Production, Purification and Characterization of Antimicrobial Biomolecules from Potential Probiotic Lactic Acid Bacteria

University dissertation from Department of Biotecnology

Abstract: Development and optimization of the production of lactic acid bacteria (LAB) and their attendant 'natural' inhibitors as biopreservatives to control undesirable bacteria currently remains a primary focus of several laboratories involved in research concerning food safety and quality. Strategies utilized to study incorporation of biopreservatives into food include either direct use of LAB-strains or use of a biopreservative preparation in the form of previously fermented product or use of semipurified, purified, or chemically synthesized bacteriocins. Modern concepts or perspectives of the application of LAB include the following selections: i) the best adapted and safely performing LAB strains, ii) strains with probiotic effects and/or health-promoting effects and finally iii) strains with food protective activities. In the study presented in this thesis, three different strains, Lactobacillus acidophilus DSM 20079, Pediococcus damnosus 2.6 and Lactobacillus sakei CCUG 42685, have been chosen to study their biotechnological properties according to one or several of the previously mentioned concepts. Structural and functional characterization of a novel antimicrobial peptide (acidocin 20079) from an LAB with interesting characteristics as producer strain (Lactobacillus acidophilus DSM 20079), has been made, showing that the isolated bacteriocin has a potential for industrial application. Furthermore, the functional properties of selected bacteriocin producing strains (L. acidophilus DSM 20079 and Pediococcus damnosus 2.6) as probiotic have been studied. The strains showed a high tolerance to the main obstacles of the GI-tract in experiments simulating native conditions at 37 °C. Finally, improvements within the field of purification of bacteriocins from non-clarified crude homogenate are needed as the currently published protocols are often cumbersome. An economically reliable process hence has to be developed for bacteriocin purification. A chromatographic matrix based on continuous supermacroporous cryogel was successfully used for the purification of sakacin P (produced by L. sakei CCUG 42685) directly from non-clarified crude. The bound bacteriocin was eluted with 81-87 % recovery in particulate free eluate with more than 60%purity after one purification step.

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