I hereby declare that the research findings of this thesis are the result of research work carried out by me in the Department of Biosciences and Bioengineering, Indian Institute. According to the general norms for reporting research findings, proper acknowledgments have been made, wherever the research findings of other researchers have been cited in this thesis.
INDIAN INSTITUTE OF TECHNOLOGY GUWAHATI DEPARTMENT OF BIOSCIENCES AND
BIOENGINEERING
CERTIFICATE
A CKNOWLEDGEMENT
I am thankful to Department of Biotechnology (DBT) and Science and Engineering Board (SERB), Department of Science and Technology (DST) for supporting my research through grants. I would like to express my sincere gratitude to Dr. Barun Kumar Dutta and Dr. Abhijit Gogoi, Department of Chemistry, IIT Guwahati for scientific collaboration. I would like to thank B. Arun Kumar Maity, Department of Biotechnology, Haldia Institute of Technology, for giving me an opportunity to explore the field of microbiology, which was an initial for me into the subject.
Nandan Bhattacharyya, Department of Biotechnology, Haldia Institute of Technology for his continuous support and encouragement. I would also like to thank my school teachers dr. Sandip Sen for their dedication to teaching. which served as a primer for my curiosity and helped me develop a penchant for science at the school level. I would also like to thank my seniors in the department, Dr. I would also like to thank Mr. Bablu Das for all the stimulating discussions and knowledge imparted to me.
Analysis of bacterial adhesion to HT-29 cells by single-color flow cytometry (FCM) and the plating method. Bactericidal activity of pediocin-loaded milk protein nanocomposite (Ped-MNC) in a simulated gastric transit experiment.
A BBREVIATIONS
L IST OF T ABLES
Secondary structure analysis of pepsin in SGF, MP, pediocin, MP in SGF and pediocin in SGF.
L IST OF F IGURES
The ability of probiotic LAB to beneficially affect human health is generally brought about by three main mechanisms (Lebeer et al., 2010). A third mechanism is through immunomodulation of the host's immune responses, leading to upregulation of anti-inflammatory genes (Rieu et al., 2014). Bacteriocins are ribosomally synthesized antimicrobial peptides (AMPs), which are secreted into the extracellular medium ( Cotter et al., 2005 ).
This class is further subdivided into Ia (lanthipeptides), Ib (head-to-tail cyclized peptides), Ic (actibiotics), Id (linear azoline-containing peptides- LAC), Ie (glycoins) and If (lasso-peptides) (Alvarez ) -Sieiro et al., 2016). This class is further subdivided into IIa (pediocin-like . bacteriocins), IIb (two-peptide bacteriocins), IIc (leaderless bacteriocins) and IId (non-pedocin-like, single-peptide bacteriocins) (Alvarez-Sieiro et al., 2016) ). These usually work by lytic (enterolysin A) or non-lytic (casein) mechanism (Alvarez-Sieiro et al., 2016).
Bacteriocin was found to cause loss of K+ ions in susceptible cells (Bhunia et al., 1991). Among the different classes of bacteriocins, nisin and pediocin PA-1 have been commercialized as food additives (Alvarez-Sieiro et al., 2016).
MOTIVATION AND OBJECTIVES OF THE PRESENT INVESTIGATION
Based on the existing literature reports as highlighted in Chapter 1 of the thesis, it is clear that probiotic lactic acid bacteria (LAB) and its metabolites, specifically bacteriocins, have come to the fore as potential therapeutic agents for selective elimination of pathogens together with minimal effect on the beneficial gut microbes. In particular, the high adhesion propensity of probiotic LAB to host intestinal cells may prevent pathogen adhesion by occluding host cell receptors or extracellular matrix (ECM) molecules. Given this background, there is an urgent need to devise alternative therapeutic approaches, which can adequately address the dual issue of developing a selection pressure that favors resistance development in pathogens and the collateral damage to the commensal microbes.
The recent unraveling of the biomedical potential of LAB has positioned them as potential candidates for anti-pathogenic therapy. In view of the enormous scope of exploring probiotic LAB and its bacteriocin for niche-specific antibacterial therapy, the essential goals of the Ph.D. Determination of the potential of probiotic LAB to prevent pathogen adhesion on extracellular matrix (ECM).
Evaluation of the potential of probiotic LAB to prevent pathogen adhesion in an in vitro cell culture model. Evaluation of the combinatorial effect of bacteriocin-loaded nanocomposite and probiotic LAB for potential anti-adhesion intervention in an in vitro model.
ABSTRACT
The solution fluorescence of a 100 µL aliquot of the cFDA-SE-labeled LAB strains was measured in a multiple reader (Infinite M200, Tecan, Austria) and considered as total fluorescence (FT). Schematic representation of the adhesion process of LAB cells on ECM molecule, collagen or mucin. Schematic representation for the experimental protocol for adhesion inhibition of the model pathogen by LAB cells on ECM.
In the present study, cFDA-SE labeling made a convenient analytical tool and enabled the detection of the adhesion of L . Furthermore, the dissociation constant of the tested LAB strains for the adhesion process on collagen. Principal component analysis (PCA) of the adhesion inhibition assays was performed using a standard method ( Fernandes et al., 2014 ).
Dot plots for the LAB strains and the pathogens indicated that the median cell population shifted for the attached cells compared to the total cells. In competition mode: the degree of inhibition of pathogen adhesion on HT-29 cells, shown by L. Schematic representation of the protocol for assessing Ped-MNC activity in a simulated gastric transit experiment.
Fluorescent labeling of target pathogens with cFDA-SE was performed as previously described (Singh et al., 2012a). Fluorescence of a 100 µl aliquot of cFDA-SE-labeled LAB strains of different cells. In the adhesion assay, a primary observation was that the presence of Ped-MNC led to a relatively higher elimination of E attached to collagen.
Despite the promising results obtained in the short-term adhesion assay, the potential of the combination of Ped-MNC and L. To acquire a nuanced understanding of the process of adhesion inhibition by Ped-MNC and LAB cells, the number of cells of different of E.
