Improvement of Methods for Safety Control of Microbial Producers and Food Produced Therewith.

The study substantiates the necessity to implement the algorithm of molecular-genetic assessment of biosafety of the genetically modified microorganisms (GMM) and to develop standardized methods to test the genetically modified strains producing enzymes, bioactive substances, and other products of microbial synthesis prior to their use in food industry. Analysis of microbial producers and related food products for the presence of GMM-associated DNA revealed high incidence of the marker genes amp and lacZ in enzyme preparations and in mycelium of industrial genetically modified producer of Aspergillus genus. The procedure of extraction of DNA from mycelium of mold fungi is optimized by including the stage of additional purification of the extracts, assessment of their purity by PCR with universal ITS primers, and determination of effective DNA concentration in the samples prior to conduction of the molecular genetic assay. For identification and genotyping of mold fungi (the biotechnological producers of enzyme preparations), the Sanger sequencing method was adapted. Using this modified method, we determined the species of five equivocally identified strains of Aspergillus genus. To identify the closely-related micromycetes of Ascomycota division, a genotyping algorithm was developed based on amplification of total DNA with expanded panel of primers and DNA sequencing by capillary electrophoresis.

[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.

[Amaranth, quinoa and buckwheat grain products: role in human nutrition and maintenance of the intestinal microbiome].

Pseudocereals such as amaranth, quinoa and buckwheat have been used as food since ancient times and in recent years there has been an increasing focus on their ability to have positive health effects. Moreover, some of the functional effects of pseudocereals could be mediated by effects on the gut microbiota. The review aims to assess the features of the chemical composition of amaranth, quinoa and buckwheat grain that determine their potential for maintaining the optimal composition of the intestinal microbiota, as well as to analyze the results of published studies evaluating the effects of pseudocereals on the intestinal microbiota. Material and methods. Scopus, Web of Science, PubMed, RSCI databases, and food composition databases were used for collection and analysis of scientific information. Results. The research presents an overview of the chemical composition of amaranth, quinoa and buckwheat grain regarding their influence on the intestinal microbiota. Compared to traditional cereals, the grain of these pseudocereals has high content of soluble dietary fiber, which could have a prebiotic effect in the gut stimulating the growth of protective microbiota populations and increasing production of short-chain fatty acids (SСFA), which play a crucial role in maintaining gut homeostasis and health in general. Amaranth, quinoa and buckwheat grain, as well as some grain fractions such as proteins and polysaccharides, may have positive effects on the gut microbiota, and the biologically active substances metabolized by them have a positive effect on the body's metabolism. The results of in vitro (by cultivation using model media) and in vivo experiments indicate that the introduction of various grain fractions of pseudocereals into the diet contributes to an increase in the content of SCFA, in alpha microbiota diversity indices, and also prevents the development of dysbiotic disorders caused by a high-fat diet. Conclusion. Pseudocereals' grain is promising raw material for the development of products that can have a positive effect on the intestinal microbiota.

A Method of Direct Quantitation of Lactobacillus spp. in Intestinal Contents Based on Real-Time PCR.

The method of direct quantitation of Lactobacillus spp. and L. acidophilus in intestinal contents based on real-time PCR was developed. It does not require culturing and allows estimating the number of living lactobacilli cells (measured in lg CFU) in absolute quantitative PCR format.

[Gut microbiome: from the reference of the norm to pathology].

А systemic assessment of the state of the human intestinal microbiome was carried out in relation to its function in the macroorganism, aimed at providing the nutriome, and the factors that determine the adequate nutritional status. A new concept of "reference gut microbiome of a healthy person" was postulated and the requirements to it were formulated: interaction with the host according to the principle of mutualism, provision of immune balance with the macroorganism due to the correct formation of mucosal immunity, implementation of metabolic and regulatory functions without losses for the nutriome. A set of characteristics and biomarkers reflecting the taxonomic composition and population properties of the microbial community, as well as the state of its essential immune and metabolic functions, was proposed as a criterion for its assessment in healthy adults who consume a diet balanced in nutritive and energy value, appropriate for age and energy spending. The influence of alimentary factors on the formation of the human intestinal microbiome in early ontogenesis, the nature of dysbiotic shifts, including those under common non-infectious alimentary-dependent diseases (obesity, food allergy, urolithiasis), in Russians were studied, the ways of their correction and maintenance of the intestinal microbiota in the process of life were substantiated taking into account modern knowledge.

[Peroral toxicological assessment of bentonite nanoclay used in the food industry].

Bentonite nanoclay (NC) manufactured from the natural sedimentary mineral bentonite contains more than 90% montmorillonite. Currently, it is widely used in food industry as processed aids - adsorbents for the purification of vegetable oils and beverages. Clay minerals have also applications as food additives and components in composite package materials. In vitro studies have shown that various forms of NC exerted cytotoxicity in many cell lines, whereas in vivo evidence of NC oral toxicity is contradictory. Therefore, this study aimed to assess the acute oral toxicity of NC and to evaluate its toxicological characteristics in a subacute 92-day experiment on Wistar rats with a daily oral administration in doses of 1, 10, and 100 mg/kg body weight (bw). Material and methods. The NC acute toxicity was evaluated in 8 male and 8 female rats with the initial bw 236±10 and 203± 10 g, respectively. NC was administered as an aqueous dispersion intragastrically at a dose of 5 g/kg bw. On the 14th day (end of the experiment), an autopsy of the chest and abdominal organs was performed. The subacute experiment was carried out on 64 male rats with an average initial bw of 117±7 g. During the experiment the levels of anxiety and memory function were evaluated using the test "Conditional reflex of passive avoidance". On the 90th day of the experiment, diurnal urinary excretion of creatinine and selenium was evaluated. At the end of the experiment, the integral parameters, the state of the intestinal wall permeability were assessed. Hematological and biochemical parameters were examined in blood, the content of non-protein thiols and the number of cells in apoptosis were determined in liver, and the state of cultivated microbiome populations was studied in cecum. Results. The results of the determination of NC acute toxicity showed the absence of rat's mortality and specific pathological changes in the internal organs at a dose as large as 5000 mg/kg bw, which allowed attributing NC to the V hazard class. Nevertheless, under the conditions of the 92-day experiment, NC caused some adverse biological effects on rat's organism. So, even at an NC dose of 1 mg/kg bw, there was a sharp inhibition of the symbiotic bifidobacterium growth, an increase in platelet count, in LDL and the LDL/HDL ratio, together with the presence of hypertriglyceridemia. At a dose of 10 mg/kg bw, an increase in spleen mass and a decrease in the de Ritis coefficient (AsAT/AlAT) were established. At a dose of 100 mg/kg bw there were shifts in the leukocyte blood count, an excessive enterococci growth in the cecum, significantly increased animal bw, along with the decrease of AsAT/AlAT and the level of serum nitrogen metabolites, indirectly indicating inhibition of catabolic processes. However, at the highest dose of NC, intestinal absorption of the protein antigen - ovalbumin, was apparently completely blocked. Conclusion. The data obtained have shown that NC has potentially adverse effects on the rats mainly at a dose of 100 mg/kg bw, nevertheless, its NOAEL in the 92-day daily oral exposure experiment is probably less than 1 mg/kg bw.

[The study of the raw milk contamination by bacteria of the genus Campylobacter using traditional microbiological methods and quantitative PCR assay].

Gastroenterocolitis caused by Campylobacter bacteria are the most common acute infectious zoonotic foodborne diseases. One of the important factors for the transmission of infection is contaminated dairy products, so the assessment of contamination of raw milk with Campylobacter is necessary to develop effective measures to suppress the growth of the pathogen and ensure the safety of products. The aim of the study was to assess the microbial characteristics of raw milk samples and the nature of their contamination with thermophilic bacteria of the Campylobacter genus. Material and methods. A total of 60 samples of raw milk from the central regions of the Russian Federation and 48 experimentally infected samples of raw milk were studied. To assess the microbial contamination of milk, the number of extraneous microflora was determined, including coliform bacteria (CFB). The identification and quantification of bacteria of the genus Campylobacter was carried out by cultural methods in comparison with quantitative PCR assay. For PCR, primers were used that detected the speciesspecific sequence of C. jejuni 16s rRNA, the presence of the cytotoxic toxin gene cdtB and the invasion gene ciaB. Results and discussion. A significant part of the samples of raw milk (31.6%) was characterized by high levels of microbial contamination exceeding 106 CFU/cm3. Gram-negative bacteria were the dominant type of bacterial microflora, their levels were comparable with the detected values of the total number of microorganisms. Coliform bacteria were found in all studied samples, and their content in 90% of the samples reached 105 CFU/cm3, and in some samples - 107 CFU/cm3. Campylobacter spp. detection rate in raw milk was 8.3%, and their number ranged from 0.1 to 100 CFU/cm3 (average 2.0×10 CFU/cm3). The isolated strains of campylobacters were identified as a C. jejuni species. In the study of the microbial background of the examined samples of raw milk, a comparative analysis of their contamination by campylobacters by rti-PCR was simultaneously carried out. The majority of samples (over 60%) were positive for the presence of 16s rRNA genomic sequence, and they were characterized by the highest values of total bacterial contamination and the amount of coliforms. The use of a multi-primer approach (simultaneous testing for the presence of 16s rRNA and the gene of cytoletal toxin cdtB C. jejuni) reduced the number of positive cases of Campylobacter DNA detection to 16.6%, which suggests a greater informative value of the cdtB gene for the detection of viable, including uncultivated cells. An indicative assessment of the results in a quantitative format showed levels of 104-106.5 genomic equivalents of the DNA in 1 cm3, suggesting the possible presence of viable Campylobacter cells in the tested probes with a significantly greater frequency than that established by cultural method. Conclusion. At low levels of Сampylobacter contamination the microbiological methods do not provide reliable detection of the pathogen due to massive contamination of raw milk by extraneous microflora. Campylobacter spp. were detected by the culture method in 8.3% of cases, while the use of multi-primer PCR assay with cdtB and ciaB genes can double the detection of C. jejuni in raw milk samples.

Studying the Phenotypic and Genotypic Expression of Antibiotic Resistance in Campylobacter jejuni under Stress Conditions.

Specific features for the development of resistance in Campylobacter jejuni strains were studied after treatment with antibiotics of 6 pharmacological groups. Populations of 18 native strains of C. jejuni (isolated from raw poultry products) and their subcultures (obtained after 2-3-fold stress exposures to antimicrobial agents in subinhibitory doses) were examined to evaluate the expression of phenotypic antibiotic resistance. Genotypic properties of strains were studied by the PCR with primers that detect the presence of genes for resistance to aminoglycosides (aphA-1, aphA-3, and aphA-7), tetracyclines (tetO), and quinolones (GZgyrA). The majority of test strains of C. jejuni exhibited a high resistance to nalidixic acid, ciprofloxacin, and tetracycline, which reached the maximum value after numerous passages. The expression of antibiotic resistance was greatest in the presence of nalidixic acid and tetracycline. Ciprofloxacin resistance of 33% strains, which were initially resistant to this antibiotic, was increased after 2-3-fold treatment. We revealed a high degree of correspondence between phenotypic and genotypic profiles of antibiotic resistance in food isolates of Campylobacter. One, two, or more genes of aphA were identified in 85% strains phenotypically resistant to aminoglycosides. The tetO gene was found nearly in all strains resistant to tetracycline. Studying the biofilm matrix in C. jejuni after culturing with antibiotics in subinhibitory doses showed that quinolones (particularly nalidixic acid) and tetracyclines potentiate the formation of biofilms and increase the tolerance of Campylobacter to stress exposures. The intensity of biofilm growth was shown to depend little on the effect of macrolides and aminoglycosides. Therefore, the presence of these agents in residual concentrations is associated with a lower risk for the development of antibiotic resistance in C. jejuni populations.

Formation of Biofilms by Foodborne Pathogens and Development of Laboratory In Vitro Model for the Study of Campylobacter Genus Bacteria Based on These Biofilms.

We analyzed the formation of biofilms by 7 strains of Campylobacter genus bacteria and 18 strains of Enterobacteriaceae genus bacteria that were isolated from plant and animal raw materials, from finished products, and swabs from the equipment of the food industry. Biofilm formation on glass plates, slides and coverslips, microtubes made of polymeric materials and Petri dishes, and polystyrene plates of different profiles were analyzed. When studying the process of films formation, different effects on bacterial populations were simulated, including variation of growth factor composition of culture media, technique of creating of anaerobiosis, and biocide treatment (active chlorine solutions in a concentration of 100 mg/dm3). The formation of biofilms by the studied cultures was assessed by the formation of extracellular matrix stained with aniline dyes on glass and polystyrene surfaces after incubation; 0.1% crystal violet solution was used as the dye. The presence and density of biomatrix were assessed by staining intensity of the surfaces of contact with broth cultures or by optical density of the stained inoculum on a spectrophotometer. Biofilms were formed by 57% Campylobacter strains and 44% Enterobacteriaceae strains. The intensity of the film formation depended on culturing conditions and protocols, species and genus of studied isolates, and largely on adhesion properties of abiotic surfaces. In 30% of Enterobacteriaceae strains, the biofilm formation capacity tended to increase under the influence of chlorine-containing biocide solutions. Thus, we developed and tested under laboratory conditions a plate version of in vitro chromogenic model for evaluation of biofilm formation capacity of C. jejuni strains and studied stress responses to negative environmental factors.

[Subacute oral toxicity in vivo of multi-walled carbon nanotubes].

Multiwall carbon nanotubes (MWCNTs) are regarded as environmental pollutants with increased risk. Recently MWCNTs have attracted attention as a promising component of packaging materials for food products, as carriers for agricultural plant growth stimulants, agrochemicals components and advanced pesticides, which creates the possibility of their exposure through the gastrointestinal tract. Objective of the research is assessment of sub-acute oral toxicity to rats of MWСNTs in an experiment lasting 100 days. MWCNTs preparation «Taunit-M®¼ was preliminary characterized by electron microscopy and Raman light scattering. Nanomaterial was administered to animals as sonicated dispersion in water with 1% by weight of nonionic surfactant Tween 20. The experiment was performed on 80 growing male Wistar rats with initial body weight (b.w.) 86±2 g. Rats of experimental groups (from 2nd to 5th) received MWCNTs dispersion instead of drinking water, the animals of the 1st control group - a carrier solution (Tween 20). Doses of MWCNTs consumed were, respectively, in groups 1-5: 0; 0.01; 0.1; 1.0 and 10 mg/kg b.w. Hematological and biochemical indices of blood were determined together with the activity of glutathione peroxidase of erythrocytes, the content of non-protein thiols in the liver, excretion of 8-hydroxy-2'-deoxyguanosine (8-oxo-G) and content of the main and transient components of gastrointestinal microbiocenosis in the cecum contents. Apoptosis of hepatocytes was studied by flow cytometry. As a result MWCNTs led to increase of blood glucose and creatinine in rats in group 2, a significant decrease in the number of neutrophils and monocytes by increasing the number of lymphocytes, decreased platelet volume, the most pronounced also in group 2, receiving the lowest dose of MWCNTs. There were no signs of oxidative DNA damage identified. At the lowest dose MWCNTs caused a significant decrease in the number of bifidobacteria, increase - citrate-assimilating Enterobacteriaceae, hemolytic aerobic microorganisms and yeast. These changes in the microbiota should be considered as adverse, apparently leading to disturbances in the immune function.

[Optimization of microbiological methods for food control based on the differential-diagnostic media for the isolation and cultivation of bacteria of the genus Campylobacter].

А screening study on the detection of campylobacteria in raw food products, semi-finished products and objects in the external environment in the poultry processing industry was conducted. The highest level of detection of campylobacteria is set for raw poultry products, including carcasses of broilers, turkeys and quail. A general accordance of getting Campylobacter in raw materials and food products with inadequate sanitary treatment of separate areas of production has been established: in most cases Campylobacter spp. was extracted from the samples, contaminated with coliform bacteria and Salmonella. It is shown that the frequency of contamination of raw poultry with pathogens is largely dependent on the cooling of the carcasses. When using the immersion method, the conditions for cross contamination with pathogens through water bath cooling are present (45% of samples infected with Campylobacter spp.). Under combined use of super-cooled water and aerosols Campylobacter were also detected quite often in 27% of samples. Contamination by pathogens was the lowest in evaporative cooling method with the use of antimicrobial hydrospray (less than 5% positive samples), allowing to recognize this method as the most promising for the production of microbiological safe products. The work on optimization of nutrient medium composition and adaptation recommended methodological scheme of analysis for detection and species identification of bacteria of the genus Campylobacter was carried out. Formulation of traditionally used growth media was modified, and balanced composition of growth and selective components was matched in accordance with the requirements of existing standards. Given the urgency of increasing the effectiveness of the methods of control of campylobacteria in foods and the lack of domestic analogues of the culture media in the Russian Federation, an optimized method for the production of dry nutrient media for the detection, identification and storage of campylobacteria isolated from food products and clinical material was developed. The conducted study allowed to develop Technical conditions 21.20.23-006-01897222-2016 «Dry culture medium for detection of bacteria of the genus Campylobacter¼ and «Instruction for use of culture media¼. Depending on the purpose of the medium produced in the following versions: the selective broth for enrichment of campylobacteria; differential selective agar for isolating and quantifying of Campylobacter spp.; semi-solid nutrient agar for cryostorage of Campylobacter strains. The list of criteria for assessing the quality of commercially available lots of dry media included: solubility, pH, gel strength of agar, the content of amine nitrogen, specificity, selectivity, growth and inhibitory properties. The practical application of these media in terms of the national laboratories will significantly simplify the use of existing standards developed based on international ISO standards, but not adapted to the main range of commercial media and reagents used in routine food control for the presence of campylobacteria.

[Antibiotic resistance of Campylobacter jejuni strains isolated from food products].

Campylobacter jejuni is a leading member of the genus Campylobacter which cause up to 90% of laboratory confirmed cases of campylobacteriosis. The most important characteristic defining the biological features of C. jejuni is their sensitivity to antibiotics. Agricultural intensification, expansion of the range of the used disinfectants and antiseptics, uncontrolled use of antibiotics in animal production is increasingly leading to the selection of the most resistant forms of Campylobacter with antibiotic resistance and multiple virulence factors. The study of antibiotic resistance of C. jejuni isolated from food and environment need for the development of new approaches for laboratory diagnosis of campylobacteriosis and confirmation of the role of food path of transmission, for creation the system of preventive measures to reduce the risk of contamination of food by Campylobacter spp. in Russia. The aim of this study was to investigate the phenotypic profiles of antibiotic resistance of Campylobacter spp. isolated from poultry and the environment in the poultry processing industry. In the analysis of 110 samples of raw poultry products and swabs from surfaces of the equipment 55 strains of the genus Campylobacter were selected, including 46 strains of C. jejuni. For study sensitivity of these strains to 15 antimicrobials (8 classes) disk diffusion assays were done using the EUCAST protocol. The following antibiotics were used: nalidixic acid, ciprofloxacin, erythromycin, gentamicin, amikacin, kanamycin, tetracycline, oxytetracycline, doxycycline, clindamycin, lincomycin, ampicillin, chloramphenicol, florfenicol, cefotaxime. All C. jejuni strains were resistant to cephalothin, which confirms their belonging to this species. 89% of the strains were insensitive to nalidixic acid, which indicates the reduction of informativeness of this test, traditionally used in the standard scheme of species identification of Саmpylobacter spp. Most of the investigated isolates were resistant to ciprofloxacin (96.3%) and tetracycline (88.6%), 34% of strains had resistance to erythromycin; 40% of tested C. jejuni were multi-resistant to four or more antibiotics. The data indicate a high prevalence of antibiotic-resistant strains among campylobacteria, contaminated poultry products during the processing of raw materials.

[The study of influence of stresses on virulence genes expression in foodborne pathogens Campylobacter jejuni].

The study of the responses to cold exposure in Campylobacterjejuni (C. jejuni)--one of the most common foodborne pathogens is important for elucidating the mechanisms of acquisition of products contaminated with campylobacter, hazardous properties. These data are also necessary to create effective systems of microbiological controls at all stages of production and storage of food. 5 pairs of oligonucleotide primers were selected for detecting of genes cadF, cdtB, ciaB, flaA, iamA, encoding the main factors of pathogenicity of foodborne pathogens Campylobacter jejuni--adhesion and invasion of epithelial cells, production of CDT-toxin and mobility. To quantify the expression levels of target genes of C. jejuni a comparative method of determining the amount of amplification products of genes encoding pathogenicity factors of Campylobacter spp. has been developed using real-time PCR with intercalating dyes. To calculate and quantify gene expression the mathematical models have been obtained that allow extrapolation of threshold cycles of amplification to the initial number of copies of RNA/DNA in the tested samples. It has been established that exposure of C. jejuni at low temperatures +4 degrees C did not lead to increased levels of expression of genes cdtB and ciaB. However, in the populations of C. jejuni subjected to freezing, followed by incubation at optimum for the pathogen temperature of +42 degrees C, the increase in expression of mRNA encoding protein subunit B of CDT-toxin and antigenic marker of invasion took place. The number of copies of RNA in C. jejuni after stress exposure increased by 1.14-2.6 lg in comparison with intact cultures. CdtB and ciaB gene expression in C. jejuni can serve as an indicator of cell response to stress and helps to restore the functions of the bacterial cells after the termination of cold exposure and return of the pathogen in conditions favourable to the realization of its pathogenic potential.

[Toxicological evaluation of colloidal nano-sized silver stabilized polyvinylpyrrolidone. IV. Influence on intestinal microbiota, immune indexes].

Nano-sized colloidal silver (NCS) stabilized with polyvinylpyrrolidone (PVP) containing nanoparticles (NPs) of silver with a diameter of 10-80 nm was administered to growing male rats (body weight 80±10 g) during the first 30 days by intragastric gavage and then for 62 days with diet consumed in doses of 0.1, 1.0 and 10 mg/kg of body weight per day based on silver (Ag). The control animals received deionized water and PVP. The composition of microbiota from the cecum was studied using standard microbiological methods with determination of the main and transient components, together with antagonistic activity of symbiotic bifidobacteria. Expression of antigens CD45RA, CD3, CD4, CD8, CD161a on lymphocytes (Ly) of peripheral blood was determined by flow cytometry; blood serum levels of cytokines IL10, IL13, TNFα were examined by ELISA. It was shown that subacute administration of colloidal Ag in all studied doses did not lead to significant changes in the composition of the main components of normal microbiota, providing, however, the inhibitory effect on the growth of some transitory components probably including opportunistic species of microorganisms. Among the studied immunological parameters decreased amount of B-Ly was noticed at the highest dose of the NCS, while changes in the other parameters of the immune system were depended ambiguously on the dose of the product. The results were analyzed in conjunction with the data of previous publications concerning the impact on the NCS on integrated, morphological, hematological, biochemical and enzymological indexes of animals in the 92-day experiment. It was concluded that significant symptoms of NCS sub-acute oral toxicity manifested starting from a dose of 1 mg/kg body weight of Ag, and the maximum not observed adverse effect dose (NOAEL) can be estimated as 0.1 mg/kg body weight.

[The study of the contamination and the levels of Campylobacter spp. during the processing of selected types of foods].

The purpose of the work was to study the nature of the Campylobacter spp. contamination during the processing of food products of plant and animal origin (raw poultry and beef meat, raw milk, leafy salads, sliced raw vegetables). In the study of 148 samples 50 strains of Campylobacter spp. (33.8%) were found. For the main phenotypic characteristics they were identified as C. jejuni spp. jejuni and C. jejuni spp. doylei (over 75%). The highest level of detection of campylobacteria (over 45%) was set for raw poultry, including the carcasses of chickens broilers, quails, turkeys and their semi-finished products. 19 of the 27 strains from poultry were identified as C. jejuni. Among the strains isolated from the environment, including swabs from equipment surfaces, 91% of the isolates were also presented by C. jejuni. It was found that the investigated foodstuffs were characterized by high levels of contamination with bacteria of the family Enterobacteriaceae, the content of which was comparable with the identified values of total viable bacteria (cfu). Salmonella was detected in 19% of the investigated poultry samples and in 14.3% of raw cow milk. In the study of swabs from surfaces of poultry processing equipment, the frequency of detection of Campylobacter strains was 38.7%, Salmonella - 12.9%. Most commonly Campylobacter and Salmonella were detected in the zones of primary processing of poultry: the frequency of isolation of Salmonella in slaughter corner was 25%, Campylobacter - 43%. When testing the swabs taken in the cooking zone of «fast food¼ restaurants Campylobacter and Salmonella were not detected. For studying the swabs from equipment surfaces and the environment for the presence of Campylobacter spp. a modified technique of sampling was developed. The method includes a comprehensive analysis in the test area with the use of three types of media for transportation and incubation of Campylobacter spp. (Preston broth with blood, Brucella broth, Cary-Blair medium), that increase the probability of detection of these pathogens.

[Condition of protective intestinal microbiota populations under stress exposure in rats received different diets with bioactive food components].

The evaluation of the levels of major colon microbiota populations (lactobacilli, bifidobacteria, enterobacteria) was carried out in two 15-days experiments on Wistar rats, exposed to stress factor (electric shock) and fed with different diets with the addition of biologic active micronutrients [extract from the leaves of Serratula coronata L. and Enzymatic hydrolyzate of the mussels meat (EHMM)]. In the first experiment animals were fed with a common vivarium diet. In the experimental group the water extract from leaves of Serratula coronata L. as a phytoecdysteroid source (5 mg per 1 kg body weight) was added to water. In the second experiment rats received balanced semisynthetic diet. In the diet of the experimental group the part of the protein (casein) was replaced by the peptides from EHMM. During the experiment the animal body weight was measured. On the 14th day of the experiment the animals were subjected to stress stimulation [electrodermal stimulation on paws (electric current 0.4 mA for 8 seconds)]. On the last day of the experiment the animals were euthanized by decapitation and micro-ecological research of protective microbiota populations in the cecal contents was carried out. The relative body weight increase was recorded in both experiments. In the second experiment in animals receiving EHMM this index (68.2 ± 3.0%) was considerably higher than in the control group and in the experimental group receiving no EHMM (57.2 ± 4.0 and 59.7 ± 2.8% respectively). The results of the microecological study showed different effect of diets with biologically active micronutrients on the population levels of lactobacilli. In the experiment with common vivarium diet no significant changes of the levels of the studied colon microbiota populations had been recorded in the rats of control group compared with rats of experimental group, exposed to stress factor but received no extract from Serratula coronata L. The decrease of the levels of lactobacilli by the end of the experiment was observed in the experimental group of rats received water extract from the leaves of Serratula coronata L (content of lactobacilli 7.76 ± 0.17 lg CFU/g) compared to those in control group and experimental group of rats received no extract (8.4 ± 0.09 and 8.69 ± 0.07 lg CFU/g respectively). Feeding with the balanced semisynthetic diet with the addition of EHMM or without it had a positive effect on the levels of lactobacilli and their balance with the aerobic component of the Enterobacteriaceae. There was a trend toward increased levels of lactic acid bacteria in the experimental group received EHMM (9.16 ± 0.12 lg CFU/g) compared with the contents in the control group and in the experimental group exposed to stress factor without adding EHMM in the diet (8.74 ± 0.34 and 8.79 ± 0.23 lg CFU/g, respectively). The conclusion about the positive (protective) effect of a semisynthetic diet enriched with peptides from EHMM was made based on the comparison of indicators that reflect the status of non-specific resistance of the organism: the integral criterion of weight gain and the levels of major colon microbiota populations of laboratory animals.

[Assessment of the impact of vitamin and dietary fiber content in the diet on the characteristics of protective colon microbiota populations of rats].

The content of lactobacilli and enterobacteria in the experiment in rats with varying levels of vitamins and dietary fiber was studied. The study was performed on 48 male weanling Wistar rats randomized into 8 groups, with the creation of vitamin deficiency (30 d.) and its further compensation (5 d.). Vitamin content in the semisynthetic diet in rats of the control group N 1 corresponded to 100% of a daily adequate intake. In the similar composition of the diet of the control group N 2 wheat bran was added in amount of 5% of the weight of the diet. In groups N 3­8 rats received a diet with the reduced amount of vitamin mixture by 5 times (20% of the adequate intake) and the total exclusion of tocopherol, thiamine and riboflavin from the mixture. The wheat bran (5% of diet mass) was added to the diets in Groups N 4, 6, 8. At the stage of compensation of deficiency rats were fed with the diets with increased content of vitamin mixture: Group 5­6 to 80% 7­8 to 200% (100 and 220% of the adequate intake, respectively), and the groups N 3­4 continued to receive deficient diet with or without wheat bran until the end of the experiment. After 35 days rats were anesthetized with ether, decapitated, necropsied and the cecum segments were selected for quantitative microbiological analysis of its contents. It has been shown that the addition of wheat bran to vitamin deficient diet lead to the reduction of the manifestation of physical sign of hypovitaminosis. It also eliminated the differences in the integrated index of growth and development of rats in comparison with the group without vitamin deficiency. It was found that the vitamin deficiency in the diet, regardless of the presence or absence of wheat bran, led to a significant reduction of the number of lactobacilli in the intestinal contents, but almost did not affect the number of normal and opportunistic pathogenic enterobacteria. The compensation of deficiency during 5 days lead to the increased number of lactobacilli, but the physiological levels and levels in control animals it reached only in rats received 220% of the vitamins with the addition of wheat bran. In the lactobacilli population in all rats received different doses of vitamins (including reduced to 20%), regardless of the presence of wheat bran, prevailing culturable representatives were 3 kinds of Lactobacillus spp. ­ acidophilus, fermentum, paracasei. These species showed stable presence in the intestine even in conditions of prolonged vitamin deficiency (35 days). L. acidophilus was the dominated lactoflora representative in all rats, its' content was average 91.7% of all culturable lactobacilli. With less constancy and in lower amounts were detected L. plantarum and representatives of coccal flora (Leuconostoc lactis, Lactococcus lactis).

[Toxicological assessment of nanostructured silica. III. Microecological, hematological indices, state of cellular immunity].

This paper is the third in a series of publications on the experimental study of subacute oral toxicity of nanostructured silicon dioxide (SiO2). We used commercial nanostructured SiO2, obtained by hydrolysis of tetrachlorosilane in the gaseous phase, with the size of the primary nanoparticles (NPs) of 5-30 nm. The aqueous dispersion of SiO2 after treatment with ultrasound was administered to rats with initial weight of 80 +/- 5 g for the first 30 days by intragastric gavage and further for 60 days with diets in doses of 0.1; 1.0; 10 and 100 mg/kg body weight per day. Animals of the control group were treated with deionized water. The amount of basic and transient populations of gut microbiocenosis, hematological indexes were measured using standard methods. Specific content of the B-lymphocytes (CD45RA+), total T-lymphocytes (CD3+), T-helper cells (CD4+), T-cytotoxic cells (CD8+), NK-cells (CD161a+) in general population of lymphocytes was evaluated byflow cytometry; serum cytokine levels of TNF-alpha, IL-6, IL-10 were determined by ELISA. No significant changes in the qualitative and quantitative composition of the intestinal microbiota populations regardless of the dose of administered nanomaterial have been found. This gave reason to believe that the postulated mechanism of the toxic effects of the NPs of SiO2, mediated by modification of the composition of the intestinal microflora and the corresponding changes in its functional activity, apparently, is not realized. The main target of nanostructured SiO2 was the T-cellular system of the immune system of animals, that was manifested in the significant decrease of the number of leukocytes (33%), number of T-helper cells (13%), CD4/CD8 ratio (27%) and increasing the number of cytotoxic lymphocytes (19%) and the level of TNF-alpha (590%). The value of the maximum dose (NOAEL) of nanostructured SiO2, has no effect on T-cell immunity was not more than 100 mg/kg body weight per day.