Molecular interactions of probiotics with oligosaccharides, plant phenolics, and mucin: Subproteome, adhesion, and protein function analyses
Başlık çevirisi mevcut değil.
- Tez No: 403437
- Danışmanlar: Prof. BIRTE SVENSSON, Prof. MAHER ABOU HACHEM
- Tez Türü: Doktora
- Konular: Biyoteknoloji, Mikrobiyoloji, Moleküler Tıp, Biotechnology, Microbiology, Molecular Medicine
- Anahtar Kelimeler: Belirtilmemiş.
- Yıl: 2016
- Dil: İngilizce
- Üniversite: Technical University of Denmark
- Enstitü: Yurtdışı Enstitü
- Ana Bilim Dalı: Belirtilmemiş.
- Bilim Dalı: Belirtilmemiş.
- Sayfa Sayısı: 186
Özet
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Özet (Çeviri)
The role of human diet in human health is getting more attention day by day as the environmental changes, especially food contaminations can lead to serious diseases such as cancer. Thus, research is focusing on functional foods such as probiotics and prebiotics in order to promote human health and prevent diseases. Probiotics are microorganisms that exert positive health benefits to the host when administered in sufficient amount and prebiotics are indigestible food ingredients that are selectively fermented by gastrointestinal microbiota, resulting in positive health effects. Furthermore, in recent years, phenolic compounds derived from plant sources have become a very interesting and important subject in human nutrition and health research because they confer health benefits, especially in prevention of cancer and chronic diseases such as cardiovascular diseases and diabetes. This PhD project aims to investigate the interactions between probiotic Lactobacillus acidophilus NCFM (NCFM), approved or emerging prebiotic carbohydrates, plant phenolic compounds, and mucin by i) in vitro analysis of adhesion to mucin and human intestinal cells, ii) comparative wholecell and surface proteomics, and iii) protein functional characterization. In vitro adhesion studies have been used for simulating the in vivo adhesion as adhesion is a crucial property for probiotics to exert their health benefits relating to their residing in the gastrointestinal tract (GIT). Adhesion analysis was performed after growth using nine carbon sources including approved and emerging prebiotics, mucin-supplemented culture, and supplementation with five phenolic compounds. Raffinose, cellobiose, and polydextrose notably increased the adhesion to mucin and to HT-29 cells. Fructooligosaccharides did not change adhesion to mucin, but adhesion to HT-29 cells was increased. Mucin-supplemented growth increased adhesion to HT-29 cells, without changing adhesion to mucin. Adhesion changes by phenolics were dependent on which phenolic was used, as well as their concentration. At 100 μg/mL, resveratrol increased the adhesion to mucin and HT-29 cells; tannic acid increased the adhesion to HT-29 cells; ferulic acid, however, decreased the adhesion to mucin. At 250 μg/mL, resveratrol increased adhesion to HT-29 cells; tannic acid decreased adhesion to mucin; caffeic acid increased the adhesion to mucin; and ferulic acid increased the adhesion to mucin and HT-29 cells. At 500 μg/mL, ferulic acid increased the adhesion to mucin. A 2-D based proteomics strategy was used to identify differentially abundant proteins in response to carbohydrates, mucin, or phenolic compounds. Surface layers (S-layers) are outermost proteinaceous layer of lactobacilli that are responsible for bacteria-host interactions, including adhesion and immune response. Comparison of surface proteomes of NCFM revealed higher abundant surface proteins under different conditions (i.e. growth on raffinose, cellobiose, or mucin-supplemented media) suggesting these proteins are connected with the increased adhesion. Furthermore, whole-cell proteome comparisons of NCFM grown in the presence of plant polyphenols showed that bacteria respond to the polyphenols by altering the metabolism such as the glycolysis and nucleotide metabolism, as well as stress response. Surface proteome comparisons of NCFM grown in the presence and absence of plant polyphenols revealed that the adhesion changes triggered by the polyphenols are correlated with the alterations in relative abundancies of the surface proteome. Four surface proteins were selected for cloning and recombinant production based on differential surface proteome analyses: phosphate starvation inducible protein; pyruvate kinase; elongation factor G (EF-G); and fructose bisphosphate aldolase. Among them, pyruvate kinase and EF-G are previously defined as moonlighting proteins for other bacteria, while the other two are considered as emerging moonlighting proteins for NCFM. Successful cloning and recombinant productions of these proteins were achieved in E. coli; however, over-production of phosphate starvation inducible protein and fructose bisphosphate aldolase has not yet been successful. Pyruvate kinase was over-produced but the yield in purification was not high probably due to the His-tag being inaccessible. Even though the yield was not high, the recombinant pyruvate kinase was used in mucin binding assays and the result showed that the recombinant pyruvate kinase can bind to mucin. In conclusion, the present PhD project adds to the understanding at the molecular level of synbiotic interactions involving NCFM and defined or emerging prebiotics, as well as dietary plant polyphenols. This contributes to understanding of potential positive effects of these combinations on the host originating from alteration in the metabolism and adhesion properties of NCFM.
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