[Microbiological and parasitological risk analysis of novel food sources obtained with the use of insects].

The problem of food safety being an important component of the country's food security, provides not only for continuous improvement of the methodology of hygienic standardization, but also for the formation of requirements for novel food, in particular, those obtained from non-traditional sources. The accumulated practical and theoretical competence in the food hygiene area, as well as knowledge of current trends of the food base broadening, allow us to analyze the risks associated with novel food obtained of insects. The purpose of the research was to analyze the microbiological and parasitological risk of novel food sources obtained with the use of insects, suggest the effective risk management measures. Material and methods. The analytical part of the work included literature search, collection of information and statistical materials published in domestic and foreign scientific editions. The search was carried out using the Google Academy retrieval system and electronic databases (PubMed, MEDLINE, EMBASE, Scopus, Web of Science, eLIBRARY), mainly in the last 25 years, using the keywords: Hermetia illucens, Tenebrio molitor, Acheta domesticus, insects, parasite, nematode, pathogen, cysts. Results. Based on the published materials' analysis, a systematization of microbiological and parasitological factors potentially capable of colonizing edible insects has been carried out. There were identified representatives of 24 groups of pathogenic and 18 opportunistic microorganisms and helminths related to microbial and parasitic pathogens, the spread of which is significantly influenced by inappropriate conditions of feeding and keeping insects. As there are currently no veterinary requirements for insect breeding and farming conditions, contamination of end products with infectious and parasitic pathogens can vary over a very wide range. Conclusion. The use of native insect biomass carries certain risks associated with its microbial and parasitic contamination, and the development of measures to prevent them requires significant resources. The possibility of deep processing of such raw materials can be considered as one of the solutions to mitigate these risks. For use in the food industry, insects should be subjected to processing similar to that currently used for soybean seeds, which includes separation of protein (entomoprotein), fat and chitin fractions, each of which would have an independent use. Thus, at present, insects should be considered as a source of novel food ingredients, first of all, complete protein of animal origin.

[Impact of black soldier fly (Hermetia illucens) larvae biomass on the immune status of rats].

The improvement of the novel foods' safety assessment algorithms is currently one of the food hygiene significant areas. Within the studying of Hermetia illucens insects' effect, the standard in vivo allergological research integrated in the protocol of medical and biological evaluation of genetically modified food has been used. The protocol was supplemented with cytokine profile indicators and pathomorphologic characteristics of immunocompetent organs' lymphoid tissue. The purpose of the research was to study the effect of black soldier fly (Hermetia illucens) larvae biomass on the rats' immune status in the experiment on the induced anaphylactic shock model. Material and methods. The effect of black soldier fly (Hermetia illucens) larvae biomass was studied in a 29-day experiment on growing (43-72 days of life) male Wistar rats fed with Hermetia illucens biomass - main group (n=29) and semi-synthetic casein diet - control group (n=29). The complex assessment of allergenic potential of Hermetia illucens biomass was carried out in the experiment on the induced anaphylactic shock model in Wistar rats. An expanded pool of immune status indicators was studied including active anaphylactic shock severity (lethality, number of severe anaphylaxis reactions, anaphylactic index); cytokine profile (content of proinflammatory and anti-inflammatory cytokines, as well as regulators of cellular and humoral immune response); IgG1 and IgG4 level before and after administration of ovalbumin permissive dose (4 mg/kg b.w.). In addition to this pathomorphologic characteristics of lymphoid tissue of the main immunocompetent organs (thymus, spleen, Payer's patches) have been obtained. Results. The significant systemic anaphylaxis reaction decrease in the main group has been shown. Comparative assessment of the serum cytokines (GM-CSF, IFN-γ, IL-10, IL-12(p70), IL-13, IL-1α, IL-1ß, IL-2, IL-4, IL-5, IL-6, TNF-α) as well as the level of immunoglobulins of the IgG1, IgG4 class before and after administration of ovalbumin permissive dose did not reveal significant differences in rats of the control and main groups. In the main group, there was a decrease in blood serum proallergic cytokines: the level of IL-4 reduced by 1.3 fold, IL-10 - 1.1 and IL-13 - 1.2 fold (p>0.05), and in animals with mild anaphylactic reaction - by 1.8, 1.4 and 1.4 times, respectively (p>0.05). The morphologic studies of the immune system organs showed no intergroup differences. Conclusion. Thus, allergological studies of black soldier fly (Hermetia illucens) larvae in the experiment with the use of systemic anaphylaxis rat model and determination of immune status indicators (anaphylactic shock severity, cytokine profile, IgG1 and IgG4 level, morphologic structure of immunocompetent organs) did not reveal any allergenic effect of the studied product.

[Alternative protein sources: bacteria Methylococcus capsulatus concentrate, characteristic of composition and biological value].

A promising growth vector of food protein production in the context of the Russian Federation's food sovereignty security is the use of microbial synthesis. Taking into consideration the proven promising use of biotechnological processes in the production of alternative protein sources, modern scientific research is focused, among other issues, on improving the technology of obtaining food microbial protein using a variety of substrates and strains-producers, as well as evaluating the consumer properties, food, biological value and safety of such products. The purpose of the research was to study and comparatively evaluate protein concentrate (PC) from bacteria Methylococcus capsulatus and basic food of animal and plant origin within the development of the technology of optimal in nutritional and biological value PC production. Material and methods. Analysis of the nutritional and biological value of PC obtained from denucleinized and purified from cell walls biomass of methanoxidizing bacteria Methylococcus capsulatus (strain GSB-15) was carried out on 46 indicators, including estimation of protein content and amino acid composition, fat content and fatty acid composition, ash and moisture. Biological studies based on measuring of net protein ratio / net protein utilization were performed on 28 growing (between 25-50 days of life) male Wistar rats. Rats in the control group (n=14) received a semi-synthetic casein diet with a protein content of ~12% in calories, the test group (n=14) received a diet including an equivalent amount of PC protein. Body weight, feed intake, and fecal and urine nitrogen losses were measured during the experiment. The biological value and digestibility of protein were judged by coefficients of protein efficiency ratio, net protein ratio, true protein digestibility, true protein biological value, true net protein utilization. Results. The nutritional value study of PC showed high protein content - 69.0%, the share of fat, moisture and ash, accounted for 0.17, 9.5 and 14.4%, respectively. The carbohydrate content was 7.0% (of which mono- and disaccharides were <0.1%). The results of a comparative assessment of Methylococcus capsulatus protein amino acid profile and basic food of animal and plant origin showed a balanced content of the most amino acids, the level of which is comparable with the protein of chicken egg, which is traditionally a standard of quality of complete protein. At the same time, the content of the essential amino acid tryptophan in PC was an order of magnitude lower than in chicken egg protein; the content of this amino acid in PC is comparable with incomplete plant proteins (sunflower, flax, rapeseed). The results of the biological value evaluation of the Methylococcus capsulatus protein in the experiment on rats indicate a relatively low biological value of the microbial synthesis protein, that is caused, most likely, by tryptophan deficiency. Rats of the test group had a significant decrease in body weight gain, feed/protein intake, coefficient of protein efficiency ratio, coefficient of net protein ratio, true protein biological value, true net protein utilization. Conclusion. The results of a comparative evaluation of PC from methanotrophic bacteria Methylococcus capsulatus denucleinized biomass and basic food of animal and plant origin indicate its relatively high nutritional value. However, the characteristics of this PC sample were not optimal in regard of protein biological value by reason of tryptophan deficiency. A single amino acid deficiency is not a valid argument for not using microbially synthesized protein in human nutrition, considering the capabilities of the modern food industry, including ways to enrich foodstuffs with missing components. In addition, there is every cause to believe that adjusting the hydrolysis technology used in the production of PC will allow to eliminate the essential amino acid loss, thereby increasing the biological value of this product.

[Monitoring methods for novel insect-derived food: the PCR protocol for the detection and identification of Hermetia Illucens insects based on the HEI-COI probe and primer system].

Forwarding development of identification methods for novel foods, derived from edible insects, is necessary to ensure control over their marketing within the framework of the current legislation's requirements. The purpose of the research was the development and validation of a monoplex TaqMan-PCR assay protocol (a real-time polymerase chain reaction with TaqMan technology) for the insect Hermetia Illucens' taxon-specific DNA detection and identification in food raw materials and foods. Material and methods. Studies were performed using samples containing the target DNA sequence (dried whole larvae of H. Illucens as well as H. Illucens in oilcake meal and powdered capsule forms) and inherently not containing the target DNA sequence (other insect species, mammals, plants, microorganisms as well as multicomponent food: meat, dairy and plant food). DNA extraction and purification were performed by CTAB methods [commercial kits "Sorb-GMO-B" (Syntol, Russia) and "DNeasy mericon Food Kit" (QIAGEN, Germany)]. For amplification of the target sequence, which was a fragment of the cytochrome c oxidase subunit I mitochondrial gene, we used primers and the probe: Hei-COI-F (CCTGAGCTGGTATAGTGGGAAC); Hei-COI-R (AATTTGGTCATCTCCAATTAAGC); Hei-COI-P (FAM-CGAGCCGAATTAGGTCATCCAGG-BHQ-1). PCR conditions were optimized using CFX96TM Real-Time PCR System (Bio-Rad, USA) and Rotor-Gene Q (QIAGEN, Germany) amplifiers by empirical selection of primer and probe concentrations and amplification of the time/temperature profile. Specificity and limit of detection were evaluated as part of method validation. Results and discussion. The optimized reaction mixture included 2.5-fold of Master Mix B [KCl, TrisCl (pH 8.8), 6.25 mM MgCl2], SynTaq DNA-polymerase, dNTP, glycerol, Tween 20, of each primers - 550 nM, probe - 100 nM. The time/temperature profile of the reaction: 95 °C - 180 s (95 °C - 15 s, 57 °C - 60 s), 40 cycles. The detection limit of the method was 0.19 ng of H. illucens DNA per reaction. The specificity of primer system and probe were experimentally confirmed in studies with DNA of other insects, animals, plants and microorganisms. Conclusion. A protocol of a monoplex TaqMan-PCR assay for the taxon-specific DNA of insect Hermetia Illucens' detection and identification in food raw materials and foods has been developed. Validity of the method has been confirmed by laboratory tests which allows to recommend it for use in surveillance of Hermetia Illucens-derived raw materials.

Systemic Biomarkers and Liver Morphology in Rats during Chronic Low-Dose Toxicant Administration against the Background of Vitamin Deficiency.

Liver morphology, intensity of apoptosis, and activity of xenobiotic metabolism enzymes were studied in a chronic model experiment in rats receiving a mixture of 6 pesticides against the background of life-long diets with adequate and insufficient supply of water-soluble vitamins. The dose of each pesticide in the mixture did not exceed the acceptable daily intake (1 ADI). It was found that chronic exposure to low doses of anthropogenic toxicants in combination with permanent vitamin deficiency provokes a number of liver changes, such as increased apoptosis activity, cytochrome P450 system depletion, steatosis, and inflammatory infiltration, which is a potential health risk factor.

[Rats' liver morphological features under conditions of different supply with vitamins and minerals].

The current practice of novel food safety assessment in the Russian Federation involves toxicological studies on the alimentary model of adaptation potential reduction of laboratory animals. Since vitamin and mineral deficiency can affect the size of structural elements of tissues, an objective estimation of the results obtained using this model is possible when determining the range of fluctuations of the studied morphometric parameters under conditions of different essential substances' supply, as well as under conditions of simulated toxic effects on the background of the corresponding supply. The purpose of the research was to investigate the morphological and morphometric features of the liver under the influence of reduced intake of vitamins and mineral elements in the combination with toxic effects of various nature, during growth and puberty of male Wistar rats. Material and methods. The article analyzed data of 4 model experiments on 140 animals that received semi-synthetic casein diet with different supply of vitamins B1, B2, B3, B6 and mineral elements Fe3+ and Mg2+, as well as data of 2 experiments on 180 animals with simulated toxic load of cadmium (Cd2+) salts and carbon tetrachloride. The animals were ~95 days old at the time of sampling, the duration of the experiments was ~65 days. For the analysis we used data on rats' body weight on the day of material sampling, absolute and relative liver weight, hepatocyte diameter, nucleus diameter and hepatocyte cytoplasm size in the central and peripheral zones of hepatic lobules. A total of 200 cells were analyzed in each group of animals. In accordance with the study design, all quantitative traits of the groups that received diets with an essential nutrient supply ranging from 75 to 2% were compared with the group that received a complete diet (100%). Results. Morphometric examination of hepatocytes revealed a linear decrease in the size of cell structural elements in the series of reducing the content of essential micronutrients in the diet. Under the conditions of 2-4% vitamin and mineral supply, cell and nucleus diameters as well as cytoplasm size were by ~16.8, 12.6 and 21.1% (p<0.05) lower respectively than in rats with optimal supply of these substances; under the conditions of 9-19% supply were by ~9.2, 9.7 and 8.7% lower (p<0.05); higher levels of supply caused reduction of hepatocyte, nucleus and cytoplasm sizes in a range not exceeding 5% (p>0.05). When comparing the size of hepatocytes of rats subjected to toxic load with the hepatocytes of rats referred to the reference standard, an increase in the size of hepatocytes under the action of carbon tetrachloride by 17.4% (p<0.05) on average and under the action of cadmium salts by 4.6% (p<0.05) was noted. Conclusion. Based on the analysis of liver morphological and morphometric studies' data, there were established sizes of hepatocytes structural elements in the rats kept on diets with decreasing supply with B group vitamins, iron and magnesium salts; the linear decrease in the sizes of structural elements of hepatocytes in the series of reduction of B group vitamins, iron and magnesium intake was revealed. Toxic exposures to carbon tetrachloride and cadmium salts against the background of a 19-30-75% supply with essential substances led to an increase in the hepatocytes size, the correlation between the degree of toxicant exposure and the supply level is not significant.

[The comprehensive studies of Hermetia illucens larvae protein's biological value].

A high protein content in the insect biomass allows to classify this product as a very promising source of protein, comparable in nutritional and biological value with proteins of animal origin. Despite a long history of safe use, in some countries insects are considered a new type of food which safety must be proven before entering the food market. The long-term Russian experience in novel food's research allows to identify the crucial stages, among which, along with toxicological and allergological tests, the protein's biological value determination takes an important place. The conclusion about the biological value of protein is formed on the basis of integrated use of chemical and biological methods, which gist comes down to the study of the nitrogen balance in the growing organism (biological method) and the calculation of the amino acid score (chemical method). The aim of the research was the comprehensive assessment of Black soldier fly (Hermetia illucens) larvae protein's biological value using chemical and biological methods. Material and methods. Biological studies based on measuring of net protein ratio/net protein utilization were performed on 28 growing (between 25-50 days of life) male Wistar rats, with an initial body weight of ~65.5±1.2 g. Rats in the control group (n=14) received a semi-synthetic casein diet with a protein content of ~12% in calories, the test group (n=14) received a diet including an equivalent amount of H. illucens protein. The diet's ingredients were replaced with the consideration of the proteins, fats, and carbohydrates content in the included product following the principle of isocaloricity and isonitrogenicity (by mass fraction of total nitrogen). H. illucens biomass and casein in the test and control groups, respectively, were the main significant sources of nitrogen in the diet. Body weight, feed intake, and fecal and urine nitrogen losses were measured during the experiment. The biological value and digestibility of protein were judged by coefficients of protein efficiency ratio, net protein ratio, true protein digestibility, true protein biological value, true net protein utilization. Chemical studies included studies of the amino acid composition of H. illucens biomass protein and calculation of the digestible indispensable amino acid score (DIAAS). Results. The general condition of animals of the both groups during the whole experiment was satisfactory, the weekly body weight increase corresponded to the level of growth typical for Wistar rats, intergroup differences were not detected. Despite the fact that in a number of indicators the test group animals differed from the control [there were noted a decrease of the net protein ratio (by 5%, p>0.05), true protein digestibility (by 11%, p<0.05), net protein utilization (by 13%, p<0.05), caused by increased excretion of nitrogen with urine (by 8%, p>0.05) and feces (by 186%, p<0.05), with the same amount of nitrogen intake], the test rats' growth rate and the nitrogen's retention degree indicate a relatively high biological value of insect protein. According to the DIAAS, H. illucens protein is characterized by high content of histidine, threonine, valine, isoleucine and leucine (DIAAS=100 and more), and is also a source of sulfur-containing amino acids - methionine and cysteine (DIAAS=86) and lysine (DIAAS=97). Conclusion. The comprehensive studies of Hermetia illucens larvae protein's biological value demonstrated a high protein content, its balanced amino acid composition and high biological value, which allows to consider Hermetia illucens as a potential source of complete dietary protein.

[Nutritional and biological value of Hermetia illucens larvae biomass].

Recent years a worldwide interest in the use of alternative sources of protein, in particular, protein from insects, has increased. Edible insects for thousands of years have been a part of the human diet in Asian-Pacific region and South America, while in the European Union, the USA and Canada the use of insects for food purposes is a modern trend that is determined by the care of the environment, global warming combating, etc. Thus, the legal rules governing the food use of insects have significant differences among countries. In the Eurasian Economic Union requirements to food are regulated by the Customs Union Technical Regulations. Since none of the Technical Regulations contains the name of such food as "products obtained with the use of insects", these products may be classified as "food products of novel type" which are subjected to state registration. Fundamental and applied research should be conducted as a part of this novel food safety assessment system, that include the determination of nutritional and biological value of food raw materials derived from insects, and toxicological, reprotoxicological, allergological experiments in vivo on several generations of laboratory animals. The aim of the research was studying and comparing the nutritional and biological values of Hermetia illucens larvae dry biomass and basic foodstuffs of animal and plant origin. Material and methods. Nutritional and biological value analysis of H. illucens minced dry larvae biomass, dried at 110-120 °C, was carried out on 83 indicators, which included determination of protein content and amino acid composition, determination of fat level and fatty acid composition, determination of the content of carbohydrates, vitamins, minerals and trace elements, ash and moisture. Results. A study of the nutritional value of dry larvae biomass showed high levels of protein and fat (39 and 38%, respectively), while ash, dietary fiber and carbohydrate accounted for less than 20%. The amino acid profile had a balanced content of essential amino acids and was comparable to the protein of a hen's egg, as well as other animal products. The fatty acid composition of the biomass was characterized by a relatively high content of lauric acid (39.9% of the total fatty acid content), also found in some fruits and seeds of tropical plants; the ratio of other acids was more consistent with the fatty acid profile of fish oil. The dry larvae biomass contained carotenoids (0.23 mg/100 g), tocopherol (3.1 mg/100 g) and thiamine (53 µg/100 g) in amounts significantly lower to those of foods, which traditionally are sources of these vitamins. Based on the analysis of the mineral composition, the H. illucens biomass can be attributed to the sources of calcium, iron, copper and chromium. In terms of the content of the above elements, as well as magnesium and zinc, dry biomass significantly exceed the main food products of animal origin (beef, eggs, fish and seafood), and in terms of the content of potassium and phosphorus, it was comparable to them. Conclusion. The results of dry larvae biomass comparative evaluation with the basic foodstuffs of animal and plant origin evidence to its high nutritional and biological value, allowing to consider H. illucens as a promising source of complete protein, lauric acid, minerals and trace elements.

[Novel food sources: from GMO to the broadening of Russia's bioresource base].

The modern strategy of humanity food providing is aimed at finding the exit from the food crisis in the shortest possible time, by the end of XXI century food and feed production should increase by at least 70%. These tasks solution implies not only the use of science-oriented technologies, but also the expansion of the food base by means of novel food sources, which don't have a history of safe use. In the Russian Federation the formation of novel food's safety assessment approaches is regulated at the state level and is the most important requirement for the possibility of usage. Russian experience of the second half of the XX century in the area of novel food sources' biomedical research unites two stages. The first of them dates back to the middle of the 1960s', when the Soviet scientists, in particular, the workforce of the Institute of Nutrition of the USSR Academy of Medical Sciences, under the leadership of Academician A.A. Pokrovskii, have developed the evaluation approaches of the biological value and safety of microbial synthesized protein. The second stage of the safety assessment research development was the work with the genetically modified organisms of plant origin (GMO), that begun in the middle of the 1990s'. Since the moment of formation in 1995-1996, 9 methodical guidelines that regulate methods of safety assessment and control over GMO have been developed. Comprehensively formed by 2020, safety assessment system has been used in the framework of 27 GMO lines state registration that passed a whole cycle of medical and biological research and were allowed for use in nutrition of the population of the Eurasian Economic Union. Within the framework of these research a considerable amount of factual material has been accumulated, a regulatory and methodological basis has been built, and a substantial background for further fundamental and applied scientific research in the field of development and safety assessment of novel food has been created.

[Multiplex polymerase chain reaction for genetically modified potato event AV43-6-G7 quantification. Proof of efficiency].

One of the ways to improve the laboratory control methodology of genetically modified organisms of plant origin (GMO) is to use multiplexing - an approach that allows you to increase the number of targets and enlarge the number of simultaneously processed samples, maximizing the potential of polymerase chain reaction in real time (PCR-RT). The aim of the study is to develop a quantitative identification protocol for genetically engineered (GE) potato event AV43-6-G7 in the format of duplex PCR-RT with the use of TaqMan® PCR technology. Material and methods. The duplex system included 2 types of specific DNA-primers and fluorescence-labeled probes: the first one is for detection of transformation event AV43- 6-G7 DNA sequence, the second is for detection of Stp23 taxon-specific potato gene. PCR parameters were chosen by empirical selection of concentrations of primers and probes, Mg2+ ions, deoxyribonucleotides, stabilizing agent for polymerase, as well as primer annealing temperature and incubation duration for each stage of the cycle. Results. As a result of these studies, the composition of the reaction mixture was optimized for the detection and quantification of GE potato event AV43-6-G7 in food. Oligonucleotide primers and fluorescent probes were selected. The compositions of reaction mixtures and temperature-time parameters of PCR were tested: 2.5-fold reaction buffer for PCR-RT in the presence of ROX (carboxy-X-rhodamine), specific to the GE component primers (AV43-6-G7-f/AV43-6-G7-r) and target taxon (GRF3/ GRR3) at 300 nM/300 nM and 100 nM/100 nM, probes at 200 nM and 200 nM, respectively; bovine serum albumin - 0.04%; MgCl2 - 3.5 mM, deoxynucleoside triphosphates - 0.3 mM, as well as the temperature-time profile of the reaction (initial denaturation of 95 °C - 5 min, followed by 45 cycles: 95 °Ð¡ - 20 sec, 58 °Ð¡ - 20 sec, 62 °Ð¡ - 40 sec). Conclusion. The validity of the developed method is confirmed by laboratory studies and testifies to its reliability.

[Multiplex PCR for detection and quantification of GM potato event EH92-527-1 in food].

Aim - the elaboration of the protocol for the quantitative detection of genetically modified (GM) potato event EH92-527-1 in the format of duplex real-time polimerase chain reaction (RT-PCR) with TaqMan® PCR technology. Material and methods. The duplex system included two types of specific DNA primers and fluorescent probes: the 1st was for identifying of the event-specific EH92-527-1 DNA, the 2nd was for identifying of the taxon-specific potato gene Stp23. The selection of PCR parameters was carried out by empirical assortment of primers and probes, Mg2+-ions, deoxyribonucleotides and polymerase stabilizing agent, primers annealing temperature and incubation time for the each cycle stage. The results of this research was the optimization of the reaction mixture composition for EH92-527-1 event and Stp23 gene fragment identification in the duplex system: 2.5× RT-PCR buffer in the presence of ROX, primers specific for the GM potato (EH92- f/EH92-r) and target taxon (GРF3/GРR3) in the amount of 250/250 and 100/100 nM, probes - 200 and 200 nM, respectively; bovine serum albumin - 0.04%; MgCl2 - 3.5 mM, deoxynucleoside triphosphates - 0.3 mM, as well as the temperature-time reaction profile (initial denaturation at 95°C - 5 minutes, the next 45 cycles: 95 °C - 20 seconds, 58 °C - 20 seconds, 62 °C - 40 seconds). Conclusion. The method for the quantitative detection of genetically modified (GM) potato event EH92-527-1 in the format of duplex RT-PCR has been elaborated. The linearity, precision, accuracy and limit of the method definition are confirmed with in vitro studies, that results testify to the method's reliability.

Analysis of the Intensity of Apoptosis in Rat Organs at Various Stages of Ontogeny.

Activity of apoptosis in the thymus, liver, and kidneys on days 20, 22, 35, 50, 80, 110, and 140 of ontogeny was studied in the experiments on rats using the alkaline gel electrophoresis and flow cytofluorometry. Changes in apoptosis intensity depended on animal age. The maximum level of this parameter was observed on day 20 of ontogeny with the following reduction in this parameter to the minimum value on days 35-50. Then it gradually increased up to relatively high levels on day 110 and significantly reduced by day 140.

[Molecular and genetic studies of genetically engineered potato: transformation event PH05-026-0048].

Expert evaluation of genetically engineered organisms (GMO) identification methods is aimed at confirmation their adequacy with the tool and methodological base used in the institutions of the Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing to control market turnover and labelling of genetically engineered food. The primer system's specificity was experimentally confirmed in studies with other GM potato lines, as well as with the results of the BLAST-analysis. The efficiency, linearity and correctness of the method meet the requirements of the European Union Reference Laboratory for GM Food and Feed. Limit of detection and limit of quantification of GM potato line PH05-026-0048 genomic DNA were 0.019% (11 copies of the GM potato genomic DNA) and 0.06% (36 copies of the GM genomic DNA of the potato) per 100 ng of total DNA, respectively.

[Research of the cadmium intoxication effect on the model of vitamin-mineral deficiency in rats].

The article presents the results of the study aimed at confirmation of the effectiveness of the rats' adaptive potential reduction under conditions of cadmium salt toxic effects. The 65-days experiment was conducted in male and female Wistar rats. Animals were divided into 6 groups of 3 control and 3 experimental, 30 males and females in each. In total 360 rats were used in the experiment (180 females and 180 males). Rats of the 1st control group received a diet with optimal (75% of the standard semisyntethic diet content) dosage of vitamins B1, B2, B3, B6 and mineral substances, Fe3+ and Mg2+, the rats of the 2nd and the 3rd control group - diets with marginal (30% for males and 28% for females) and submarginal (19% for males and 18% for females) doses of essential micronutrients. Animals of the 1-3th experimental groups received Cd2+ on the background of optimal, marginal and submarginal providing of essential micronutrients. The hematological, biochemical and morphological parameters and the antioxidant status of rats have been studied. The obtained results allowed to identify patterns of cadmium toxic effect strengthen on the background of essential nutrients reducing (in the row from optimal to submarginal). These changes showed erythrocyte and platelet blood profiles, and a set of indicators of the antioxidant defense system and lipid peroxidation of blood and liver. Thus, the activity of erythrocyte antioxidant enzymes - glutathione reductase, glutathione peroxidase, catalase and superoxide dismutase in rats of the 1st experimental group were on average by 23% higher than in animals of the 1st control group, the rats of the 2nd and the 3rd experimental groups by 62 and 67% higher, respectively. The content of lipid peroxidation products in blood and liver of male and female rats showed a similar trend: an increase by 5% in the 1st experimental group by 9 and 25% in the 2nd and 3rd experimental groups respectively. Thus, the modification of the diets' vitamin-mineral composition may be used as a model of adaptive potential reduction in rats in the toxicological research of objects with unknown toxicity, in particular novel food products.

[Characteristic of the rats' immune status during vitamin and mineral deficiency].

The article presents the results of rats' immune status evaluation in conditions of different levels of B vitamins (B1, B2, B3, B6) and minerals (Fe3+ and Mg2+) supplying. Male Wistar rats (with initial body weight 90-110 g) within 65 days received model diets with 75, 30 and 19% content of the essential substances, females (with initial body weight 56-76 g) received diets with 75, 28 and 18% content. Some indicators of humoral and cellular immunity, including leukocyte count, lysozyme serum activity, IgG and IgE level, cytokine profile of blood serum and of the masses of the immune system internal organs have been examined. It has been shown that the decrease in the content of B vitamins, iron and magnesium in the diet didn't cause complex changes of the rats' immune status, therefore, the proposed model of adaptive potential change is not optimal for studying of immune system condtion. The extensive research of the immune status of rats (18 studied indicators, a sample of 90 males and 90 females) allows to set the range of physiological fluctuations of these indexes in Wistar rats of the corresponding gender and age.

[Control over geneticallymodified sources of plant origin in food: scientific basis and methodical maintenance].

The article presents data on global production of genetically modified organisms of plant origin (GMO), as well as on basic principles of organizing and methodical maintenance of the GMO control system in the framework of worldwide trend to increasing usage of such products. The results of GMO turnover monitoring, that was conducted by Rospotrebnadzor in 2003-2016 (there were made more than 300 000 analysis), indicate a decrease of such products usage: from 10-12% in 2003-2004 to less than 0.1% to in 2016.

Modification of vitamin-mineral diet composition as a model of adaptive potential reducing in laboratory animals.

This publication presents the results of research that was aimed at elaboration of adaptive potential reducing model, intended for toxicological experiments. Two series of research (with a duration of 70 days each) were conducted on Wistar rats. In the 1st series five groups of animals received diets with 100, 75,50,25 and 0% of vitamins B1, B2, B3, B6 and minerals(Fe3+ and Mg2+); in the 2nd series four groups of animals received diets with 21.37, 9.94, 4.62, 2.15% of this vitamins and minerals. In the 1st series of studies the intervals of maximum, medium and minimum content of essential nutrients in the diet was established; in the 2nd series the range of the lowest possible concentrations of these elements that provided the lowest level of adaptive potential and not causing the pathology development was determined. The certain set of hematological, biochemical, immunological and other indicators were investigated, this article analyzes the results of zoometric studies, mortality of animals, as well as the results of antioxidant status (activity of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and malondialdehyde content in red blood cells) studies. Based on the evaluation of the data which were obtained in the 1st series, it follows that a dose reduction of relevant essential nutrients to 25% didn't significantly affect the values of the studied indicators, and the complete elimination of these substances resulted in massive death of animals. In the 2nd series a significant differences between the groups were observed from the range of increased mortality (groups with 2.15 and 4.62% content of essential nutrients) to the range of deviations from central tendency of some parameters (group with 21.37% content). The data allowed to trace the dependence of these differences on the levels of vitamins and minerals in the diet. The results were used to determine threshold values of vitamins and minerals that provided the necessary reduction of the adaptive potential level in male and female rats. Taking into account the risk of pathology development, three dosages of essential substances have been established - optimal, marginal and submarginal, which provide consistent decline of adaptive potential of laboratory animals: 75, 30 and 19% for males and 75, 28 and 18% for females, respectively. The modification of vitamin and mineral composition of the diet can be used as a model of adaptive potential reduce in toxicological research.

[Investigation of the liver DNA methylation profile of rats under the influence of hepatotoxicants of different nature].

The functional importance of DNA methylation, which is a special case of epigenetic variation, is meant for regulation of many biological processes, ranged from tissue specific gene expression to remodeling of chromatin structure. Disorders of the DNA methylation can cause changes in the cell's phenotype, providing a significant impact on the development of pathology. Both exogenous and endogenous factors are able to cause disruption of DNA methylation, while epigenetic changes usually precede the emergence of clinical and morphological symptoms of pathological process development, consequently the parameters of DNA methylation can be used as sensitive biomarkers to detect adverse effects on the organism. The purpose of the study was to identify genes of the liver, the methylation profile of which changes under the influence of hepatotoxicants of different nature. The experiment was carried out on 60 male Wistar rats with initial body weight (b.w.) 83.3±1.5 g. Animals were randomly divided into 6 groups - 1 control and 5 test groups, with 10 rats in each group. During the first two weeks of the experiment the rats of the 1-5th test groups were administered to aflatoxin B1 (200 Mg/kg b.w.), cadmium chloride 2,5-hydrate (2 mg/kg), monosodium glutamate (1000 mg/kg), epigallocatechin gallate (EGCG) (1000 mg/kg), paracetamol (150 mg/kg), accordingly. Methylation of the liver genes in rats was determined by using high-performance methods, based on bisulfite sequencing of reduced representation. For each sample from 12 to 30 million pairs of reads were received, genes which demonstrated significant changes in methylation when exposed to toxic factors were identified: aflatoxin B1 caused changes in the methylation of 57 genes; cadmium - 54 genes; monosodium glutamate - 39 genes; EGCG -198 genes; paracetamol - 167 genes. The comparison of genes with altered methylation in the experimental groups revealed that none of the genes repeatedly occurred under the influence of each toxicant out of five, the highest number of repeats accounted 3. As a result of the present analysis 7 genes have been selected: methylation change in Fan1 gene was observed when exposed to cadmium, monosodium glutamate, EGCG; gene Lppr2 - under the influence of aflatoxin B1, EGCG, paracetamol; gene Mlh3 - under the influence of aflatoxin B1, cadmium, paracetamol; Sirt7 gene - under the influence of cadmium, EGCG, paracetamol; gene Fbxo15 - when exposed to cadmium, monosodium glutamate, paracetamol; gene E2f1 - when exposed to cadmium, EGCG, paracetamol; gene Mrps16 - when exposed to cadmium, EGCG, paracetamol. On the basis of the received data the project of the panel of genes-biomarkers of toxic effect, including genes Fan1, Lppr2, Mlh3, Sirt7, Fbxo15, E2f1, Mrps16 has been formed.

Assessment of the impact of genetically modified LibertyLink® maize on reproductive function and progeny development of Wistar rats in three generations.

This publication presents the assessment of the impact of genetically modified (GM) LibertyLink® maize on reproductive function, prenatal and postnatal progeny development of Wistar rats over three generations. The animals were divided into two groups, which were fed with rodent diet with inclusion of GM LibertyLink® ('test' group) and non-GM near-isogenic counterpart ('control' group) maize varieties. The maize was included into the diet at maximum possible level (between 32 and 33%) not causing nutritional imbalance or metabolic disturbance for the experimental animals. Data analysis showed no impact of LibertyLink® maize on the animals' fertility: the observed mating efficiency in both groups was within the normal expected range values under the given experiment conditions. The comparison of progeny prenatal development in the generations F0-F2 has not shown any differences between the groups. Analysis of the physical development of the F0-F2 progeny or pups body weight and length progress did not show any abnormalities. The average number of pups per litter in the control and test groups was within the expected range of variations. Therefore, the results should be considered as direct evidence of the lack of any reproductive toxicity of LibertyLink® maize (a.k.a. T25 maize).

Transfer of Silver Nanoparticles through the Placenta and Breast Milk during in vivo Experiments on Rats.

Silver nanoparticles (NPs), widely used in the manufacture of various types of consumer products and for medical applications, belong to novel types of materials that pose potential risks to human health. The potential negative effects of the influence of these NPs on reproduction are insufficiently researched. A quantitative assessment of the transfer of metallic silver nanoparticles through the placenta and breast milk was carried out during an in vivo experiment. We used 34.9 ± 14.8 nm in size silver NPs that were stabilized by low-molecularweight polyvinylpyrrolidone and labeled with the (110m)Ag radioactive isotope using thermal neutron irradiation in a nuclear reactor. [(110m)Ag]-labeled NPs preparations were administered intragastrically via a gavage needle to pregnant (20(th) day of gestation) or lactating (14-16th day of lactation) female rats at a dose of 1.69-2.21 mg/kg of body weight upon conversion into silver. The accumulation of NPs in rat fetuses and infant rats consuming their mother's breast milk was evaluated using a low-background semiconductor gamma-ray spectrometer 24 and 48 hours following labeling, respectively. In all cases, we observed a penetration of the [(110m)Ag]-labeled NPs through the placenta and ther entry into the mother's milk in amounts exceeding by 100-1,000 times the sensitivity of the utilized analytical method. The average level of accumulation of NPs in fetuses was 0.085-0.147% of the administered dose, which was comparable to the accumulation of the label in the liver, blood, and muscle carcass of adult animals and exceeded the penetration of NPs across the hematoencephalic barrier into the brain of females by a factor of 10-100. In lactating females, the total accumulation of [(110m)Ag]-labeled NPs into the milk exceeded 1.94 ± 0.29% of the administered dose over a 48 h period of lactation; not less than 25% of this amount was absorbed into the gastrointestinal tract of infant rats. Thus, this was the first time experimental evidence of the transfer of NPs from mother to offspring through the placenta and breast milk was obtained.