Potencijalne terapijske biomolekule probiotika kao živih lijekova

doctoral thesis

• Title (english): Potential therapeutic biomolecules of probiotics as living drugs
• Author: Nina Čuljak
• Mentor: Andreja Leboš-Pavunc (mentor)
• Committee member: Blaženka Kos (predsjednik povjerenstva)
• Committee member: Jadranka Frece (član povjerenstva)
• Committee member: Nada Oršolić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Biochemical Engineering); (Laboratory for Antibiotic, Enzyme, Probiotic and Starter Cultures Technology); Zagreb
• Defense date and country: 2024, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology
• Universal decimal classification (UDC): 54 - Chemistry. Crystallography. Mineralogy

Abstract

Cilj ovog rada bio je istražiti pet odabranih sojeva bakterija mliječne kiseline (BMK), izoliranih iz majčinog mlijeka, selekcioniranih producenata potencijalnih terapijskih biomolekula – S-proteina i egzopolisaharida (EPS-a). Proteomička analiza sojeva Levilactobacillus brevis MB1, MB2, MB13 i MB20 je potvrdila prisutnost površinskih S-proteina, čija je tercijarna struktura određena primjenom I-TASSER servera. Dokazana je funkcionalna uloga S-proteina u preživljavanju simuliranih uvjeta gastrointestinalnog trakta, adheziji na Caco-2 stanice te kompetitivnoj ekskluziji patogena. U genomu soja Limosilactobacillus fermentum MC1 su detektirani geni uključeni u Wzx/Wzy-ovisan put biosinteze EPS-a, a strukturna analiza EPS-a provedena NMR (engl. nuclear magnetic resonance) spektroskopijom je pokazala da se sastoji od dva polisaharida. Dokazana je uloga EPS-a u adheziji na Caco-2 stanice te kompetitivnoj ekskluziji Escherichia coli 3014. Ustanovljeno je antimikrobno djelovanje supernatanata ispitivanih sojeva BMK prema patogenim bakterijama te antioksidacijsko djelovanje ispitivanih sojeva. Povećanje ekspresije JAM-A, okludina, ZO-1, MUC2 i ZG16 te smanjenje ekspresije proupalnih citokina IL-6, IL-8 i IL-1β in vitro ukazuju na regeneracijsko i imunomodulacijsko djelovanje sojeva BMK i njihovih biomolekula. Pozitivan učinak soja L. fermentum MC1 i njegovog EPS-a na upalne procese u crijevima miševa, regeneraciju crijevne sluznice i mikrobiotu, ukazuje na potencijalnu primjenu soja kao probiotika i njegovih EPS-a kao prebiotika. Sojevi BMK su zaštićeni mikroinkapsulacijom u alginatu, sa i bez dodatka galakto- ili frukto-oligosaharida, te nanoinkapsulacijom, što se očituje velikom stopom preživljavanja nepovoljnih uvjeta (> 10⁸ CFU/mL).

Abstract (english)

The aim of this study was to investigate five lactic acid bacteria (LAB) strains, isolated from breast milk, selected as producers of potential therapeutic biomolecules – S-proteins and exopolysaccharides (EPSs). Proteomic analysis of strains Levilactobacillus brevis MB1, MB2, MB13 and MB20 confirmed the presence of surface S-proteins, whose tertiary structure was determined using the I-TASSER server. The functional role of S-proteins was demonstrated in terms of survival under simulated gastrointestinal tract conditions, adhesion to Caco-2 cells and competitive exclusion of pathogens. Genes involved in the Wzx/Wzy-dependent pathway of EPSs biosynthesis were detected in the genome of Limosilactobacillus fermentum MC1, and nuclear magnetic resonance (NMR) structural analysis revealed that EPSs consist of two polysaccharides. The role of EPSs in adhesion to Caco-2 cells and competitive exclusion of Escherichia coli 3014 was demonstrated. The antimicrobial activity of the supernatants of the tested LAB strains against pathogenic bacteria and the antioxidant activity of the tested strains were confirmed. The increase in the expression of JAM-A, occludin, ZO-1, MUC2 and ZG16, and the decrease in the expression of the proinflammatory cytokines IL-6, IL-8 and IL-1β indicate the regenerative and immunomodulatory effects of the LAB strains and their biomolecules. Positive effects of L. fermentum MC1 and its EPSs on inflammatory processes in the intestine of mice, the regeneration of the intestinal mucosa and the microbiota indicate potential application of the strain L. fermentum MC1 as probiotic and its EPSs as prebiotics. The LAB strains were protected by microencapsulation in alginate, with and without the addition of galacto- or fructo-oligosaccharides, and by nanoencapsulation, as evidenced by a high survival rate under unfavourable conditions (> 10⁸ CFU/mL).

• Keywords: probiotici; S-proteini; egzopolisaharidi; imunomodulacija; mikrobiota; inkapsulacija
• Keywords (english): probiotics; S-proteins; exopolysaccharides; immunomodulation; microbiota; encapsulation
• Language: croatian
• URN:NBN: urn:nbn:hr:159:778656
• Promotion: 2024
• Study programme: Title: Postgraduate PhD Study in Nutrition; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor/doktorica znanosti, područje biotehničkih znanosti, polje nutricionizam (doktor/doktorica znanosti, područje biotehničkih znanosti, polje nutricionizam)
• Type of resource: Text
• Extent: 161 str. ; 30 cm
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2025-02-14
• Created on: 2024-08-27 10:02:14