Comparative proteomic study of pig muscle proteins during growth and development of an animal

The production of high-quality pork is closely related to the growth and development of muscle tissue. The present article provides a comparative proteomic research of l. dorsi, b. femoris, m. brachiocephalicus during the pigs’ growth and development (at age of 60 days and 180 days). This work was supported by data of electrophoretic methods: one-dimensional electrophoresis according to Laemmli with densitometric assessment in the ImageJ software and two-dimensional electrophoresis according to O’Farrell method with its further processing on the software ImageMaster. The mass spectrometric identification was conducted with the help of the high-performance liquid chromatography (HPLC) system connected to a mass spectrometer; further the data were interpreted by search algorithm Andromeda. When comparing frequency diagrams of one-dimensional electrophoregrams of all three muscle tissues of weaned pigs, the greatest difference was observed for the muscle sample l. dorsi. Comparison of diagrams of muscle tissue samples taken for mature pigs showed a great similarity of all three studied muscles samples. Within the framework of the research, the Fold indicator was calculated. The exceeding its value by more than 2 units is generally considered to be a statistically significant difference. When analyzing two-dimensional electrophoretograms of weaned pigs’ muscles, 18 protein fractions were revealed with Fold > 2. When examining the muscle tissue of mature pigs, 15 of those proteins were found; the differences were mostly detected in the minor protein fractions. The mass spectrometric analysis of the cut bands with well-pronounced differences from the onedimensional electrophoretogram revealed 214 proteins involved to a greater extent in cellular and metabolic processes, physical activity and localization. Growth and development protein — semaphorin‑6B (96.78 kDa) — was revealed in muscle tissue of l. dorsi, a. Also in l. dorsi and b. femoris the growth and development proteins were found: cadherin‑13 (78.23 kDa), cadherin‑7 (87.01 kDa), the F‑actin-cap protein beta subunit (30.66 kDa), and two uncharacterized proteins at 65.60 kDa and 63.88 kDa

Comparison of the proteomic profile of pork byproducts during their storage

In this article, the proteomic profiles of pork by-products (snout, tongue, liver, kidney, spleen) were studied by comparative method on the first day and the fifth day of their storage. Two-dimensional electrophoresis according to O’Farrell was used for the aims of this article, while the results were further processed in ImageMaster software. Proteomic maps of by-products showed clear changes in protein composition after visualization and images analysis. There was a decrease and increase in manifestation intensity of some proteins. The study of the obtained electrophoregrams with the help of references resources allowed identifying various compounds in the by-products. 9 protein fractions with various intensity of manifestation were found on the day 1st and 5th. On the 1st day the following substances were intensively manifested: in the liver — glutathione peroxidase 4 (22.3 kDa), LEAP-2 (8.8 kDa); in the kidneys — quinone oxidoreductase (34.9 kDa); in the spleen — glycoprotein CD59 (13.7 kDa), in the patch — protein flint (49.07 kDa). It is noted that these proteins play their role in stopping certain processes in cells, like oxidation, microbial activity, and accumulation of toxic substances. These processes can worsen the quality of raw materials, and further lead to spoilage of the food product. On the 5th day of storage the highest intensity of manifestation of glyceraldehyde-3-phosphate dehydrogenase (35.8 kDa) in the liver was observed; superoxide dismutase [Cu-Zn] (15.8 kDa) was noted in the kidneys, colony-stimulating factor (16.2 kDa) was observed in the spleen and glutaredoxin –1 (11.8 kDa) in the tongue. In its turn, on the fifth day these chemical processes manifested themselves more intensely, as the fatty acids and glucose broke down. To obtain more accurate results, the proteins were compared by their volume. Among the identified fractions the highest expression was observed in LEAP 2 (8.8 kDa) on the first day, and in glyceraldehyde-3-phosphate dehydrogenase (35.8 kDa) on the fifth day. The least change in the intensity of manifestation was noted for superoxide dismutase [Cu-Zn] (15.8 kDa), which volume increased during storage by 13% for 5 days. The analysis of the obtained electrophoregrams allowed identifying various compounds, tracing the changes in the qualitative composition of protein in by-products during various periods of their storage. The obtained data demonstrate the transformation of protein molecules during storage, which makes it possible to determine the changes and quality of the food products.In this article, the proteomic profiles of pork by-products (snout, tongue, liver, kidney, spleen) were studied by comparative method on the first day and the fifth day of their storage. Two-dimensional electrophoresis according to O’Farrell was used for the aims of this article, while the results were further processed in ImageMaster software. Proteomic maps of by-products showed clear changes in protein composition after visualization and images analysis. There was a decrease and increase in manifestation intensity of some proteins. The study of the obtained electrophoregrams with the help of references resources allowed identifying various compounds in the by-products. 9 protein fractions with various intensity of manifestation were found on the day 1st and 5th. On the 1st day the following substances were intensively manifested: in the liver — glutathione peroxidase 4 (22.3 kDa), LEAP-2 (8.8 kDa); in the kidneys — quinone oxidoreductase (34.9 kDa); in the spleen — glycoprotein CD59 (13.7 kDa), in the patch — protein flint (49.07 kDa). It is noted that these proteins play their role in stopping certain processes in cells, like oxidation, microbial activity, and accumulation of toxic substances. These processes can worsen the quality of raw materials, and further lead to spoilage of the food product. On the 5th day of storage the highest intensity of manifestation of glyceraldehyde-3-phosphate dehydrogenase (35.8 kDa) in the liver was observed; superoxide dismutase [Cu-Zn] (15.8 kDa) was noted in the kidneys, colony-stimulating factor (16.2 kDa) was observed in the spleen and glutaredoxin –1 (11.8 kDa) in the tongue. In its turn, on the fifth day these chemical processes manifested themselves more intensely, as the fatty acids and glucose broke down. To obtain more accurate results, the proteins were compared by their volume. Among the identified fractions the highest expression was observed in LEAP 2 (8.8 kDa) on the first day, and in glyceraldehyde-3-phosphate dehydrogenase (35.8 kDa) on the fifth day. The least change in the intensity of manifestation was noted for superoxide dismutase [Cu-Zn] (15.8 kDa), which volume increased during storage by 13% for 5 days. The analysis of the obtained electrophoregrams allowed identifying various compounds, tracing the changes in the qualitative composition of protein in by-products during various periods of their storage. The obtained data demonstrate the transformation of protein molecules during storage, which makes it possible to determine the changes and quality of the food products

Methodology of adipose tissue type detection in mammals

Nowadays, an interest in studying the composition, properties and functions of adipose tissue (AT) is growing among researchers, which is conditioned by its important role in the normal functioning of the body. Due to different types of adipose tissue (AT) in mammals (white, beige, brown and pink) and different physiological tasks performed by each type of AT, rapid, correct and effective detection of an AT type is highly topical. Methods used today are labor consuming and in the case of NMR and CT expensive, which limits possibilities of scientists. In this connection, the aim of this research was to develop a methodological approach allowing rapid and effective detection of an adipose tissue type. A methodology was formed based on the concept, formalized requirements for the method, step-wise structure of investigations and interpretation of results. The concept is based on differences in the structure of the adipose cell (adipocyte) of different AT types. The method is based on extraction of heme containing proteins. To this end, solvents and parameters of extraction that facilitate their better extraction have been chosen. An AT type has been determined by the total content of iron contained in the cytochrome fragment. Our own modification was selected. This modification includes preliminary mincing of a sample with the ice-cold TES buffer (pH 8.5) in a ratio of 1:5 (g: mL), homogenization at 9,000 rpm for 2 min with the following centrifugation at 10,000 g and 4 °C for 15 min. Effectiveness of the proposed method was confirmed by the histological and electrophoretic analyses. Therefore, the new methodology of identification and differentiation of adipocytes was proposed for rapid and effective detection of an adipose tissue type.Nowadays, an interest in studying the composition, properties and functions of adipose tissue (AT) is growing among researchers, which is conditioned by its important role in the normal functioning of the body. Due to different types of adipose tissue (AT) in mammals (white, beige, brown and pink) and different physiological tasks performed by each type of AT, rapid, correct and effective detection of an AT type is highly topical. Methods used today are labor consuming and in the case of NMR and CT expensive, which limits possibilities of scientists. In this connection, the aim of this research was to develop a methodological approach allowing rapid and effective detection of an adipose tissue type. A methodology was formed based on the concept, formalized requirements for the method, step-wise structure of investigations and interpretation of results. The concept is based on differences in the structure of the adipose cell (adipocyte) of different AT types. The method is based on extraction of heme containing proteins. To this end, solvents and parameters of extraction that facilitate their better extraction have been chosen. An AT type has been determined by the total content of iron contained in the cytochrome fragment. Our own modification was selected. This modification includes preliminary mincing of a sample with the ice-cold TES buffer (pH 8.5) in a ratio of 1:5 (g: mL), homogenization at 9,000 rpm for 2 min with the following centrifugation at 10,000 g and 4 °C for 15 min. Effectiveness of the proposed method was confirmed by the histological and electrophoretic analyses. Therefore, the new methodology of identification and differentiation of adipocytes was proposed for rapid and effective detection of an adipose tissue type

Biotechnological techniques for intensification of protein extraction from the porcine pancreas

Processing of secondary products after slaughter of farm animals is in demand. The pancreas is a rich source of bioactive protein substances, effective extraction of which is a serious problem today due to their aggregation. The aim of the work was to assess the extractivity of protein substances of the porcine pancreas using sodium chloride, trehalose, arginine, and combination of glycine and proline. The protein concentration was determined in the obtained extracts by the biuret reaction and their protein composition was assessed by densitometry of two-dimensional electropherograms using software ImageMaster™ 2D Platinum powered by Melanie 8.0. The results showed a positive effect of anti-aggregation agents on the release of protein substances into a solution. The highest protein concentration (33.36±0.64 g/l) was observed when adding 1М L-arginine; however, it was conditioned mainly by an increase in the content of three major protein fractions rather than by diversity of the protein composition. In general, the use of 0.9% NaCl as an extractive agent was quite effective, but selectivity to certain protein groups was observed for anti-aggregation agents such as sodium chloride, trehalose, arginine, glycine and proline, as well as their combination. The obtained results are important for intensifying extraction of protein substances including target ones with the subsequent application in different fields

Study of the functional product’s protein compounds digestion features

The aim of the study was to investigate the transformation of meat product’s proteins from pig hearts and aortas during enzymatic hydrolysis in an in vitro model of the gastrointestinal tract. The model consisted of three phases simulating digestion processes: “oral cavity” phase (a-amylase, pH 7.0; 2 min), “stomach” phase (pork pepsin, pH 3.0; 120 min), “intestine” phase (pork pancreatin, pH 7.0; 130 min). The product was sequentially subjected to hydrolysis, at the end of each phase, samples were taken to determine the protein concentration (biuret method) and visualize the protein fractions (one-dimensional electrophoresis). A significant increase in protein concentration at the “stomach” phase was revealed by 3.2 times, and the absolute content by 4.6 times. At the “intestine” phase, a decrease in the number of peptide complexes with copper ions by 1.8 times, the absolute protein content by 8.5% was re‑ vealed. The noted tendency was confirmed by electrophoretic studies — at the stage, simulating digestion in the stomach, the prod‑ ucts of meat product’s proteins hydrolysis were visualized; at the “intestine” phase, a low expression of protein fractions in the range of more than 10 kDa is shown. The maximum hydrolysis of protein compounds at the “stomach” phase to poly- and oligopeptides was confirmed, continuing at the “intestine” stage with the accumulation of free amino acids. This methodology makes it possible to visualize the products of hydrolysis of proteins in a meat product at all stages of the model and to monitor changes in protein concentration in the system

Comparison of the proteomic profile of pork byproducts during their storage

In this article, the proteomic profiles of pork by-products (snout, tongue, liver, kidney, spleen) were studied by comparative method on the first day and the fifth day of their storage. Two-dimensional electrophoresis according to O’Farrell was used for the aims of this article, while the results were further processed in ImageMaster software. Proteomic maps of by-products showed clear changes in protein composition after visualization and images analysis. There was a decrease and increase in manifestation intensity of some proteins. The study of the obtained electrophoregrams with the help of references resources allowed identifying various compounds in the by-products. 9 protein fractions with various intensity of manifestation were found on the day 1st and 5th. On the 1st day the following substances were intensively manifested: in the liver — glutathione peroxidase 4 (22.3 kDa), LEAP-2 (8.8 kDa); in the kidneys — quinone oxidoreductase (34.9 kDa); in the spleen — glycoprotein CD59 (13.7 kDa), in the patch — protein flint (49.07 kDa). It is noted that these proteins play their role in stopping certain processes in cells, like oxidation, microbial activity, and accumulation of toxic substances. These processes can worsen the quality of raw materials, and further lead to spoilage of the food product. On the 5th day of storage the highest intensity of manifestation of glyceraldehyde-3-phosphate dehydrogenase (35.8 kDa) in the liver was observed; superoxide dismutase [Cu-Zn] (15.8 kDa) was noted in the kidneys, colony-stimulating factor (16.2 kDa) was observed in the spleen and glutaredoxin –1 (11.8 kDa) in the tongue. In its turn, on the fifth day these chemical processes manifested themselves more intensely, as the fatty acids and glucose broke down. To obtain more accurate results, the proteins were compared by their volume. Among the identified fractions the highest expression was observed in LEAP 2 (8.8 kDa) on the first day, and in glyceraldehyde-3-phosphate dehydrogenase (35.8 kDa) on the fifth day. The least change in the intensity of manifestation was noted for superoxide dismutase [Cu-Zn] (15.8 kDa), which volume increased during storage by 13% for 5 days. The analysis of the obtained electrophoregrams allowed identifying various compounds, tracing the changes in the qualitative composition of protein in by-products during various periods of their storage. The obtained data demonstrate the transformation of protein molecules during storage, which makes it possible to determine the changes and quality of the food products

Study of the functional product’s protein compounds digestion features

The aim of the study was to investigate the transformation of meat product’s proteins from pig hearts and aortas during enzymatic hydrolysis in an in vitro model of the gastrointestinal tract. The model consisted of three phases simulating digestion processes: “oral cavity” phase (a-amylase, pH 7.0; 2 min), “stomach” phase (pork pepsin, pH 3.0; 120 min), “intestine” phase (pork pancreatin, pH 7.0; 130 min). The product was sequentially subjected to hydrolysis, at the end of each phase, samples were taken to determine the protein concentration (biuret method) and visualize the protein fractions (one-dimensional electrophoresis). A significant increase in protein concentration at the “stomach” phase was revealed by 3.2 times, and the absolute content by 4.6 times. At the “intestine” phase, a decrease in the number of peptide complexes with copper ions by 1.8 times, the absolute protein content by 8.5% was re‑ vealed. The noted tendency was confirmed by electrophoretic studies — at the stage, simulating digestion in the stomach, the prod‑ ucts of meat product’s proteins hydrolysis were visualized; at the “intestine” phase, a low expression of protein fractions in the range of more than 10 kDa is shown. The maximum hydrolysis of protein compounds at the “stomach” phase to poly- and oligopeptides was confirmed, continuing at the “intestine” stage with the accumulation of free amino acids. This methodology makes it possible to visualize the products of hydrolysis of proteins in a meat product at all stages of the model and to monitor changes in protein concentration in the system