THE STuDY OF THE INFLuENCE OF MODEL MEAT SYSTEMS ON THE ALLERGIC IMMuNE RESPONSE IN VIVO

This article presents the results of studying the effect of homogeneous model meat systems produced using enzyme preparation containing fungal protease and microbiological starter culture of Lactobacillus plantarum on the allergic reactions within specific immunity in vivo. According to the results, it is established that experimental products have no negative effect on the clinical parameters of laboratory animals. During the experiment, with the introduction of experimental products into diet, the dynamics of body weight changes in all groups of animals was positive. At the end of the experiment, there were smaller increase in the weight of rats and lower values of weight gain (Group 1 — 14.0 %, Group 2 — 15.9 %, Group 3 — 20.2 %). This is possibly due to the adaptation processes occurring in response to introduction of meat systems into the diet, which confirms the leveling of the daily weight gain of experimental and intact animals since the 16th day of the experiment. According to the results of clinical blood analysis of the animals consuming experimental products, an increase is detected in leukocytes and lymphocytes by up to 18 %; in granulocytes by up to 35 %; and in monocytes by up to 8 %; in hemoglobin concentration, hematocrit and mean corpuscular hemoglobin concentration by more than 3 %; in red cell distribution width and mean corpuscular volume by up to 2 %, in comparison with intact animals. The correlation of these data with ELISA parameters for serum of experimental animals (histamine and immunoglobulin E) allowed to suggest the expression of reaginic antibodies and interaction on the surface of basophils and mast cells, which led to the degranulation and release (increase) of histamine, as a vasoactive factor, by 40 % compared with intact animals. The overall conclusion of the studies is that experimental model meat systems may trigger the activation of specific immune responses in laboratory animals. This is possibly due to proteasemediated formation of greater amount of indigestible polypeptides and peptides that invoke local adaptation responses. Аннотация В данной статье представлены результаты изучения влияния гомогенных мясных модельных систем, полученных с применением ферментного препарата грибной протеазы и микробиологической стартовой культуры Lactobacillus plantarum, на аллергическую реакцию специфического иммунитета in vivo. По результатам исследований установлено, что опытные продукты не оказывают негативного влияния на клиническое состояние лабораторных животных. На протяжении эксперимента динамика изменения массы тела животных всех групп была положительной, при введении опытных образцов в рацион, отмечены меньший прирост веса крыс и меньшие значения привесов животных в конце эксперимента (у крыс 1 группы — 14,0 %, 2 группы — 15,9 %, у животных 3 группы — 20,2 %). Возможно это связано с адаптационными процессами, происходящими в ответ на введение в рацион мясных систем, что подтверждает нивелирование ежесуточного привеса опытных и интактных животных начиная с 16-х суток эксперимента. По результатам общего клинического анализа крови животных, потреблявших опытные продукты, выявлено увеличение лейкоцитов и лимфоцитов до 18 %, гранулоцитов до 35 % и моноцитов до 8 %, концентрации гемоглобина, уровня гематокрита и средней концентрации гемоглобина в эритроците свыше 3 %, ширины распределения эритроцитов и среднего объема эритроцита до 2 % по сравнению с интактными животными. Соотношение этих данных с анализом иммуноферментных показателей сыворотки крови опытных животных (гистамин и иммуноглобулин Е) позволило высказать предположение об экспрессии реагиновых антител и взаимодействии на поверхности базофилов и тучных клеток, приводящих к дегрануляции и высвобождению (увеличению) гистамина, как вазоактивного фактора, на 40 % в сравнении с интактной группой. Общий вывод исследований указывает на то, что опытные мясные модельные системы могут вызывать активацию специфических иммунных реакций у лабораторных животных. Возможно, это связано с образованием под действием протеаз большего количества сложноусваеваемых полипептидных и пептидных соединений, вызывающих местные адаптационные реакции. уДК/uDC: 637.5:57.083.32:57.084.1 DOI 10.21323/2414-438X-2017-2-1-57-68


Abstract
This article presents the results of studying the effect of homogeneous model meat systems produced using enzyme preparation containing fungal protease and microbiological starter culture of Lactobacillus plantarum on the allergic reactions within specific immunity in vivo.According to the results, it is established that experimental products have no negative effect on the clinical parameters of laboratory animals.During the experiment, with the introduction of experimental products into diet, the dynamics of body weight changes in all groups of animals was positive.At the end of the experiment, there were smaller increase in the weight of rats and lower values of weight gain (Group 1 -14.0 %, Group 2 -15.9 %, Group 3 -20.2%).This is possibly due to the adaptation processes occurring in response to introduction of meat systems into the diet, which confirms the leveling of the daily weight gain of experimental and intact animals since the 16th day of the experiment.According to the results of clinical blood analysis of the animals consuming experimental products, an increase is detected in leukocytes and lymphocytes by up to 18 %; in granulocytes by up to 35 %; and in monocytes by up to 8 %; in hemoglobin concentration, hematocrit and mean corpuscular hemoglobin concentration by more than 3 %; in red cell distribution width and mean corpuscular volume by up to 2 %, in comparison with intact animals.The correlation of these data with ELISA parameters for serum of experimental animals (histamine and immunoglobulin E) allowed to suggest the expression of reaginic antibodies and interaction on the surface of basophils and mast cells, which led to the degranulation and release (increase) of histamine, as a vasoactive factor, by 40 % compared with intact animals.The overall conclusion of the studies is that experimental model meat systems may trigger the activation of specific immune responses in laboratory animals.This is possibly due to proteasemediated formation of greater amount of indigestible polypeptides and peptides that invoke local adaptation responses.
Одним Конечно, данный перечень не является исчерпывающим и как правильно отмечено -это наиболее распространенные компоненты, так как у детей, important for young children, because child's body is not always able to split food substrates into safely metabolized semi-elemental and elemental nutrients [1], due to immaturity of the enzyme systems of gastrointestinal tract.Most children, especially those who live in cities, had at least one allergic reaction to food ingredient, macro-or micro-component, and in 30 % of children allergic problems become chronic.
One of the regulative documents confirming relevance and prevalence of food allergy is the adopted Technical Regulations of the Customs Union TR TS 022/2011 «Food products in terms of its labeling.»Article 4 «Requirements for labeling of food products» of this document establishes requirements for manufacturer to indicate components of food products (including food additives, flavoring agents), bioactive additives, which consumption may cause allergic reactions or is contraindicated in certain types of diseases.Moreover, the number of potential allergens should be indicated regardless of their number.
According to TR TS 022/2011, the most common components, which consumption may cause allergic reactions or is contraindicated in certain types of diseases, include the following: • peanuts and related processed products; • aspartame and aspartame-acesulfame salt; • mustard and related processed products; • sulfur dioxide and sulfites, if their total content is greater than 10 milligrams per kilogram or 10 milligrams per liter based on sulfur dioxide; • cereals containing gluten and related processed products; • sesame and related processed products; • lupine and related processed products; • shellfish and related processed products; • milk and related processed products (including lactose); • nuts and related processed products; • crustaceans and related processed products; • fish and related processed products (excluding fish gelatin used as a base in preparations containing vitamins and carotenoids); • celery and related processed products; • soybeans and related processed products; • eggs and related processed products.
Animal meat contains two main potential allergens -serum albumin and gamma globulin [3].Acute sensitivity to these substances in children may cause gastrointestinal disorders (vomiting, diarrhea, indigestion), rash, itching at different parts of the body, allergic rhinitis and even asthma attack (anaphylaxis) [4,5].Serum albumin is the most widely studied and most abundant blood protein (70 % of total protein composition).Its plasma concentration is 35-55 mg/ml.Bovine serum albumin (BSA) is a globule in the form of oblate spheroid with 17 semiaxes to 42 angstroms consisting of 607 amino acid residues.BSA has the complex spatial structure of three domains, each of which, in turn, is divided into two subdomains (A-B and C).The molecular weight of BSA is ~69 kDa.
Food allergy, as a rule, affects 15-40 % of children starting from an early age.In recent years, the percentage of children suffering from food allergy to cow's milk and beef has increased significantly, since these products have antigenic affinity for proteins in their composition [6].Restricting or excluding these foods from the nutrition of children creates great difficulties to meet physiological need for animal protein, which is extremely important for their normal growth and development.
In this regard, the range of products with reduced allergenic potential is currently represented mainly by artificial mixtures of milk and soy proteins [7,8,9,10,11].
• filtering, i.e. removing the allergen (production of dairy products, juices); • combination of different methods (preparation of mixtures based on dairy and vegetable proteins).
In this context, an extremely urgent task arises in terms of creating a new technology for baby foods based on meat raw materials with hypoallergenic properties.In this case, given the characteristics of meat raw materials and processing technologies, combined approaches to reduce allergenic potential, including fermentation and heat treatment, are becoming fairly promising.
Thus, the aim of this study was to investigate the influence of homogenized meat products treated with the enzyme preparation on the allergic immune response in laboratory animals.

Materials and methods
The study was carried out by Тhe V.M. Gorbatov All-Russian Meat Research Institute at the Experimental Clinic-Laboratory of Biologically Active Substances of Animal Origin.
The experiment was carried out on clinically healthy, sexually naive laboratory Wistar rats of spf-category with weight of 348 ± 17 g obtained from the Center for Genetic Resources of Laboratory Animals in Federal Research Center of the Cytology and Genetics Institute, Siberian Branch of the Russian Academy of Sciences, Novosibirsk.
Before the experiment, the animals underwent adaptation to the laboratory conditions for 14 days.Throughout the experiment, the rats were kept in a system of individually ventilated cages (IVC) as part of the VENT II ventilation unit and Type III Bio.A.S. cage rack (EHRET, Germany) ensuring optimal microclimate in each individual cage.The conditions of rats in IVC were similar as for temperature (22 ± 3 °С), humidity (50-60 %), illumination 12/12 (6.00 to 18.00 light day).
Throughout the experiment, rats were fed ad libitum with all-in-one mixed feed balanced by protein (Assortiment-Agro, Russia) according to GOST R 50258-92.Nutritional value of mixed feed is presented in Table 1.
Иммуноферментные показатели (иммуноглобулин Е, гистамин) определяли на анализаторе Immunochem The experiment lasted 28 days.Observation of the animals was carried out daily throughout the experiment.Clinical parameters and behavior of animals, neuromuscular functions, hair condition, feed intake, and water consumption were recorded.Prior to the study and every 3 days of the study, animals were weighed on an electronic laboratory scale (Ohaus, USA) to plot weight gain chart.
At the end of the experiment, the animals were euthanized in an euthanasia cage (VetTech, UK) with carbon dioxide followed by blood sampling and general autopsy.
Animal management, nutrition, care, manipulations, removal from the experiment were carried out in accordance with the requirements of the Ministry of Health of the Russian Federation Order No. 267 dated 19.06.2003 «On the approval of the laboratory practice rules», the requirements of the Ministry of Health of the USSR Order No. 742 dated 13.11.1984«On the approval of the rules for work using experimental animals», the International Rules for animal protection -Directive 2010/63/EU of the European Parliament and the Council of the European Union.
STATISTICA 10 software was used to analyze the results.The results were presented as weighted average ± standard deviation.Statistical significance was calculated using one-way ANOVA test and Duncan test.The probability of 0.05 was chosen as a significant level.

Results and discussion
Previous studies have shown that the enzyme preparation of fungal protease containing the complex of peptidases and proteases obtained by targeted fermentation with selected strain of Aspergillus oryzae followed by purification possesses the greatest species-specific activity with respect to studied substrate (BSA) [15,16].This preparation hydrolyses protein substances to amino acids and peptides with lower molecular weight [17].However, the products obtained after enzymatic treatment had low sensory parameters.To improve sensory quality, as well as to obtain more complete biotransformation of BSA in meat raw materials, lactic acid microorganisms were used.The experimental data obtained for the fractional composition of proteins indicated the absence of bovine serum albumin fraction in the samples [18].Thus, the use of two-stage processing of meat raw materials allowed reducing the allergenic potential of beef due to biotransformation of the main allergenic protein -BSA.
The condition of the animals prior to the experiment beginning was physiologically normal.The rats were mobile and active; muscles were in tonus; tactile response was active; hair tightly adhered to body, not ruffled, smooth, clean, shiny; the skin was elastic, without compromising integrity; visible mucous membranes were pale pink, without efflux and other signs of inflammatory reactions.Eyes were bright red.Urination and defecation were within normal limits.Rats actively ate feed, which confirms full consumption of the proposed samples, both in control and experimental groups.Animal survival throughout the experiment was 100 %.
The maximum weight gain was observed in the intact animals of Group 3 and accounted for 20.2 %; the animals in Group 2 consuming experimental meat products increased their weight by 15.9 %, and rats in Group 1 -by 14.0 % compared to initial weight of the animals.
Analysis of hematological test results (Table 2) for the rats in Group 2 consuming experimental feed showed a significant increase in leukocytes and lymphocytes by up to 18 %, granulocytes by up to 35 % and monocytes by up to 8 % (P> 0.05) compared to the intact animals in Group 3. In Group 1 there was a decrease in the relative content of lymphocytes and monocytes, 4.9 % and 35.0 % respectively, while the proportion of granulocytes increased (up to 30 %) in comparison to the animals in Group 3 (P <0.05).
ELISA analysis of rat serum showed that consumption of the control meat products by animals in Group 1 did not mediate a significant change in the immunoglobulin E concentrations compared to the intact animals in Group 3. It was noted that introduction of the experimental meat product into the diet of the animals in Group 2 resulted in an insignificant increase in the immunoglobulin E concentration by 4.5 % (P <0.05) compared to the intact animals in Group 1 and by 4.9 % (P> 0 , 05) compared to the intact animals in Group 3.There was a significant histamine increase in rat serum in Group 1 (27 %) and in Group 2 (up to 40 %) (P <0.05) compared to the intact animals in Group 3. While the histamine level in serum of the animals in Group 2 was 11.1 % (P <0.05) higher compared to the rats in Group 1 (Table 3).
According to the autopsy results and macroscopic study of animal organs, differences between the animal groups receiving different diets have not been established.

Conclusion
During the experiment, there were no significant changes in the clinical parameters of the laboratory animals in the experimental group.Throughout the experiment, the dynamics of body weight changes was positive.With the introduction of the experimental products into the diet, a smaller increase in the weight and smaller values of animal weight gain at the end of the experiment were noted (14.0 % for the rats in Group 1, 15.9 % for the rats in Group 2, 20.2 % for the rats in Group 3).This is probably due to the adaptation processes occurring in response to the introduction of meat systems into the diet.This assumption is confirmed by the fact that, starting from the 16th day, the rats in Groups 1 and 2 increased their weight in average by 3.4-3.7 g/day (reaching daily weight gain of the intact rats in Group 3).
Based on the results of the clinical blood analysis for the animals consuming the experimental product, a significant change in the concentration of immune blood cells was revealed (an increase in leukocytes and lymphocytes by up to 18 %, granulocytes by up to 35 % and monocytes by up to 8 %); an increase in the functional activity of erythrocytes (an increase in hemoglobin concentration, hematocrit and mean corpuscular hemoglobin concentration by more than 3 %, the red cell distribution width and mean corpuscular volume by up to 2 %) compared with the intact animals in Group 3. The correlation of these data with serum ELISA analysis (histamine and immunoglobulin E) allows suggesting the expression of reaginic antibodies (including IgE, increasing in rats of Group 2 by up to 4.5 %) and interactions on the surface of basophils and mast cells (increase in leukocytes due to lymphocytes and granulocytes) leading to degranulation and release of histamine as a vasoactive factor (an increase in histamine by up to 40 %) [19].
Thus, based on the results, it can be concluded that model meat systems produced using the enzyme preparation of fungal protease and microbial starter culture of Lactobacillus plantarum may lead to the activation of specific immune responses in laboratory animals.This is possibly due to protease-mediated formation of a greater amount of indigestible polypeptides and peptides that invoke local adaptation responses [20,21].
For more accurate information on the possible sensitizing or hypoallergenic properties of the model meat systems being developed, studies will be conducted to analyze the proteomic and peptide composition and bioavailability in vitro, to detect anti-nutritional substances, and to search for specific antibodies to serum albumin in vivo.
The technological flow chart for model meat product manufacturing is shown in Fig.1.The dashed arrows indicate the process of control samples production.After the adaptation period, the animals were individually labeled and randomly assigned to different groups: Group 1 -control animals consumed control samples of meat products; Group 2 -animals consumed experimental meat products treated with enzyme preparation and lactic acid bacteria; Group 3 -intact animals consumed mixed feed.