Selection methods for probiotic microorganisms with high adhesive properties

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Introduction
The Russian Biotechnological University is working to expand the collection of industrially valuable microorganisms. One of the most important areas is the selection of crops with probiotic potential. Probiotics are preparations based on living microorganisms designed to correct the autochthonous microflora of humans and treat a number of diseases.
The classification of probiotics includes three groups [30]:
• Medical probiotics are microbiological preparations that contain strains of live or inactivated microorganisms. In this case, we are talking only about medicines that have clearly defined indications for use.
• Probiotics-biologically active additives are complex preparations based on living microorganisms, manufactured at pharmaceutical and other enterprises, which are used as biologically active additives (hereinafter – dietary supplements) to food and, as a rule, are distributed through the pharmacy network.
• Alimentary probiotics are cultures of microorganisms or products enriched with them that play the role of a supplement to nutrition.
Selection of probiotics is based on safety, functionality and manufacturability.
Requirements for the safety of microorganisms used as probiotics:
• must belong to the same species as microorganisms – representatives of the microflora of the gastrointestinal tract (hereinafter referred to as the gastrointestinal tract) of a healthy person;
• must be non-pathogenic and non-toxic;
• should not carry transmissible antibiotic resistance genes.
The selection of preferred probiotic microorganisms includes the following functional properties of the cultures under consideration (shown in Fig. 1): resistance to digestive juices, enzymes and bile of the gastrointestinal tract; adhesion to epithelium and engraftment in the human digestive tract; immunomodulation and immunostimulation of the body; optimization of metabolic processes; antagonism to pathogenic and conditionally pathogenic microorganisms; antimutagenic properties.
When selecting starter cultures and starter cultures with probiotic properties, including those used in the food industry, such technological aspects as a high degree of survival in the technological process, antagonistic activity in relation to sanitary-indicative microflora, stability in products and viability during storage should be taken into account.

Figure 1. Positive effect of probiotics on human health [20]

Selection of probiotic microorganisms is carried out in accordance with the following regulatory legal documentation:
• Methodological guidelines of MU 2.3.2.2789–10 "Food raw materials and food products. Methodological guidelines for the sanitary and epidemiological assessment of the safety and functional potential of probiotic microorganisms used for food production";
• Methodological guidelines of MUC 4.2.2602–10 "System of pre-registration preclinical study of drug safety. Selection, verification and storage of production strains used in the production of probiotics";
• OFS.1.7.1.0008.15 Probiotics;
• Methodological guidelines of MUC 4.2.1890–04 "Control methods. Biological and microbiological factors. Determination of the sensitivity of microorganisms to antibacterial drugs".
Lactic acid bacteria are used as probiotics, starter cultures and starter cultures with probiotic properties in most cases. Lactobacilli are non–spore-forming, gram-positive, immobile, rod-shaped bacteria, differing in facultative anaerobic processes: they are able to grow in an anaerobic and aerobic environment, producing lactic acid as the final product of fermentation [13]. The human gastrointestinal tract is colonized by several Lactobacillus species, including Lactobacillus acidophilus, L. brevis, L. casei, L. fermentum, L. gasseri, L. johnsonii, L. paracasei [14, 15]. Some lactobacilli have been approved by FAO/WHO as living microorganisms that, when introduced into the body, benefit human health [16]. This is what distinguishes living microorganisms directly used as probiotics from those used in fermentation to produce food products.
Lactobacilli affect the human gastrointestinal tract in several ways, for example, inhibit the growth of pathogenic microorganisms due to the ability to synthesize lactic, propionic and acetic acids. The accumulation of the above organic acids reduces the pH of the medium, as a result of which there is a suppression of the growth and development of pathogenic microorganisms [12, 21, 22]. Another mechanism is the competitive prevention of attachment of pathogens to the gastrointestinal epithelium [9, 23-26].
The gut microbiome is the main site of innate and acquired immunity [2-6]. It is covered with mucus (mucin), which contains most of the microbiota and is the site of adhesion, whether probiotic, opportunistic or pathogenic microorganisms. Mucus is a gel layer necessary for hydration and lubrication, as well as a barrier against pathogenic microorganisms and toxins [7-9]. It consists mainly of water, glycoproteins, salts and lipids. Mucins are large extracellular proteins that are highly glycosylated (about 80% of salts and lipids) [10, 11]. The scheme of the gastrointestinal mucosa and mucin is shown in Fig. 2.

Figure 2. The scheme of the gastrointestinal mucosa and the structure of mucin [10]

Protein fragments of mucins have a central glycosylated region consisting of sequence repeats rich in serine, threonine and proline, N- and C-terminal regions, and also contain residues of cysteine and oligosaccharides [27-29].
Probiotic bacteria play a potential protective role against enteropathogens through various mechanisms, including the production of antimicrobial compounds, reduced adhesion of pathogenic bacteria, and competition for host cell binding sites. Competitive displacement by probiotic bacteria has a beneficial effect not only on the intestines, but also on the urogenital tract and oral cavity. In vitro studies with various intestinal cell lines have been widely used in recent decades to assess the ability of probiotic bacteria to adhere and antagonism to pathogens.
Adhesion, that is, attachment to the epithelial cells of the gastrointestinal tract, is one of the important properties of probiotic strains, therefore, the determination of adhesive properties is considered a necessary step for the study of probiotic microorganisms. It has been established that the adhesive ability is a strain-specific feature, which should be taken into account when selecting probiotic cultures. In this regard, an effective method for determining the adhesive properties of the probiotic strains under study is required.
The purpose of this study was to develop a comprehensive method of selection of highly active probiotic microorganisms capable of proliferation and complementation of the autochthonous intestinal microflora of an individual.
To achieve this goal , the following tasks were formulated:
1. To develop a scheme for selecting promising probiotics according to the level of adhesive activity of strains belonging to the most commonly used types of microbial cultures in clinical practice.
2. To determine the adhesive activity of probiotic cultures on red blood cells.
3. To determine the adhesive activity of probiotic cultures on the cell line.
Materials and methods.
The object of the study was cultures of lactic acid bacteria and yeast with probiotic potential, including the main species used in the food industry and clinical practice: Lactococcus lactis, Lactiplantibacillus plantarum, Enterococcus faecium, Streptococcus thermophilus, Saccharomyces cerevisiae var.boulardii.
Indicators of probiotic cultures of microorganisms, such as the degree of adhesion, were determined according to standard methods described in the Guidelines of the MUC 4.2.2602–10 "System of pre-registration preclinical study of drug safety. Selection, verification and storage of production strains used in the production of probiotics".
When determining the adhesive activity of lactic acid bacteria on cell cultures, the cell culture was grown on a 6-well plate under conditions of 5% CO2 until the formation of a monolayer for 5-7 days. The resulting monolayer of CACO2 cells was inoculated with 1 ml of a bacterial culture of a certain concentration. After inoculation, the cultures were kept for 1 hour at 37 ° C, then washed with saline to remove unbound bacterial cells. Removal of the monolayer with associated bacterial cells was carried out with solutions of 0.02% Versene and 0.25% trypsin in a ratio of 3:1. To count the associated bacterial cells, the dilution method was used when growing cells on a dense MRS medium.
Discussion and results.
The most noticeable point of interaction between microorganisms and humans is located on the mucous membranes, which indicates that mucus adhesion is the main target for controlling the settlement of probiotics. The methodology for quantifying bacterial adhesion to mucus consists in quantifying the proportion of residual bacteria associated with functional mucus surfaces after washing. Quantification of residual bacteria can range from simple cell counting to surface plasmon resonance or atomic force microscopy. The most common method of quantifying mucus adhesion is the use of a fluorescent indicator as a correlate of cell concentration. Surfaces are often modified by incubation with mucus or by culturing intestinal epithelial cells/organ tissues [31].
However, this method is quite expensive and impractical for use in the initial screening of lactic acid bacteria and yeast with probiotic potential.

About the authors

Marya S. Kanochkina

Russian Biotechnological University; Research Organization “Microbial Nutrients Immunocorrectors” LLC

Email: kanoch@yandex.ru
ORCID iD: 0000-0001-6077-5957
SPIN-code: 2584-6474

Cand. Sci. (Tech)

Russian Federation, 11 Volokolamsk Highway, 125080 Moscow; Moscow

Ivan A. Fomenko

Russian Biotechnological University

Email: fomencoia@mgupp.ru
ORCID iD: 0000-0003-2478-1705
SPIN-code: 5861-2838

Cand. Sci. (Tech)

Russian Federation, 11 Volokolamsk Highway, 125080 Moscow

Irina M. Chernukha

Federal Research Center for Food Systems

Email: imcher@inbox.ru
ORCID iD: 0000-0003-4298-0927
SPIN-code: 3423-3754

Doc. Sci. (Tech)

Russian Federation, Moscow

Natalia G. Mashentseva

Russian Biotechnological University

Author for correspondence.
Email: natali-mng@yandex.ru
ORCID iD: 0000-0002-9287-0585
SPIN-code: 9791-5806
Scopus Author ID: 57060483400
ResearcherId: R-8014-2016
https://mgupp.ru/obuchayushchimsya/instituty-i-kafedry/teacher.php?CODE=27797

Doc. Sci. (Tech), Professor

Russian Federation, 11 Volokolamsk Highway, 125080 Moscow

References

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