Effects of Amino Acid Additives on Protein Stability during Electrothermal Supercharging in ESI-MS.

The surprising formation of highly charged protein ions from aqueous ammonium bicarbonate solution is a fascinating phenomenon referred to as electrothermal supercharging (ETS). Although the precise mechanism involved is not clearly understood, previous studies predominantly suggest that ETS is due to native protein destabilization in the presence of bicarbonate anion inside the electrospray ionization droplets under high temperatures and spray voltages. To evaluate existing hypotheses surrounding the underlying mechanism of ETS, the effects of several additives on protein charging under ETS conditions were investigated. The changes in the protein charge state distributions were compared by measuring the ratios between the intensities of highest intensity charge states of native and unfolded protein envelopes and shifts in the lowest and highest observed charge states. This study demonstrated that source temperature plays a more important role in ETS compared to spray voltage, especially when using a nebulized microelectrospray ionization source. Moreover, the effect of amino acids on ETS were generally in good agreement with the extensive literature available on the stabilization or destabilization of proteins by these additives in bulk solution. Among the natural amino acids, protein supercharging was significantly reduced by proline and glycine; however, imidazole provided the highest degree of noncovalent complex stabilization against ETS, outperforming the amino acids. Overall, our study shows that the simple addition of stabilizing reagents such as proline and imidazole can reduce the extent of apparent protein unfolding and supercharging in ammonium bicarbonate solution and provide evidence against the roles of charge depletion and thermal unfolding during ETS.

Thermal Inactivation Kinetics of Salmonella and Enterococcus faecium NRRL-B2354 on Whole Chia Seeds (Salvia hispanica L.).

ABSTRACT: Intervention technologies for inactivating Salmonella on whole chia seeds are currently limited. Determination of the thermal inactivation kinetics of Salmonella on chia seeds and selection of an appropriate nonpathogenic surrogate will provide a foundation for selecting and optimizing thermal pasteurization processes for chia seeds. In this study, chia seed samples from three separate production lots were inoculated with a five-strain Salmonella cocktail or Enterococcus faecium NRRL-B2354 and equilibrated to a water activity of 0.53 at room temperature (25°C). After equilibration for at least 3 days, the inoculated seeds were subjected to isothermal treatments at 80, 85, or 90°C. Samples were removed at six time points, and surviving bacteria were enumerated. Whole chia seeds were diluted in a filter bag at 1:30 because bacterial recovery with this method was similar to that obtained from ground seeds. Survivor data were fitted to consolidated models: one primary model (log linear or Weibull) and one secondary model (Bigelow). E. faecium had higher thermal resistance than did Salmonella, suggesting that E. faecium may be a suitable conservative nonpathogenic surrogate for Salmonella. The Weibull model was a better fit for the survivor data than was the log-linear model for both bacteria based on the lower root mean square error and corrected Akaike's information criterion values. Lipid oxidation measurements and fatty acid concentrations were significantly different from those of the control samples, but the overall magnitude of the differences was relatively small. The thermal inactivation kinetics of Salmonella and E. faecium on chia seeds may be used as a basis for developing thermal pasteurization processes for chia seeds.

A Comparison of Three Methods for Determining Thermal Inactivation Kinetics: A Case Study on Salmonella enterica in Whole Milk Powder.

ABSTRACT: Different methods for determining the thermal inactivation kinetics of microorganisms can result in discrepancies in thermal resistance values. In this study, thermal resistance of Salmonella in whole milk powder was determined with three methods: thermal death time (TDT) disk in water bath, pouches in water bath, and the TDT Sandwich system. Samples from three production lots of whole milk powder were inoculated with a five-strain Salmonella cocktail and equilibrated to a water activity of 0.20. The samples were then subjected to three isothermal treatments at 75, 80, or 85°C. Samples were removed at six time points and cultures were enumerated for survivors. The inactivation data were fitted to two consolidated models: two primary models (log linear and Weibull) and one secondary model (Bigelow). Normality testing indicated that all the model parameters were normally distributed. None of the model parameters for both consolidated models were significantly different (α = 0.05). The amount of inactivation during the come-up time was also not significantly different among the methods (α = 0.05). However, the TDT Sandwich resulted in less inactivation during the come-up time and overall less variation in model parameters. The survivor data from all three methods were combined and fitted to both consolidated models. The Weibull had a lower root mean square error and a better fit, according to the corrected Akaike's information criterion. The three thermal treatment methods produced results that were not significantly different; thus, the methods are interchangeable, at least for Salmonella in whole milk powder. Comparisons with more methods, other microorganisms, and larger varieties of food products using the same framework presented in this study could provide guidance for standardizing thermal inactivation kinetics studies for microorganisms in foods.