Synergistic inactivation of bacteria based on a combination of low frequency, low-intensity ultrasound and a food grade antioxidant.

This study evaluated a synergistic antimicrobial treatment using a combination of low frequency and a low-intensity ultrasound (LFU) and a food-grade antioxidant, propyl gallate (PG), against a model gram-positive (Listeria innocua) and the gram-negative bacteria (Escherichia coli O157:H7). Bacterial inactivation kinetic measurements were complemented by characterization of biophysical changes in liposomes, changes in bacterial membrane permeability, morphological changes in bacterial cells, and intracellular oxidative stress upon treatment with PG, LFU, and a combination of PG + LFU. Combination of PG + LFU significantly (>4 log CFU/mL, P < 0.05) enhanced the inactivation of both L. innocua and E. coli O157:H7 compared to PG or LFU treatment. As expected, L. innocua had a significantly higher resistance to inactivation compared to E. coli using a combination of PG + LFU. Biophysical measurements in liposomes, bacterial permeability measurements, and scanning electron microscope (SEM)-based morphological measurements show rapid interactions of PG with membranes. Upon extended treatment of cells with PG + LFU, a significant increase in membrane damage was observed compared to PG or LFU alone. A lack of change in the intracellular thiol content following the combined treatment and limited effectiveness of exogenously added antioxidants in attenuating the synergistic antimicrobial action demonstrated that oxidative stress was not a leading mechanism responsible for the synergistic inactivation by PG + LFU. Overall, the study illustrates synergistic inactivation of bacteria using a combination of PG + LFU based on enhanced membrane damage and its potential for applications in the food and environmental systems.

Inactivation of foodborne pathogens based on synergistic effects of ultrasound and natural compounds during fresh produce washing.

Ultrasound has potential to be used for disinfection, and its antimicrobial effectiveness can be enhanced in presence of natural compounds. In this study, we compared the antimicrobial effects of ultrasound at 20 kHz (US 20 kHz) or 1 MHz (US 1 MHz) in combination with carvacrol, citral, cinnamic acid, geraniol, gallic acid, lactic acid, or limonene against E. coli K12 and Listeria innocua at a constant power density in water. Compared to the cumulative effect of the individual treatments, the combined treatment of US 1 MHz and 10 mM citral generated >1.5 log CFU/mL additional inactivation of E. coli K12. Similarly, combined treatments of US 1 MHz and 2 mM carvacrol (30 min), US 20 kHz and 2 mM carvacrol, 10 mM citral, or 5 mM geraniol (15 min) generated >0.5-2.0 log CFU/mL additional inactivation in L. innocua. The synergistic effect of citral, as a presentative compound, and US 20 kHz treatment was determined to be a result of enhanced dispersion of insoluble citral droplets in combination with physical impact on bacterial membrane structures, whereas the inactivation by US 1 MHz was likely due to generation of oxidative stress within the bacteria. Combined ultrasound and citral treatments improved the bacterial inactivation in simulated wash water in presence of organic matter or during washing of inoculated blueberries but only additive antimicrobial effects were observed. Findings in this study will be useful to enhance fresh produce safety and shelf-life and design other alternative ultrasound based sanitation processes.

CYSTATHIONINE GAMMA-SYNTHASE activity in rice is developmentally regulated and strongly correlated with sulfate.

An important goal of rice cultivar development is improvement of protein quality, especially with respect to essential amino acids such as methionine. With the goal of increasing seed methionine content, we generated Oryza sativa ssp. japonica cv. Taipei 309 transgenic lines expressing a feedback-desensitized CYSTATHIONINE GAMMA-SYNTHASE from Arabidopsis thaliana (AtD-CGS) under the control of the maize ubiquitin promoter. Despite persistently elevated cystathionine gamma-synthase (CGS) activity in the AtD-CGS transgenic lines relative to untransformed Taipei, sulfate was the only sulfur-containing compound found to be elevated throughout vegetative development. Accumulation of methionine and other sulfur-containing metabolites was limited to the leaves of young plants. Sulfate concentration was found to strongly and positively correlate with CGS activity across vegetative development, irrespective of whether the activity was provided by the endogenous rice CGS or by a combination of endogenous and AtD-CGS. Conversely, the concentrations of glutathione, valine, and leucine were clearly negatively correlated with CGS activity in the same tissues. We also observed a strong decrease in CGS activity in both untransformed Taipei and the AtD-CGS transgenic lines as the plants approached heading stage. The mechanism for this downregulation is currently unknown and of potential importance for efforts to increase methionine content in rice.

Improving the nutritive value of rice seeds: elevation of cysteine and methionine contents in rice plants by ectopic expression of a bacterial serine acetyltransferase.

With the aim of increasing the cysteine level in rice (Oryza sativa L.) and thus improving its nutritional quality, transgenic rice plants were generated expressing an Escherichia coli serine acetyltransferase isoform (EcSAT), the enzyme synthesizing O-acetylserine, the precursor of cysteine. The gene was fused to the transit peptide of the Arabidopsis Rubisco and driven by a ubiquitin promoter to target the enzyme to plastids. Twenty-two transgenic plants were examined for transgene protein expression, and five lines with a high expression level and enzymatic activity, respectively, were selected for further analysis. In these lines, the contents of cysteine and glutathione increased 2.4-fold and 2-fold, respectively. More important is the increase in free methionine and methionine incorporated into the water-soluble protein fraction in seeds. Free methionine increased in leaves up to 2.7-fold, in seeds up to 1.4-fold, and bound to seed proteins up to 4.8-fold, respectively, while the bound methionine level remained constant or even decreased in leaves. Notably, the transgenic lines exhibited higher isoleucine, leucine, and valine contents (each up to 2-fold depending on tissue, free, or bound), indicating a potential conversion of methionine via methionine γ-lyase to isoleucine. As the transgenic rice plants overexpressing EcSAT had significantly higher levels of both soluble and protein-bound methionine, isoleucine, cysteine, and glutathione in rice they may represent a model and target system for improving the nutritional quality of cereal crops.