Changes In Protein Molecules Research Articles

Ligand-induced conformational changes in protein molecules detected by sum-frequency generation

We present the first demonstration of ligand-induced conformational changes in a biological molecule, a protein, by sum-frequency generation (SFG). Constructs of KRasG12D protein were prepared by selectively deuterating residues of a single amino acid type using isotope-labeled amino acids and cell-free protein synthesis. By attaching labeled protein to a supported bilayer membrane via a His-tag to Ni-NTA-bearing lipids, we ensured that single layers of ordered molecules were formed while preserving the protein’s native structure. Exceptionally large SFG amide I signals were produced in both labeled and unlabeled proteins, demonstrating a high degree of orientational order upon attachment to the bilayer. Deuterated protein also produced SFG signals in the CDx spectral region, which were not present in the unlabeled protein. The CDx signals were measured before and after binding a peptide inhibitor, KRpep-2d, revealing shifts in SFG intensity due to conformational changes at the labeled sites. In particular, peaks associated with CDx stretching vibrations for alanine, valine, and glycine changed substantially in amplitude upon inhibitor binding. By inspection of the crystal structure, these three residues are uniquely colocated on the protein surface in and near the nucleotide binding site, which is in allosteric communication with the site of peptide inhibitor binding, suggesting an approach to identify a ligand’s binding site. The technique offers a highly sensitive, nonperturbative method of mapping ligand-induced conformational changes and allosteric networks in biological molecules for studies of the relationship between structure and function and mechanisms of action in drug discovery.

Effect of terahertz radiation on drug activity in bacterial cells

Terahertz (THz) waves have been reported to change membrane permeability and induce conformational changes in protein molecules. Drugs action on cells involves membrane permeability, and we therefore investigated the effect of THz waves on the activity of the cytotoxic drug bleomycin on Escherichia coli. 0.46 THz radiation with an average power of 2.5 W/cm2 was noncytotoxic to E. coli cells. However, 0.46 THz radiation enhanced the cytotoxic activity of bleomycin in E. coli cells, and the drug-enhancing effect depended on the power density of the THz waves. Then, the activity of the drug remaining in the culture medium after THz radiation did not differ from that remaining after non-radiation. This indicates that THz radiation does not affect the bacterial cell-membrane permeability to bleomycin. Thus, these results suggest that 0.46 THz radiation enhances the cytotoxicity of bleomycin to E. coli cells and may influence the mechanism of bleomycin action within cells rather than affecting drug uptake.

Mechanism of enhancing rehydration capacity and foaming property of spray-dried egg white powder by lactic acid fermentation

Spray-dried egg white powder has limited applicability as a raw material in food production due to its deteriorative rehydration capacity. Lactic acid fermentation is a promising technique for protein denaturation and has been widely used in the food industry. In this work, we investigated the effects of 4, 8, and 12-h lactic acid fermentation on protein structure, powder characteristics, rehydration capacity, and foaming property of spray-dried egg white powder. Results show that the fermentation treatment significantly reduced the surface hydrophobicity of egg white powder samples, while 8 and 12 h of fermentation significantly increased their zeta potential. During the 12 h of fermentation, the particle size of the powder gradually decreased from 2.59 μm to 1.69, 1.23, and 0.76 μm, respectively. Scanning electron microscope result shows the same and also indicates a concave hole structure in fermented egg white powders. These changes in protein molecules result in the significantly improved wettability, dispersibility, and solubility of the powder, thus enhancing its rehydration capacity. The foaming ability and foam stability of egg white powder also significantly improved after fermentation. Furthermore, the fermentation process altered the secondary structure, moisture content, bulk densities, flowability, and color of the powder. This work demonstrates the usefulness of lactic acid fermentation in enhancing the quality of egg white powder. The findings have significant implications for the development of better-quality egg white powder products in the food industry.

Comparison of effects of potassium iodide and potassium chloride on structure and physicochemical properties of proteins

The effects of KI (1.25, 6.25, 12.50 mM) on the structure and physicochemical properties of proteins (bovine serum albumin, ovalbumin, and lysozyme protein) at the corresponding pH values were investigated. The results showed that the interaction of I− with protein molecules was significantly stronger than that of Cl− under different concentration conditions. The higher the concentration, the more pronounced the interaction was by turning potassium iodide into potassium iodate, which bound to proteins in a more stable form, and the conformational changes of protein molecules were more remarkable. At high concentrations, the hydrodynamic radii of the bovine serum albumin and ovalbumin proteins increased significantly, up to 20.97 % and 157.95 %, respectively, and affected the microrheological behavior of the system, weakened the tracer particle motion and the elasticity of the system, changed the microenvironment in which the particles were located, with significant changes in the secondary structure of their amide I bands. Overall, there was no significant effect on lysozyme proteins. This study offers new directions for physiological mechanisms and promotes the development and application of food products.

Screening of the differentially expressed proteins in malignant transformation of BEAS-2B cells induced by coal tar pitch extract.

Coal tar pitch extract (CTPE) was carcinogenic and could cause occupational lung cancer. Hence, we explored the changes of protein molecules during CTPE-induced malignant transformation (MT) of immortalized human bronchial epithelial (BEAS-2B) cells and provided clues for screening early biomarkers of CTPE-associated occupational lung cancer. The MT model of BEAS-2B cells induced by CTPE with 15.0μg/mL. Subsequently, the MT of the BEAS-2B cells was verified by morphological observation, cell proliferation test, plate colony formation assay, and cell cycle assay. At the end of the experiment, we explored the differentially expressed proteins (DEPs) by total protein tandem mass tags quantitative proteomics technique between DMSO40 cells and CTPE40 cells. It was found that the proliferation ability, and colony formation rate were enhanced, and the cell cycle was changed. Then, bioinformatics analysis showed that a total of 107 DEPs were screened between CTPE40 and DMSO40 cells, of which 74 were up-regulated and 33 were down-regulated. As a result, 6 hub proteins were screened by protein-protein interaction network analysis. The expression levels of COX7A2, COX7C, MT-CO2, NDUFB4, and NDUFB7 were up-regulated as well as the expression of RPS29 protein was down-regulated. In summary, we established an MT model in vitro and explored the changes in protein molecules. As a result, this study suggested that changes of protein molecules, including COX7A2, COX7C, NDUFB7, MT-CO2, NDUFB4, and RPS29, occurred at the stage of BEAS-2B cell malignancy following CTPE exposure, which provided key information for screening biomarkers for CTPE-related occupational lung cancer.

The time-zero HSQC method improves the linear free energy relationship of a polypeptide chain through the accurate measurement of residue-specific equilibrium constants.

EXSY (exchange spectroscopy) NMR provides the residue-specific equilibrium constants, K, and residue-specific kinetic rate constants, k, of a polypeptide chain in a two-state exchange in the slow exchange regime. A linear free energy relationship (LFER) discovered in a log k versus log K plot is considered to be a physicochemical basis for smooth folding and conformational changes of protein molecules. For accurate determination of the thermodynamic and kinetic parameters, the measurement bias arising from state-specific differences in the R1 and R2 relaxation rates of 1H and other nuclei in HSQC and EXSY experiments must be minimized. Here, we showed that the time-zero HSQC acquisition scheme (HSQC0) is effective for this purpose, in combination with a special analytical method (Π analysis) for EXSY. As an example, we applied the HSQC0 + Π method to the two-state exchange of nukacin ISK-1 in an aqueous solution. Nukacin ISK-1 is a 27-residue lantibiotic peptide containing three mono-sulfide linkages. The resultant bias-free residue-based LFER provided valuable insights into the transition state of the topological interconversion of nukacin ISK-1. We found that two amino acid residues were exceptions in the residue-based LFER relationship. We inferred that the two residues could adopt special conformations in the transition state, to allow the threading of some side chains through a ring structure formed by one of the mono-sulfide linkages. In this context, the two residues are a useful target for the manipulation of the physicochemical properties and biological activities of nukacin ISK-1.

Grass pea (Lathyrus sativus L.) protein yield and functionality as affected by extraction method: Alkaline, ultrasound‐assisted, and ultrasound pretreatment extraction

AbstractBackground and ObjectivesPlant protein products are now considered sustainable substitutes for animal proteins in new product formulations, and various methods such as ultrasound have been used to improve their properties. The aim of this study was to investigate the physicochemical and functional properties of grass pea (Lathyrus sativus L.) protein isolate was extracted, as a promising source of plant‐based protein, using conventional (CP), pre‐ultrasonic (PUP) and ultrasound‐assisted (UAP) treatments.FindingsExtraction yield, protein content, and protein yield of ultrasound treatments were increased. Ultrasound treatments improved the functional properties of pea protein. Thermal and surface properties were also altered due to the conformational changes in protein molecules during sonication. According to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, there were no differences in protein fractions of different samples in terms of molecular weight, and protein fractions between 23 and 100 kDa were observed. However, Fourier transform infrared spectroscopy results revealed changes in different treatments suggesting that sonication changed protein secondary structures. Correspondingly, optical and fluorescence microscopy images of protein solutions and foams revealed changes in the structure of proteins and CP presented larger protein aggregates and larger foam bubble sizes than PUP and UAP.ConclusionsUltrasound treatment could increase extraction and improve the functionality of proteins which was attributed to changes in the secondary structure of proteins.Significance and NoveltyGrass pea protein isolate can be considered a novel source of valuable protein and ultrasound treatment could enhance the functionality of the protein increasing potential applications of this ingredient in the food industry.

Sample Flow Rate Scan in Electrospray Ionization Mass Spectrometry Reveals Alterations in Protein Charge State Distribution.

Sample flow rate is one of the parameters that influence the sensitivity of electrospray ionization (ESI) mass spectrometry. By varying the sample flow rate, initial droplets of different sizes can be generated. Protein molecules in small droplets may form gas-phase ions earlier than the ones in large droplets. Here, we have systematically studied the influence of sample flow rate on the ESI charge state distributions (CSDs) of model proteins. A dedicated programmable sample flow rate scanner was used to infuse protein samples at different flow rates into a mass spectrometer. The synergistic influence of sample flow rate and various electrolytes (ammonium acetate, ammonium bicarbonate, ammonium formate, and piperidine) was studied. Significant alterations to the CSDs with increasing flow rate were observed. For example, in the presence of ammonium acetate, at low flow rates, lower charge states of proteins showed high intensities, while at high flow rates, ions related to higher charge states of proteins dominated the spectra. On the other hand, in the presence of piperidine, a significant reduction in the ion currents of all charge states was observed during the flow rate scan. Our observations suggest that at low flow rates the protein molecules follow a charged residue model of ionization mechanism, and at high flow rates-due to structural changes in protein molecules in large ESI droplets-the charged residue and chain ejection models can possibly coexist. We propose the use of sample flow rate scan as a way to reveal the influence of flow rate on the CSDs of the studied proteins.

Effect of CPEB4 on Migration and Cycle of Chronic Myeloid Leukemia Cell

To investigate the effects of CPEB4 on the migration and cycle of K562 cells and the changes of protein molecules that may be involved in the regulatory mechanism. Western blot was used to detect the expression of CPEB4 in normal leukocytes and K562 cells. The overexpression plasmid pcDNA3.1(+)-His-CPEB4, silencing plasmid pPLK+Puro-CPEB4 shRNA were transfected into K562 cells by electroporation so as to change CPEB4. The transfection efficiency was detected by Western blot. Finally, the migration and cycle of different cells were detected by Transwell chamber and flow cytometry.Western blot was used to detect the expression changes of MMP2, MMP9, CDK4, CyclinD1 and P21 proteins. Compared with normal white blood cells, the expression of CPEB4 protein in K562 cells was significantly enhanced (P＜0.01); Compared with the control group, CPEB4-silenced K562 cells showed that the cell migration ability was significantly enhanced (P＜0.01); G0/G1 phase cell ratio reduced, G2/M phase cell ratio increased, and cell cycle progression accelerated（P＜0.01）, The expression levels of MMP2 (P＜0.05), MMP9 (P＜0.05), CDK4 (P＜0.01), CyclinD1 (P＜0.01) proteins increased significantly. The expression level of P21 protein significantly decreased (P＜0.01). The migration ability of K562 cells after CPEB4 overexpression was decreased (P＜0.01), the cell ratio of G0/G1 phase in the cell cycle increased, the cell proportion of S phase decreased and the cell cycle progression was arrested at G0/G1 phase (P＜0.01). The expression of P21 protein increased, MMP2 , MMP9, CDK4, CyclinD1 protein expression decreased significantly（P＜0.05-0.01）. CPEB4 can inhibit the migration of K562 cells and arrest cell cycle progression at G0/G1 phase. Its mechanism may be related with regulating the exprossion of MMP2, MMP9, CDK4, CyclinD1 and P21 proteins.

Numerical study on mechanisms of soy protein as a functional modifier for polymer materials

In the past decades, to meet the increasing demands for protecting the environments, taking bio-degradable green materials as alternatives to traditional petroleum-based polymer materials has become a global interest. From the previous studies, it is demonstrated that soy protein-based biomaterial is one of the promising candidates to be used as functional modifier for polymers, due to the complex structures of protein and diverse interactions with polymers. However, the underlying mechanisms remain unclear. Molecular dynamic (MD) simulation is a power tool and can provide insights in understanding the conformational changes of protein molecules, as well as the interactions between proteins and polymers. In this study, 11S molecule of soy protein is chosen as a representative, and three different denaturation processes are applied via MD simulation, including heat denaturation at two temperatures and the breaking of disulfide bonds. It is observed that by controlling the denaturation conditions, the structures and properties of the protein molecules can be manipulated. Low temperature condition has limited impacts on the structures and properties of 11S molecules; high temperature process, on the other hand, is found to lead to an intensive secondary phase transition of the molecules; moreover, by breaking the disulfide bonds, the hydrophobic residues of the molecules can be largely exposed, potentially forming strong interactions with the hydrophobic poly(vinylidene fluoride) (PVDF) polymer. Besides, different protein-polymer interactions could result in various property-modification effects on PVDF polymer. For instance, due to the strong interaction between PVDF and the protein molecule denatured without disulfide bonds, the dipole moment of PVDF at the interface is largely enhanced, and the polarizability under external electric fields is also improved, which is in agreement with experimental result.

Redox State of PDA Directs Cellular Responses through Preadsorbed Protein

Polydopamine (PDA) is capable of adhering on nearly all kinds of surfaces and shows good biocompatibility. Moreover, its surface state could be switched between oxidation and reduction states under different electrical stimulation. In this work, the effects of PDA redox states on protein adsorption, as well as the subsequent effects on cellular responses were characterized and evaluated. It was found that the electrical treatment changed the redox states of PDA, which in turn changed the state of preadsorbed protein molecules and eventually affected the cellular responses. BSA preadsorbed PDA film was found to be beneficial for cell proliferation when PDA was changed into reduction state (RPDA), while BMP-2 preadsorbed PDA film showed promotion on cell differentiation when PDA was changed into oxidation state (OPDA). It was found that the transitions of PDA to RPDA and OPDA greatly changed the secondary structure of protein preadsorbed on it. This work provides a deeper insight on changes of protein molecules between cells and PDA surfaces with different redox states, which is important for both optimization of cell-material interactions and application of PDA as a functional coating for biomedical engineering.

Ubiquitin and regulation of apoptosis in Jurkat cells

Introduction.One of the crucial tasks in medicine is studying the molecular mechanisms of selective management of tumor cell apoptosis following conformational changes in protein molecules (ubiquitination).The purpose of the study. The aim of the project is to establish the role of ubiquitin and ubiquitinligase in dexamethasone-induced apoptosis in Jurkat cells.Materials and methods.The study was carried out on the Jurkat tumor cell line (intact cells and cells cultured in the presence of an apoptosis inducer dexamethasone in the final concentration of 10 µmol. In intact and dexamethasone-affected Jurkat cells, implementation of apoptosis and the amount of FAS-, TNF Receptor 1 and cells with reduced mitochondrial membrane potential were assessed by flow cytometry using FITC-conjugated Annexin V and Propidium Iodide. The levels of NF-κB, Apaf-1, ubiquitin and ubiquitin ligase were determined by Western blot analysis. The activity of caspase-3 was measured by spectrofluorometry.Results.When adding the apoptosis inducer dexamethasone to the Jurkat cell culture, we registered a fall in the concentration of ubiquitin and a rise in the level of ubiquitinligase against the backdrop of activated receptor(an increase in the amount of Annexin V positive cells, FASand TNF Receptor 1) and mitochondrialmediated (an increase in the number of cells with reduced mitochondrial membrane potential and elevation of Apaf-1 level) pathways of apoptosis, as opposed to the intact cell culture. We estimated the completion of apoptosis by determining the activity of caspase-3 in the investigated tumor cells.Conclusion.The obtained findings allow the conclusion that ubiquitination of regulatory and effector proteins in programmed cell death is one of the molecular mechanisms that regulates and selectively controls apoptosis in Jurkat cells.

Methodical aspects of determining the oxidative modification of the protein

Introduction. To date, a large amount of data has been accumulated on oxidative stress, its inducers and its effect on the human body. In this article, the main changes in protein molecules under the influence of products of oxidative stress are reviewed, and a brief review of the main methods for detecting protein’s modification was made. Aim of investigation - selection of some methodical approaches to determination of level of oxidative modification of protein of blood serum. Materials and methods. A modification of the Reznick et al. method to determine the oxidative modification of the protein in a microversion, the essence of which is a proportional decrease in the volumes of reagents participating in the reaction and in the replacement of certain reagents with their analogues in chemical nature. Results. The concentration of serum ketone-dinitrophenylhydrazones of neutral type (kDNPn) of healthy peripheral blood serum was 1.24±0.15, the ketone-dinitrophenylhydrazones of basic type (kDNPb) were 0.74±0.11, and the aldehyde-dinitrophenylhydrazones of neutral type (aDNPn) 0.37±0.07 nmol/mg protein. The total total concentration of oxidized proteins of peripheral blood serum of healthy donors was 2.35±0.11 nmol/mg protein. It was revealed that aDNPn amount for 16%, for kDNPb - 31.5%, for kDNPn - 52.5% of oxidized proteins. Thus, the ratio of oxidized proteins of blood serum of healthy donors to aDNPn : kDNPb : kDNPn is 1 : 2 : 3.3. Conclusion. During the research it was found that when the level of oxidized protein is determined by a micro-method, the level of oxidized proteins corresponds to the results obtained both in the classical variant of the reaction and in the literature data.

Effects of extrusion conditions on the physicochemical properties of soy protein/gluten composite

Soybean protein-gluten blend was extruded using a co-rotating twin-screw extruder. Effects of different extrusion conditions on the textural properties of extrudates were analyzed by central composite design through the evaluation of texturization index, water holding capacity, and hardness of extruded protein products. Extrusion process variables including feed moisture content (40%-60%), screw speed (12-20 Hz), extrusion temperature (120°C-180°C), and gluten content (16%-32%) were studied by the nonlinear regression equations analysis. The extrusion process was also optimized using response surface methodology (RSM). The influence of moisture content on the hardness of extrudate was the most significant. The optimized results, including feed moisture content, extrusion temperature, screw speed and gluten content were 49.18%-50.15%, 155.24°C-159.86°C, 16.41-16.73 Hz, 20.00%-20.03%, respectively. The microstructures of TISP products were analyzed by scanning electron microscopy (SEM) and the changes of protein molecules before and after extrusion were also analyzed by SDS-PAGE electrophoresis. Keywords: soybean protein, gluten, extrusion, response surface methodology (RSM), SDS-PAGE electrophoresis, Microstructural properties DOI: 10.25165/j.ijabe.20181104.4162 Citation: Wu M, Sun Y, Bi C H, Ji F, Li B R, Xing J J. Effects of extrusion conditions on the physicochemical properties of soy protein/gluten composite. Int J Agric & Biol Eng, 2018; 11(4): 230-237.

Physical driving force of actomyosin motility based on the hydration effect.

We propose a driving force hypothesis based on previous thermodynamics, kinetics and structural data as well as additional experiments and calculations presented here on water-related phenomena in the actomyosin systems. Although Szent-Györgyi pointed out the importance of water in muscle contraction in 1951, few studies have focused on the water science of muscle because of the difficulty of analyzing hydration properties of the muscle proteins, actin, and myosin. The thermodynamics and energetics of muscle contraction are linked to the water-mediated regulation of protein-ligand and protein-protein interactions along with structural changes in protein molecules. In this study, we assume the following two points: (1) the periodic electric field distribution along an actin filament (F-actin) is unidirectionally modified upon binding of myosin subfragment 1 (M or myosin S1) with ADP and inorganic phosphate Pi (M.ADP.Pi complex) and (2) the solvation free energy of myosin S1 depends on the external electric field strength and the solvation free energy of myosin S1 in close proximity to F-actin can become the potential force to drive myosin S1 along F-actin. The first assumption is supported by integration of experimental reports. The second assumption is supported by model calculations utilizing molecular dynamics (MD) simulation to determine solvation free energies of a small organic molecule and two small proteins. MD simulations utilize the energy representation method (ER) and the roughly proportional relationship between the solvation free energy and the solvent-accessible surface area (SASA) of the protein. The estimated driving force acting on myosin S1 is as high as several piconewtons (pN), which is consistent with the experimentally observed force.

Ultrasound assisted intensification of enzyme activity and its properties: a mini-review.

Over the last decade, ultrasound technique has emerged as the potential technology which shows large applications in food and biotechnology processes. Earlier, ultrasound has been employed as a method of enzyme inactivation but recently, it has been found that ultrasound does not inactivate all enzymes, particularly, under mild conditions. It has been shown that the use of ultrasonic treatment at appropriate frequencies and intensity levels can lead to enhanced enzyme activity due to favourable conformational changes in protein molecules without altering its structural integrity. The present review article gives an overview of influence of ultrasound irradiation parameters (intensity, duty cycle and frequency) and enzyme related factors (enzyme concentration, temperature and pH) on the catalytic activity of enzyme during ultrasound treatment. Also, it includes the effect of ultrasound on thermal kinetic parameters and Michaelis-Menten kinetic parameters (km and Vmax) of enzymes. Further, in this review, the physical and chemical effects of ultrasound on enzyme have been correlated with thermodynamic parameters (enthalpy and entropy). Various techniques used for investigating the conformation changes in enzyme after sonication have been highlighted. At the end, different techniques of immobilization for ultrasound treated enzyme have been summarized.

Changes of protein composition and its relevance with textural properties during processing of fermented solid beef

In this paper, fermented solid beef inoculated with two starter cultures (Lactobacillus curvatus and Pediococcus pentosaceus) in different inoculums concentration, fermentation time, and fermentation temperature was evaluated for change of protein composition (water-soluble proteins, insoluble protein, and salt-soluble proteins) and nonprotein nitrogen during fermentation and the relevance with textural properties was also investigated. The amount of water-soluble and salt-soluble proteins decreased while nonprotein and insoluble proteins increased as fermentation progressed. With the increase of inoculums concentration from 0.5% to 2.5%, the amount of water-soluble and salt-soluble proteins decreased from 23.8 to 19.6 mg/g and 51.2 to 33.5 mg/g, respectively, and the amount of nonprotein and insoluble proteins increased from 17.8 to 24.4 mg/g and 20.3 to 49.5 mg/g, respectively. During 0 to 32 hr of fermentation, the water-soluble and salt-soluble proteins decreased from 24.4 to16.0 mg/g and 54.0 to 22.0 mg/g, respectively, and the amount of nonprotein and insoluble proteins increased from 16.3 to 30.8 mg/g and 19.6 to 70.5 mg/g, respectively. With the fermentation temperature increased from 20°C to 40°C, the water-soluble decreased from 34.6 to 21.8 mg/g; the amount of salt-soluble proteins decreased from 60.0 to 21.2 mg/g; the nonprotein and insoluble proteins increased from 16.2 to 24.3 mg/g and 29.7 to 70.4 mg/g, respectively. From SDS-PAGE analysis of water-soluble and salt-soluble proteins, solid beef protein tended to degrade to smaller protein, peptide, and amino acid with protease and peptidases enzymes, and partially form large polymers. Hardness, elasticity, gumminess, and chewiness were significantly negatively correlated with water-soluble and salt-soluble proteins, and were significantly positively correlated with insoluble proteins. The optimum inoculum population, fermentation time, and fermentation temperature were 2%, 32 hr, and 35°C, respectively. Practical applications Our paper studied the effects of fermentation conditions including inoculums concentration, fermentation temperature, and fermentation time on the change of protein composition of fermented solid beef and nonprotein nitrogen. Meanwhile, explored the relationship between the protein composition and texture properties. Fermentation degrades proteins into smaller protein, peptides, and free amino acids. It is beneficial for absorption and good for health. And now the fermentation meat products become more and more popular. However, the quality of beef products is related to muscle proteins that the changes of protein molecules can be effectively affected the properties of products. Our preliminary observation provides a theoretical basis for further researches on quality evaluation of fermentation meat products, and hope to improve the quality of fermentation meat products.

The effect of cryomechanodestruction on activation of heteropolysaccaride-protein nanocomplexes when developing nanotechnologies of plant supplements

The regularities and mechanisms of the effect of deep processing of plant raw materials were established, such as finely dispersed grinding in developing nanotechnology of obtaining frozen nanopuree and nanopowders on the transformation of bound amino acids of protein to free soluble form by меchanolysis of molecules of protein (by 45…55 % to separate α­amino acids). We discovered the mechanism of mechanodestruction of protein molecules and its nanocomplexes with other biopolymers and BAS, which is linked to mechanocracking. In the deep processing of plant raw materials, in particular, Jerusalem artichoke, which is based on the comprehensive action of cryogenic «shock» freezing, freeze drying and finely dispersed grinding processes when obtaining nanopowders, the processes of cryodestruction, mechanodestruction and mechanochemistry occur that lead to the fuller extraction of BAS from the raw material (by 1.8…2.3 times more than is in the original raw material) and destruction of biopolymers (inulin, proteins) to their monomers. It was found that the freezing and cryomechanodestruction lead to the transformation of chemical substances of Jerusalem artichoke (cryomechanochemistry) and transformation, in particular, conformational changes of protein molecules: reduction of radius of the volume of a protein molecule, radius of its nucleus, and also to a decrease in the indicator of filling the nucleaus with hydrophobic remains of amino acids. In addition, the shape of protein molecules changes. We proposed and designed cryogenic nanotechnology of finely dispersed frozen nanopuree and nanopowders from Jerusalem artichoke with prebiotic properties. It was shown that nanosupplements exceed the known world analogues in the content of BAS and dispersed composition. In addition, a large part of the substances (both BAS and biopolymers) is in the nanodimensional form.

DEVELOPMENT OF THE NEW METHOD OF THE MELTED CHEESE PRODUCTS WITHOUT SALT-MELTERS USING CRYOMECHANOLYSIS

The aim of the work is elaboration of the principally new method of deep processing of rennet cheeses to the melting using the complex action of freezing and cryomechanolysis on the raw material that gives a possibility to destruct the hardly soluble biopolymers and to transform them into soluble form. The principally new method of the deep processing of rennet cheeses for receiving the melt cheese products without salts-smelters was elaborated. It differs from the traditional ones by the complete exclusion of the salts-smelters. This method is based on the use of the influence of freezing and fine-dispersed comminution on the raw material. It allows open biological potential of the rennet cheeses more fully and to extract the hidden (bound) protein forms from nanocomplexes of lipids and mineral substances. It allows destruct the proteins of rennet cheeses to the separate polymers and dipeptides and tripeptides. The used technological methods gave a possibility to exclude the salts-smelters at the rennet cheeses manufacturing. They favor the transformation of lipid-proteins paracaseinate calcium phosphate complexes to the separate amino acids and peptides and allow receive homogenous plastic mass. It was established, that at the complex action of freezing and fine-dispersed comminution on the rennet cheese the destruction of hardly soluble lipid-protein nanocomplexes and release of protein from the bound state into free one – nanoform (by 33,5…35 % more) takes place. The mechanisms of this process, connected with cryomechanodestruction of connections between lipids and proteins and non-fermented catalysis of nanocomplexes were described. It was established, that cryomechanodestruction and non-fermented catalysis of protein to the separate monomers – α-amino acids (by 55…60 %) takes place at freezing and fine-dispersed comminution of rennet cheese before melting. The mechanism of freezing and non-fermented analysis, connected with cryomechanocracking of protein molecules at the expanse of peptide protein connections destruction to the separate α-amino acids and their transformation into the free form was described. It was also demonstrated, that the conformational changes of protein molecules take place synchronously. The offered and elaborated nanotechnology of melt cheese products on the base of rennet cheeses without salts-smelters includes complex action of freezing and fine-dispersed comminution. The mechanisms of processes, connected with cryomechanodestruction of connections between lipids and protein to the separate α-amino acids are described. The cheese fillings for “Pancake” confectionary and cheese snacks – falafels were manufactured on the base of cheese mass, received using the new method and enriching vegetable nanoadditives. They exceed the well-known analogs by chemical composition and are remarkable for the storage life, increased in 2 times. At the same time the significant part of substances (BAS and biopolymers) in cheese filings is in nanodimensional form (55…60 % of protein), especially, free α-amino acids, easily assimilated by the human organism. The recipes and technologies of sauces-dressings, sauces-deeps, cheese snacks and so on are also elaborated on the base of cheese mass, received by the new method.

Analysis of binding ability of two tetramethylpyridylporphyrins to albumin and its complex with bilirubin

An interaction between 5,10,15,20-tetrakis-(N-methyl-x-pyridyl)porphyrins, x=2; 4 (TMPyPs) with bovine serum albumin (BSA) and its bilirubin (BR) complex was investigated by UV–Viz and fluorescence spectroscopy under imitated physiological conditions involving molecular docking studies. The parameters of forming intermolecular complexes (binding constants, quenching rate constants, quenching sphere radius etc.) were determined. It was showed that the interaction between proteins and TMPyPs occurs via static quenching of protein fluorescence and has predominantly hydrophobic and electrostatic character. It was revealed that obtained complexes are relatively stable, but in the case of TMPyP4 binding with proteins occurs better than TMPyP2. Nevertheless, both TMPyPs have better binding ability with free protein compared to BRBSA at the same time. The influence of TMPyPs on the conformational changes in protein molecules was studied using synchronous fluorescence spectroscopy. It was found that there is no competition of BR with TMPyPs for binging sites on protein molecule and BR displacement does not occur. Molecular docking calculations have showed that TMPyPs can bind with albumin via tryptophan residue in the hydrophilic binding site of protein molecule but it is not one possible interaction way.