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Project titleApplying the biopotential of adaptogenic BAS from plant raw materials to create new functional foods with probiotic effects for active longevity and health.

Keywordsbiologically active substances, gastrointestinal microbiota normalisation, fermented products, adaptogens, probiotics, in vitro models

Annotation
The biotechnology of functional foods is developing intensively, due to the high social demand for foods with pronounced biological properties. The importance of the generally accepted concept of "food" has expanded and now it is not only a source of energy to maintain metabolism and homeostasis of the body, but also a source of compounds that exhibit therapeutic effect in relation to the prevention of diseases of various etiologies. One of the ways to correct human health is to consume functional ingredients as part of the daily diet. In the food industry, it is particularly popular to create specialised and functional plant-based foods, both fermented and non-fermented. This is due to the diversity, availability and reproducibility of raw materials. The development of this area will also help to address the issue of resource conservation and reduce the scarcity of raw materials of animal origin. The growing demand for products based on vegetable raw materials can also be explained by the individual characteristics of some population groups, in particular lactose intolerance and malabsorption, allergies to milk proteins, environmental, ethical and religious aspects. It should be noted that the inclusion of functional plant foods and foods with minimal processing in the diet helps to strengthen the human immune system, which is very important in the current epidemiological situation. In connection with changes in the rhythm of human life, unspecific reactions of the organism to external stimuli - stress, high production loads, epidemics of viral diseases, antibiotic therapy, exposure to ionising radiation and unfavourable ecological situation - are topical problems. Examples of pathological conditions are neuroendocrine and immune system disorders (chronic fatigue syndrome, reduced physical performance, increased fatigue, acute/chronic stress conditions, multiple sclerosis, autism, Alzheimer's and Parkinson's diseases), as well as disorders associated with changes in the quantity and quality of intestinal microflora, leading to digestive disorders, type 2 diabetes, obesity, colorectal cancer, infections caused by Clostridium difficile bacteria. The use of herbal preparations, in particular substances with adaptogenic properties, is widely used in the prevention of negative conditions of the body. Substances belonging to this group exhibit a pronounced neuroprotective, antidepressant, stimulating activity, improve the ability to work under stress and fatigue, increase tolerance to mental exhaustion, and are used in rehabilitation correction to eliminate the reversible symptoms of maladaptation. The use of probiotic microorganisms biopotentially possessing high adaptability and stability in conditions of GIT promotes normalization of natural intestinal microflora of a person which plays the major role in immunomodulation of organism and increase of resistance to infectious and bacterial infections. In addition, there is a proven correlation between the state of the intestinal microflora and the quality of human life, in particular the presence of depressive conditions. The main objective of the food industry is to meet society's demand for food. Since society is a heterogeneous society, it is necessary to take into account the demands of all social groups and modernize the market for basic foodstuffs. Therefore, the project provides for the development of plant-based products with both adaptogenic and probiotic effects in order to broaden the range of products. Within the framework of solving the above-mentioned problems, functional fermented products based on plant raw materials and ice cream will be developed, applicable to maintaining the balance of the intestinal microbiota and increasing the tolerance of the immune and central nervous systems due to the presence of biologically active substances of plant origin possessing adaptogenic properties in their composition. As substances stimulating physical and mental activity, as well as increasing the body's resistance to external negative factors, BAS extracted from plants growing in the Russian Federation with adaptogenic properties will be used: lemongrass, levzea, rhodiola, eleuterococus, echinacea, garlic, rose hips, sea buckthorn. The probiotic effect of the developed product will be due to the symbiosis of probiotic bacteria of the genus Lactobacillus and the genus Bifidobacterium. As a result of this research the growth patterns of probiotic microorganisms of genera Lactobacillus and Bifidobacterium (Lactobacillus acidophilus; Lactobacillus delbrueckii subsp. Bulgaricus; Bifidobacterium bifidum; Bacillus coagulans MTCC 5856, Bifidobacterium longum) in plant raw material will be obtained, adaptation of probiotic microorganisms will be done. During development of a complex of biologically active substances with adaptogenic properties, modern methods of extraction will be applied, optimum processing parameters will be determined. A rational method of drying will be determined and temperature treatment regimes at various methods of drying of BAS obtained will be worked out. Regularities of adaptation of bacteria of genera Lactobacillus and Bifidobacterium to plant substrate will be investigated, biochemical and cultural properties will be studied, and rational quantitative and qualitative composition of plant raw materials will be established. Based on this research, innovative products with adaptogenic and probiotic properties will be developed on the basis of raw materials of plant origin and their structural-mechanical, functional and organoleptic characteristics, as well as storability will be studied. The biofunctional properties of the products obtained will be investigated in in vitro model systems. By results of researches the complete set of the project of technical documentation for a vegetative basis for reception of the fermented functional drink and an ice-cream on the basis of vegetative raw material possessing adaptogenic and probiotic actions will be developed. The results of scientific research will expand the range of innovative plant-based functional food products, which is a key trend in the Russian and global FoodTech markets.

Expected results
As a result of the project, a new approach to creating alternative functional food products based on vegetable raw materials, enriched with biologically active substances of adaptogenic effect and having a probiotic effect will be proposed. Implementation of the project involves obtaining a set of scientific results: -the growth patterns of probiotic microorganism populations of the genus Lactobacillus and Bifidobacterium (Lactobacillus acidophilus; Lactobacillus delbrueckii subsp. Bulgaricus; Bifidobacterium bifidum; Bacillus coagulans MTCC 5856, Bifidobacterium longum) in plant raw materials for production of fermented plant-based drinks and ice cream (soya, rice, oats, wheat, peas, buckwheat, pumpkin, lentils, topinambur); -the process of adaptation of a consortium of probiotic microorganisms of the genus Lactobacillus and the genus Bifidobacterium (Lactobacillus acidophilus; Lactobacillus delbrueckii subsp. Bulgaricus; Bifidobacterium bifidum; Bacillus coagulans MTCC 5856, Bifidobacterium longum) to plant matrix and its rational quantitative and qualitative macro- and microelement composition to provide microbial cell survival in necessary quantity for probiotic effect as well as for intensification of acidification process was determined; -Will be obtained and characterized BAS of adaptogenic action from plant raw materials, such as lemongrass, leuzea, rhodiola, eleuterococus, echinacea, garlic, rosehip, sea buckthorn; -Sonochemical treatment regimes for extracting herbal raw materials and preserving the BAA adaptogenic properties in the process of their extraction will be established; - Data will be obtained on the effect of natural plant adaptogens on the growth of probiotic microorganisms of the genus Lactobacillus and Bifidobacterium; - Recommended drying regimes for BAS of adaptogenic action, which ensure the preservation of these BAS, will be established; - Data on biologically active metabolites produced by microorganisms of genus Lactobacillus and genus Bifidobacterium during fermentation of vegetable raw materials will be obtained; - Data will be obtained on the effect of low-temperature exposure parameters (temperature range +4ºC to -18ºC) on the BAA complex of adaptogenic properties and probiotic microorganisms during storage of vegetable raw materials and fermented vegetable base; On the basis of obtained data the composition and production technology of functional fermented drink and ice cream based on vegetable raw materials that provide probiotic and adaptogenic effects at systematic use will be developed; -A project of normative and technical documentation will be developed - technological instruction (TI) and technical specifications (TS); - The technology of BAS complex enrichment of products based on vegetable raw materials will be proposed; -Functional properties of obtained products will be studied in model systems in vitro; -A patent application will be filed for a plant basis for obtaining fermented functional beverage and ice cream based on plant raw materials with adaptogenic and probiotic activity. The possibility of practical use of the expected results of the project in the economy: - The results of successful research will be available to both large companies and small food processing companies producing plant-based products. - The introduction of the obtained framework for the production of fermented products on the food market will establish a mutually beneficial relationship between the links of the socio-economic system "consumer" - "enterprise". At the same time, the producer's efficiency and demand will increase in the global aspect, in particular, in the sphere of national economic importance. - Creation of food products from plant raw materials of domestic origin will support the policy of import substitution and resource-saving that is in effect in the Russian Federation. - The increase in assortment of products on vegetable raw materials and consumer interest in it will lead to reduction of deficiency of animal raw materials that will be positively reflected in ecological situation. - Optimization of technology of adaptation of probiotic microorganisms to plant raw materials and BAS of adaptogenic property will lead to the development of new food products in various sectors of the food industrial complex. - The intensive development of biotechnology of fortified and specialized foods, which is promoted by the goal and objectives of this project, will lead to the isolation of a new market segment, aimed at meeting the needs of people for a healthy diet. The possibility of practical use of the expected results of the project in the social sphere: - The results of the project can be used to provide the population with safe, nutritious and healthy products to increase the functional reserves of the human body and maintain health, in particular to maintain the health of people with weakened immunity, people exposed to various adverse influences in order to increase the overall resistance of the body, prevent fatigue during physical and mental stress and increase performance capacity. - The results of the project are applicable not only to the needs of people who do not wish to switch completely to vegetarian and vegan diets but add more plant-based products to their diet, but also to people with hypersensitivity to components of animal origin (food allergies to protein), lactose intolerance and people following a low-cholesterol diet, sportsmen, and people who have given up animal products for ecological, ethical and religious reasons.

Annotation of the results obtained in 2023
In the second year of research, the survival of probiotic microorganisms genus Lactobacillus, genus Bifidobacterium, genus Propionibacterium, and genus Streptococcus during the fermentation of plant dispersions was studied. It has been shown that the biomass increase depends on the plant dispersion type and bacterial strain. The influence of plant dispersions on the fermentation process by Str. thermophilus bacteria was studied, which made it possible to characterize the fermentation process and identify differences in the acid accumulation between dairy raw materials and plant dispersions. Str. thermophilus has been shown to be capable of fermenting various types of plant dispersions and plant mixtures. The influence of the use of probiotic bacteria on the antioxidant activity changes of plant dispersions from soy beans, rice grains, oats, wheat grains, pea beans, buckwheat seeds, pumpkin seeds, lentils, and Jerusalem artichoke tubers after fermentation and during storage was studied. The results showed that changes in antioxidant activity depend on both the type of plant dispersion and the probiotic culture used. The ability of probiotic microorganisms to ferment carbohydrates such as glucose, galactose, fructose, sucrose, maltose, melibiose, and stachyose has been determined. It has been shown that the most omnivorous are B. bifidum, B. coagulans, P. schermanii, Str. thermophilus. L. acidophilus showed the greatest selective ability for fermentation. Data were obtained on changes in the concentration of biologically active substances when drying extracts of Rhodiola, Garlic, Eleutherococcus, Rose hips, and Sea buckthorn in various ways. It has been shown that for all types of extracts, from the point of view of the safety of biologically active substances, freeze-drying is recommended. It has been established that ultrasonic cavitation has both positive and negative effects on the growth of biomass of probiotic microorganisms during the fermentation process and depends on the type of plant material and strain of bacteria. The effect of adding dry extracts from Schisandra fruits, Leuzea roots, Rhodiola roots, Eleutherococcus roots, Echinacea herb, Garlic cloves, Rosehips, and Sea buckthorn fruits on the increase in biomass of probiotic bacteria was studied. It has been proven that the addition of dry extracts to the plant mixture generally has a positive effect on the increase in the biomass of probiotic microorganisms and the antioxidant activity of the fermented plant mixture compared to the controls without extracts. It has been experimentally shown that the addition of a mixture of dry extracts with adaptogenic properties leads to an increase in the amount of biomass growth of microorganisms such as Bifidobacterium, Streptococcus thermophilus, Lactobacillus acidophilus and does not reduce the amount of Lactobacillus delbrueckii subsp. bulgaricus. The developed fermented plant drink and frozen plant dessert were shown to be natural sources of molybdenum and manganese. Technological parameters and a production scheme have been developed with critical control points for a fermented plant drink and a fermented plant frozen dessert based on soy, lentils, wheat, and pumpkin seeds with probiotic bacteria and dry extracts of Rhodiola, Eleutherococcus, Garlic, Rosehips, and Sea buckthorn. It is shown that standard equipment for the production of fermented plant beverages and desserts is used to produce these products. It has been proven that the stability of thixotropic properties is maintained during the storage of beverages and frozen desserts. Storing a fermented frozen dessert increases its resistance to melting during storage. The fatty acid composition of the lipid composition of the fermented drink and dessert was determined during the shelf life. It has been shown that polyunsaturated fatty acids, represented by omega-3, omega-6, omega-7, and omega-9, predominate in the beverage and frozen desserts. It has been shown that all essential amino acids are present in the developed fermented products. Calculation of the biological value showed that the limiting amino acids are methionine and cysteine in total. During storage of the fermented plant beverage, the content of methionine total leucine, and isoleucine increase. When storing a fermented vegetable dessert, the content of all amino acids increases to varying degrees. The survival of probiotic microorganisms and changes in antioxidant activity were assessed in vitro system simulating the digestion process at various stages. It has been shown that the amount of probiotic microorganisms in the ileum is sufficient to positively affect the human body. It has been shown that there is an increase in this antioxidant activity in both plant products, which is possibly due to the release of peptides, amino acids, and low molecular weight compounds with antioxidant properties. It has been experimentally demonstrated that salidroside and allicin have the best absorption capacity. Eleutherosides are the worst absorbed. The vitamin composition, content of biologically active substances, and changes in quality indicators of a fermented plant beverage and a fermented frozen dessert during storage were determined. The fermented plant drink has been shown to be a source of eleutherosides and allicin. It has been proven that during the storage of developed fermented products, there is an increase in antioxidant activity and a slight decrease in viable probiotic bacteria.

Publications

1. Gelazov R.Kh., Iakovchenko N.V., Bayazitov K.R., Shiriaev V.A. Перспективы применения пробиотических микроорганизмов при производстве ферментированных продуктов на гречневой основе Аграрная наука, 381(4): 138-145 (year - 2024) https://doi.org/10.32634/0869-8155-2024-381-4-138-145

2. Iakovchenko N.V. Antsyperova M.A. Перспективы применения пробиотических микроорганизмов при производстве ферментированных продуктов на соевой основе Аграрная наука, 380(3): 149-156 (year - 2024) https://doi.org/10.32634/0869-8155-2024-380-3-149-156

3. Lavrentev FV, Baranovskaia DA, Shiriaev VA, Fomicheva DA, Iatsenko VA, Ivanov MS, Ashikhmina MS, Morozova OV, Iakovchenko NV. Influence of pre-treatment methods on quality indicators and mineral composition of plant milk from different sources of raw materials Journal of the Science of Food and Agriculture, 104: 967-978. (year - 2023) https://doi.org/10.1002/jsfa.12992

Annotation of the results obtained in 2022
In the first year of research the influence of pre-treatment on plant raw materials for the production of plant-based milk was assessed. Obtained data highlights the correlation of different types of processing raw materials (ultrasonic treatment, roasting, freezing under various temperature) with the content of physicochemical composition (solids, protein, fat, carbohydrates, fiber, ash, mineral composition, pH, titratable acidity) and organoleptic characteristics. Various types of plant-based milk obtained from soybeans, rice, oats, wheat, peas, buckwheat, pumpkin seeds, lentils, Jerusalem artichoke tubers were examined.The results reveal the potential of using the preliminary processing of plant-origin raw materials with the purpose of adjustment requiredphysicochemical and organoleptic characteristics of various types of plant-based milk. The correlation between acid accumulations andthe duration of fermentation forprobiotic bacteria of the genus Lactobacillus and Bifidobacterium in various types of plant-based milk was determined. Strains of probiotic bacteria provided a stable increase of biomass during fermentation and efficiency of obtaininghigh quality organoleptic characteristics fermented products from soybeans, rice, oats, wheat, peas, buckwheat, pumpkin seeds, lentils, Jerusalem artichoke tubers were selected. Optimal technological parameters for using ultrasonic treatment for obtaining most advantageous extracts from plant sourcessuch as lemongrass, leuzea, rhodiola, eleutherococcus, echinacea, garlic, rosehip, sea buckthorn have been consented. Based on the experimental data of the particle size, solubility, wettability, mass fraction of moisture, phenol content and antioxidant activity, the most efficient modes of drying (spray and lyophilic) of the extracts were determined that provided products with high quality characteristics. Research results presents that all extracts with the exception of lemongrass and sea buckthorn extracts can be spray-dried and lyophilic-dried without the use of a carrier. The influence of adding various typesof extract (lemongrass, leuzea, rhodiola, eleutherococcus, echinacea, garlic, rosehip, sea buckthorn) on the process of acid accumulation, the survival of probiotic microorganisms during the fermentation of the plant mixture was assessed. Optimal amounts of dry powders of extracts for the developed plant mixture have been selected. Research data provided the feasibilityto obtain a fermented product with specifichigh-quality indicators. Selectedstrains of probiotic microorganism of the genera Lactobacillus and Bifidobacterium in combination with extracts make it possible to obtain a plant mixture with optimal consumer properties. Thus, an innovative mixture based on various types of plant milk was modified. Moreover a technology for an enrichment with an extract composition with adaptogenic properties and a consortium of probiotic microorganisms for the production of a fermented drink and ice cream was developed.

Publications

1. Iakovchenko N.V., Popova N.V. Влияние предварительной обработки сырья на функционально-технологические свойства растительного молока Вестник ЮУрГУ. Серия «Пищевые и биотехнологии», Т. 11, № 1. С. 46-54 (year - 2023) https://doi.org/10.14529/food230105

2. Naumenko N.V., Iakovchenko N.V., Radkevich A.V., Piven' N.G. Современные подходы в получении сырьевых ингредиентов на основе растительного сырья Сборник научных статей и докладов IX Международной научно-технической конференции "ПРОДОВОЛЬСТВЕННАЯ БЕЗОПАСНОСТЬ: НАУЧНОЕ, КАДРОВОЕ И ИНФОРМАЦИОННОЕ ОБЕСПЕЧЕНИЕ" (Воронеж, 15-17 декабря 2022), С. 304-309 (year - 2023)