RESUMO
The interest in probiotics has increased rapidly the latest years together with the global market for probiotic products. Consequently, establishing reliable microbiological methods for assuring the presence of a certain number of viable microorganisms in probiotic products has become increasingly important. To assure adequate numbers of viable cells, authorities are enquiring for information on viability rates within a certain shelf-life in colony forming units (CFU). This information is obtained from plate count enumeration, a method that enables detection of bacterial cells based on their ability to replicate. Although performing plate count enumeration is one manner of assessing viability, cells can still be viable without possessing the ability to replicate. Thus, to properly assess probiotic viability, further analysis of a broader group of characteristics using several types of methods is proposed. In addition to viability, it is crucial to identify how well the cells in a probiotic product can survive in the gastrointestinal tract (GIT) and thus be able to mediate the desired health benefit while passing through the human body. A broad spectrum of different assay designs for assessing probiotic gastric tolerance have been used in research and quality control. However, the absence of any consensus on how to assess these qualities makes it difficult to compare between laboratories and to translate the results into in vivo tolerance. This review presents and discusses the complexity of assuring that a probiotic is suitable for beneficial consumption. It summarizes the information that can be subtracted from the currently available methods for assessment of viability and stress tolerance of a probiotic, hereby altogether defined as "activity." Strengths and limitations of the different methods are presented together with favorable method combinations. Finally, the importance of choosing a set of analyses that reveals the necessary aspects of probiotic activity for a certain product or application is emphasized.
RESUMO
Advanced glycation end products are formed by non-enzymatic reactions between proteins and carbohydrates, causing irreversible lysine and arginine alterations that severely affect protein structure and function. The resulting modifications induce inflammation by binding to scavenger receptors. An increase in advanced glycation end products is observed in a number of diseases e.g. atherosclerosis and cancer. Since advanced glycation end products also are present in healthy individuals, their detection and quantification are of great importance for usage as potential biomarkers. Current methods for advanced glycation end product detection are though limited and solely measure total glycation. This study describes a new epitope-mapped single chain variable fragment, D1-B2, against carboxymethyllysine, produced from a phage library that was constructed from mouse immunizations. The phage library was selected against advanced glycation end product targets using a phage display platform. Characterization of its binding pattern was performed using large synthetic glycated peptide and protein libraries displayed on microarray slides. D1-B2 showed a preference for an aspartic acid, three positions N-terminally from a carboxymethyllysine residue and also bound to a broad collection of glycated proteins. Positive immunohistochemical staining of mouse atherosclerotic plaques and of a tissue microarray of human pancreatic tumors confirmed the usability of the new scFv for advanced glycation end product detection in tissues. This study demonstrates a promising methodology for high-throughput generation of epitope-mapped monoclonal antibodies against AGE.
