Potencijalne terapijske biomolekule probiotičkih sojeva autohtonih bakterija mliječne kiseline

doctoral thesis

• Title (english): Potential therapeutic biomolecules of probiotic strains of autochthonous lactic acid bacteria
• Author: Martina Banić
• Mentor: Blaženka Kos (mentor)
• Mentor: Jurica Žučko (komentor)
• Committee member: Jagoda Šušković (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: 2021-07-23, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology
• Universal decimal classification (UDC): 57 - Biological sciences. Physical anthropology. Biodiversity

Abstract

Iz 30 uzoraka majčinog mlijeka i fecesa dojenčadi, izolirana je DNA te je provedeno sekvenciranje V1-V3 regije 16S rRNA gena. Taksonomskom analizom dobivenih sekvenci, utvrĎena je korelacija u sastavu mikrobioma mlijeka majki i fecesa njihove dojenčadi. Hijerarhijskom klaster analizom RAPD-PCR elektroforetskih profila 100 bakterijskih sojeva izoliranih iz majčinog mlijeka, odabrano je 28 genetički različitih sojeva. SDS-PAGE analizom je po prvi puta ustanovljena prisutnost S-proteina kod 4 soja izolirana iz majčinog mlijeka, a PCR analizom je kod 6 Lactobacillus plantarum sojeva potvrĎena prisutnost 3 gena (plnJ, plnA i plnEF) koji sudjeluju u biosintezi bakteriocina. Detaljno su okarakterizirani i probiotički sojevi producenti potencijalnih terapijskih biomolekula, Lb. brevis SF9B i Lb. plantarum SF9C, izolirani iz rasola kiselog kupusa. Analizom cijelog genoma soja SF9B, pronaĎena su 3 gena (slpB, slp i slpD) koji kodiraju S-proteine. LC-MS analizom proteinskih vrpci dobivenih SDS-PAGE i 2D-EF, ustanovljena je ekspresija samo jednog, slpB gena. Sekundarna struktura izoliranog S-proteina je pretpostavljena PSIPRED metodom, a tercijarna pomoću I-TASSER servera. PotvrĎeno je da soj SF9C sadrţi 9 gena koji kodiraju za bakteriocine. Tercijarne strukture plantaricina PlnJK i PlnEF pretpostavljene su pomoću SWISS-MODEL servera, a fizikalno-kemijska svojstva njihovih α-uzvojnica HeliQuest serverom. DGGE analizom i analizom fekalnog mikrobioma, potvrĎen je in vivo utjecaj primjene zdruţene kulture sojeva SF9B i SF9C na sastav fekalnog mikrobioma 18 štakora.

Abstract (english)

DNA was isolated from 30 samples of breast milk and infant feces, and sequencing of the V1-V3 region of the 16S rRNA gene was performed. By taxonomic analysis of the obtained sequences, a correlation in the composition of the microbiome of breast milk and infant feaces was found. 28 genetically distinct strains were selected by hierarchical cluster analysis of RAPD-PCR electrophoresis profiles of 100 strains isolated from breast milk. The SDS-PAGE analysis revealed for the first time the presence of S-proteins in 4 strains isolated from breast milk, and PCR analysis confirmed the presence of 3 genes (plnJ, plnA and plnEF) involved in bacteriocin biosynthesis in 6 Lactobacillus plantarum strains. Lb. brevis SF9B and Lb. plantarum SF9C, probiotic strains producing potential therapeutic biomolecules isolated from sauerkraut brine, were also characterized in detail. WGS analysis of the SF9B strain revealed 3 genes (slpB, slpC and slpD) encoding S-proteins. LC-MS analysis of the protein bands obtained by SDS-PAGE and 2D-EF revealed the expression of only one, the slpB gene. Prediction of the secondary structure of the S-protein was performed using the PSIPRED method, and that of the tertiary structure using the I-TASSER server. It was confirmed that the strain SF9C contains 9 bacteriocin-encoding genes. The SWISS-MODEL server was used to predict the tertiary structure of PlnJK and PlnEF plantaricins, whereas physicochemical properties of their α-helices were determined using the HeliQuest server. DGGE analyses and faecal microbiome analyses confirmed the in vivo impact of SF9B and SF9C co-culture application on the faecal microbiome composition of 18 rats.

• Keywords: probiotici; S-proteini; bakteriocini; mikrobiom majčinog mlijeka; mikrobiom fecesa dojenčadi
• Keywords (english): probiotics; S-proteins; bakteriocins; breast milk microbiome; infant faecal microbiome
• Language: croatian
• URN:NBN: urn:nbn:hr:159:547802
• Promotion: 2021
• 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: 198 str. : 30 cm
• File origin: Born digital
• Access conditions: Open access
• Created on: 2021-11-24 14:14:26