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Bifidobacteria are able to utilize a diverse range of host-derived and dietary carbohydrates, the latter of which include many plant-derived oligo- and polysaccharides. Different bifidobacterial strains may possess different carbohydrate utilization abilities. These metabolic abilities can be studied using classical bacterial growth assessment methods, such as measurement of changes in optical density or acidity of the culture in the presence of the particular carbohydrate to generate growth and acidification curves, respectively. Scientists may also be interested in the growth rate during the exponential growth phase, and the maximum OD that is reached on a particular sugar, or the length of the lag phase. Furthermore, high-performance liquid chromatography (HPLC) and high-performance anion exchange chromatography coupled to pulsed amperometric detection (HPAEC-PAD) are extensively used in carbohydrate and metabolic end-product analysis due to their versatility and separation capabilities.This chapter describes some of the available methods to assess EPS production in bifidobacteria, being largely based on those developed for the same purpose for members of the lactic acid bacteria group. The first step is detection of putative EPS-producing bifidobacteria based on a mucoid and/or ropy phenotype. Next, a basic procedure is described for the isolation of the glycan polymer based on the release from bifidobacterial cells grown and collected from the surface of agar-MRSc ("crude EPS"), followed by a purification procedure intended to remove other bacterial macromolecules (DNA and proteinaceous material) to generate "purified EPS." Finally, several methods used for quantification and physical-chemical characterization of isolated/purified polysaccharide are outlined.The biological significance of conjugated fatty acids (CFAs) has been linked to positive health effects based on biomedical, in vitro, and clinical studies. Of note, conjugated linoleic acids (CLAs) are the most widely characterized fatty acids as geometric isomers cis-9,trans-11 and trans-10,cis-12 CLA occur naturally in ruminant fats, dairy products, and hydrogenated oils. Concerning CLAs, it is known that bacterial biohydrogenation, a process whereby ruminal bacteria or starter cultures of lactic acid bacteria have the ability to synthesize CLA by altering the chemical structure of essential fatty acids via enzymatic mechanisms, produces a multitude of isomers with desirable properties. Bifidobacterium species are classed as food grade microorganisms and some of these strains harness molecular determinants that are responsible for the bioconversion of free fatty acids to CLAs. https://www.selleckchem.com/products/cetuximab.html However, molecular mechanisms have yet to be fully elucidated. Reports pertaining to CLAs have been attributed to suppressing tumor growth, delaying the onset of diabetes mellitus and reducing body fat in obese individuals. Given the increased attention for their bioactive properties, we describe in this chapter the qualitative and quantitative methods used to identify and quantify CLA isomers produced by bifidobacterial strains in supplemented broth media. These approaches enable rapid detection of potential CLA producing strains and accurate measurement of fatty acids in biological matrices.Bifidobacteria are important early colonizers of the human intestinal tract. The relative abundance of bifidobacterial species may be modulated, in part, by bacteriophage activity. Metagenomic studies of these populations is a crucial step in understanding this important interaction. This chapter outlines the technical instructions required to analyze the virome of a bifidobacteria-rich sample, for example, an infant fecal sample.Bifidobacteria are commensal microorganisms able to colonize several ecological niches. Since their discovery, culture-dependent methods combined with the most modern next-generation sequencing techniques have contributed to shed light on the ecological, functional and genomic features of bifidobacteria, purporting them as microorganisms with probiotic traits. Thanks to their acclaimed health-promoting effects, several members of the Bifidobacterium genus have been included in a variety of functional foods and drugs. In this context, the functional relevance of bifidobacteria in the gut explains ongoing efforts to isolate novel and potentially beneficial strains. For this purpose, development of effective and selective isolation protocols in concert with knowledge on the physiological characteristics of bifidobacterial are fundamental requirements for their recovery and discovery from their natural environments, in particular from fecal samples.At present, only a limited number of Bifidobacterium species are amenable to genetic manipulation using mutagenesis. This lack of genetic accessibility among the majority of bifidobacterial strains represents a significant roadblock for the study of gene function and expression in these potential probiotics. Genetic tools for generating mutants are difficult to develop for bifidobacteria, as they require workarounds for obstacles such as low transformation efficiencies, and the presence of differing and sometimes multiple restriction modification systems, in different strains. Site-directed mutagenesis is a frequently applied molecular strategy for the generation of targeted mutations, resulting in gene deletion or disruption, or alteration of their expression, thereby revealing information regarding their function. This strategy has been employed as a molecular tool in some Bifidobacterium strains and is typically achieved using a nonreplicating vector, harboring a DNA fragment corresponding to an internal part of the gene to be mutated. This vector is introduced into a bifidobacterial cell of the strain in question by electroporation. Through homologous recombination, this vector is integrated into the genomic DNA of said cell, disrupting the coding region of the targeted gene, thus preventing the expression of a functional protein product. Such mutant versions of Bifidobacterium strains may then be assessed for alterations in their phenotype or gene expression.
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