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Evaluation of Leuconostoc bacteriocins as food preservatives

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dc.contributor.advisor Von Holy A en
dc.contributor.advisor Roberts RF en
dc.contributor.author Swarts AJ en
dc.date.accessioned 2016-09-22T11:18:11Z
dc.date.available 2016-09-22T11:18:11Z
dc.date.created 1994 en
dc.date.submitted 2002 en
dc.identifier.uri http://hdl.handle.net/20.500.11892/109489
dc.description.abstract The objective of this study was the application of flow cytometry to detect cell death and injury caused by exposure to preservation stress. This technology was compared with preexisting determinative measures for bacterial biomass determination. Leucocin B-TAlla may be used as biological preservative. Bacteriocin activity in the culture supernatant of the producer was insufficient to perform preservation experiments. Several methods for concentrating the specific activity of leucocin B-TAlla was assessed to optimize conditions for the ultimate large scale production. The level of preservatives heat, lysozyme, EDTA, lactic acid, sodium lactate, nisin and leucocin B-TAlla, required to inhibit Listeria monocytogenes was determined by monitoring changes in optical density. Listeria monocytogenes was subjected to varying levels of each factor. Bacteria can exist in viable but non-culturable states when subjected to stress which cannot be detected by culturing on solid media. The use of flow cytometry was investigated to detect non-culturable states. Rhodamine 123 (RH) combined with Propidium Iodide (PI), Acridine Orange (AO), Fluorescein Diacetate (FDA), or the Baclight Bacterial Viability Kit<sup>TM</sup> (Baclight) were used to distinguish viable from non-viable cells. Active Listeria monocytogenes cells were subjected to mild heat (60&#186;C for 7 minutes) and EDTA treatments. The growth response was determined by standard plate counts, optical density measurements, and flow cytometry with FDA staining. Mixed proportions of dead and live control populations of Listeria monocytogenes were analyzed by flow cytometry to determine detection limits of the Dead/Live Baclight Bacterial Viability Kit<sup>TM</sup>. The Baclight bacterial viability kit T was then used to assess injury and death of Listeria monocytogenes exposed to lysozyme, lactic acid, heat, and nisin on their own, as well as in combination with leucocin B-TAlla. Listeria monocytogenes was grown in batch culture the presence of these preservatives. Samples were taken at 0, 6, 12, 18, 24, 48 and 72 hours and stained with the Baclight bacterial viability kit for analysis by flow cytometry. The changes in physiological status of each population exposed to the different stress factors were compared with changes within a control population without the addition of stress factors. The substitutions of the media components yeast and beef extracts, or Tween 80, led to the development of a suitable partial purification procedure. Leuconostoc mesenteroides DSM20343 culture supernatant preparation did not interfere with the growth of Listeria monocytogenes. At a level of 800 AU of leucocin B-TAlla and nisin, listerial growth was inhibited for up to 8 hours. Heat at 60&#186; C at 7 minutes delayed growth for 8 hours. Lactic acid and sodium lactate reduced growth as observed by a difference in slope of the growth curve, compared with the control. The antimicrobial effect of lactic acid was found to be due to more man just a reduction in pH. EDTA retarded growth in a concentration dependent manner. Lysozyme inhibited growth at a concentration of 1 mgml<sup>-1</sup>. It was concluded that each factor should be applied at a level which inhibits growth for 24 hours or less, in order to detect synergistic effects should they occur, when determining the effect of combinations of factors. Leucocin B-TAlla was therefore proposed for use at the level of 800 AUml<sup>-1</sup>, heat at 7 minutes at 60 &#186;C, lactic acid at a pH of 5.5, sodium lactate at 3%, EDTA at 0.2% and lysozyme at lmgml<sup>-1</sup>. Rhl23/PI or AO was unable to distinguish live and dead cells. For the FDA study, reproducible dead and live controls were successfully analyzed. FDA staining showed that leucocin B-TAlla at 800 activity units (AUlml<sup>-1</sup> exhibited greater antilisterial activity than EDTA or heat treatment on their own. Sublethal heat treatment on its own showed a short term inhibitory effect which was similar but less pronounced than that resulting from EDTA treatment alone. Leucocin B-TAlla in combination with heat or EDTA, showed enhanced antilisterial activity when compared to any of these stress factors on their own. However, no distinction between live and stressed populations was possible. The itaclight kit was found to be a reliable and rapid measure of viability oi Listeria monocytogenes, since it could distinguish live, dead and stressed cells. Flow cytometry using the ifczclight kit can therefore be used to quantify the effect of sublethal preservation stresses on Listeria monocytogenes and other food borne pathogens and spoilage organisms. High correlations for flow cytometric detection of defined proportions of live or dead cells in mixtures between 10 and 100% of dead (r<sup>2</sup>=0.97) or live (r<sup>2</sup>=0.99) cells were obtained. However, mixtures containing less than 10% of either live or dead control cells gave correlations below 0.72. The growth of L. monocytogenes in the absence and presence of leucocin B-TAlla, was analyzed by flow cytometry using Baclight, plate counts and optical density measurements. Although leucocin B-TAlla initially inhibited listerial growth, the uptake of both Baclight dyes suggested that cells remained viable but became leaky, possibly indicating bacteriocin induced pore formation in the target membranes. It was possible to differentiate live, dead and stressed populations of Listeria monocytogenes exposed to the different stress factors, relative to the non-stressed control population. Cell wall damage caused by lysozyme was evident from the observation of a stressed population of variable physiological character upon exposure to lysozyme. Exposure to leucocin gave a stressed population of a more uniform physiological character. Nisin has a similar effect to leucocin. Heat caused cell lysis, with little stress in heat resistant cells. Different mechanisms of antibiosis were therefore distinguishable. Lysozyme, lactic acid and heat reduced bacterial growth. It was found that of all the treatments, nisin was the single most effective preservative used against Listeria monocytogenes. Combining acid or nisin with leucocin B-TAlla was most effective, compared with combinations involving heat or lysozyme. By considering associations between time of exposure to each stress factor, the level of stress factor applied, and the physiological response detected by flow cytometry, the effect of preservative stress factors on the growth of Listeria monocytogenes was modeled (using the loglinear model). The significance of each interaction was compared with bacterial counts and optical density measurements. Trends observed were in agreement with traditional methods, but flow cytometry gave more specific responses and differentiated dead, live and stressed cells. Flow cytometry proved to be a sensitive and rapid tool for microbiological analysis. en
dc.language English en
dc.title Evaluation of Leuconostoc bacteriocins as food preservatives en
dc.type Masters degree en
dc.description.degree MSc en


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