Linsey Garcia-Gonzalez

Influence of type of microorganism, food ingredients and food properties on high-pressure carbon dioxide inactivation of microorganisms

High pressure carbon dioxide (HPCD) treatment is currently considered as an attractive non-thermal process for preserving food. Industrial application of this technique requires, among others, systematic (quantitative) data on the inactivation of food relevant pathogenic and spoilage microorganisms, and in-depth information on the effect that the composition and the properties of a food matrix have on the inactivation efficacy. The first objective of this study, therefore, is to evaluate and compare the HPCD susceptibility of several food pathogens and spoilage microorganisms under the same treatment conditions. In the second part, the influence of different food components (NaCl, oil, starch, whey protein and emulsifier) and food properties (pH, fluid viscosity and water activity) on the inactivation efficacy of HPCD was determined. For the first aim, a range of Gram-negative and Gram-positive bacteria, yeasts and spores were treated with pressurized CO(2) at 10.5 MPa and 35 degrees C during 20 min. Bacterial susceptibility towards HPCD treatments followed the sequence Gram-negative approximately Gram-positive>yeasts>spores and appeared to be related to the acid resistance of the organisms. To study the effect of different food compounds on HPCD inactivation, the reduction degree of Pseudomonas fluorescens was determined in media with and without these components at 10.5 MPa and 35 degrees C after 5 or 20 min, depending on the tested component. NaCl and the emulsifiers Tween 80 and sucrose stearate enhanced bacterial reduction, while oil reduced the bactericidal efficacy of HPCD. Starch and whey proteins did not influence inactivation. Finally, the influence of pH, fluid viscosity and water activity was investigated by determining the reduction of P. fluorescens at 10.5 MPa and 35 degrees C in suspensions from which the pH, viscosity and water activity were adjusted with respectively NaOH or HCl, gelatin or polyethylene glycol, and sucrose, NaCl or glycerol. Treatment time depended on the studied food property with 5 min for the pH experiments, while other experiments lasted 20 min. The results indicated that P. fluorescens cells became more sensitive to HPCD treatments at low pH and viscosity. Not water activity but the kind of soluble solute used to lower water activity influenced inactivation. High NaCl-concentrations lead to total inactivation, while sucrose and glycerol strongly protected the cells against inactivation.

Membrane permeabilization and cellular death of Escherichia coli, Listeria monocytogenes and Saccharomyces cerevisiae as induced by high pressure carbon dioxide treatment.

In this study, the relationship between (irreversible) membrane permeabilization and loss of viability in Escherichia coli, Listeria monocytogenes and Saccharomyces cerevisiae cells subjected to high pressure carbon dioxide (HPCD) treatment at different process conditions including temperature (35-45 degrees C), pressure (10.5-21.0 MPa) and treatment time (0-60 min) was examined. Loss of membrane integrity was measured as increased uptake of the fluorescent dye propidium iodide (PI) with spectrofluorometry, while cell inactivation was determined by viable cell count. Uptake of PI by all three strains indicated that membrane damage is involved in the mechanism of HPCD inactivation of vegetative cells. The extent of membrane permeabilization and cellular death increased with the severity of the HPCD treatment. The resistance of the three tested organisms to HPCD treatment changed as a function of treatment time, leading to significant tailing in the survival curves, and was dependent on pressure and temperature. The results in this study also indicated a HPCD-induced damage on nucleic acids during cell inactivation. Transmission electron microscopy showed that HPCD treatment had a profound effect on the intracellular organization of the micro-organisms and influenced the permeability of the bacterial cells by introducing pores in the cell wall.

Individual and Combined Effects of pH and Lactic Acid Concentration on Listeria innocua Inactivation: Development of a Predictive Model and Assessment of Experimental Variability

ABSTRACT In food technology, organic acids (e.g., lactic acid, acetic acid, and citric acid) are popular preservatives. The purpose of this study was to separate the individual effects of the influencing factors pH and undissociated lactic acid on Listeria innocua inactivation. Therefore, the inactivation process was investigated under controlled, initial conditions of pH (pH0) and undissociated lactic acid ([LaH]0). The resulting inactivation curves consisted of a (sometimes negligible) shoulder period followed by a descent phase. In a few cases, a tailing phase was observed. Depending on the conditions, the descent phase contained one or two log-linear parts or had a convex or concave shape. In addition, the inactivation process was characterized by a certain variability, dependent on the severity of the conditions. Furthermore, in the neighborhood of the growth/no growth interface sometimes contradictory observations occurred. Overall, the individual effects of the influencing factors pH and undissociated lactic acid could clearly be distinguished and were also apparent based on fluorescence microscopy. Appropriate model types were developed and enabled prediction of which conditions of pH0 and [LaH]0 are necessary to obtain a predetermined inactivation (number of decimal reductions) within a predetermined time range.

The development of Escherichia coli and Listeria monocytogenes variants resistant to high-pressure carbon dioxide inactivation.

Aims:  The objective of this study was to investigate whether bacterial cells could develop resistance (as a part of their adaptation strategy) to high‐pressure CO2 (HPCD) inactivation.

Targeted poly(3-hydroxybutyrate-co-3-hydroxyvalerate) bioplastic production from carbon dioxide

A microbial production process was developed to convert CO2 and valeric acid into tailored poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) bioplastics. The aim was to understand microbial PHBV production in mixotrophic conditions and to control the monomer distribution in the polymer. Continuous sparging of CO2 with pulse and pH-stat feeding of valeric acid were evaluated to produce PHBV copolyesters with predefined properties. The desired random monomer distribution was obtained by limiting the valeric acid concentration (below 1 gL-1). 1H-NMR, 13C-NMR and chromatographic analysis of the PHBV copolymer confirmed both the monomer distribution and the 3-hydroxyvalerate (3HV) fraction in the produced PHBV. A physical-based model was developed for mixotrophic PHBV production, which was calibrated and validated with independent experimental datasets. To produce PHBV with a predefined 3HV fraction, an operating diagram was constructed. This tool was able to predict the 3HV fraction with a very good accuracy (2% deviation).

Poly-β-hydroxybutyrate administration during early life: effects on performance, immunity and microbial community of European sea bass yolk-sac larvae

The reliable production of marine fish larvae is one of the major bottlenecks in aquaculture due to high mortalities mainly caused by infectious diseases. To evaluate if the compound poly-β-hydroxybutyrate (PHB) might be a suitable immunoprophylactic measure in fish larviculture, its capacity to improve immunity and performance in European sea bass (Dicentrarchus labrax) yolk-sac larvae was explored. PHB was applied from mouth opening onwards to stimulate the developing larval immune system at the earliest possible point in time. Larval survival, growth, microbiota composition, gene expression profiles and disease resistance were assessed. PHB administration improved larval survival and, furthermore, altered the larva-associated microbiota composition. The bacterial challenge test using pathogenic Vibrio anguillarum revealed that the larval disease resistance was not influenced by PHB. The expression profiles of 26 genes involved e.g. in the immune response showed that PHB affected the expression of the antimicrobial peptides ferritin (fer) and dicentracin (dic), however, the response to PHB was inconsistent and weaker than previously demonstrated for sea bass post-larvae. Hence, the present study highlights the need for more research focusing on the immunostimulation of different early developmental stages for gaining a more comprehensive picture and advancing a sustainable production of high quality fry.