Results phase I/2012

Discovery of lactic acid bacteria strain probiotic effect determined during last years the need for understanding the mechanisms involved in the beneficial impact on human health especially because these effects are strain specific. In order to achieve the project’s objectives, the first phase aimed to obtain a mini-collection of 224 new lactic acid bacteria strains isolated from specific ecological niches, their characterization and preliminary taxonomic classification.

The sources of isolation were varied and covered a broad ecological spectrum, represented by: newborn feces, milk, fermented food and fermented vegetable. In order to characterize and to identify, newly isolated and purified lactic acid bacteria strains were analyzed in terms of morphology (macroscopic or microscopic appearance), physiology (growth at various temperatures and halotolerance) and biochemical traits (catalase production, organic acids production, capacity to metabolize different carbon sources). Subsequently, to confirm the taxonomic status, strains were analyzed using standardized API kits and BIOLOG system.

The results have shown variations in morpho-physiological and biochemical traits of the isolates according to isolation niche. In terms of cell morphology, strains isolated from fermented products are generally cocci and strains isolated from newborn feces presented mainly the bacillary and coccobacillary forms. Strains from human gastrointestinaly tractus have lower capacity for biosynthesis of organic acids and low resistance to temperature changes compared with strains isolated from fermented products. After all microbiological and biochemical determinations carried out, newly isolated lactic acid bacteria strains were identified as belonging to the genera Lactobacillus, Lactococcus, Enterococcus, Bifidobacterium, Streptococcus and Pediococcus.

All isolated and characterized strains were cryopreserved and stored in the collection of the Department of Genetics, Faculty of Biology.


Results phase II/2013

The main objective of this phase was to select the lactic acid bacteria (LAB) strains with probiotic potential from the stock of newly isolated strains (224 strains) using conventional and modern methodology.

The studies focused on: in vitro determination of the safety use as probiotics for human health of the selected strains; qualitative and quantitative evaluation of the antimicrobial activity against enteropathogenic microorganisms; analysis of the interactions between lactic acid bacteria strains and eukaryotic epithelial cells; characterization of the biologically active compounds produced by LAB strains  and in vitro testing of the resistance of LAB strains under conditions similar to those of the gastrointestinal tract.

An important analyzed aspect was to establish if the selected strains does not affect human health and comply with FAO/WHO guide criteria. 30% of the studied LAB strains showed reduced hemolytic activity after 48 hours and its were excluded from the study. Due to the risk of the antibiotic resistance gene transfer to pathogenic strains, LAB strains were tested in order to determine the antibiotic sensitivity spectrum. It has been found that the majority of the analyzed strains presented sensitivity to chloramphenicol and resistance to kanamycin. In addition, 50% of the strains presented no resistance to antibiotics such as ampicillin, tetracycline, erythromycin and gentamicin, resistance known to be determined by a number of genes located in transposable elements or plasmids.

Some of the benefic effects of lactic acid bacteria are based on the synthesis of biologically active compounds such as compounds with antimicrobial activity. The results revealed that the majority of the selected strains show antimicrobial activity against pathogenic strains involved in production of gastrointestinal infections, for three of them (strains 19.3, 41.2, 26.1) this activity had been correlated with the biosynthesis of bacteriocins. It should be noted that the broadest spectrum of activity was presented by the bacteriocin isolated from strain 19.3. 

Regarding the LAB strains capacity for synthesis of important active compounds such as organic acids our analysis had shown that all strains of lactic acid bacteria presented high concentrations of lactic and acetic acid. Only one strain (34.1) biosynthesizes a broad range of organic acids (butyric acid, fenillactic acid, hidroxifenilllactic acid, succinic acid, malic acid and formic acid).

A criteria for the selection of probiotic strains is the assessment of their ability to adhere and interact with the host cells, allowing probiotic microorganisms to colonize the digestive tract and to perform their beneficial effects for a longer period of time. The studies carried out on cell line HTC-8 had shown that the LAB strains isolated from the digestive tract have a high capacity of adherence compared to the ones obtained from fermentated products. The majority of strains showed no cytotoxic effects and did not induced morphological changes of the epithelial cells.

Withal, the strains of lactic acid bacteria must withstand to the environmental conditions of the gastrointestinal tract. For this reason, the selected strains were tested in a unicameral system for gastrointestinal simulation (GIS1) in order to establish the resistance to different pH values, bile salts and digestive enzymes. The results showed that the resistance to digestive tract conditions depends on strain and on the conditions existing at each level of the digestive tract.

In conclusion, all the objectives of this stage were fully accomplished and some of the results were included in three articles published in ISI quoted journals.

It should be noted that the system used for testing the resistance of the lactic acid bacteria strains in the gastrointestinal tract conditions, developed by partner P3-USAMVB (team leader Professor Dr. Emanuel Vamanu) gained international recognition by winning the gold medal at the International Exhibition of Inventions, Geneva, 2013.

The aims proposed for this project stage have been completely attained and results have been included in 4 papers published in ISI journals, 3 papers published in IDB journals and, respectively, in presentations to international congresses / symposia, published as abstracts.


Results phase III/2014

In order to increase the beneficial effects of the probiotic microorganisms over the host organism and for an optimal use of such microbes, accurate understanding of their action mechanisms is needed. Thus, microbial strains from probiotic products should improve the health state of the host organism without changing the intestinal homeostasis; this in turn, requires rigorous testing of the microbial strains prior to include them in a potential probiotic product.
To attain the major aim of this step of the PROLAB project, that is obtaining of lactic bacteria consortia with probiotic effect, we have continued several research activities started in 2013 and we have also performed preliminary analyses that will be completed in 2015. The main research activities performed in this stage were: analyses of the interactions between lactic bacterial strains and human cell lines, respectively; analyses on the immunomodulating effect of the lactic bacterial strains / microbial fractions; analyses of the active biological compounds and microbial resistance to the transit through the human gastro-intestinal tractum; growth conditions influence upon the biosynthesis of antimicrobial compounds; evaluation of the growth inhibition between microbial strains within the consortia; evaluation of a potential synergism of the lactic bacteria beneficial effects as consortia; influence of the microbial strains to be used in consortia upon the human microbiota simulated in GIS1 system and chemical analyses of samples collected from GIS1. Our scientific results have lead to several microbial consortia consisting in selected lactic bacterial strains, that will be further analyzed in order to complete the final composition of the consortium having an optimal probiotic effect.
Results phase IV/2015

In last decades studies regarding probiotic products for improving the health of the consumer, were focused on obtaining new probiotic strains and highlighted the complexity of interactions between probiotic microorganisms and the human host (including microbiota of the gastrointestinal tract), finding that  positive effects were usually strain specific. The major goal of this project was to obtain strains and consortia of lactic acid bacteria with enhanced probiotic effect.

The operational objectives of phase IV-2015 were focused on: characterization of antimicrobial compounds produced by lactic acid bacteria and influence on culture conditions; the influence of consortia on human microbiota simulated in GIS2 system; chemically analysis of samples taken from GIS2 system; quantitative analysis of the compounds released by the epithelial cells in presence of lactic acid bacteria consortia, the cytotoxicity assay and the taxonomic identification of lactic acid bacteria strains by molecular techniques.

All objectives proposed in this phase have been achieved and 3 consortia were selected out of the 10 consortia obtained in the previous stage. The 3 selected consortia presented antimicrobial activity and enhanced immunomodulatory effect.

Some of the results were disseminated through publication in 4 ISI papers (3 of them were accepted and one was published), 2 BDI papers, 1 patent and 4 participation in international conferences.
It should be noted that the GIS2 system was awarded with the gold medal at the salon EUROINVENT 2015.


Results phase V/2016

The main objective of this phase was to assess the functionality of probiotic lactic acid bacteria consortium.

To attain this major aim of the project, the research activities performed were focused on: influence of consortium on human microbiota simulated in GIS2 system, chemical analysis of samples taken from GIS2 system, qualitative and quantitative analysis of consortium’s influence on adherence ability of pathogenic microorganisms to human gastrointestinal tract tumors, taxonomic identification of consortium’s lactic acid bacteria strains by molecular techniques, evaluation of biological parameters and stability of probiotic characteristics in various conditions of preservation and storage, improving the experimental model in order to obtain complex probiotic products and to prepare in vivo tests for probiotic effect.

In conclusion, all the objectives of this stage were fully accomplished and one consortium was selected out of the 3 consortia obtained in the previous stages. The probiotic lactic acid bacteria consortium consisting of Lactobacillus (L.) casei, L. fermentum şi L. plantarum was retested for probiotic effect and to establish the optimal conditions of storage and preservation.

The results were disseminated through publication in 4 ISI papers (2 of them were published and 2 were accepted), 1 BDI paper, 2 participation in international conferences and 2 national patents.