Comparative study of technologies for extraction of biologically active substances from the raw material of animal origin

Technologies of isolation and concentration of biologically active substances, developed in the middle of the 20th century, need adjustment and adaptation to modern conditions both to increase the activity of substances and for greater economic efficiency. The aim of the research is the comparison of dynamics of biologically active compounds extraction from porcines pancreas in two methods: the saline method based on 0.9% sodium chloride solution, and the acidic method based on 2.4% trichloroacetic acid solution. Also the purpose of research is to assess the possibilities for further optimization of technologies. The total protein concentration based on the biuret reaction in the samples taken during the extraction, as well as the calculation and analysis of the point degrees and rates of extraction are chosen as the controlled parameters. Local maxima of the protein yields into the extractant media at the 60th, 135th and 255th minute were recorded during saline extraction; and at the 75th and 135th minute during acid extraction. Also the proteomic profile of the extracts was studied. Wide range of compounds with molecular weight of less than 52 kDa was found in extracts based on physiological saline solution, and protein substances of whole presented range of molecular weights in trichloroacetic acid based extracts were considered. The predominance of low molecular weight protein fraction of interest was noted also in this method of extraction in comparison with the other methods of extraction. According to the UniProt database, we assume availability of probable compounds with a molecular weight of less than 30 kDa in the purified acidic extract. The presence of some proteins absent in the final saline extract was noted. The acidic erythrograms showed a weak degrading effect of both types of extracts on the membranes of rat erythrocytes, as well as the cytoprotective effect of acidic ultrafiltrates (less than 3 kDa). The obtained results prove a better efficiency of trichloroacetic acid extraction method used for obtaining a mixture of a wide range of compounds, including biologically active substances of low molecular weight

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

Evolution of in vitro digestibility techniques: a systematic review

The inability to reproduce certain digestive processes in vivo, high research costs and ethical aspects have led to the development of a large number of in vitro digestion models. These models allow us to take into account various factors of modeling complex multistage physiological processes occurring in the gastrointestinal tract, which makes them promising and widely used. A significant part of in vitro methods includes assessment by enzymatic digestion and are based on the calculation of nitrogen remaining after digestion in relation to the initial total nitrogen (according to the Dumas, Kjeldahl method, spectrophotometric or chromatographic method). There are also a number of titrometric methods (pH‑stat), which are mainly used to assess the digestibility of feed, most successfully for aquatic animals due to the simplicity of their digestive tract. Methods for assessing the digestibility of food products by enzymatic digestion have undergone various stages of evolution (since 1947) and have been widely modified by including various enzymes (pepsin, trypsin, pancreatin, erepsin, etc.) in model systems, indices for various products have been determined on their basis (pepsin-digest-residue (PDR) index, 1956; pepsin pancreatin digest (PPD) index, 1964; pepsin digest dialysate (PDD), 1989). As a result, a single protocol was formed to study the digestibility of food — INFOGEST (2014–2019), which includes three stages of digestion (oral, gastric and intestinal). It allows researchers to accurately reproduce the conditions of the human gastrointestinal tract and is widely used by scientists around the world

Водно-солевая экстракция как метод получения смеси биологически активных соединений белковой природы из поджелудочной железы свиньи

A relevant solution to the problem of processing meat industry waste in Russia is to obtain useful biologically active compounds from abundant organs. The aim of this study was to examine the effectiveness of the saline extraction as a method for extracting a mixture of promising biologically active compounds from the porcine pancreas, as well as to determine the optimal time for the process. The study consisted of extraction of the porcine pancreas with 0,9% sodium chloride solution for 5 h 30 min with further determination of the total protein concentration and proteomic profile of the samples taken throughout the process. Based on the analysis of the dependence of the total protein content in the extractant on time, the optimal extraction time was determined to be 135–150 minutes. When studying the results of electrophoresis and the data of their processing, the optimal extraction time for the targeted isolation of the low-molecular fraction of compounds was also determined to be 90 min. At the same time, 13 protein bands with a molecular weight of 52 kDa and below were found on the electropherograms. Saline should be considered applicable for obtaining extracts rich in biologically active substances, incl. hormones, enzymes and other physiologically active compounds.Актуальным решением проблемы переработки отходов мясной промышленности в России является получение полезных биологически активных соединений из богатых ими органов. Целью настоящего исследования было изучение эффективности метода экстракции физиологическим раствором как способа извлечения смеси перспективных биологически активных соединений из поджелудочной железы свиньи, а также определение оптимального времени процесса. Исследование заключалось в проведении экстракции поджелудочной железы 0,9% раствором натрия хлорида в течение 5 ч 30 мин с дальнейшим определением общей концентрации белка биуретовым методом. Также получен протеомный профиль образцов, отбираемых на протяжении всего процесса, методом одномерного денатурирующего электрофореза по Лэммли в 12,5% полиакриламидном геле. На основе анализа зависимости содержания общего белка в экстрагенте от времени определено оптимальное время экстракции, которое составило 135–150 мин. По результатам электрофореза и данных биоинформационного анализа оптимальное время экстракции для целенаправленного выделения низкомолекулярной фракции соединений составляло 90 мин. На электрофореграммах обнаружены 13 белковых полос с молекулярной массой 52 кДа и ниже. Таким образом, 0,9% раствор натрия хлорида применим для получения экстрактов, богатых биоактивными веществами, в том числе гормонами, ферментами и другими физиологически активными соединениями

Применение метода тонкослойной хроматографии для анализа антиоксидантной активности

Plants are a rich source of effective non-toxic biologically active substances. Various physicochemical methods of analysis are used for evaluation of plant antioxidant activity. Composition of ethanol extracts of red, yellow and white onion husks, dried rosemary, basil, and chaga were studied by high performance thin layer chromatography (HPTLC) method. The antioxidant activity of the obtained fractions on a chromatographic plate was assessed by subsequent DPPH screening. The extracts red and yellow onion husk and rosemary demonstrated the highest antioxidant activity, variability of the qualitative composition and similarity of antioxidant profiles, while extract of white onion husks did not contain any antioxidant classes. Intensive spots with Rf of 0.13-0.97 were observed along the whole chromatogram track corresponding to red onion husks. It was also found that all tested extract, excepting white onion husk and chaga, contained spots with varying degrees of intensity in the Rf range of 0.96-0.98, which corresponded quercetin Rf value.Для исследования антиоксидантной активности растительного сырья, которое является богатым источником эффективных нетоксичных биологически активных веществ, используются различные физикохимические методы анализа. В данной работе был применен метод высокоэффективной тонкослойной хроматографии (ВЭТСХ) для разделения биологически активных веществ, входящих в этанольные экстракты шелухи красного, желтого и белого лука, сушенных розмарина, базилика и чаги. Антиоксидантную активность полученных фракций на хроматографической пластине оценивали последующим DPPH-скринингом. Наибольшими показателями антиоксидантной активности и вариабельностью качественного состава характеризовались экстракты шелухи красного и желтого лука, а также розмарина, причем для этих образцов была отмечена схожесть антиоксидантных профилей. В экстракте белого лука не было обнаружено каких-либо классов антиоксидантов. Для шелухи красного лука было отменено наличие значительно выраженных пятен по всему треку, значения Rf которых расположены в диапазоне 0,13-0,97. Доказано, что все экстракты, кроме шелухи белого лука и чаги, в разной степени интенсивности имеют пятна с коэффициентом замедления в диапазоне 0,96-0,98, что соответствует полученному значению Rf кверцетина

Ассоциация полиморфных вариантов генов ферментов биотрансформации ксенобиотиков с восприимчивостью к заболеваемости туберкулезом легких

The objective: to investigate the relationship of polymorphic variants of genes of xenobiotic biotransformation metabolism enzymes (NAT2 (590G>A (rs1799930)), CYP2E1 (9896C>G (rs2070676)), ABCB1 (3435T>C (rs1045642)), GSTM1 (E/D), GSTT1 (E/D) with the risk of pulmonary tuberculosis.Subjects and Methods. Within the framework of this study, a population sample of unrelated 1081 individuals of Slavic nationalities (mainly Russians) living in the territory of Kursk Oblast was used.Results. The del/del (D/D) GSTM1 genotype was associated with an increased risk of developing tuberculosis, while carrying the del/del (D/D) GSTT1 genotype was associated with a lower risk of developing the disease. Polymorphism 3435T>C ABCB1 (TC genotype) was associated with increased susceptibility to pulmonary tuberculosis in the codominization model, while TT+TC genotypes had the same association in the dominance model.Цель исследования: исследовать взаимосвязь полиморфных вариантов генов ферментов метаболизма биотрансформации ксенобиотиков (NAT2 (590G>A (rs1799930)), CYP2E1 (9896C>G (rs2070676)), ABCB1 (3435T>C (rs1045642)), GSTM1 (E/D), GSTT1 (E/D) с риском развития туберкулеза легких.Материал и методы. В рамках исследования использовалась гомогенная по этническому составу популяционная выборка неродственных индивидов славянских национальностей (преимущественно русских), проживающих на территории Курской области, общей численностью 1 081 индивид.Результаты. Генотип del/del (D/D) GSTM1 ассоциировался с повышенным риском развития туберкулеза, тогда как носительство генотипа del/del (D/D) GSTT1 было ассоциировано с пониженным риском развития болезни. Полиморфизм 3435T>C ABCB1 (генотип TС) ассоциировался с повышенной восприимчивостью к туберкулезу легких в рамках модели кодоминирования, а генотипы ТТ+ТС в рамках модели доминирования

Structure of Metaphase Chromosomes: A Role for Effects of Macromolecular Crowding

In metaphase chromosomes, chromatin is compacted to a concentration of several hundred mg/ml by mechanisms which remain elusive. Effects mediated by the ionic environment are considered most frequently because mono- and di-valent cations cause polynucleosome chains to form compact ∼30-nm diameter fibres in vitro, but this conformation is not detected in chromosomes in situ. A further unconsidered factor is predicted to influence the compaction of chromosomes, namely the forces which arise from crowding by macromolecules in the surrounding cytoplasm whose measured concentration is 100–200 mg/ml. To mimic these conditions, chromosomes were released from mitotic CHO cells in solutions containing an inert volume-occupying macromolecule (8 kDa polyethylene glycol, 10.5 kDa dextran, or 70 kDa Ficoll) in 100 µM K-Hepes buffer, with contaminating cations at only low micromolar concentrations. Optical and electron microscopy showed that these chromosomes conserved their characteristic structure and compaction, and their volume varied inversely with the concentration of a crowding macromolecule. They showed a canonical nucleosomal structure and contained the characteristic proteins topoisomerase IIα and the condensin subunit SMC2. These observations, together with evidence that the cytoplasm is crowded in vivo, suggest that macromolecular crowding effects should be considered a significant and perhaps major factor in compacting chromosomes. This model may explain why ∼30-nm fibres characteristic of cation-mediated compaction are not seen in chromosomes in situ. Considering that crowding by cytoplasmic macromolecules maintains the compaction of bacterial chromosomes and has been proposed to form the liquid crystalline chromosomes of dinoflagellates, a crowded environment may be an essential characteristic of all genomes

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