Probiotics of Georgia and “Caucasian Longevity” Phenomenon
Investigation of Probiotics, Isolated from Traditional Fermented Milk Products and Ecological Isolates in the Regions of Georgia with Phenomenon of “Caucasian Longevity”: Genetic Typing and Creation of Live Cultures’ Bank
Tech Area / Field
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
3 Approved without Funding
Tbilisi State Medical University, Georgia, Tbilisi
- Biophage Pharma Inc., Canada, QC, Quebec City\nQuanta Medical, France, Rueil-Malmaison
Project summaryPrincipal biological feature of the bacteria of the genus Lactobacillus, that allows them to colonize various biotypes of human and animal bodies and successfully compete with other representatives of microbial biocenosis, are their fermentative, adhesive and antagonistic activity, resistance towards highly acidic environment and resistance towards digestive enzymes. Antagonistic features of lactobacilli are conditioned by the production of organic acids, lysozime, hydrogen peroxide and formation of bacteriocins. Antagonistic activity and adhesive features determine the value of the strain and possibility of its use for production of probiotics. These characteristics are strain-specific and may significantly differ among the representatives of the same genus.
Owing to their characteristic features, bacteria of the genus Lactobacillus frequently represent basic components of pharmaceutical preparations of probiotics, biologically active food supplements and functional food products. Consumption of food enriched with lactobacilli contributes to normalization of intestinal microbiocenosis and gastrointestinal function, and consequently – to enhancement of immune system and maintenance of homeostasis if human body.
Numerous data indicate that fermented milk products have been used since ancient times in Georgia. Products of fermentation of the milk of different animals (cows, goats, sheep, buffalos etc.) differ not just by milk origin, but also by methods of preparation, physical, chemical and organoleptic indices and microbial flora.
One of the most ancient fermented milk products being used in Georgia is matsoni. Georgians were familiar with its positive effects at various gastrointestinal diseases, liver diseases, skin diseases at least five centuries ago (Georgian medicinal book “Karabadini”). Matsoni was used in the form of complex compresses (moist packs) etc.
In early 1980ies researchers of Gerontology Department of Institute of Morphology of Georgian Ministry of Education have initiated investigation of microbial profile of fermentation starters used for production of so called “farmer’s matsoni”, produced in the remote regions, far away from milk factories. The latter condition seems particularly important to us, since such approach allows making a collection of endemic strains of lactobacilli. All samples of starter cultures were collected in populations characterized with high longevity index.
It has been proved by standard microbiological and genetical methods (PCR-analysis) that dominating microbial flora of investigated starter cultures is represented by Lactobacillus delbrueckii ssp. bulgaricus JCM,1002T and Streptococcus thermophilus ATCC 19258T. Beside above-mentioned cultures, enterococci and bifidobacteria have been isolated, although their species identification has not been carried out because of lack of financing.
On the basis of dominant microbial flora, starter culture for fermented milk product “Mariami” has been prepared out of highly active strains, and technology of milk fermentation has been developed (Patent # 254,09.07.1993, Georgia).
In proposed project we refer to the phenomenon of longevity in number of ethnical groups of population of certain regions (e.g. Caucasus), were active longevity of inpiduals correlates with certain stability of the main link of intestinal microbial flora. In this case, number of lactobacteria does not decline with age and remains on high level even in inpiduals above 95 years of age. Investigation of species composition of fermentation starters used for preparation of national fermented milk products, isolation of bacterial cultures and investigation of their characteristics is timely issue for identification of new strains of lactic acid microorganisms, which could be promising for production of functional food products for specific categories of consumers (age, illness (including allergies), occupation). Introduction of bacterial cultures isolated from national fermented milk products into the range of industrial starter cultures will lead to persification of fermented milk products, characterized by positive impact on human health and improvement of immune status. One of the most topical objectives of the project is assessment of anti-allergic properties of lactic acid bacterial cultures.
Genotyping and pasportization of lactic acid bacteria, isolated from national fermented milk products of functional food consumed in the regions of Georgia with prevalence of active longevity, will be carried out in the frames of this project. The appropriate sample of lactic acid bacteria will be selected out of the number of investigated isolates of probiotics, on the basis of valuable properties and biological activity, for further use in technology of functional food production for maintenance and correction of microbial ecology of human body.
In this regard, certain regions of Georgia, differing from each other by above-mentioned aspects and at the same time, distanced only by tens of kilometers, represent a convenient model for observation. Physical and geographical conditions of these regions vary from subalpine zones to semideserts and subtropical climate zones. At the same time, nutritional specifics of above-mentioned regions (constant prevalence of milk products in the diet) allows to conduct concurrent search for local strains of obligatory representatives of intestinal microflora both inside the body and in typical food products. Diversity of subjects of observation presumably should precondition persity of the components of intestinal microbiocenosis on species, subspecies and strain level. However, comprehensive comparative investigation of “profile” biological properties of isolates is virtually impossible without prior creation of live cultures’ collection. After passportization and detailed molecular and biological characterization of isolates, investigation of the mechanisms of their biological activity (including probiotic activity) will open new prospects from the practical point of view, as well.
The Goal of the Project is creation of the basic collection of live cultures of obligatory representatives of human and animal intestinal microflora (lactobacteria, bifidobacteria, enterococci), isolated in various regions of Georgia. The sources for culture isolation can be represented by local healthy elderly inhabitants, home-made fermented milk products, or cattle – as milk producer.
Project presents an opportunity for eight qualified weapon scientists/specialists, experienced in weapon specialty as well as in the sphere of medical and biological studies. All of them are doctors of science. Activities planned in the frames of the project, undoubtfully will rise significant interest among foreign specialists from number of countries, and therefore, will promote integration of Georgian scientists into international research community.
Presented project envisages support of fundamental investigations solely for peaceful purposes and has a great significance not only for the future, but for the present as well, since in the conditions of current crisis, caused by deep political, economical and ecological changes during past decade, elderly population became one of the groups with highest risk of morbidity and mortality, which requires design of revolutionary preventive approach to the issues of health of population in general, and prolongation of life and maintenance of health on the late stage of ontogenesis, in particular.
Following technical provision of the project, during the first year collection of natural isolates of lactic acid bacteria (lactobacteria, bifidobacteria and enterococci) will be carried out in 8 regions of Georgia, from the various sources: milk products and human and animal intestine,. Bifidobacteria will be isolated only from human/animal intestines. Collection of 10 strains of each genus of bacteria in each of the regions is planned for the first year (about 80 strains for each genus, about 240 strains in total). Genotyping of all strains will be conducted (ex MLST). Their taxonomic identity will be determined by means of classical microbiological tests, based on physiological and biochemical properties of bacteria. For bifidobacteria, their resistance towards oxygen will be evaluated, taking into consideration that selected bifidobacteria will not be strict anaerobes and will tolerate effects of oxygen. After physiological and biochemical characterization of the strains of lactic acid bacteria, the following steps of characterization will be taken: testing for in vitro survivability in gastrointestinal tract, determination of resistance towards antibiotics (determination of MIC levels in accordance with European standards), confirmation of species identity by sequence analysis of 16S рRNA, adhesive ability and anti-microbial activity towards test-strains of opportunistic microorganisms of human gastrointestinal tract.
Second year of the project implementation will be dedicated to collection of natural isolates from above-mentioned sources and physiological, biochemical and genetical characterization of lactic acid bacteria in 12 regions of Eastern Georgia. Collection of 10 strains of each bacterial genus is planned from each of the regions during the second year (about 120 strains for each bacterial genus, 360 strains in total).
Throughout the project implementation process, during collection of natural isolates, parallel with characterization and passportization of lactic acid bacterial cultures, the database of probiotic strains will be created. At the end of the third year, the most promising strains will be selected from the collection of lactic acid bacteria based on their probiotic and other properties, potentially valuable for the industry.
Regular exchange of information with foreign collaborators will be carried out throughout project implementation process. They will participate in crosscheck of obtained results, will offer advice, remarks and comments in the process of preparation of technical and reporting materials (quarterly, annual, final etc.) that will be regularly sent to ISTC, which will undoubtfully improve quality of outcomes. Joint symposia and workshops will be organized.
Assessment of health status of older adults will focus on functional assessment, including physical, cognitive/mental (e.g. thinking and remembering), psychological, and social aspects of a person’s life, as well as "quality of life" - combination of all above-mentioned functional aspects of life. This approach considers rapid screening of several spheres by asking screening questions, related to various areas of health and function. Whenever a potential problem is found, it will be evaluated more completely. Answers to screening questions are based on the older inpidual’s day-to-day activities. In case of necessity, another person, familiar with the inpidual being assessed (e.g. a caregiver or family member), will be asked to provide (or verify) additional information about his/her daily functions.
Bacteriological methods will be employed for isolation of lactobacteria, bifidobacteria and enterococci from the feces of healthy long-livers and from the food products.
Isolated cultures may be subjected to molecular and biological typing. The goal of the molecular-biological group is precise identification of investigated probiotics by means of PCR, using species-specific primers. Besides, one of the main objectives of the group is molecular genotyping of the strains of lactobacilli, bifidobacteria and enterococci, isolated in various regions of Georgia from various fermentation starters of milk products. Passportization of these strains and creation of database represent the most important stage of our study.
The following two methods of genotyping are most acceptable for above-mentioned bacteria:
- Randomly amplified polymorphic DNA (RAPD);
- Pulsed Field Gel Electrophoresis (PFGE).
RAPD is widely used for genotyping of various species of lactobacilli and can be used in case of bifidobacteria and enterococci, too. PFGE is a universal method of genotyping of bacteria, embracing entire bacterial genome and, in contrast with many other methods of typing, has strong differentiating ability.
Much interest is aroused by study of plasmid spectrum of lactobacilli, as well, as they carry some of the genes playing significant role in functioning of bacteria. Investigation of gene composition of plasmids in endemic species of lactic acid bacteria is one of the main objectives of this project.
The International Science and Technology Center (ISTC) is an intergovernmental organization connecting scientists from Kazakhstan, Armenia, Tajikistan, Kyrgyzstan, and Georgia with their peers and research organizations in the EU, Japan, Republic of Korea, Norway and the United States.
ISTC facilitates international science projects and assists the global scientific and business community to source and engage with CIS and Georgian institutes that develop or possess an excellence of scientific know-how.