NOVEL FUNCTIONAL FOOD FOR PREVENTION OF NON-COMMUNICABLE DISEASES

© 2019 V. V. Bati et al. Published by the Ivan Franko National University of Lviv on behalf of Біологічні Cтудії / Studia Biologica. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://www.budapestopenaccessinitiative.org/ and Creative Commons Attribution 4.0 License), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. UDC: 579.676

This work is devoted to a coverage of our new approaches in a design of functional food products (FFPs). In our opinion, the necessary requirement for the construction of such new FFPs is a use of sequenced microorganisms with (clinically) proven beneficial biological properties (as probiotic components or microbial fermentation, starters of fermentation) and edible plants including berries which in turn are a source of prebiotic and biologically active substances, whose content largely depends on the conditions of cultivation and methods of technological food processing.
We have developed and proposed for production of FFP with the expected (prognostic, predefined) specific properties. This is a sour milk drink consisting of pre-selected with biological properties established in vitro and in vivo [3,4] the probio tic composition of authors strains of microorganisms Lactobacillus casei ATCC 27139, Lactobacillus plantarum JCM 1149, L. casei MGB63-1, L. plantarum and prebiotic component presented by extracts (freshly dried juice) of сherry plum, blueberries, pomegranate, persimmon and сornelian cherry. These plant extracts (berries) are rich in biologically active substances (BAS) and are characterized by the ability to modulate the microbiocenosis of human intestine causing its correction towards a reduction of pathogenic and opportunistic pathogens and growth of beneficial microorganisms, antioxidant and antiinflammatory properties [13,26].
Functional product possesses antibacterial activity against pathogens of the intestinal infections of Escherichia coli (EPEC), Salmonella enterica subspecies enterica serovar Enteritidis (S. enteritidis), Shigella dysenteriae until complete inhibition of their growth after 72 hours of co-cultivation. Also, an essential antibacterial activity of sour milk beverage with blueberry extract was established in relation to a number of the opportunistic microorganisms such as Morganella morganii, Staphylococcus aureus,

INTRODUCTION
Modern studies indicate that human microbiome is the most accurate (sensitive) indicator of it's physiological health. According to experts from the World Health Organization (WHO), the health of human depends on nutrition as the most important factor regulating human microbiome [11]. Changes in microbiota are closely related to functioning of all macroorganism systems and associated with metabolic diseases [8], including obesity, type 2 diabetes, cardiovascular diseases, etc. Today, noncommunicable diseases (NCDs) rank first in the mortality of the population without exception of all countries of the world.
The most important factor that regulates intestinal microbiota of human is nutrition [10]. Today, food is considered not only as a set of essential nutrients for growth and development, but as an important component of optimal human health [9]. For a correction of human microbiota, it is often recommended to prescribe pro-, pre-or synbiotics. However, their use does not lead to a prevention of the NCD's due to a lack of probable criteria for assessing their efficacy in clinical trials and sporadic and non-personified use [18].
In in vivo studies, researchers found that cow's non-fermented milk could cause an inhomogeneous (negative) reaction to the intestinal microbiota. However, the use of fermented cow's milk, such effects were absent due to fermentation [24].
The most promising method of designing innovative functional food products of vegetable origin, and primarily fermented FFP, with their proven ability to exert a positive influence on human health is to carefully select an original microbial leaven, to obtain complete reliable information on a course of enzymatic processes and the ability of their components to directed correction of intestinal microbiota [18] (oppression of conditionally pathogenic representatives of microbiota and neutral or stimulatory action against beneficial microorganisms) and presence of proven clinical anti-inflammatory properties.
According to classical views [13,19], functional foods include food and beverages whose use leads to regulation of a number of physiological processes of the human body to prevent the emergence of human diseases including (partially) caused by ISSN 1996- immune-metabolic disorders of the body. Modern studies of human microbiota and its functional properties (so-called function) open up today the possibilities for constructing a new generation of FFPs [24]. In this regard the most promising is a creation of FFP capable of direct and regulate intestinal microbiota of the individual, in particular for the prevention of obesity, type 2 diabetes, cardiovascular diseases and other noncommunicable diseases (NCDs) [6].
The aim of our study was to select in vitro and in vivo probiotic microorganisms, components of berries with predetermined properties. We developed on their basis a functional new generation food for prevention of NCD associated with the disturbed exchange of substances with probative use for regulating the intestinal microbiota. Besides, we developed the technological scheme of its production and study its properties.

MATERIALS AND METHODS
The basis for a compositional functional fermented drink was a pasteurized cow milk leavened by (L. casei ATCC 27139, L. plantarum JCM 1149, L. casei MGB63-1, L. plantarum in an amount not less than 10 7 CFU/ml each strain). Previously, their high and selective antagonistic activity with respect to a number of opportunistic bacteria (namely, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli enteropathogenic (EPEC), Enterobacter cloacae, Morganella morganii, Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Staphylococcus hominis, Proteus mirabilis, Salmonella enterica subsp. enterica serovar Enteritidis, Shigella flexneri) has been demonstrated [3]. It was determined by the method of delayed antagonism and agar blocks behind Yegorov [3]. In addition, these strains of lactobacilli are characterized by specific immunomodulatory properties studied earlier in experiments on mice of the BALB / C line [3] and synergistic properties [6]. In similar studies, we have shown the ability of probiotic microorganisms to stimulate the local immune response and mechanisms that provide it [15,19].
To make a sour-milk drink, we chose the native homogenates of the above-mentioned edible plants -dill (Anethum graveolens) and pomegranate (Púnica granátum), blueberries (Vaccinium murtillus L.) and cherry plum (Prunus divaricata) obtained using the Grindomix™ electric mixerin provided sterile conditions by using nylon nano-filters with a pore width of 44 µm (BD Falcon , USA).
We used the dairy product prepared in the laboratory conditions. The manufacture of this drink was carried out under sterile conditions using classical technologies, according to the technological scheme ( Fig. 1). Physico-chemical, organoleptic and sanitary-hygienic indices were determined according to DSTU 2661: 2010. The process of fermentation was controlled for acidity 75-90 °Т.
Testing of the pro-and antimicrobial activity of end functional product was carried out by in vitro compatible culture experiment by placing 10 ml of suspension of test strains of microorganisms (24-hour cultures) in a concentration of 0.5 according to Mc-Farland (1.5×10 8 CFU/ml) in test tubes. After 24, 48 and 72 hours. Their co-incubation was determined by the number of microorganisms by sowing 10 µl in a triple repetition on an appropriate nutrient medium.
The tested microorganisms belonged to different phylogenetic groups, and their etiological role was also distinct. These strains included: 1) museum strain test cultures (collectable strains) E. coli ATCC 25922 (F-50), S. aureus ATCC 25923 (F-49), P. aeruginosa ATCC 27853 (F-51)); 2) clinical isolates -pathogens of opportunistic infections (P. mirabilis, S. aureus and MRSA, E. cloacae, K. pneumoniae, P. aeruginosa), food For mathematical analysis of data, IBM SPSS Statistics V. 22.0 software was used; graphic processing of the data was done using Origin 10.

RESULTS AND DISCUSSION
Nutrition is undoubtedly a major modifiable determinant of a disease. At the recent United Nations general assembly on the NCD prevention and control, the importance of establishing preventative health strategies was widely acknowledged [17]. One of the main factors was proposed changes in food choices and the increased use of functional foods to prevent of NCD [17,23,25]. Modulating the intestinal microbiota via dietary interventions is becoming a promising strategy for verifying a cause of chronic inflammation involved in obesity and metabolic syndrome [25].
When creating a new generation FFPs one of the main steps is to select and justify a choice of ingredients that should provide new expected properties of the composite product.
Based on the results of our previous studies [3,15], we have proposed the principles of creating a new generation FFP based on plant components and microorganisms for the purpose of their prognostic and personified application for preventing the emergence of noncommunicable diseases [6], through correction of microbial cenosis in the intestines of the macroorganism .
We produced a functional sour milk drink based on the lactobacilli with the desired functional properties -the ability to provide antimicrobial action of opportunistic microorganisms and to modulate the intestinal microbiota. Other scientists proved in vivo that if an insufficient number of lactobacilli observed a significant decrease in the number of bifidobacteria [3].
Our task was to choose natural plant fillers rich in BAS to enhance the qualities of the functional product, that would have the ability to synergize and provide the desired effect on the intestinal microbe. For this purpose, we have selected previously extracted edible extracts of various edible plants with known effects on different groups of microorganisms [13,30].
Our studies have shown that selected extracts not only inhibited the main representatives -test and clinical strains, pathogenic and opportunistic bacteria, but were capable of stimulating useful commensal microorganisms of the intestine. Such actions were characterized by the extracts of blueberries (V. murtillus L.), aliche (P. divaricata), pomegranate (P. granátum), dill (A. graveolens) [13]. Prebiotic plant components rich in BAS are able to regulate a balance of microbiota in the gastrointestinal tract -shown in experiments in vivo, previously investigated and characterized by an expressive antioxidant and anti-inflammatory properties [13,30]. Thus, created by their combination in the form of a new functional beverage can potentially prevent the emergence of both infectious and human NCD, caused by respectively pathogenic and opportunistic microorganisms or microbial disorders and associated chronic inflammation [3,12].
A brief technological process is described here: pasteurized milk (T = 80 °С, 10-15 min) was cooled to T = 40 ±2 °C, introduced with the strains of microorganisms and mixed. The function of the starter cultures is to ferment lactose (milk sugar) to produce lactic acid. An increase in lactic acid decreases pH and causes the milk to clot, or form a soft gel that is characteristic of sour milk drink [5]. By using a thermostat method, milk is held at 40±2 °С until a pH 4.7 is reached. This allows the fermentation to form a soft gel and the characteristic flavor of sour milk drink. This process can take 6-8 hours after which the half of product was cooled and stored at 3-5 °C for 3-7 days. In order to prevent the destruction of BAS just before the use in the end intermediate product, homogenates of blueberries / cherry plum / pomegranate or fresh dill were added in the ratio of 5-7 percent. A technological scheme of making a sour-milk drink is shown in Fig.1. Throughout a period of storage of the half-finished product, the number of authored microorganisms were determined by seeding serial dilutions of the FFP on the medium with added marker antibiotics for indicating each of strains of lactobacilli. During the first 7 days, high titres of lactobacilli above 10 8 CFU/ml (according to DSTU 2661: 2010, not less than 10 7 CFU/ml), on the 8th day of storage, the number of all lactobacilli decreased significantly that substantiates the suitability of the product within 3-7 days (Fig. 2).  The proposed sour-milk product with author›s strains and various edible plant extracts were characterized by a selective antimicrobial ability in addition to the anti-inflammatory effect that was proven earlier.
Data listed in Table 1 indicate the ability of this semi-finished product (without plant extracts) after 48 hours of compatible cultivation to delay the growth of the opportunistic and pathogenic strains of microorganisms: EPEC, Salmonella enterica and Shigella dysenteriae. For 72 hours of co-cultivation, it was found at least a smaller but still significant inhibitory effect of this semifinished product on the comensal Escheri chia stains -E. coli 058 and E. coli (Shaedler's strain) was less significant, the micro-organism titers were sown in amount (2.0 ± 0.1)×10 2 CFU/ml and (2.5 ± 0.3)×10 2 CFU/ml. A decrease in concentration of E. coli strains ATCC 25922 and E. faecalis was observed at a later date and to a lesser extent.
At the same time, we established the pro-bacterial property of this semi-product with a respect to the representatives of commensal microbiota, such as L. acidophilus, L. salivarius, as well as probiotic microorganisms B. subtilis 8130 and S. cerevisiae cultured throughout the study in a large number (10 8 -10 9 ) CFU/ml. However, this intermediate product did not exhibit bactericidal action against P. mirabilis, P. aeruginosa, E. cloacae, in contrast to the author strains characterized by such action.
Examples of the antagonistic activity of the developed half-product are also shown in Fig. 3  The same method of co-cultivation determined the spectrum of anti-and pro-microbial properties of the final functional product already with the blueberry extracted (homogenate) added ( Table 2). The end product, similar to the previous half-product, had an ability to inhibit the pathogens of intestinal infections of EPEC, Salmonella enterica subsp. enterica serovar Enteritidis, S. dysenteriae until a complete inhibition of their growth after 72 hours of co-cultivation. Also, the essential antibacterial activity of the sour milk beverage with blueberry extract was established in relation to a number of the opportunistic microorganisms such as M. morganii, S. aureus, MRSA, E. coli. The same effect was observed for E. coli ATCC 25922 collection strain.  At the same time, a new generation of FFP stimulated a growth of representatives of useful microbiota. So the amount of lactobacilli isolated from the mucous membranes of the mouth and intestines of healthy individuals were stable for 72 hours of compatible cultivation -up to 10 8 -10 9 CFU / ml, and the content of the probiotic L. fermentum strain was also high.
The content of representatives of E. coli, E. faecalis, E. coli Sсhaedler's strain, E. coli 058 also decreased somewhat, and the strains did not exceed the amount of 10 2 -10 3 CFU / ml on the third day of testing.
Despite the previously established ability of blueberries extract to completely suppress the growth of clinical strains P. mirabilis and P. aeruginosa, the end product was not characterized by such properties. Figure 4 presents examples of the antagonistic activity of a end sour-milk drink with a native extract of blueberries using a co-cultivating method in relation to EPEC and E. coli ATCC 25922; M. morganii; Salmonella enterica subsp. enterica serovar Enteritidis.
The results of the combined culturing effects of the tested microorganisms in the developed fermented milk product with cherry plum are shown in Table 3.
The antibacterial activity in vitro of the sour-milk product with pomegranate for a prevention and treatment of food-borne toxic infections and non-infectious human pathologies caused by the excited metabolism of the following strains of microorganisms: For the third day of testing a number of K. pneumoniae also decreased, and did not exceed the amount of 10 7 -10 4 CFU / ml. However, it has been noted that suppressive action has increased with the addition of native extracts (blueberries / cherry plum / dill / pomegranate). Cherry plum and pomegranate had a greater antagonistic effect on K. pneumoniae and Klebsiella oxytoca.  The obtained data are relevant because it proves a possibility of creating (constructing) a new generation of FFP with predetermined properties, namely those that are capable of selectively adjusting the balance of microbiota of the gastrointestinal tract and preventing the emergence of infectious or associated with inadequate nutrition of somatic pathologies.
We used new approaches for production of FFP. They are characterized primarily by the patient-oriented (personified) purpose, as they allow adjusting their composition based on individual indices of intestinal microbial patients or healthy individuals. It is the basis of targeted diet and prevention of diet and diagnosis of human diseases by supporting and / or correction pro-or anti-inflammatory processes of mucous membranes of the body [6] associated with the colonization of mucous membranes by the microorganism.

CONCLUSIONS
We proposed new principles and approaches in a design of new generation of FFPs developed on the basis of pre-selected plant components and microorganisms for the purpose of their prognostic and personalized use to prevent the emergence of NCD through targeted correction of microbial cenosis in the gut of the macroorganism and effective also in the treatment of a certain number food poisoning.
The antibacterial activity in vitro of the sour-milk product for the prevention and treatment of food-borne toxic infections and non-infectious human pathologies caused by the excited metabolism of the following strains of microorganisms: E. coli ATCC 25922, EPEC, Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis), S. dysenteriae was also established in in vitro studies. The dairy drink with cherry plum showed antibacterial activity against strains of microorganisms: E. coli ATCC 25922, EPEC, S. aureus, MRSA, S. Enteritidis, S. dysenteriae, P. aeruginosa, E. cloacae; The dairy product with blueberries was suppressed by: P. aeruginosa, M. morganii, L. monocytogenes, S. Enteritidis, S. dysenteriae, S. aureus, E. coli ATCC 25922, EPEC, MRSA, and also comensal strains of E. coli 058 and E. coli Schaedler's strain. It was noted that a suppressive action has increased with the addition of native extracts (blueberries / cherry plum / dill / pomegranate). Cherry plum and pomegranate had a greater antagonistic effect on K. pneumoniae and Klebsiella oxytoca.
A probacterial activity of sour-milk drink in relation to representatives of useful microbiota of functional product with a pomegranate/ blueberries / cherry plum / dill in relation to strains L. fermentum and L. acidophilus.
A new sour-milk product of variable composition of components with synergistic action provides a simultaneous normalization of intestinal microbiota and suppresses growth of opportunistic and pathogenic microorganisms. The added value of such product is the presence of BAS of edible plants and berries of local origin characterized by a proven anti-inflammatory action.

ACKNOWLEDGMENTS
This work is funded under the EU FP7 Theme 2: "Food, agriculture, fisheries, and biotechnology", Project BaSeFood ; Grant Agreement no: 227118. [http://www.basefood-fp7.eu/www.basefood-fp7.eu/index.html'] and State governmental budget of Ministry of Education and Science, Topic: "Implementation of new approaches to the creation and applying of modern pharmabiotics" Registration number 0117U000379.