9960±79,355,6400±12,7290±18,927,5
1:20025,0±0,323,0±0,28,0720±52,8400±10,644,4720±14,5500±32,330,5

The method of determining the quantitative content of food proteins, including consistently pursued mixing of sample substrate and enzymatic substances in connection with the stabilizing solution, incubating the formed mixture at a temperature of 37°C, centrifugation formed after incubation of enzyme-substrate complex to obtain pure liquid fraction and determining the quantitative content of food proteins by calculation, characterized in that the mixing of prototypes PR is upset with enzymatic substance in the form of pancreatic juice, pre-diluted stabilizing solution of 50%concentration when the ratio of the mass of the prototype of the substrate 1:10, incubating the prepared mixture is conducted within 5-15 min before carrying out the quantification of food proteins by calculation obtained by centrifugation net liquid fraction is diluted with a stabilizing solution in the ratio 1:100-200, the number of food proteins is defined as equal to the interest expense protease enzymes in comparison with the control sample solution pancreatic juice.



 

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