The thesis is devoted to the development of biotechnological basis for the production of selenium-enriched lactic acid bacteria.
It was shown that lactic acid bacteria of the genera Lactobacillus and Enterococcus represented that normobiota of the intestinal tract of older age groups women. The quantitative composition of isolated microorganisms varied with the age, nutritional, and health status of the examined women.
The probiotic properties of isolated strains, their morphological, cultural, physiological and biochemical properties have been studied. The only strain to meet all the WHO requirements (criteria) for probiotics was the strain Lactobacillus sp. 55, which displayed a wide spectrum of probiotic properties. This strain demonstrated antagonistic activity against clinically important strains of Staphylococcus aureus (inhibition zone diameter 18 mm), Staphylococcus epidermidis (12 mm), Bacillus cereus (10 mm), Proteus vulgaris (8 mm), Pseudomonas aeruginosa (10 mm), Shigella flexneri (12 mm), Salmonella enterica var. abony (7 mm), Klebsiella pneumonia (7 mm). Іts antibacterial effect against S. epidermidis (27 mm), S. aureus (22 mm), S. flexneri (7 mm), P. vulgaris (30 mm), P. aeruginosa 17 mm) was due to the production of substances other than lactic acid. Strain Lactobacillus sp. 55 possessed high adhesion ability, exhibited sensitivity to most antibiotics except gentamicin, polymyxin and ciprofloxacin.
Strain Lactobacillus sp. 55 was identified as Lactobacillus gasseri based on 16S rRNA gene sequencing. The nucleotide sequence of the 16S rRNA gene was submitted to the database GenBank (NCBI) with the accession number KT 314159.
The red color of biomass indicated the presence of elemental selenium Se (0) which was confirmed by scanning electron microscopy of the samples. Thus, for the first time the ability of lactic acid bacteria of the species L. gasseri to accumulate elemental selenium in their biomass in the form of nanoparticles ranging in the size from 49 to 113 nm has been demonstrated.
The optimization of selenium-containing medium composition and cultivation parameters of the strain L. gasseri 55 was conducted using the mathematical methods of experimental design. The following medium composition (g/l) is proposed for industrial cultivation: corn extract – 30; peptone – 5; glucose – 10; MgSO4•7H2O – 1.2; MnSO4•4H2O – 0.3; Na2SeO3– 0.008; KH2PO4 - 2; K2HPO4•3H2O – 2; CH3COONa – 5; Twin-80 - 1; agar-agar - 1. Under conditions of periodic cultivation in the optimized selenium-containing medium supplemented with agar-agar the cell number reached 4×109 CFU/ml in 18 h of cultivation, the substrate was completely consumed while bacterial strain retained its viability and physiological activity.
The obtained results indicate that selenium-enriched strain L. gasseri 55 is resistant to gastric juice (85% cells remain viable), 70% cells survived in the presence of bile salts which was twice the survival rate of the native strain.
The probiotic selenium-enriched strain L. gasseri 55 demonstrated antimicrobial activity against a wide range of clinically important strains of opportunistic microorganisms, the inhibitory effect of selenium against S. enterica, K. pneumoniae, S. flexneri, P. vulgaris and S. epidermidis was shown for the first time. It has been established that selenium enrichment of the strain L. gasseri 55 resulted in the 2.5-9-fold increase of its inhibitory effect against opportunistic microorganisms compared to the native strain.
The virulence study of viable cells of L. gasseri 55 showed that the strain is avirulent and incapable of penetrating internal organs. Selenium enrichment of the strains has been shown not to affect its virulence and the strain L. gasseri 55 may be recommended as a dietary source of non-toxic selenium for human and animal nutrition.
Based on the obtained results the technological scheme for the production of selenium enriched L. gasseri strain 55 was developed, which could in turn serve as the basis of the technology for the production of functional food products and medical products for humans and animals.
