ISSN 2415-3060 (print), ISSN 2522-4972 (online)
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УЖМБС 2020, 5(4): 240–247

Gel Chromatographic Fractionation of Metabolic Complexes of L. rhamnosus GG and S. boulardii and their Antimicrobial Activity

Isaуenko O. 1, Babych E. 1, Gorbach T. 2, Semenchenko O. 3, Kotsar O. 2

The rapid increase in antibiotic-resistant microorganisms encourages the development of alternative antimicrobial drugs, in particular on the basis of antimicrobial peptides. The purpose of the work was to study the composition of metabolic complexes of Lactobacillus rhamnosus GG and Saccharomyces boulardii by chromatographic fractionation and antimicrobial activity against antibiotic-resistant pathogens to create a new generation of antimicrobial polyfunctional candidate drugs. Material and methods. Disintegrates, which are structural components, are obtained by low-frequency ultrasonic treatment of probiotic cells of L. rhamnosus GG or S. boulardii. The metabolic complexes were obtained by the author's method of cultivation of lactobacteria and saccharomycete in their own disintegrates, saccharomycetes and a combination of saccharomycetes with lactobacteria – in disintegrate of lactobacterias. The fractionation of the substances of L. rhamnosus and S. boulardii was performed by gel permeation chromatography. Antimicrobial activity against multidrug-resistant strains of Staphylococcus epidermidis PR, Enterobacter cloacae PR, Enterococcus faecalis PR was studied by a qualitative method. Results and discussion. The study established multicomponent nature of all experimental samples with the predominant content of peptide components (~ 87% – ~ 98%) and lower specific weight of fractions with molecular weights ≥ 12 kDa (~ 2% – ~ 13%). We revealed differences in the composition of experimental substances. This indicates that in the process of culturing cells in ultrasonic disintegrates is the production of metabolites, which is accompanied by changes in the components of metabolic complexes compared with disintegrates. The difference in molecular weights and specific gravity of the fractions of the metabolic complex of lactobacilli and its combination with saccharomycetes proves that the addition of saccharomycetes to lactobacilli allows obtaining excellent substances with their own composition. Gel chromatographic separation of metabolites of L. rhamnosus and S. boulardii, obtained by the same method of production, but from different microorganisms, have different composition of fractionated compounds. This proves that the difference in the composition of the fractionated components also depends on the producer strain (lactobacilli or saccharomycetes). The most effective antimicrobial action against pathogens are lactobacteria. They contain an overwhelming number of fractions with molecular weights ~ 4,2 – 5,7 kDa. Saccharomycetes samples are characterized by lower antibacterial activity and dominant components with masses of ~ 1,1 kDa. Perhaps less pronounced effect of saccharomycetes was on multidrug-resistant pathogens due to the dominant number of fractions with lower molecular weight. The largest molecular weight fraction ≥ 12 kDa is contained in close amounts in metabolic complexes with different antimicrobial activity, which indicates the absence of influence of this component on multidrug-resistant opportunistic pathogens. Conclusion. The fractional composition of the metabolic complexes of L. rhamnosus LGG and S. boulardii presented in the work will be useful in the construction of antimicrobial polyfunctional candidate-medications based on them.

Keywords: gel permeation chromatography, bacteriocins, antimicrobial activity, multidrug-resistant microorganisms, lactobacteria, saccharomycetes

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  1. Kang HK, Kim C, Seo CH, Park Y. The therapeutic applications of antimicrobial peptides (AMPs): a patent review. J Microbiol. 2017; 55(1): 1-12.
  2. Azimova VT, Potaturkina-Nesterova NI, Nesterov AS. Endogennyye antimikrobnyye peptidy cheloveka [Endogenic antimicrobial peptides of human]. Sovr probl nauki i obraz. 2015; 1(1). [Russian]
  3. Mishra B, Reiling S, Zarena D, Wang G. Host defense antimicrobial peptides as antibiotics: design and application strategies. Curr Opin Chem Biol. 2017; 38: 87-96.
  4. Isayenko OY, Knysh OV, Babych YM, Ryzhkova TN, Dyukareva GI. Effect of disintegrates and metabolites of Lactobacillus rhamnosus and Saccharomyces boulardii on biofilms of antibiotic resistant conditionally pathogenic and pathogenic bacteria. Regul Mech Biosyst. 2019; 10(1): 3-8.
  5. Isayenko OYu. Protydyfteriyni vlastyvosti strukturno-metabolitnykh kompleksiv probiotychnykh shtamiv laktobakteriy i sakharomitsetiv v testakh in vitro ta in vivo [Anti-diphtheria properties of structural-metabolites complexes of Lactobacteria and Saccharomyces probiotic strains]. Fiziol Zh. 2019; 65(6): 51-60. [Ukrainian].
  6. Isayenko OY, Knysh OV, Babych EM, Kivva FV, Balak OK, Naboychenko OA. Vplyv produktiv metabolizmu Lactobacillus rhamnosus GG na test-kulʹtury stafilokokiv ta korynebakteriy [The influence of metabolic products of Lactobacillus rhamnosus GG on the test-culture of staphylococcus and corynebacterium]. Visn probl bioloh i medyts. 2017; 2(136): 246-51. [Ukrainian].
  7. Pithva S, Ambalam Р, Dave JM, Vyas BRM. Antimicrobial peptides of probiotic Lactobacillus strains. In: Science against microbial pathogens: communicating current research and technological advances. Editors: A. Mendez-Vilas. FORMATEX; 2011. p. 987-91.
  8. Bermudez-Brito M, Plaza-Díaz J, Muñoz-Quezada S, Gómez-Llorente C, Gil A. Probiotic Mechanisms of Action. Ann Nutr Metab. 2012; 61: 160-74.
  9. Sobolev AV, Kolobov AA, Krasovskaya IE, Grishina TV. Podbor metodov vydeleniya antimikrobnykh peptidov iz kul'tury Lactobacillus plantarum 8pa-3 [Selection of methods for the isolation of antimicrobial peptides from the culture of Lactobacillus plantarum 8pa-3]. Russian immunological journal. 2014; 8(17): 873-5.
  10. Rybal'chenko OV, Orlova OG, Bondarenko VM. Antimikrobnyye peptidy laktobatsill [Antimicrobial peptides of lactobacilli]. Zh Mikrobiol Epidemiol Immunobiol. 2013; 4: 89-100. [Russian]
  11. Ali MAE, Abdel-Fatah OM, Janson JC. Antimicrobial potential of Saccharomyces boulardii extracts and fractions. JASR. 2012; 8(8): 4537-43.
  12. Venkateswarulu TC, Krupanidhi S, Indira М, Bobby N. Estimation of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of antimicrobial peptides of Saccharomyces boulardii against selected pathogenic strains. Karbala International Journal of Modern Science. 2019; 5(4): 8.
  13. Niamah АК, Al-Manhel AJ, Al-Shawi MJ. Isolation, purification and characterization of antimicrobial peptides produced from Saccharomyces boulardii. IJPR. 2018; 24(3): 455-61.