[Influence of lactoferrin and enzymatiс hydrolysates of cow's and mare's milk proteins on anaphylactic sensitivity and cytokine profile of rats].

The development of innovative ingredients of specialized formula for children with intolerance to cow's milk proteins requires accounting the influence of the protein component on the allergic sensitivity. The aim of the research was to study the effect of lactoferrin (LF) from cow colostrum, cow's milk protein hydrolysate (CMPH) and mare's milk protein hydrolysate (MMPH) on the severity of the systemic anaphylaxis reaction, the levels of specific IgG antibodies and cytokines in rats parenterally sensitized with ovalbumin (OVA). Material and methods. The experiment was carried out on 4 groups of 26 male Wistar rats, which were sensitized intraperitoneally with chicken egg OVA and a systemic anaphylaxis reaction was induced on the day 29 by intravenous administration of a challenge dose of the antigen (6 mg per kg body weight). LF, CMPH and MMPH were introduced into the diet in doses of 1.4-2 g/kg body weight per day (on an average 1.59±0.04, 1.53±0.05 and 1.48±0.05 g per kg body weight respectively). The content of IgG antibodies in the blood serum was determined by an indirect ELISA; the levels of cytokines IL-1α, IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12(p70), IL-13, GM-CSF, IFN-γ and TNF-α were detected by multiplex immunoassay. Results. Dietary LF did not have a significant effect on the severity of active anaphylactic shock (AAS), concentrations of antibodies and cytokines in the blood of animals. As a result of CMPH consumption, there were no significant changes in AAS severity and IgG antibodies to OVA but significant increase in TNF-α level was observed as well as a significant decrease in IL-1α (p<0.05). In animals receiving MMPH, there were also no significant changes in the severity of AAS, but a 1.9-fold decrease in the level of IgG antibodies to OVA (p<0.001) was noticed along with a significant increase in IL-12(p70) (p<0.05) and IL-10 (p<0.10) level. Conclusion. Cosumption of LF by sensitized rats didn't significantly affect their anaphylactic sensitivity and cytokine profile, while CMPH intake induced some signs of pro-inflammatory processes. Consumption of MMPH was accompanied by the formation of an anti-inflammatory cytokine profile, which corresponds to a decrease in the intensity of the humoral immune response to the model allergen. Differences in the effects of two hydrolisates, which have similar degrees of hydrolysis, may be associated with the specific composition of glycopeptides formed during the enzymatic cleavage of milk protein produced by these two species of dairy animals.

[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.

[Effect of L-carnitine and resveratrol complex on the profile of cytokines and regulatory proteins in normal and obese rats].

Chronic inflammation in adipose tissue, peripheral organs and some compartments of the brain are among pathogenetic factors in obesity. The use of bioactive compounds with anti-inflammatory and hypolipidemic activity in the composition of specialized products and dietary supplements is considered as an approach in the diet therapy of obesity and related conditions. The aim of the research was to study the effect of a complex supplement containing resveratrol and L-carnitine (RC supplement) on the immunological parameters of inflammation (the profile of cytokines and regulatory proteins) in rats fed a balanced or hypercaloric diet. Material and methods. Male Wistar rats received for 63 days a standard balanced diet (SD) or a high-carbohydrate-high-fat diet (HFCD) with an excess of total fat and fructose, as well as RC supplement at a low (25 mg/kg body weight as Res and 300 mg/kg body weight as L-Car) or high (50 and 600 mg/kg body weight, respectively) dose. The content of leptin, ghrelin, cytokines and chemokines in blood serum (BS), lysates of white adipose tissue (WAT) and spleen, amygdala and hippocampus of the brain, the content of regulatory proteins Akt, IRS-1, GCK-3a/b, p70/S6, BAD, m-TOR, PTEN and S6 ribosomal protein in the amygdala and hippocampus have been studied the by multiplex immunoassay. Results. In rats that consumed RC as part of SD, there was a decrease in the levels of leptin and its ratio with ghrelin, the levels of proinflammatory cytokines (TNF-α, IL-1α, IL-12p40 and IL-12p70, IFN-γ) in BS and WAT, chemokines (MCP-1, M-CSF, MIP-2) in WAT. Some of these effects were more pronounced at a low dose of RC than at a large dose, and some of them were also canceled or changed in direction in animals treated with HFCD. In the amygdala, RC consumption increased the content of both pro- and antiinflammatory cytokines; the most significant was the increase in IL-7 levels in animals fed SD, and RANTES in animals fed HFCD. In the hippocampus of rats, the RC intake had an insignificant effect on the levels of cytokines and chemokines. Akt-1 kinase and the substrate of the insulin receptor IRS-1 were the main targets of RC action in the regions of the brain. Conclusion. The complex dietary supplement RC exerted a hypoleptinemic effect, revealed certain anti-inflammatory effects and modulated a number of the brain factors influencing behavioral responses in obesity. However, the synergistic effect of resveratrol and L-carnitine in the composition of the supplement wasn't not observed, and the effectiveness of its action decreased in conditions of a hypercaloric diet consumption.

[Methods for the identification and quantification of microplastics in foods (a review)].

The adverse effects of microplastics (MP) found in food on the health have recently been recognized as a new source of human health risks. In order to evaluate and minimize them, it is necessary to evaluate the exposure using sensitive and specific methods. The aim of the research was the substantiation of methodological approaches to the identification and quantification of microplastics in food based on the analysis of literature data. Material and methods. Literature selection was carried out using the PubMed international reference database for the period from 2014 to 2023 using keywords corresponding to the context of the research theme. A total of 159 sources were selected, of which 94 original and review papers were included in the review according to the criteria of their relevance to the problem under consideration, scientific reliability and completeness. Results. At present, various approaches have been developed that make it possible to isolate MPs from complex bioorganic matrices (such as, for example, seafood), classify them by chemical composition, and quantify their content through the mass or number of particles. Among the most developed physic-chemical methods for the analysis of MPs are Fourier transform IR spectrometry and Raman microspectrometry, pyrolysis gas chromatography - mass spectrometry, thermogravimetric analysis, as well as approaches based on liquid chromatography, microfluorimetry, analytical scanning and transmission electron microscopy and others. Unsolved problems in the field of MP research in food include the high laboriousness and low performance of the identification technologies used, the lack of reference and standard samples of MP, the complexity of the equipment used, which makes it difficult to use it for routine hygienic control. The issue of the influence of MP aging degree on the results of its qualitative and quantitative determination has not been sufficiently studied. Some hopes in the field of development of rapid analysis of MTs are pinned on the use of aptamers. Conclusion. Existing analytical methods make it possible to determine the content of MPs in environmental objects, but further improvement and validation of these methods is required in relation to the assessment of the content of MPs in various types of food.

[Effect of microand nanoplastics on the gastrointestinal mucosa and intestinal microbiome].

Worldwide production and use of the polymers has led to intensive environmental pollution with micro- and nanoplastics (MP and NP). Accumulating in ecosystems, MP are transmitted through food chains and enter the human body. The associated health risks are of grave concern and require assessment. The main entry gate for MP/NP supplied with food is the gastrointestinal tract (GIT). Despite the well-established concept of MP/NP toxicity, information about their actual effects on the GIT is contradictory. The aim of the research was to establish the nature and mechanisms of NPs and MPs action on the gastrointestinal mucosa and intestinal microbiome, basing on the literature data. Material and methods. The review was compiled after selecting of 90 documents from major databases including Web of Science, PubMed, Scopus, Elsevier, Springer and Google Scholar (up to March 2023). Results. In animal studies and in vitro models, it was shown that MP/NP affect mucus secretion, its rheological characteristics, and can cause an increase in the permeability of tight junctions of epithelial cells by reducing the expression of zonula occludens protein 1 (ZO-1), occludin and claudin-1, which promotes the penetration of MP through the intestinal wall. Various adsorption layers (coronas) formed on the surface of MPs both abiotically and during transit through the GIT can lead to both increased and reduced toxicity of MPs. Biofilms formed on MP/NP surface create favorable conditions for the activity of pathogenic bacteria and horizontal gene exchange between the components of the biofilm and the intestinal microbiome. Animal experiments have shown an unfavorable effect of MP/NP on the intestinal microbiota and its key metabolites, contributing to the development of dysbiosis. Conclusion. Most data on the effect of MP on the GIT have been obtained using a model object - polystyrene microspheres, which are rarely found in practice. A frequent limitation of the in vitro studies is the discrepancy between used doses (concentrations) of MP and those that may occur when MP are consumed with food. Data on the potential impact of MP/NP on the GIT protective barrier and intestinal microbiota obtained under various experimental conditions are contradictory. Thus, evidence of the impact of MP/NP on the GIT and intestinal microbiota of humans needs further confirmation, which will allow us to move on to the development of a set of measures that can reduce the risks of MP exposure to human health.

[Nanocellulose in the food industry and medicine: structure, production and application].

Cellulose is the most abundant polymer in the biosphere and has many technical applications, including food production. The development of cellulose technology in the 21st century has led to the emergence of nanocellulose (NC), which has widely varying chemical and physical properties and, therefore, has fundamentally new areas of application in biomedicine and the food industry. The aim of this work is to review the literature on the structure, sources, methods for obtaining nanocelluloses, including methods for their chemical modification, current and prospective applications in the food industry, packaging materials, biomedicine, etc. Material and methods. For the analysis, sources were selected mainly for the period from 2014 to 2022, contained in the international databases PubMed, WoS and Scopus and meeting the requirements of scientific reliability and completeness. Results. Among the main types of NC there can be identified nanofibrous cellulose (NFC), consisting of fibers longer than 500 nm and about 10-20 nm thick, and nanocrystalline cellulose (NCC) with particles 100-500 nm long and less than 100 nm in diameter. A special group of materials includes bacterial NC (BNC) produced by microorganisms and representing entangled coils or layers of cellulose fibers with a thickness of less than 100 nm. Significant changes in the physical, chemical and functional properties of NC can be achieved by its physical and chemical modification, which leads to a change in swelling, an increase in the mechanical strength and stability of hydrogels, and compatibility with synthetic polymers. NFC, NCC and BNC are offered as food ingredients for inclusion both in mass consumption products and in specialized foods for dietary and therapeutic uses, as well as in the so-called «functional products¼, for which manufacturers declare the ability to influence actively on the state of intestinal microflora and digestive function. In biomedical applications, of great interest is the biocompatibility of BNC with various cell types in combination with biodegradability, which makes it possible to create new types of materials for reconstructive surgery, effective and safe dressings. When used as packaging materials, NC products successfully compete with synthetic polymers without causing long-term environmental pollution. Factors hindering the introduction of NC products into practice are the gaps of knowledge of NC biological action, combined with the risks caused by possible residual amounts of chemicals and biochemical reagents, bacterial toxins, enzyme preparations and microorganisms-producers in the composition of NC. Conclusion. There is an important task to develop a system for regulating NCs and products with its content, in accordance with the technical regulations of the Customs Union of the EAEU.

[Assessment of the influence of an enzymal preparation - a complex of glucoamylase and xylanase from Aspergillus awamori Xyl T-15 on the intestinal microbiom and immunological indicators of rats].

The requirements for the safety of food products obtained by microbial synthesis are including as obligation for to conduct toxicological studies - the study of various biochemical and immunological markers of toxic effects. The necessity of these studies is explained by a possible change in the structure of food ingredients produced by a microbial cell and, consequently, a change in their biological properties, as well as the possible presence of living forms and/or DNA of producer strains or of their toxic metabolites in these ingredients. At the same time, it is well known that the nutrient composition of foods has a significant impact on the composition and properties of microorganisms that make up the gut microbiome, which, in turn, determines the immune status. The purpose of the research was to justify the analyses of gut microbiocenosis composition for inclusion in the protocol of safety investigation of foods obtained by microbial synthesis [on the example of an enzyme preparation (EP) - a complex of glucoamylase and xylanase from a genetically modified strain of Aspergillus awamori Xyl T-15]. Material and methods. In experimental studies carried out for 80 days, Wistar rats (males and females) were used. The study of the effect of EP (a complex of glucoamylase and xylanase from a genetically modified Aspergillus awamori Xyl T-15 strain) in dozes 10, 100 and 1000 mg/kg body mass on the cecum microbiome and the immune status (content of cytokines and chemokines: IL-1a, IL-4, IL-6, IL-10, IL-17A, INF-γ, TNF-α, MCP-1, MIP-1a and Regulated on Activation Normal T-cell Expressed and Secreted - RANTES) was carried out. Results. It has been shown that EP - a complex of glucoamylase and xylanase from A. awamori Xyl T-15 at doses of 100 mg/kg or more causes mild disturbances in the composition of gut microbiocenosis. At the same time, these disorders have a significant immunomodulat ory and immunotoxic effect on the body, which manifests itself in a dose-dependent change in the profile of pro-inflammatory cytokines and chemokines in blood and spleen. The adverse effect of EP on the body is probably due to the formation of metabolites that are not formed during usual digestive processes in the gastrointestinal tract. The minimum effective dose (LOAEL) of EP was 100 mg/kg body weight In accordance with established requirements, the activity of the EP should not appear in ready-to-use food. Subject to this requirement, amount of EP entering the body cannot exceed the established LOAEL level. Therefore, a complex of glucoamylase and xylanase can be used in food industry, subject to the establishment of regulations «for technological purposes¼ for A. awamori Xyl T-15 strain. Conclusion. The data obtained on the relationship between the state of the microbiome and the immune status upon the introduction of EP indicate the need to include indicators of the state of gut microbiocenosis in the test protocol of safety.

[Effect of resveratrol, carnitin, quercetin and aromatic amino acids on the xenobiotic metabolising and antioxidant enzymes in the liver during obesity in rats with different genotypes].

The use of minor biologically active substances (BAS) in specialized products is one of the promising areas in the diet therapy for obesity and other alimentary-dependent diseases (metabolic syndrome, type 2 diabetes mellitus, etc.). The effects of the BAS in patients are often ambiguous, depending on a number of factors, one of which is the state of the organism enzyme systems (enzymes of xenobiotic metabolism and antioxidant defense), the patient's genotype, and many others. The aim was to study the effect of BAS [quercetin (Q), L-carnitine (L-Car), resveratrol (Res), aromatic amino acids tyrosine (Tyr) and tryptophan (Trp)] on the activities of phase I and II of xenobiotic-metabolising enzymes and antioxidant enzymes in rats using various in vivo models of obesity and with impaired dopamine transport. Material and methods. The activities of cytochrome P450 enzymes (CYP1A1 and CYP3A), glutathione transferase (GT), UDPglucuronosyltransferase (UDP-GT), hemoxygenase-1 (HO-1), and quinone reductase (QR) were determined by fluorimetric, spectrophotometric methods and HPLC in microsomes and cytosolic fraction of rat's liver. We used rats of outbred Wistar line, Zucker ZF line with hereditarily determined obesity and DAT-KO line with a knockout of the DAT dopamine transporter gene, which for 62 days were fed a standard balanced control or high-carbohydrate high-fat diet (30% fat by weight and 20% fructose solution instead of water) supplemented with BAS, such as Q, Res, L-Car, Tyr and Trp in doses 50, 25, 300, 1250 and 250 mg/kg of body weight respectively. Results and discussion. The presence of a DAT knockout led to a small but statistically significant decrease in the activity of GT in the liver in both homozygous and heterozygous animals. The CYP1A1 activity was significantly decreased in all carriers of the DAT knockout gene, while HO-1 activity, on the contrary, was increased, independently of the composition of the diet used. In Zucker ZF rats of all groups, in comparison with Wistar rats fed the corresponding diets, the activities of GT, UDP-GT, CYP1A1, CYP3A and QR were significantly reduced in terms of the total protein content. HO-1 activity was reduced in Zucker ZF rats in comparison with Wistar rats to a lesser extent, however, the addition of Q significantly influenced the difference between the two lines. Trp consumption led to a significant increase in GT activity in Wistar rats. In DAT-KO homozygotes this effect was insignificant, while in heterozygotes it was absent. Similarly, consumption of Trp resulted in a significant increase in CYP1A1 activity only in Wistar rats, but not in DAT-KO rats. The activity of UDP-GT under Trp intake increased only in DAT heterozygotes. The genotype significantly influenced the response of QR activity to Trp consumption, but in an ambiguous way - there was an increase in activity in heterozygotes and a decrease in homozygotes for DAT knockout. CYP1A1 activity was significantly increased in rats treated with Tyr. Conclusion. The data obtained indicate that the effect of various dietary supplements used in the treatment of obesity and metabolic syndrome on the xenobiotic-metabolising enzymes and antioxidant enzymes can have a different nature and direction depending on the genotype and the level of spontaneous physical activity and energy expenditure determined by it, which should be taken into account when approaches to personalized diet therapy of alimentary-dependent diseases are developing.

[Liver gene expression in normal and obese rats received resveratrol and L-carnitine].

The use of nutraceuticals with anti-inflammatory and hypolipidemic activity in the composition of foods for special dietary uses and dietary supplements is one of the effective methods of dietary therapy of alimentary obesity and related diseases. The aim was to study the effect of the combined intake of resveratrol and L-carnitine (RC) on the expression of genes responsible for the metabolism of carbohydrates, fats and inflammatory reactions in the liver and kidneys of rats in normal conditions and with diet-induced obesity. Material and methods. Male Wistar rats received for 63 days a standard balanced diet or a high-fat-high-carbohydrate diet (HFCD) with an excess of total fat (30%) and fructose (20% solution instead of drinking water), or the same diets supplemented with RC in a low (25 mg/kg body weight as resveratrol and 300 mg/kg as L-carnitine) or high (50 and 600 mg/kg body weight, respectively) doses. The expression of genes (Khk, Gck, Pklr, Acaca, Acacb, Fasn, Scd, Srebf1, Mlxipl, Ppara, Pparg, Actb, Gapdh) in liver cells was studied by the method of real-time reverse transcription polymerase chain reaction (RT-PCR). The distribution of tyrosine aminotransferase (TAT), nuclear factor erythroid 2-related factor 2 (NRF-2) and intercellular adhesion molecule type 2 (ICAM-2) in the liver and kidneys was assessed by confocal laser microscopy and immunohistochemistry. Results. Increased expression of Fasn (fatty acid synthase) in rats treated with high-fat high-carbohydrate diet (HFCD) decreased under RC intake. RC consumption caused a decrease in the number of TAT-, NRF-2- and ICAM-2-positive cells in the liver of rats treated with HFCD, but had the opposite effect in the kidneys. The consumption of RC at the low dose by rats fed HFCD caused changes in the expression profiles of the studied marker genes, indicating a possible hypolipidemic effect. However, observed increased expression of lipogenic genes in the liver and elevated level of NRF-2 and ICAM-2 in kidney against the background of consumption of RC with the standard balanced diet cannot be assessed as unambiguously positive. Conclusion. Thus, possible negative effects caused, most likely, by the interaction of nutraceuticals with various mechanisms of action should be taken into account when developing formulations of dietary supplements and foods for special dietary uses for dietary therapy of obesity.

[Influence of the L-carnitine and resveratrol complex on physiological, biochemical and morphological indicators of normal and obese rats].

Specialized products and dietary supplements, enriched with complexes of minor biologically active substances (BAS), are often offered as components of therapeutic diets in the treatment of obesity and metabolic syndrome. At the same time, the possible effects of the interactions of BAS when consuming a multicomponent product have not been studied enough. The aim - to study the action on rats' organism of a complex supplement (RС), containing resveratrol (Res) and L-carnitine (L-Car), when consumed with a standard balanced or hypercaloric diet. Material and methods. Male Wistar rats received for 63 days a standard balanced diet (SD) or a high-fat-high-carbohydrate diet (HFCD) with an excess of total fat (30%) and fructose (20% solution instead of drinking water), or the same diets supplemented with RС in a low (25 mg/kg body weight as Res and 300 mg/kg body weight as L-Car) or high (50 and 600 mg/kg body weight, respectively) doses. The muscle grip strength, behavioral reactions in tests of the conditioned passive avoidance reflex (CPAR) and elevated plus maze (EPM) were studied. At the end of the experiment, the mass of adipose tissue and internal organs was determined together with the activity of microsomal and cytosolic liver enzymes for specific substrates, plasma biochemical parameters, liver morphology by lightoptical microscopy, accumulation of lipofuscin-like granules (LLG) in the liver and kidneys by laser confocal microscopy. Results. In the rats fed HFCD, compared with SD, there was an increase in the mass index of liver, total inguinal and retroperitoneal white adipose tissue, in the levels of glucose and triglycerides, in the activity of hepatic CYP1A1 and CYP3A monooxygenases, UDPglucuronosyltransferase, heme oxygenase, and simultaneous decrease of high and low density lipoprotein cholesterol, and quinone oxidoreductase activity. The RС intake stimulated the locomotor activity of rats in EPM, however, this effect was less pronounced against the background of HFCD consumption. In rats consuming SD (but not HFCD), the addition of RС caused an increase in search activity and anxiety according to the EPM and CPAR data. The effect on short- and long-term memory retention was statistically insignificant. RС intake did not have hypolipidemic and hypoglycemic properties but caused in low dose an increase in the ratio of the activity of transaminases AST/ALT in animals fed HFCD. The liver CYP3A activity increased in rats supplemented with RС in high dose fed HFCD. In the kidneys of animals, the consumption of RС resulted in increased accumulation of LLG. Conclusion. When studying the effect of the complex supplement RС on normal and obese rats according to the studied physiological, morphological and biochemical indexes, no positive effects were revealed, that would not have manifested themselves for Res and L-Car separate intake. No evidence of synergistic action of L-Car and Res were found, and some of the effects of the complex supplement can be considered as adverse. This requires careful assessment when combined using these substances in complex diet therapy of metabolic disorders in humans.

[Risk assessment of glucoamylase and xylanase complex from Aspergillus awamori Xyl T-15].

The introduction of methods for food production using microbial synthesis, including those obtained with the help of genetically modified (GM) microorganisms, at the present stage, allows to increase production volumes and reduce the cost of food. At the same time, such products in accordance with TR CU 021/2011 "On food safety" are classified as a "novel food"¼ and can be placed on the market only after its risk estimation for health. The emergence of new data and research methods in the last years has made it necessary to improve the risk assessment system for this category of food. The aim of the research is to develope risk assessment approaches of food obtained by microbial synthesis on the example of the GM strain Aspergillus awamori Xyl T-15 and the enzyme preparation (EP) (a complex of glucoamylase and xylanase) produced by it. Material and methods. Outbred ICR mice (CD-1) and Wistar rats (males and females) were used in the experimental studies. Investigations of GM strain A. awamori Xyl T-15 virulence and its ability to disseminate internal organs have been carried out. Acute and subacute (during 80 days) toxicity of EP (a complex of glucoamylase and xylanase) have been studied. Results. The presented experimental data allow us to make a conclusion about the avirulence of the A. awamori Xyl T-15 strain, the lack of ability to disseminate internal organs (invasiveness). At the same time, the strain is characterized by the ability to produce mycotoxins (ochratoxin, fumonisin B2, T-2 and HT-2 toxins). The EP, a complex of glucoamylase and xylanase from A. awamori Xyl T-15, has a low oral acute toxicity for rats (LD50>5000 mg/kg). I ntragastric EP administration at doses of 10, 100 and 1000 mg/kg of body weight during 80 days had not revealed adversely affect on the rate of weight gain in animals, indicators of anxiety and cognitive function, and some studied biochemical indicators. At a dose of 100 mg/kg b.w. or more, there were changes in the relative mass of organs (lungs, kidneys, adrenal glands), small shifts in the parameters of erythropoiesis and leukocyte formula, at a dose of 1000 mg/kg b.w. - an increase in oxidative DNA destruction. T he most pronounced and dose-dependent was the effect of the EP on hepatocyte apoptosis. According to this indicator, the not observed adverse effect level (NOAEL) for EP is not more than 100 mg/kg b.w. in terms of protein. The main target organ for the toxic effect of EP is the liver. Conclusion. The data obtained demonstrate the necessity to conduct an additional analysis of the risks of possible negative effects of EP, namely, to study its impact on the gut microbiocenosis and the immune status of experimental animals, to analyze the presence of determinants of pathogenicity and antibiotic resistance, DNA of selective marker genes of A. awamori Xyl T-15 strain by PCR analysis and DNA sequencing methods.

[Chemical food safety: development of methodological and regulatory base].

The review presents the results of studies carried out in the Federal Research Centre of Nutrition and Biotechnology in the direction of food toxicology, the purpose of which was to improve the risk assessment methodology, substantiate hygienic regulations for the content of chemical contaminants in foodstuffs and develop methods for their detection and quantitative determination. New challenges and problems associated with the control and regulation of chemical contaminants in foods are associated, firstly, with the identification of previously unrecognized chemical factors harmful to human health, and, secondly, are caused by the progress of technologies, accompanied by the emergence of new sources of nutrients and methods of processing foodstuffs, which, along with many benefits and advantages, creates new potential risks to consumer health. Among the priority chemical pollutants, which should be mentioned currently as objects of improved regulation and control methods, are toxic elements (organic and inorganic forms of arsenic, mercury, nickel), veterinary drugs, phycotoxins, phytotoxins, new mycotoxins, various forms of polychlorinated biphenyls and polycyclic aromatic hydrocarbons, biologically active substances of plant origin, concentrated during the production of extracts, as well as so-called technological contaminants, food additives, residual amounts of technological aids. An independent problem is the assessment of risks from nanoparticles and nanomaterials used in the production of foodstuffs, as well as enzyme preparations and food ingredients produced with the help of genetically modified microorganisms. The system of toxicological and hygienic assessment and control of chemical contaminants in foodstuffs operating in Russia is constantly being improved on the basis of new scientific data to substantiate the permissible levels of their content in products and new methods of analysis. The results obtained are reflected in the regulatory documents of the Russian Federation and the Eurasian Economic Union.

[The content of essential and toxic microelements in the organs of mice of various lines receiving a high-carb high-fat diet and supplemented with quercetin].

Quercetin (Q) is known to be a powerful chelating agent for metal ions. Due to this property, Q, when ingested, is able to intervene actively in microelement homeostasis. The assessment of the possible significance of these effects for the dietary use of Q in obesity is complicated by the fact that in obese patients there are numerous changes in mineral metabolism and microelement homeostasis due to the pathogenesis of the disease. Thus, it is of considerable interest to identify the systemic effects of biologically active substances, including Q, on mineral metabolism in biological in vivo models (due to the limited choice of biosubstrates in clinical observations - blood plasma, urine, hair). The aim was to study the possible effects of Q on the levels of essential, non-essential and toxic elements in mice of three lines: db/db with knockout of the leptin receptor gene, prone to spontaneous development of obesity, inbred line C57Bl/6J, relatively resistant to the development of nutritional obesity, and complex hybrid of the 2nd generation DBCB, genetically more prone to developing obesity and fatty hepatosis when consuming diets with an excess quota of fat and simple carbohydrates. Material and methods. The content of 16 chemical elements (Fe, Mg, Cu, Mn, Co, Se, Zn, Cr, Al, Cd, As, Pb, Ni, Ag, V, Cs) was determined by mass spectrometry with inductively coupled plasma in the liver, kidneys, brain of genetically obese db/db mice (8-10 weeks old) receiving standard semisynthetic ration (SSD) and Q for 47 days at doses of 25 and 100 mg/kg body weight, C57Bl/6J mice treated with SPR or a high-fat high-carb diet (HFCD - 30% fats and 20% fructose solution instead of water) and Q supplementation at the same doses, DBCB tetrahybrid mice treated with SSD, HFCD and HFCD supplemented with Q for 63 days (25 mg/kg body weight). The compounds of Fe, Zn, Cu, Mn, Mg, Cr, Ni, Se, and V were included in the salt mixture in the form of inorganic salts or oxides in amounts close to physiological needs; Al, Cd, As, Pb, Ag, Cs, Co - were present in the diets in background amounts. Results and discussion. In db/db mice, in comparison with C57Bl/6J, a decreased content of Pb in the liver and increased in the kidneys and brain, decreased Co in the kidneys, increased Cs and As in the brain, which could not be explained by differences in the consumption of these elements with feed. The consumption of Q decreased the content of Mn, Cs, V, Ni, As in the liver in db/db mice and increased Cu and did not significantly affect the level of trace elements in the liver in animals C57Bl/6J. In kidneys of C57Bl/ 6J receiving Q, the content of As and Al increased. In brain of db/db mice, Q supplementation caused a decrease in the content of As, Pb, Cs and Se. In tetrahybrids DBCB, Q increased Pb levels in liver and brain and decreased in kidneys; increased the level of V in liver and brain; decreased As content in kidneys and increased in liver. Multiple correlations were noted between the organ content of elements in various valence forms, as well as between the content of trace elements and biochemical indicators of the intensity of catabolic and anabolic processes. Conclusion. Quercetin has an effect on the homeostasis of microelements, depending both on the animal genotype and on the diet, and not having unambiguous physiological significance. Indicators of the status of essential and toxic trace elements are recommended to be included in the protocols of preclinical trials of the efficacy and safety of minor biologically active food substances.

[Influence of the DAT gene knockout on exchange of essential and toxic trace elements in rats].

The maintenance of energy homeostasis of the body according to modern data is carried out with the active participation of dopaminergic neurons of the central nervous system. The synthesis and metabolism of dopamine (DA) occurs both in the brain and in peripheral tissues. Violation of the synthesis and metabolism of DA is considered as a link in the vicious cycle which it formed during the development of diet-induced obesity. According to modern data, a number of essential and toxic trace elements, such as Cd, Al, As, Mn, Fe, Cu, Zn, are actively involved in the exchange of DA in the brain and peripheral organs and tissues. One way to assess this relationship is to compare changes in the microelement status of the organism when consuming hypercaloric diets in animals with normal and impaired DA transport. The latter can be animals with a knockout of the DAT transporter gene, which performs DA reabsorption with subsequent storage in the composition of secretory granules. The aim is a comparative study of the content of a number of essential and toxic elements in the brain, liver, and kidneys of rats that differ in the allelic variants of the DAT gene fed balanced diet and the diet with an excess of energy value. Material and methods. The study was carried out on 30 male rats of the DAT-KO knockout line (homozygotes DAT-/- and heterozygotes DAT+/-), 8-10 weeks old, and 13 males rats of the outbred Wistar line (DAT+/+) of the same age. For 62 days the animals (6 groups) received a semi-synthetic diet containing essential elements in the salt mixture or a similar high-fat-highcarbohydrate diet (HFCD) with 30% fat and 20% fructose solution instead of drinking water. The content of 16 trace elements (Fe, Mg, Cu, Mn, Co, Se, Zn, Cr, V, Cs, Ag, Al, Cd, As, Pb, Ni) were determined by inductively coupled plasma mass spectrometry in the liver, kidneys, and brain of rats with a knockout of the dopamine DAT transporter gene: homozygotes (DAT -/-) and heterozygotes (DAT+/-), as well as wild-type rats (DAT+/+) of the Wistar strain. Results and discussion. In the liver, DAT knockout led to an increase in the content of As, Cd, Co, and Cs and a decrease in Fe; in the kidneys - to an increase in the levels of Pb, As, Cd and Se, in the brain - an increase in the content of most of the studied trace elements, including Pb, As, Cs, Al and Cu. Conclusion. Against the background of consumption of HFCD, the effect of DAT knockout on the content of a number of elements was more pronounced compared with the consumption of the control diet. The revealed changes in the trace element content in DAT knockout rats are considered in terms of the effect of DA metabolism in the central nervous system and in peripheral tissues on the status of trace elements.

Comparative Whole-Transcriptome Profiling of Liver Tissue from Wistar Rats Fed with Diets Containing Different Amounts of Fat, Fructose, and Cholesterol.

Differential expression of 30,003 genes was studied in the liver of female Wistar rats fed with isocaloric diets with the excess of fat, fructose, or cholesterol, or their combinations for 62 days using the method of whole-transcriptome profiling on a microchip. Relative mRNA expression levels of the Asah2, Crot, Crtc2, Fmo3, GSTA2, LOC1009122026, LOC102551184, NpY, NqO1, Prom1, Retsat, RGD1305464, Tmem104, and Whsc1 genes were also determined by RT-qPCR. All the tested diets affected differently the key metabolic pathways (KEGGs). Significant changes in the expression of steroid metabolism gene were observed in the liver of animals fed with the tested diets (except the high-fat high fructose diet). Both high-fat and high-fructose diets caused a significant decrease in the expression of squalene synthase (FDFT1 gene) responsible for the initial stage of cholesterol synthesis. On the contrary, in animals fed with the high-cholesterol diet (0.5% cholesterol), expression of the FDFT1 gene did not differ from the control group; however, these animals were characterized by changes in the expression of glucose and glycogen synthesis genes, which could lead to the suppression of glycogen synthesis and gluconeogenesis. At the same time, this group demonstrated different liver tissue morphology in comparison with the animals fed with the high-fructose high-fat diet, manifested as the presence of lipid vacuoles of a smaller size in hepatocytes. The high-fructose and high-fructose high-fat diets affected the metabolic pathways associated with intracellular protein catabolism (endocytosis, phagocytosis, proteasomal degradation, protein processing in the endoplasmic reticulum), tight junctions and intercellular contacts, adhesion molecules, and intracellular RNA transport. Rats fed with the high-fructose high-fat or high-cholesterol diets demonstrated consistent changes in the expression of the Crot, Prom1, and RGD1305464 genes, which reflected a coordinated shift in the regulation of lipid and carbohydrate metabolisms.

[Immunological markers of alimentary-induced hyperlipidemia in Wistar rats].

Changes in plasma levels of the main groups of cytokines and adipokines may correlate with the severity of metabolic disorders in hyperlipidemia and obesity. The aim of the study was to assess the significance of ghrelin, leptin, their ratio (L/Gh), and the cytokine profile as biomarkers at dietary-induced hyperlipidemia. Material and methods. We used 48 female Wistar rats with an initial body weight of 123±1 g, which were divided into 6 groups. Group 1 (control) received a balanced semi-synthetic diet according to AIN93; group 2 - diet with excess fat (30% by weight); group 3 - a diet with the addition of 20% fructose solution instead of drinking water, group 4 - a diet with excess fat and fructose, group 5 - a diet with added cholesterol (0.5%), group 6 - a diet with cholesterol and fructose. On the 64th day of the experiment, the mass of internal organs was determined; the levels of cytokines and adipokines in blood plasma were measured by multiplex immunoassay. Results and discussion. A decrease in the level of leptin was found in group 5 compared with the control and with groups 2, 4 and 6 groups (p<0.05). The lowest level of ghrelin was found in group 2 (p<0.05) against the background of high concentrations of leptin. Significant correlations were found between L/Gh and the total mass of animals (r=0.321; р=0.034), the relative mass of adipose tissue (r=0.439; р=0.003) and with the relative mass of the spleen (r=-0.460; р=0.003). In group 2, at the maximum L/Gh ratio, a significantly higher weight of adipose tissue was found, whereas in groups 3 and 5, at the lowest L/Gh ratio, the relative amount of total fat was the lowest. L/Gh ratio correlated with the level of monocyte chemotactic protein-1 (MCP-1), RANTES, IL-18 and macrophage colony-stimulating factor (M-CSF). The concentrations of IL-17, IL-18, IL-4, IL-5, MIP-3a, IFN-γ, M-CSF and RANTES in the experimental groups were reduced compared with the control, with the most pronounced effect in group 5 together with the lowest L/Gh ratio. Conclusion. The presence of a significant correlation between L/Gh ratio and changes in the weight of rats' body, spleen, adipose tissue, as well as levels of cytokines involved in inflammation regulation, confirms the importance of L/Gh ratio as a biomarker in an in vivo model of dyslipidemia.

[Effect of quercetin on the expression of the carbohydrate and lipid metabolism genes in the liver of rats with genetic].

Quercetin (Q; 3,3',4',5,7-pentahydroxyflavone) is considered as a promising component of specialized products for the correction of metabolic disorders in obesity and metabolic syndrome. At the same time, the results of evaluating the clinical efficacy of Q are ambiguous, and the mechanisms of its influence on lipid and carbohydrate-energy metabolism are not well understood. The aim of the work was to study the effect of quercetin (Q 3,3',4',5,7-pentahydroxyflavone) on the expression of key glycolysis and lipogenesis enzymes' genes in Zucker-Leprfa (Z) rats characterized by hereditary obesity, compared to «wild-type¼ Wistar (W) rats. Material and methods. 24 male Z rats and 32 male W rats aged 8-10 weeks were used. Animals of each line were divided into 4 groups of equal numbers. For 62 days the animals of the first groups (controls) received a balanced diet according to AIN93M, the seconds - the same diet with Q added in a dose of 50 mg/kg body weight. Animals of the third groups received a high-fat, high-carbohydrate diet (HFCD) with fat 30% by weight and with the replacement of drinking water with a 20% solution of fructose, the fourths groups - the same diet and supplementation with Q. After removing animals from the experiment, expression levels of liver carbohydrate and lipid metabolism genes Khk, Gck, Pklr, Acaca, Fasn, Scd, Srebf1, Mlxipl, Ppara and Pparg were determined by real-time polymerase chain reaction (RT-PCR) with reverse transcription using Actb and Gapdh as reference genes. The levels of triglycerides, total and HDL cholesterol, lipolytic activity and immunoreactive leptin were determined in plasma. Results and discussion. When comparing two animal lines, a significantly higher level of expression of Ppara, Pparg, Mlxipl, Acaca, Fasn, Scd was shown in Z rats compared to W rats, which is consistent with the development of dyslipidemia in the first ones and elevated levels of leptin under both types of diets used. The addition of Q caused in W rats a decrease in the expression of Scd, Mlxipl, Khk and Gck, more pronounced on the background of HFCD whereas in Z rats there were no similar effects, or they had the opposite direction. In addition, in Z rats, consumption of Q led to increased expression of Pklr, which was not observed in W rats. Conclusion. The modulating effect of Q on the expression of key genes of lipid and carbohydrate metabolism enzymes significantly differs in wild-type W rats and mutant Z rats with hereditary obesity, and this difference appears to be potentiated by the consumption of excess fat and fructose.

Identification of silver nanoparticles in the small intestinal mucosa, liver, and spleen of rats by transmission electron microscopy.

The effects of water-dispersed Ag nanoparticles on the small intestinal mucosa, liver, and spleen of rats were studied by transmission electron microscopy. Acute experiments demonstrated penetration of Ag nanoparticles injected into the isolated intestinal loop into the intestinal mucosa, liver, and splenic tissues. Ultrastructural changes (lobed nucleus, megamitochondria) were found in the studied organs. These data indicated that injection of water-dispersed Ag nanoparticles into the gastrointestinal tract was followed by their penetration through the epithelium of the small intestinal mucosa into other organs, e.g. into the liver and spleen. This fact is essential for evaluation of potential risks of the nanoparticle effects on human health and environment.

Effects of titanium dioxide nanoparticles on small intestinal mucosa in rats.

Penetration of titanium dioxide nanoparticles into enterocytes after their administration into isolated loop of rat small intestine was shown in vivo by transmission electron microscopy. Using electron diffraction, titanium dioxide nanoparticles were identified in the apical regions of the cells under plasma membranes and in deeper parts of the cytoplasm as solitary objects or small aggregations. Water dispersions of nanoparticles (3-h exposure to high concentrations) caused no appreciable morphological changes in enterocyte ultrastructure. A 28-day subacute intragastric administration of water dispersion of nanoparticles to rats led to titanium accumulation in the liver, their level was significantly higher than in the control group, which was shown by mass spectrometry with inductive-bound plasma. These data indicated the possibility of penetration of titanium dioxide nanoparticles through the gastrointestinal barrier under near-physiological conditions.

[Full transcriptome analysis of gene expression in liver of mice in a comparative study of quercetin efficiency on two obesity models].

BACKGROUND: Quercetin (Q; 3,3',4',5,7 - pentahydroxyflavone) can help alleviate the pathological effects of nutritional obesity and metabolic syndrome when taken as part of products for special dietary needs and food supplements. The mechanisms of action of Q at the genetic level are not well understood. AIMS: To study gene expression in liver tissue of mice with alimentary and genetically determined obesity upon intake of Q with diet. MATERIALS AND METHODS: During 46 days of the experiment on 32 male C57Bl/6J mice fed a diet with an excess of fat and fructose and 24 male genetically obese db/db mice the effect of Q in dose of 25 or 100 mg/kg of body weight was studied on differential expression of 39430 genes in mice livers by full transcriptome profiling on microchip according to the Agilent One-Color Microarray-Based Gene Expression Analysis Low Input Quick Amp Labeling protocol (version 6.8). To identify metabolic pathways (KEGGs) that were targets of Q exposure, transcriptomic data were analyzed using bioinformatics methods in an "R" environment. RESULTS: Differences were revealed in the nature of Q supplementation action in animals with dietary induced and genetically determined obesity on a number of key metabolic pathways, including the metabolism of lipids and steroids (Saa3, Cidec, Scd1, Apoa4, Acss2, Fabp5, Car3, Acacb, Insig2 genes), amino acids and nitrogen bases (Ngef, Gls2), carbohydrates (G6pdx, Pdk4), regulation of cell growth, apoptosis and proliferation (Btg3, Cgref1, Fst, Nrep Tuba8), neurotransmission (Grin2d, Camk2b), immune system reactions (CD14i, Jchain, Ifi27l2b). CONCLUSIONS: The data obtained help to explain the ambiguous effectiveness of Q, like other polyphenols, in the dietary treatment of various forms of obesity in humans, as well as to form a set of sensitive biomarkers that allow us to elucidate the effectiveness of minor biologically active food substances in preclinical trials of new means of metabolic correction of obesity and metabolic syndrome.