How to determine the digestibility of proteins

 

(57) Abstract:

Use: food industry, medical biochemistry, Enzymology, agriculture. The essence of the invention: sample preparation of protein applied to the strip of filter paper in several replications, part of the samples treated with a proteolytic enzyme (pepsin, papain), and the other part left as a control; after incubation, all samples stained lidocainum 10B, then the complex of the protein with the dye elute determine the protein content in the resulting colored solution by spectrophotometric method and evaluate the digestibility of the sample as a fraction of hydrolyzed protein, expressed as a percentage of the original quantity. table 2.

The invention relates to physico-chemical biology and biotechnology, namely, biological chemistry, molecular biology, Bioorganicheskaya chemistry, and can be used by scientific institutions in the study of the properties of proteins and enzymes.

Known methods for determining the digestibility of proteins, consisting in effect of proteolytic enzymes on the appropriate proteins with subsequent determination of the remaining protein in solution (Axtell et al., 1981; Salgo et al., 1985, Kaczkowski et al., digestibility of some sparingly soluble in aqueous solutions of proteins type prolamins and glutelins grain cereal. The latter proteins can be dissolved only in the presence of urea, alcohols or detergents. However, the latter compounds significantly reduce or completely inhibit the activity of proteolytic enzymes, and information about the digestibility may not be obtained or will be distorted. To eliminate this drawback of the proposed test crop to put in a package of filter paper, followed by maintaining in the solution of the enzyme (Pokhilenko, 1985; Levitsky A. P., Pokhilenko L. I., 1987). This method is registered as the invention (ed.St. N 1247750) and selected as the prototype, has a number of significant drawbacks. First, the analysis is quite long, and only the procedure enzymatic processing takes 15-24 hours secondly, using this method it is impossible to determine the digestibility of water-soluble proteins. Thirdly, enzymatic hydrolysis, are all proteins that are present in the grain, and to judge the digestibility of individual proteins is not possible. Fourth, the method is not provided by the study denaturirovannykh proteins.

The aim of the invention is to speed up diagnosis and improve efficiency when determining preparemessage, fix (denatured) by using a high temperature before or after the enzyme treatment, and then to areas containing protein, put a solution of the enzyme after incubation for the desired time (no more than 2 h) determine the remaining associated protein.

A significant characteristic of the prototype, common to him and the claimed subject of research, the treatment of proteins with a solution of the enzyme.

The proposed method differs from the prototype to the following:

analyze both water-soluble and water-insoluble proteins,

allows you to explore both native and denatured proteins,

preparations of proteins applied to filter paper,

consumption of protein assessed by linking it with the dye.

P R I m e R 1. 10 mg of commercial proteins human serum albumin (CSA) and trypsin inhibitor from soy dissolved separately in 2 ml of distilled water. On filter paper strips with a width of 2-3 cm is applied for 5 µl of each protein in the eightfold repetition and placed in the incubator for 15 min at a temperature of 110aboutC. Then the strips are washed for 5 min in 5% solution of acetic acid, slightly dry and put on 5 µl of 0.025% of n samples). Four other spots of proteins on filter paper leave for control. Strips of paper are placed over the solution of 5% acetic acid in a desiccator for 2 h at a temperature of 37aboutC. Then paint on protein for 20 min with 1% solution amidating 10B, cooked in a mixture of ethanol - trichloroacetic acid - water, taken in the ratio of 8:1:1. The excess dye is removed by washing 7% solution of acetic acid and dried paper under the dryer. Colored circles of filter paper with protein, treated with pepsin, and control samples are cut out and placed in test tubes containing 2 ml of 0.1 M solution Paon and extracted dye with shaking for 1 h, the Optical density of the colored solutions was measured on a spectrophotometer at a wavelength of 620 nm, and the protein content in each sample is calculated using a calibration curve based on a different dilutions of CSA (5 to 50 μg of protein per 5 μl of the solution). To determine the digestibility find the average content of the four parallel control sample CSA equal 14,84 µg of protein. From this value, subtract the average content of the four parallel test sample is subjected to the action of pepsin and equal to 4.87, CSA pepsin equal to 67.2 per cent . Similarly find the digestibility of trypsin inhibitor from soy, which is equal to 53.2 per cent. In General, the calculated digestibility can be represented as the following formula:

100% , where acounterand aexperiencethe protein content in control and experimental samples.

P R I m m e R 2. Corn and sorghum (corn pre-remove the germ, grind to a fine powder, weighed 600 mg of each sample into centrifuge tubes, poured 1.2 ml of 1 M NaCl solution and put in the fridge for 1 h with periodic stirring every 15 minutes Then centrifuged at 7000 g for 15 minutes a Solution containing albumin and globulins collect and operation to extract these proteins are repeated three times. Then the precipitate flour is washed with 5 ml of distilled water twice to remove salt and after centrifugation poured in 1.2 l of 65% or 70% isopropanol for corn or sorghum, respectively. After mixing, the tubes stoppered and placed in a water bath for 1 h at a temperature of 60aboutC. Centrifuged at 7000g for 15 min and collect the solution of prolamins. Then, the operation for removing these proteins are repeated three times. After that draught of flour poured 5 ml of distillator NaOH and extracted glutamine for 1 h at room temperature, stirring every 15 minutes Centrifuged at 7000g for 15 min and the supernatant collected.

Selected fractions of proteins applied to filter paper strips 5 µl in eight-fold repetition. Further, the procedure for determining the digestibility of proteins by pepsin same as described in example 1. The ratio of enzyme : substrate 1 : 20. The digestibility of proteins from corn and sorghum are presented in table.1.

P R I m e R 3. Proteins (albumins - globulins, prolamins, glutelin) from corn and sorghum distinguish, as described in example 2. Then proteins cause 5 µl on filter paper in the eightfold repetition and placed in the incubator for 15 min at a temperature of 110aboutC. After this slip of paper was incubated for 5 min in 0.2 M solution of sodium acetate, brought to pH 6.5 with acetic acid, slightly dry and put on a 3-6 µl of 0.025% solution of papain prepared 0.2 M solution of sodium acetate containing 1% 3-mercapto-1,2-propane diol to four lots of protein (prototypes, the ratio of enzyme : substrate 1 : 20). Four other spots proteins on filter paper leave for control. Strips of paper are placed over 0.2 M to krasivuyu protein dye and determining the digestibility of the same as described in example 1. Data of TRANS - remote protein fractions of grain using papain are presented in table.1.

P R I m e R 4. Proteins (albumins-globulins, prolamins and glutelin) was isolated as described in example 2. Then the squirrels put on filter paper for 5 ál in the eightfold repetition, slightly dry and for each segment add 10-15 µl of a 5% solution of acetic acid. Then four experimental plot containing protein, drip 5-10 µl of 0.025% solution of pepsin prepared in 5% acetic acid (ratio of enzyme : substrate 1 : 20), and four other spots left for control. Strips of filter paper is placed over the solution of 5% acetic acid in a desiccator for 2 h at a temperature of 37aboutC. and Then incubated for 15 min at 110aboutTo determine the protein content in the samples using amidating 10 B, as described in example 1. Data on the digestibility of Undenatured proteins are presented in table.2.

P R I m e R 5. Proteins isolated and applied to filter paper as described in example 2. Paper slightly dry and for each segment, containing protein, put on 10-15 ál of 0.2 M solution of sodium acetate, pH 6.5. Then at four sites (experimental Obrera 3 (the ratio of enzyme : substrate 1 : 20), and four other area leave for control. Strips of filter - roll of paper is placed over 0.2 M solution of sodium acetate, pH 6.5) in a desiccator for 2 h at a temperature of 37aboutC. the Further procedure is the same as described in example 4. Data on the digestibility of Undenatured proteins are presented in table.2.

The use of the proposed method for determining the digestibility of proteins in comparison with the known has the following advantages:

reduces the analysis time by 2-3 times or more;

allows you to determine the digestibility of individual proteins, both water-soluble and not soluble in aqueous solutions;

enables comparative analysis of digestibility denaturirovannykh proteins and proteins in their native state;

you can determine the digestibility microgramos amounts of protein.

How to DETERMINE the DIGESTIBILITY of PROTEINS, including the treatment of protein deposited on the matrix of filter paper, a proteolytic enzyme and evaluation of the efficiency of proteolysis by shrinkage of the substrate in the reaction zone, characterized in that the experienced and untreated enzyme control sample after incubation the dye, then the dye elusiv by the formula

100%

where Aaboutand Ato- protein content, respectively, in the experimental and control samples.

 

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