[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.

[Physiological and biochemical in vivo study of polyphenols and 20-hydroxyecdisone from quinoa grains effect on resistance to physical exercise in Wistar rats].

Increasing the ability of the human body to adapt to physical stress is relevant from the standpoint of using foods for special uses containing functional food ingredients (FFI) with effectiveness proven in vivo. The purpose of this study was to evaluate the effect of FFI from Chenopodium quinoa grains with a high content of polyphenols and phytoecdysteroids on the physical endurance of male Wistar rats. Material and methods. The experiment was carried out during 36 days using 50 weaned male Wistar rats. The animals were randomly divided into 3 groups (n=12): Control, Run and Run-FFI. Rats of the Control and Run groups received a standard semisynthetic diet during the experiment. Rats of the Run-FFI group received a semi-synthetic diet with the addition of FFI in an amount of 0.055±0.003%, containing phytoecdysteroids (50.4±0.6 mg/g) and polyphenols (212.0±2.0 mg/g). During the experiment, the rats were assessed for their neuromotor function (grip strength of front paws), memory, and behavioral reactions in the "Elevated Plus Maze" (EPM), "Conditioned Passive Avoidance Reflex" (CPAR) and "Open Field" (OF) tests. Once a week, animals from the Run and Run-FFI groups were subjected to moderate physical load on a "Treadmill". On the 36th day of the experiment, the animals of these groups were subjected to exhausting physical load. Immediately after running, the animals were placed in metabolic cages to collect daily urine. At the end of the experiment, the content of corticosterone, the activity of catalase, indicators of protein, lipid and mineral metabolism, indexes of the liver functional state and antioxidant defense system parameters were analyzed in the blood serum; the level of prostaglandin E2 and dopamine were determined in daily urine. Results. Physiological tests (CRAR, OF) showed that weekly exercise increased anxiety in laboratory animals. The FFI introduction into the diet led to normalization of the assessed parameters (EPM). As a result of 36-day consumption of FFI against the background of physical loads, a significant decrease by 22% in the main stress marker, corticosterone, was revealed in the blood of rats, as well as significant increase by 23% in the stress inhibitor - prostaglandin E2 urinary excretion, compared with animals of the Run group to the level not differed from the indicators of the control animals. There were no differences in endurance performance between the Run and Run-FFI groups on the results of the exhaustive exercise. Consumption of FFI prevented the formation of excess ammonia, significantly reducing the level of urea in the blood and normalizing its excretion to control levels in the urine, which was increased in the Run group by 19%. Conclusion. The results obtained demonstrated the adaptogenic properties of the developed FFI in response to stress caused by weekly moderate and acute exhaustive physical activity. The obtained data on the biological effect of the developed FPI on the adaptive potential of laboratory animals will serve as an experimental basis for its inclusion in the composition of specialized foods.

[Pathoanatomical algorithm for differential diagnosis of Paget's disease of the breast]. / Patologo-anatomicheskii algoritm differentsial'noi diagnostiki bolezni Pedzheta molochnoi zhelezy.

Paget's disease of the breast is a rare type of cancer that affects the skin of the nipple and usually the areola. At the same time, most patients also have one or more tumors in the immediate vicinity of the focus of mammary Paget's disease. This tumor must be distinguished from normal or atypical Toker cells, and also differentiated from diseases such as Bowen's disease of the nipple and melanocytic lesions of the nipple and areola region, including nipple melanoma and BAP1-inactivated nevus (Wiesner nevus). Currently, there is no routine pathological diagnostic algorithm for these conditions. The aim of the work is to formulate a clear clinical and morphological algorithm for diagnosing Paget's disease of the breast and Toker cells, Bowen's disease of the nipple and areola, as well as melanoma and BAP1-inactivated nevi of the above localizations. Surgical material obtained from patients with Paget's disease of the breast (18), Toker cells of the nipple (2), Bowen's disease of the nipple (6), melanoma of the nipple (1), BAP1-inactivated nevus (1) was studied. The material was examined histologically with hematoxylin and eosin staining, Alcian blue and PAS reaction, as well as immunohistochemically with the following panel of antibodies: CD138, p53, CK8, CK7, HER2/neu, EMA, HMB-45, Melan A, S-100, p63, p16 and BAP1. An easy-to-learn pathoanatomical algorithm for diagnosing Paget's cancer has been developed, which will be especially useful for pathologists who encounter pathology of the nipple and areola in their work.

[Modified method for obtaining phycocyanine concentrate of Arthrospira platensis biomass].

The biomass of Arthrospira platensis cyanobacteria is a source of bioactive compounds such as chlorophylls, carotenoids, and, particularly, phycobiliproteins: C-phycocyanin and allophycocyanin. The wide range of biological activity shown by extracts with a high content of phycocyanins determines the prospects for their use as dietary supplements and ingredients of special foods. For food purposes, the degree of purity of phycocyanin concentrates, determined by the ratio of optical densities of their aqueous solutions at two wavelengths, namely D620/D280, must be greater than 0.7. Most methods for obtaining phycocyanin concentrates include laborious steps of fractional ammonium sulphate precipitation of protein from A. platensis biomass extracts followed by removal of salts solution. The use of membrane technology, specifically microfiltration, makes it possible to significantly intensify and simplify the process of obtaining phycocyanin concentrates. The aim of this research was to modify the method for obtaining a high-purity A. platensis phycocyanin concentrate by replacing the stage of ammonium sulfate precipitation of the protein by ultrafiltration of the extract followed by microfiltration. Material and methods. A sample of dry A. platensis biomass was used as a feedstock. Extraction of A. platensis biomass was carried out at a temperature of +40 °C for 3 h, the resulting suspension was centrifuged, and the supernatant was separated from the sediment. The obtained extract was subjected to ultrafiltration (membrane with a pore diameter of 30 kDa) followed by removal of the permeate containing low molecular weight impurities. The retentate was subjected to microfiltration (membrane with a pore size of 0.2 µm), concentrated by reverse osmosis and freeze-dried. Results. The content of C-phycocyanin and allophycocyanin in the dry concentrate was 42.0±1.3 and 7.0±0.3%, respectively, the degree of purity was 1.98. Conclusion. The scheme for obtaining A. platensis phycocyanin concentrate has been modified. A concentrate was obtained with a high degree of purity, allowing its use in food.

[Innovative methods for extracting biolactive сompounds from plant materials].

Modern methods for extracting bioactive сompounds (BAC) from various raw materials are focused on efficacy and environmental awareness, involve the use of mathematical and statistical optimization methods, the choice of green solvents, and the use of additive extraction technology. The aim of this review was to present and briefly discuss up-to-date information on modern technological approaches to the production of plant BAС extracts for applying in food ingredients and foods for special dietary uses. Material and methods. For the main search of sources, the PubMed bibliographic database, Scopus and Web of Science databases, and the Google Scholar search engine were used. The search depth was 15 years. Results. The article presents a brief review of modern approaches to the extraction, concentration and purification of polyphenolic compounds from various plant materials. As an additive extraction technology aimed at destroying/increasing the permeability of the plant cell wall, a wide range of physical methods has been successfully used: ultrasound, microwave radiation, homogenization, application of a pulsed electric field, high hydrostatic pressure, cryo-crushing. A brief description of each method, its advantages and disadvantages are presented. Improving food safety and compliance with environmental regulations requires the choice of a safe, environmentally friendly and yet efficient extraction process. For these purposes enzymatic extraction, environmentally friendly solvents, such as deep eutectic solvents, supercritical fluid extraction and membrane technology are successfully used. The use of mathematical and statistical methods can reduce the total number of experimental trials and reduce the cost and time of experiments. The use of these methods together makes it possible to vary deliberately the process parameters in relation to a specific plant material and the final product, which provides the opportunity to isolate BAS concentrates from plant raw materials with a high yield. Conclusion. The introduction of innovative technological approaches for obtaining extracts of BAC of plant origin determines the prospects for the production of a wide range of foods for special dietary uses that meet high safety and efficiency requirements.

[In vivo study of the biological value of amaranth protein concentrate and its module with chicken egg protein].

Amaranth (Amaranthus L.), like other pseudocereals as quinoa (Chenopodium quinoa Willd.), chia (Salvia hispanica L.) and buckwheat (Fagopyrum sp.), is a promising source of dietary protein. Depending on the subspecies and breeds of amaranth, the protein content in its grain is estimated from 13.1 to 21.5%, and its amino acid score varies over a significant range and can be limited. The aim of this study was to obtain a protein concentrate from amaranth (Amaranthus L.) grain of the Voronezh breed, enrich it with chicken egg protein, determine the amino acid score of the obtained protein module, and experimentally evaluate in vivo its true digestibility and biological value. Material and methods. The amaranth protein concentrate was obtained from grain according to the technological scheme, including its enzymatic treatment, alkaline extraction, acid precipitation of proteins, microfiltration and lyophilization. The amino acid composition and amino acid score of the concentrate were determined. The protein module was obtained by mixing amaranth protein concentrate and chicken egg protein in a weight ratio of 58:42. The true digestibility and biological value of the protein module has been determined in vivo. The experiment was carried out on 32 Wistar male rats divided into 2 groups (n=16 rats): control group 1 with a body weight of 118.7±3.1 g and experimental group 2 with a body weight of 119.5±3.0 g. Animals of groups 1 and 2 received diets in which egg protein and a protein module were used as a protein source, respectively. Within 15 days of the experiment, individual indicators of food intake and body weight gain of each animal were determined. From the 14th to the 15th day food intake was determined and feces were collected. The amount of nitrogen in the food and feces was determined for each rat using the Kjeldahl method. The true digestibility of the protein was determined according to obtained data. Results. The resulting amaranth protein concentrate contained 70.4±0.6% of protein, 17.0±1.0% fat, 9.8±0.8% carbohydrates, 1.8±0.2% ash, its moisture content was 1.4±0.1%. There were no significant differences in food intake and body weight gain between animals of both groups. The calculated value of the true digestibility of chicken egg protein was 98.8±0.1% for the control group 1, of the protein module was 99.0±0.1% for the experimental group 2, the differences between the groups were not significant. Conclusion. The results of amino acid analysis and the in vivo study of the true digestibility of the protein module (composition amaranth protein/chicken egg protein) indicate the absence of limitation relative to the amino acid scale of the "ideal" protein (FAO/WHO, 2007) and high true digestibility. The biological value of the protein module, calculated according to PDCAAS, is 99.0±0.1%, which confirms the prospects for its inclusion in specialized foods.

[Physiological and biochemical evaluation of the effectiveness of a new food ingredient - blueberry polyphenol concentrate].

Extracts from bilberry leaves and blueberries containing a wide range of biologically active compounds, including polyphenols, are of particular interest due to their antioxidant, hypoglycemic and hypolipidemic properties. In this regard, The aim of this research was to investigate in vivo the effect of a concentrate of blueberrie polyphenolic compounds with buckwheat flour on some physiological and biochemical parameters in C57Bl/6 mice with impaired carbohydrate and lipid metabolism induced by the consumption of a high fat high carbohydrate (HFHC) diet. Material and methods. The polyphenol concentrate was obtained by sorption of blueberry extract on grinded buckwheat flour. Total polyphenol content was determined by Folin-Ciocalteu method, profiles of anthocyanins, flavonoids and easily digested carbohydrates were determined by HPLC. An in vivo experiment was carried out using 84 male mice C57Bl/6 for 109 days. Animals were divided into 3 groups: control fed standard semisynthetic diet, control treated with HFHC diet and experimental group treated with HFHC diet with addition of blueberry polyphenol concentrate (60 mg-eq. of gallic acid/kg body weight). Food intake, body weight gain and fasting blood glucose levels were measured during the experiment. Grip strength of the front paws of the animals was measured weekly. Oral glucose tolerance and insulin resistance tests were carried out twice. Common physiological tests (Elevated Plus Maze and Passive Avoidance Test) were used to assess the anxiety and memory of animals. Glycated hemoglobin level was determined in blood, plasma was collected for leptin and insulin level determination. The hepatic levels of triglycerides and cholesterol were assessed. Results. The concentrate of polyphenols extracted from blueberries and sorbed on grinded buckwheat flour was obtained under conditions that made it possible to exclude the sorption of easily digested carbohydrates - glucose, fructose and sucrose on the flour. The total concentrate content of polyphenols was 65.5±0.7 mg-eq. gallic acid/g, anthocyanins - 27.3±2.7 mg/g, flavonoids - 1.2±0.1 mg/g. The consumption of the concentrate by C57Bl/6 male mice with carbohydrate and lipid metabolism disorders induced by a HFHC diet had a significant (p<0.05) hypoglycemic and hypolipidemic effect, reducing the area under the curve in the insulin resistance test by 5.7% and decreasing the insulin and leptin levels by 31.3 and 15.9%, respectively (relative to the animals of comparison group fed HFHC diet). The consumption of the concentrate had a significant (p<0.05) anxiolytic effect, reducing the anxiety of animals by 2.2 times, as determined in the Elevated Plus Maze test. Conclusion. The results of the study indicate the prospects for using the developed blueberry polyphenol concentrate adsorbed on buckwheat flour as part of specialized foods for the prevention of such alimentary dependent diseases as metabolic syndrome, obesity, and diabetes mellitus.

[Influence of multiple insufficiency of vitamin D and B group vitamins, calcium and magnesium in the diet of rats on micronutrient sufficiency and plasma biochemical indicators].

The lack of vitamins D, B group, calcium and magnesium is common for the diet of the Russian population. It has been previously demonstrated that that the elimination of B vitamin deficiency is a necessary condition for the implementation of vitamin D biological functions. The aim of the research was to assess the effect of a combined deficiency of vitamins D and B group, calcium and magnesium in the diet of rats on biomarkers of micronutrient sufficiency and plasma biochemical indicators. Material amd methods. Male Wistar rats with an initial body weight of 66±1 g were randomly assigned to one of five groups. The rats of the 1st group (Control) were fed a standard semi-synthetic diet (SD) for 28 days. The combined deficit of vitamins D and B group, calcium and magnesium in rats of four experimental groups was caused by a 5-fold decrease in their content in the vitamin mixture and 2-fold decrease in their content in mineral mixture of the SD for 23 days. Over the next 5 days, the rats of 2nd group (+В+D+Ca+Mg) were fed a diet replenished for all missing vitamins and minerals, the rats of 3rd group (-В+D+Ca+Mg) were fed a diet with continued deficiency of B group vitamins, the rats of the 4th group (+В+D-Ca-Mg) were fed a diet with continued lack of calcium and magnesium, the diet of the 5th group (-B-D-Ca-Mg) was not replenished. Vitamins B1 and B2 in lyophilized liver and brain and urine, riboflavin in plasma and 4-pyridoxic acid in urine were determined by fluorimetric methods, 25(OH)D in plasma was determined by ELISA, the level of vitamins A and E in blood plasma and lyophilized liver, of vitamin E in whole brain - by HPLC. Biochemical parameters of blood plasma were determined using a biochemical analyzer. Results. In rats of the 5th group (-B-D-Ca-Mg), there were 3.4-fold increase (p<0.05) in iron plasma level, 1.7-fold elevation (p<0.10) in alkaline phosphatase activity, and 1.8-fold decrease (p<0.05) in alanine aminotransferase (ALT) activity on the background of the increase in α-tocopherol blood plasma level by 26.7% (p<0.05) and liver content by 2.0 fold (p<0.05) relative to the indicators in animals who hadn't passed the deficiency of micronutrients (Control) and / or from the 2nd group (+B+D+Ca+Mg). The lack of B group vitamins in the diet, which persisted during the correction of vitamin D, calcium and magnesium deficiency in rats of the 3rd group (-B+D+Ca+Mg), inhibited the recovery of diagnostically significant biochemical parameters of blood plasma (namely, an increased level of glucose, iron, triglycerides, cholesterol, α-tocopherol, increased alkaline phosphatase activity and reduced ALT activity) to the level in animals of the control group and/or rats fed the diet replenished for all missing micronutrients (+B+D+Ca+Mg). Conclusion. Combined deficiency of several micronutrients led to changes in biochemical blood parameters. Reduced intake of calcium and magnesium during the correction of the lack of vitamins D and B group in the diet can have a negative impact on vitamin B2 status. Even under normal dietary vitamin E intake the combined deficiency of several other micronutrients affected the metabolism of this vitamin (increased levels of vitamin E in the liver and blood plasma of animals). Chronic combined alimentary deficit of B vitamins, calcium and magnesium, which is characteristic in the diet of the Russian population, reduces vitamin D bioavailability, which justifies the expediency of using vitamin-mineral complexes.

Effect of Exhaustive Training or Forced Immobilization on Physiological Condition and Main Metabolic and Stress Markers of Wistar Male Rats.

For correct and reliable experimental in vivo assessment of antistress effect of various bioactive substances, appropriate biomodels reproducing stress and organism response to stress in laboratory animals should be chosen. We chose treadmill test for simulating exhaustive physical load and forced immobilization accompanied by disorders of physiological and psychological condition. Verification of the models used indicates their wide applicability for testing certain biological manifestations under reproduced stress exposure.

[Efficiency of vitamin D deficit correction depending on rats' supply with B vitamins].

Despite the presence of combined deficiency of vitamins D and group B among the population of Russia, the intake of cholecalciferol is often recommended without correcting the supply with B group vitamins, which are involved in ensuring the biological functions of vitamin D. The aim of the study was to compare the effectiveness of vitamin D deficit correction by replenishing its content in the diet to an adequate level without eliminating the deficit of B vitamins and by restoring vitamin D level in combination with B vitamins. Material and methods. The experiment was carried out on male Wistar rats (n=33) with an initial body weight of 69.5±0.8 g. Combined deficit of vitamins D and B group in rats (n=24) was caused by a 5-fold decrease in their content in the vitamin mixture of a semi-synthetic diet for 23 days. Over the next 7 days, in order to correct vitamin deficiency, 12 rats (group «-B+D¼) were fed a diet, replenished up to 100% for vitamin D with continued deficiency of B group vitamins, and 12 rats (group «+B+D¼) were fed a diet replenished for all missing vitamins. Animals of the control group (n=9) received a full semi-synthetic diet during the entire experiment. The concentration of vitamins A and E in blood plasma and lyophilized liver and whole brain was determined by HPLC, vitamins B1 and B2 in the liver, brain and urine, riboflavin in plasma and 4-pyridoxic acid in urine - by fluorimetric methods, 25(OH)D in blood plasma was determined by ELISA. The content of calcium, magnesium, iron, manganese, zinc and copper in freeze-dried liver and brain was determined by atomic absorption method, biochemical parameters of blood and urine were determined using a biochemical analyzer. Results. The only vitamin D addition to the feed with a persisting deficiency of B vitamins did not restore the concentration of 25(OH)D and osteocalcin to the level in control animals sufficiently provided with all vitamins. In animals of the "-B+D" group, 25(OH)D plasma level was reduced by 17.3% (p<0.10), osteocalcin - by 11.7% (p<0.05), the activity of aspartate aminotransferase was 1.5 fold less, alanine aminotransferase - 2.3 fold (p<0.05), lactate dehydrogenase - by 14.9% (p<0.10), while the concentration of iron exceeded 2.7 times, glucose - by 15.0%, calcium - by 8.0%, creatinine - by 8.7% (p<0.05), urea - by 32.1%, direct bilirubin - by 24.2% (p<0.10 ) compared with corresponding indicator in rats of the control group. The level of cholesterol and HDL cholesterol was 14.7% and 15.9% higher (p<0.10) than in animals of the «+B+D¼ group. Conclusions. Deficiency of B vitamins inhibits the restoration of adequate supply with vitamin D. In the presence of a lack of B vitamins in rats, vitamin D deficit and its consequences cannot be completely eliminated. Adequate supply with vitamins D and B group are synergistic factors in maintaining the level of glucose, cholesterol in blood plasma and other diagnostically significant parameters.

Effect of Vanadium Complex with Enzymatic Hydrolysate of Soy Protein on Carbohydrate and Lipid Metabolism Disorders in Male Wistar Rats.

The effects of vanadium complex with enzymatic hydrolysate of soy protein (V-EHSPI) were studied in male Wistar rats with induced disorders of carbohydrate and lipid metabolism. The content of vanadium (IV) in the studied complex was 15.8 mg/g dry product. High-lipid high-carbohydrate diet was used to induce disorders of lipid and carbohydrate metabolism. Addition of vanadium in three different doses to the diet over 100-day experiment reduced body weight gain and the levels of glucose, insulin, leptin, and triglycerides. V-EHSPI produced beneficial effects on carbohydrate and lipid metabolism even in a dose 5 µg/kg body weight/day (calculated from the mean food consumption over the entire treatment period). Significant inhibition of growth and changes in the weight of organs in animals treated with V-EHSPI attested to toxicity of vanadium in the studied dose range.

[Enzymatic hydrolysates of whey protein and chicken egg ppotein: production, physical-chemical and immunochemical characteristics].

Reducing the manifestations of food allergy by the inclusion of specialized foods in the nutrition of children and adults suffering from this disease is an important problem. The aim was to obtain and characterize in vitro food protein hydrolysates to evaluate their use in specialized foods with reduced potential allergenicity. MATERIAL AND METHODS: Whey protein concentrate (WPC) and chicken egg protein (CEP) and enzymes such as pancreatin and alkalase have been used. Proteolysis of proteins was carried out in an FA-10 fermenter for 3 hours at an enzyme : substrate ratio of 1:50 in dry matter, at optimal pH and temperature for pancreatin and alkalase. Enzymes were inactivated at +75 °C and fermentolizate was ultrafiltered. The solutions were concentrated by reverse osmosis and freeze-dried. The molecular weight distribution of the peptide fractions was evaluated by HPLC. Residual antigenicity was determined by the method of indirect enzyme-linked immunosorbent assay and expressed as the fold of antigenicity reduction relative to the original protein. RESULTS AND DISCUSSION: During WPC proteolysis with pancreatin the hydrolyzate was obtained with a fold reduction of antigenicity of 2.3×103 relative to the initial WPC. A decrease in antigenicity of 4.7×104 times was achieved with proteolysis of WPC by alkalase. The combination of WPC fermentolysis with pancreatin or alkalase followed by ultrafiltration reduced the content of high molecular weight peptides with a mass more than 8.7 kDa. The multiplicity of decrease in antigenicity with respect to the starting protein was 1.64×105 and 1.90×105, respectively. After repeated ultrafiltration the reduction in antigenicity of the obtained WPC alkalase or pancreatin hydrolysate was more than 1.0×106 and more than 5.0×105, respectively. The decrease in antigenicity of the CEP hydrolyzate obtained with proteolysis by alkalase and ultrafiltration compared to the initial CEP was 1.0×105 times, and 5.0×105 times when we used repeated ultrafiltration. CONCLUSION: A significant decrease in the content of high molecular weight peptides and a decrease in the antigenicity of peptide mixtures based on WPC and CEP to the values that permit their use in hypoallergenic products is achieved by combining proteolysis and double ultrafiltration through a UF10 membrane.

[Enzymatic hydrolysates of food proteins for specialized foods for therapeutic and prophylactic nutrition].

The review considers some issues of obtaining, as well as physic-chemical, organoleptic, immunochemical (residual antigenicity) characteristics of enzymatic hydrolysates from food proteins (EHFP) that are widely used in food products for various purposes, as well as assessing their biological activity. The results of experimental works and patents, which describe the most widely used approaches to the production of EHFP with desired properties (hydrolysates for therapeutic and prophylactic products), as well as assessments of biological activity and immunochemical properties are given. The use of various enzyme preparations (of bacterial, fungal and animal origin), as well as one- and two-stage hydrolysis schemes and options for instrumentation of fermentolysis processes are considered. It is concluded that in order to achieve the required reduction in antigenicity for hydrolysates used as part of therapeutic (hypoallergenic) foods (to values not higher than 10-5 relative to the antigenicity of the original protein) membrane ultrafiltration stages are necessary. The main disadvantage of such hydrolysates is their unsatisfactory organoleptic properties (bitterness and high osmolarity) that can be improved using a number of additional technological approaches. The use of partial hydrolysates (or hydrolysates with an average degree of hydrolysis, with a residual antigenicity of 10-4 to 10-5) with significantly better organoleptic properties compared with deep hydrolysates in therapeutic foods is considered. Of considerable interest are the issues of immunomodulatory, antioxidant and hypoallergenic properties of EHFP. It has been suggested that soybean and chicken egg hydrolysates may be promising as functional ingredients with antimicrobial, antihypertensive and immunomodulatory effects in various specialized foods, as well as in cases of food intolerance only to cow milk proteins.

[Influence of coagulated egg melange on the physical endurance of growing male Wistar rats: physiological and biochemical assessment].

Melange, is one of the most commonly used products of egg processing, however, its use for the production of functional foods is limited by its liquid consistency. The range of functional foods, including those that increase physical endurance and working ability, can be expanded by using the coagulated form of the melange. Thus, the preclinical testing of the nutritional value of coagulated melange and the effect of its consumption on the physical endurance of laboratory animals is relevant. The aim of this research was the experimental comparative physiological and biochemical in vivo evaluation of the effects of coagulated melange consumption by Wistar male rats on their growth indices and physical endurance. Material and methods. Samples of the lyophilized egg melange (EM) and coagulated egg melange (CEM) were used as objects of the research The experiment was conducted with the use of 45 growing male Wistar rats with initial body weight 80±5 g. According to the preliminary training results, 27 animals were selected for further experiment. The motor function was evaluated by means of grip test. The rats were randomized into 2 experimental groups according to their weight and grip strength. Animals were treated for 32 days with isocaloric and isonitrogenic semi-synthetic diets with EM and CEM as protein component (25% of the dry weight), consequently. The grip test was conducted once per week, two days a week animals were trained on the treadmill for 10 min. On the 31st day of the experiment, animals were exposed to exhaustive training till total exhaustion. On the 32nd day of experiment animals were decapitated under light ether anesthesia. The concentration of glucose, triglycerides, cholesterol (CHL), high-density lipoproteins (HDL) and low-density lipoproteins (LDL) was determined in the blood serum. Results and discussion. The greater consumption of food (20±1 g/rat/day) containing CEM by animals corresponded to a greater increase (204±9%) of their body weight over the entire experiment period compared with animals that received EM (13±1 g/rat/day; 160±9%, respectively, p≤0.05). An increase in the intake of food containing CEM had a pronounced positive effect on the physical endurance of the animals, assessed in the grip test. For the 21 days of the experiment, this indicator increased by 26% in rats consuming EM, whereas in rats consuming CEM, the grip strength increased more significantly - by 42%. Consumption of CEM by animals increased the run time (33.8±1.4 min) and the distance covered (557±35 m) in the treadmill test before the exhaustion in comparison with similar indicators for animals that received EM (24.8±2.5 min and 365±50 m, respectively , p≤0.05). The obtained results indicate an increased resistance of animals treated with CEM to exhausting physical loads. Conclusion. The consumption of CEM by growing Wistar male rats increased more effectively their growth and physical endurance in comparison with animals treated with non-coagulated egg melange. These results show the prospective of the use of CEM as a functional food ingredient in specialized foods, enhancing physical endurance and working ability.

[Organic source of vanadium. preparation and physicalchemical characteristic].

Antidiabetic properties of vanadium are known more than 100 years, however the researches of specific therapeutic usage of vanadium were conducted only in the last two decades. Along with, the organic vanadium compounds are more harmless in comparison with inorganic vanadium salts. Thus, the development of method of obtaining the organic source of vanadium with high bioavailability is prospective field. Aim of the work was to obtain and provide the physical-chemical characterization of vanadium complex with enzymatic hydrolysate of soy protein isolate (SPI), obtained by one-stage enzymatic hydrolysis. MATERIAL AND METHODS: The complex was obtained at room temperature: 10% water solution of SPI was mixed with 25% solution of vanadium salt (VOSO4×хH2O) in ratio 10:1 (in dry matter). The reaction was kept during 1 h at constant mixing with pH kept at 7.0- 7.1 with 1.0 M NaOH. The concentration of vanadium was determined in dry product by means of inductively coupled plasma mass-spectrometry. The chromatograms of SPI and V-SPI were obtained by means of size-exclusion high-pressure liquid chromatography, and then were integrated by weight method in the range of free till full column volume. RESULTS AND DISCUSSION: The obtained complex of vanadium with SPI enzymatic hydrolysate (V-SPI) was water-soluble and contained 15.8 mg of vanadium per gram of product dry weight. Analysis of the molecular weight distribution of the peptide fractions of the original SPI enzymatic hydrolysate and the V-SPI complex showed that more than 87% of the vanadium complex was in peptide fractions with molecular weights more than 4.1 kD, including more than 75% of vanadium contained in fractions with molecular weights from 14.6 to 4.1 kD. CONCLUSION: The experimental evaluation in vivo will be the next stage of this research. The complex bioavailability and its effects on carbohydrate and lipid metabolism of Wistar rats with obesity will be evaluated.

[New functional food ingredient - lipid module, source of astaxantine and plasmalogenes].

The aim of this work was to study the impact of modification of fatty acids composition of laboratory animals' diet in the presence of plasmalogenes (PG), astaxanthin (ASTA) and their combination on animals' adaptation potential in stress conditions. MATERIAL AND METHODS: The fatty acids composition of diet was altered with the use of lipid module, containing 88.7% of high oleic sunflower oil, 6.3% of coconut oil and 5% of micralagae Schizochytrium sp. oil. The experiment was conducted with the use of 80 male Wistar rats with initial body weight 125±5 g. Selected animals (n=50) were divided into 5 groups according to their activity in tests «Open field¼ (OF) and «Elevated plus-maze¼ (EPM). The animals of control group 1st were not exposed to physiological tests - this was an intact control group. The animals of control groups 1st and 2nd were treated with standard half-synthetic diets for 30 days [381 kcal/100 g of dry food, 20.1% of casein on calories, 10% of fat (the mixture of lard and sunflower oil in 1:1 ratio)]. In the diet of 3d group animals the sunflower oil (50% of fat in the diet) was substituted with lipid module, enriched with ASTA (4.0±0.3 mg/day/kg b.w.); in the diet of group 4 animals - with lipid module, enriched with PG (79.0±2.0 mg/day/kg b.w.); in the diet of group 5 animals - with lipid module, enriched with ASTA and PG (the same doses). The evaluation of anxiety level and total exploration activity was conducted in «elevated plus maze¼ test. On the 31st day of experiment, animals were exposed to forced swim test for evaluation of their physical endurance and capacity in the conditions of high stress. RESULTS AND DISCUSSION: The introduction into the animal diet of lipid module, enriched with PG and/or ASTA had pronounced hypolipidemic effect, lowered total serum cholesterol against a background of decrease in low-density lypoproteins (LDL) level. The concentration of ω-3 polyunsaturated fatty acid (PUFA) - docosahexaenoic acid in liver cells of animals treated with lipid module enriched with PG and/or ASTA increased more the 3 times, while ω-6 linoleic acid decreased twice. The consumption of enriched with ASTA lipid module inhibited the increase in blood corticosterone level after stress (exhausting physical exercise) and lowered it to the control animals values, thus showing adaptogenic effect. The animals treated with lipid module enriched with ASTA (group 3) spent significantly less time in open arms of the maze in comparison with the first test, what may show the increase in anxiety level. The introduction of PG into lipid module neutralized this effect. The forced swim test with load showed no increase in working ability and endurance of animals of all tested groups. CONCLUSION: The further study of adaptogenic action of PG in combination with ASTA in the composition of lipid module compared to the similar effect of traditional phospholipids is of special interest.

[The impact of bilberry leaves' polyphenols on the anxiety level, spatial learning and memory of db/db mice].

Numerous experimental and clinical studies have shown high efficiency of plant polyphenolic compounds in restoring age-related memory and learning disorders. In the present study a functional food ingredient (FFI) was obtained by sorption of an aqueous solution of bilberry leaves extract on buckwheat flour, which allowed to concentrate polyphenols and increase their storage stability. The aim of this study was to evaluate the impact of a developed FFI, enriched with bilberry leaves` polyphenols, on the anxiety level, locomotor activity, memory and spatial learning of db/db mice with genetical type 2 diabetes. Material and methods. The experiment was conducted using 10 heterozygote male db/db mice and 10 homozygote male db/+ mice as the comparison control group (7 weeks of age). According to body weight, blood glucose level, the results of insulin resistance test and elevated plus-maze (EPM) test, animals were randomized into three groups: control group C1 - db/+ animals, control group C2 and experimental group G3 - obese db/db mice. Buckwheat flour was included into the diet of C2 group in a dose 22.5 g/100 g; FFI was included into the diet of G3 group in a dose 2.5 g/100 g (that was equal to 59.2± 1.4 mg-eq gallic acid per 100 g of the diet). The anxiety level and general locomotor activity were evaluated in the EPM test. The evaluation of behavior, memory and spatial learning was performed using passive avoidance test (PAT). Glycated hemoglobin level was determined in blood, insulin and leptin levels were determined in blood plasma, general antioxidant activity was determined in liver cytosolic fraction. Results and discussion. The obtained data on biochemical parameters and insulin resistance tests showed the absence of normalizing effects of developed FFI. However, the inclusion of polyphenol-containing FFI into the diet led to beneficial changes in physiological parameters. Animals of G3 group, provided with FFI, were significantly less anxious compared to both control groups. During PAT testing of short-term memory, no animals in G3 group entered to the dark compartment (0%), what demonstrated increased learning ability and well-established memory of these animals in comparison with C1 (50%) and C2 groups (80%). Conclusion. The results prove the effectiveness of bilberry leaves` polyphenols, sorbed on the brown buckwheat flour, in the correction of central nervous system disorders in db/ db mice with genetically altered type 2 diabetes, what points at possible prospect of FFI inclusion in therapeutic products for patients with type 2 diabetes mellitus.

[A comparative evaluation of the effect of soy protein and its enzymatic hydrolysate on lipid metabolism in male Wistar rats with induced obesity].

Among various food proteins, soybean proteins have the greatest traditions of application for the dietary correction and prevention of lipid metabolism disorders and related complications. Aim. In an in vivo experiment using male Wistar rats, the lipid-lowering properties of soy protein and its enzymatic hydrolysate were tested to evaluate their possible use as ingredients of specialized foods. Material and methods. Animals were randomly divided into 3 groups: control group G1 and 2 experimental groups G2 and G3. The total duration of the experiment was 70 days. The animals of the control group G1 were fed with high-lipid semi-synthetic diet. Animals of the experimental groups G2 and G3 received the same high-fat semi-synthetic diet, but with a 50% replacement of casein with soy protein isolate (SPI) and enzymatic hydrolyzate of SPI (EHSPI), respectively. The blood glucose was measured once per 2 weeks. At the end of the experiment on the 71st day the level of glycated hemoglobin was determined in the blood; the levels of triglycerides, cholesterol, high density lipoproteins (HDL), low density lipoproteins (LDL) and the concentration of malon dialdehyde were determined in the serum. Results and discussion. Starting from the 6th week of the experiment and prior to its completion, the average food intake of animals from the G3 group was significantly (р<0.05) lower compared to animals of the G1 control group. The food intake of animals of group G2 was significantly (р<0.05) reduced compared with this indicator for animals of group G1, starting from the week 8 of the experiment and prior to its completion. The monitoring of the body weight gain did not reveal significant differences between all groups of animals, despite differences in the food intake. Replacing casein in the diet by 50% with SPI had a pronounced antioxidant and cholesterol-lowering effect. The total cholesterol content (1.65±0.05 mmol/l) decreased significantly (р<0.05) due to a decrease in LDL (0.90±0.03 mmol/l), and malon dialdehyde level lowered (3.7±0.5 µmol/l, р<0.05) in the serum of group G2 rats compared with animals of the control group G1 (2.01±0.13 and 1.12±0.09 mmol/l; 5.1±0.4 µmol/l, respectively). Replacing casein by 50% with EHSPI in the diet of G3 rats was unfavorable, significantly (р<0.05) increasing the level of total cholesterol (2.76±0.16 mmol/l) and cholesterol in LDL (1.66±0.12 mmol/l) in blood of these animals compared with animals of both comparison groups G1 and G2. Conclusion. A preclinical comparative study of the cholesterol-lowering and antioxidant properties of the SPI substantiates the prospect of its following clinical trials with the aim of including into the composition of specialized foods for prevention and diet therapy of the disorders of endogenous cholesterol homeostasis.

[Streptozotocin induced diabetes rat models].

The introduction of chemical compounds in diabetes modeling can't adequately reflect the development of the disease. However, the choice of an experimental model of diabetes type 1 or 2 is largely determined by the purpose of the research: testing of pharmacological activity, genetic research or clarifying the mechanisms of disease development. The high cost of respective genetic lines of laboratory animals, the complexity of reproduction of the model, the special conditions of care and a high degree of inbreeding determine the necessity for the development, testing, and improvement of non-genetic models. The most widely used chemical models of type 1 diabetes in modern experimental diabetology are alloxan models and of type 2 or mixed type diabetes are streptozotocin models. Sensitivity to the introduction of the diabetogenic compounds can essentially depend on the species, but also on animal genetic line and its age. The results of studies in which the injection of streptozotocin (STZ) to laboratory animals (mice and rats) simulated type 2 diabetes are shown. It is noted thatpre-treatment with nicotinamide can simulate the state more appropriate to type 2 diabetes. Taking into account the leading role of abdominal obesity as a risk factor for type 2 diabetes, considerable attention in the article is paid to the modeling of type 2 diabetes by STZ-injection and high-fat diet. As alternative models of a type 2 diabetes in rodents also induce by streptozotocin injection and high fructose diet. The combined effect of low dose STZ and high fructose diet allows in relatively short period induce the development of type 2 diabetes in rats. It is concluded that the modeling of diabetes by STZ injection are widely demand for the optimization screening of biologically active substances with antidiabetic action in experiments in vivo and is a step before their clinical trials in the composition of specialized preventive and therapeutic products.

[The complexes of copper, manganese and chromium with enzymatic hydrolysate of pig spleen: research in vitro].

This report describes the preparation and the results of physical and chemical analysis of complexes of enzymatic hydrolysate of pig spleen (EHPS) with manganese, copper and chromium. The complexes were prepared using schemes including the reaction of complexation of inorganic cations with EHPS-peptides structures and application of membrane technology. The process of microfiltration of the resulting mixtures was carried out in tangential flow and low molecular weight fractions were collected. Solutions of copper and manganese complexes with EHPS were subjected to nanofiltration to remove inorganic ions from the reaction mixture. The obtained preparations were lyophilic dried and the molecular weight distribution of the protein fractions in Cu-EHPS, Mn-EHPS and Cr-EHPS complexes was analyzed by exclusion medium pressure liquid chromatography. The percentage relation of fractions with specific molecular weight range was calculated by applying the weighted integration of chromatograms. The determination of copper, manganese and chromium levels in the complexes was performed by atomic absorption method. The content of microelements in the preparations is for copper 16.5 ± 0.3 mg/g, for manganese--24.9 ± 0.5 mg/g and for chromium--2.5 ± 0.2 mg/g.