Lactoferrin in breast milk-based powders.

This study aimed to determine lactoferrin (LF) in breast milk-based powders and formulas. Lactoferrin is an important whey protein in all mammalian milks and is responsible in large part for the known antimicrobial effects of human milk in particular. As breast feeding is not always possible, formulas based on cows milk have been developed in order to meet the nutritional needs of the newborn, while more recently human breast milk-based powders have been introduced to offer the biological functionality of human milk to pre-term and critically ill babies. In the present work, the amount of LF in commercial breast milk-based powders was tested by a validated RF-HPLC method for the determination of LF in breast milk in order to examine both the applicability of the method but at a second level the amount of LF in these commercial products. The detection of LF was possible but the complexity of the matrix lead us to the use the standard addition methodology in order to achieve quantification. The results indicated that breast milk-based powders had higher amount of LF than cows milk-based formulas, both non-fortified and fortified.

Food Microstructure and Fat Content Affect Growth Morphology, Growth Kinetics, and Preferred Phase for Cell Growth of Listeria monocytogenes in Fish-Based Model Systems.

Food microstructure significantly affects microbial growth dynamics, but knowledge concerning the exact influencing mechanisms at a microscopic scale is limited. The food microstructural influence on Listeria monocytogenes (green fluorescent protein strain) growth at 10°C in fish-based food model systems was investigated by confocal laser scanning microscopy. The model systems had different microstructures, i.e., liquid, xanthan (high-viscosity liquid), aqueous gel, and emulsion and gelled emulsion systems varying in fat content. Bacteria grew as single cells, small aggregates, and microcolonies of different sizes (based on colony radii [size I, 1.5 to 5.0 µm; size II, 5.0 to 10.0 µm; size III, 10.0 to 15.0 µm; and size IV, ≥15 µm]). In the liquid, small aggregates and size I microcolonies were predominantly present, while size II and III microcolonies were predominant in the xanthan and aqueous gel. Cells in the emulsions and gelled emulsions grew in the aqueous phase and on the fat-water interface. A microbial adhesion to solvent assay demonstrated limited bacterial nonpolar solvent affinities, implying that this behavior was probably not caused by cell surface hydrophobicity. In systems containing 1 and 5% fat, the largest cell volume was mainly represented by size I and II microcolonies, while at 10 and 20% fat a few size IV microcolonies comprised nearly the total cell volume. Microscopic results (concerning, e.g., growth morphology, microcolony size, intercolony distances, and the preferred phase for growth) were related to previously obtained macroscopic growth dynamics in the model systems for an L. monocytogenes strain cocktail, leading to more substantiated explanations for the influence of food microstructural aspects on lag phase duration and growth rate.IMPORTANCEListeria monocytogenes is one of the most hazardous foodborne pathogens due to the high fatality rate of the disease (i.e., listeriosis). In this study, the growth behavior of L. monocytogenes was investigated at a microscopic scale in food model systems that mimic processed fish products (e.g., fish paté and fish soup), and the results were related to macroscopic growth parameters. Many studies have previously focused on the food microstructural influence on microbial growth. The novelty of this work lies in (i) the microscopic investigation of products with a complex composition and/or structure using confocal laser scanning microscopy and (ii) the direct link to the macroscopic level. Growth behavior (i.e., concerning bacterial growth morphology and preferred phase for growth) was more complex than assumed in common macroscopic studies. Consequently, the effectiveness of industrial antimicrobial food preservation technologies (e.g., thermal processing) might be overestimated for certain products, which may have critical food safety implications.

Effect of microstructure and initial cell conditions on thermal inactivation kinetics and sublethal injury of Listeria monocytogenes in fish-based food model systems.

The development of more accurate predictive models that describe the microbial kinetics of mild thermal treatments of foods requires knowledge concerning the influence of food microstructure and initial cell conditions on foodborne pathogens' inactivation kinetics. The effect of food microstructure and initial cell conditions on thermal inactivation kinetics and sublethal injury (SI) of Listeria monocytogenes was investigated at 59, 64 and 69°C. Fish-based food model systems with different microstructures, possessing minimal compositional and physicochemical variations, were used. L. monocytogenes growth morphology had no significant influence on thermal inactivation kinetics. A gelled matrix resulted in a lower specific inactivation rate kmax and a higher residual cell population Nres, while the presence of fat droplets resulted in a higherkmaxand did not influenceNres. SI was higher in viscous than in gelled systems and more prominent for cells that were grown inside the matrix. Hence, predictive thermal inactivation models could benefit from the inclusion of factors related to the nature of the food matrix and fat properties. Starting inactivation from cells that were grown inside the matrix, resulted in lower (i.e., fail-safe)kmaxvalues and more uncertainty onNres as compared to starting from cells grown at optimal conditions.

Radio frequency inactivation of Salmonella Typhimurium and Listeria monocytogenes in skimmed and whole milk powder.

Milk powder is a convenient, shelf-stable food ingredient used in a variety of food products. However, pathogenic bacteria can be present and survive during prolonged storage, leading to outbreaks of foodborne diseases and product recalls. Radio frequency (RF) heating is a processing technology suitable for bulk treatment of milk powder, aiming at microbial inactivation. This study investigates the RF inactivation of Salmonella Typhimurium and Listeria monocytogenes in two types of milk powder; skimmed and whole milk powder. Specifically, the aims were to (i) examine the influence of the powder's composition on bacterial inactivation, (ii) evaluate the response of bacteria with different Gram properties (Gram positive and Gram negative) and (iii) verify the use of Enterococcus faecium as a surrogate for the two microorganisms for the specific RF process. In order to examine exclusively the influence of RF, a non-isothermal temperature profile was used, employing solely different RF energy levels to heat the product to the target temperatures. A log-linear model with a Bigelow-type temperature dependency was fitted to the experimental data. S. Typhimurium was less susceptible to RF treatments in comparison to L.monocytogenes, demonstrating a higher inactivation rate (k) and higher percentage of sublethal injury. A higher k was also observed for both microorganisms in the whole milk powder, indicating that the increased fat content and decreased levels of lactose and protein in the milk powder had an adverse impact on the microbial survival for both pathogens. The surrogate microorganism E. faecium successfully validated the microbial response of the two microorganisms to RF treatments. In general, a low heating rate RF-only process was successful in inactivating the two foodborne pathogens in skimmed and whole milk powder by 4 log(CFU/g).

Influence of microbial cell morphology and composition on radio frequency heating of simple media at different frequencies.

The effect of Listeria monocytogenes, Salmonella Typhimurium, and Saccharomyces cerevisiae on RF heating was studied in sterilized Milli-Q water and saline solution during treatments at 27.0 ± 0.6 MHz and 3.0 ± 0.02 MHz for 30 min. The presence of microorganisms caused a significant increase in temperature (maximum to 54.9 °C), with no significant decrease in cell numbers being observed for any conditions. For both media and frequencies, heating rates followed the order S. Typhimurium ≤ L. monocytogenes ≤ S. cerevisiae, except for heating at 3.0 ± 0.02 MHz in saline solution, where heating rates for S. cerevisiae and S. Typhimurium were equal. Generally, heating rates for microorganisms were significantly higher at 27.0 ± 0.6 MHz than at 3.0 ± 0.02 MHz, except for the S. cerevisiae case. Observed phenomena were probably caused by differences in the cell lipid and peptidoglycan content, with interaction effects with salt being present. This study was the first to investigate the influence of the presence of microorganisms on heating behavior of simple media. On the long term, more research on this topic could lead to finding specific RF frequencies more suitable for the heating of specific media and products for various applications.

The Inclusion of the Food Microstructural Influence in Predictive Microbiology: State-of-the-Art.

Predictive microbiology has steadily evolved into one of the most important tools to assess and control the microbiological safety of food products. Predictive models were traditionally developed based on experiments in liquid laboratory media, meaning that food microstructural effects were not represented in these models. Since food microstructure is known to exert a significant effect on microbial growth and inactivation dynamics, the applicability of predictive models is limited if food microstructure is not taken into account. Over the last 10-20 years, researchers, therefore, developed a variety of models that do include certain food microstructural influences. This review provides an overview of the most notable microstructure-including models which were developed over the years, both for microbial growth and inactivation.

A Population Balance Model to Describe the Evolution of Sublethal Injury.

The detection and quantification of sublethal injury (SI) of pathogenic microorganisms has become a common procedure when assessing the efficiency of microbial inactivation treatments. However, while a plethora of studies investigates SI in function of time, no suitable modelling procedure for SI data has been proposed thus far. In this study, a new SI model structure was developed that relies on existing microbial inactivation models. This model is based on the description of inactivation kinetics between the subpopulations of healthy, sublethally injured and dead cells. The model was validated by means of case studies on previously published results, modelled by different inactivation models, i.e., (i) log-linear inactivation; (ii) biphasic inactivation; and (iii) log-linear inactivation with tailing. Results were compared to those obtained by the traditional method that relies on calculating SI from independent inactivation models on non-selective and selective media. The log-linear inactivation case study demonstrated that the SI model is equivalent to the use of independent models when there can be no mistake in calculating SI. The biphasic inactivation case study illustrated how the SI model avoids unrealistic calculations of SI that would otherwise occur. The final case study on log-linear inactivation with tailing clarified that the SI model provides a more mechanistic description than the independent models, in this case allowing the reduction of the number of model parameters. As such, this paper provides a comprehensive overview of the potential and applications for the newly presented SI model.

Combined Effect of Cold Atmospheric Plasma and Hydrogen Peroxide Treatment on Mature Listeria monocytogenes and Salmonella Typhimurium Biofilms.

Cold Atmospheric Plasma (CAP) is a promising novel method for biofilm inactivation as log-reduction values up to 4.0 log10 (CFU/cm2) have been reported. Nevertheless, as the efficacy of CAP itself is not sufficient for complete inactivation of mature biofilms, the hurdle technology could be applied in order to obtain higher combined efficacies. In this study, CAP treatment was combined with a mild hydrogen peroxide (H2O2) treatment for disinfection of 1 and 7 day(s) old Listeria monocytogenes and Salmonella Typhimurium biofilms. Three different treatment sequences were investigated in order to determine the most effective treatment sequence, i.e., (i) first CAP, then H2O2, (ii) first H2O2, then CAP, and (iii) a simultaneous treatment of CAP and H2O2. Removal of the biofilm, induction of sub-lethal injury, and H2O2 breakdown due to the presence of catalase within the biofilms were investigated in order to comment on their possible contribution to the combined inactivation efficacy. Results indicated that the preferred treatment sequence was dependent on the biofilm forming species, biofilm age, and applied H2O2 concentration [0.05 or 0.20% (v/v)]. At the lowest H2O2 concentration, the highest log-reductions were generally observed if the CAP treatment was preceded by the H2O2 treatment, while at the highest H2O2 concentration, the opposite sequence (first CAP, then H2O2) proved to be more effective. Induction of sub-lethal injury contributed to the combined bactericidal effect, while the presence of catalase within the biofilms resulted in an increased resistance. In addition, high log-reductions were partially the result of biofilm removal. The highest overall log-reductions [i.e., up to 5.42 ± 0.33 log10 (CFU/cm2)] were obtained at the highest H2O2 concentration if CAP treatment was followed by H2O2 treatment. As this resulted in almost complete inactivation of the L. monocytogenes and S. Typhimurium biofilms, the combined treatment of CAP and H2O2 proved to be a promising method for disinfection of abiotic surfaces.

The Complex Effect of Food Matrix Fat Content on Thermal Inactivation of Listeria monocytogenes: Case Study in Emulsion and Gelled Emulsion Model Systems.

Previous studies on the influence of food matrix fat content on thermal inactivation kinetics of food pathogens have shown contradictory results due to the combined influence of fat content and other factors such as composition. Therefore, thermal inactivation of Listeria monocytogenes at 59, 64, and 69°C was systematically investigated in emulsion and gelled emulsion food model systems with various fat content (1, 5, 10, and 20%), such that the effect of fat content was isolated. Thermal conductivity and rheological properties of the model systems were quantified, as well as the effect of these properties on the thermal load of the model systems. Thermal conductivity was complexly related to fat content, the nature of the food matrix (i.e., viscous or gelled), and temperature. For the emulsions, the consistency index K increased with increasing fat content, while the flow behavior index n followed the opposite trend. For the gelled emulsions, the storage modulus G' was always larger than the loss modulus G″ (i.e., measure of elastic and viscous properties, respectively). The phase angle δ [i.e., arctan (G″/G')] was proportional with fat content, but this relation became more complex at higher temperatures. The thermal load of the model systems was not largely affected by food matrix fat content. Thermal inactivation of L. monocytogenes was investigated by means of the maximum specific inactivation rate k max, log reductions, and sublethal injury (SI). Both for emulsions and gelled emulsions, k max decreased with increasing fat content below approximately 60°C, while a more complex behavior was observed at higher temperatures. In the emulsions, log reductions were considerably lower (i.e., 2-3 log) at 1% fat than in systems with higher fat content. In the gelled emulsions, log reductions generally decreased with increasing fat content. SI decreased with increasing fat content, both in emulsions and gelled emulsions. In conclusion, the inactivation rate (i.e., k max) of L. monocytogenes was affected by a complex relation between food matrix fat content, thermal conductivity, rheological properties, and inactivation temperature. Due to the small scale of the model systems, differences in k max did not directly affect the final log reductions in a similar fashion.

Effect of food microstructure on growth dynamics of Listeria monocytogenes in fish-based model systems.

Traditionally, predictive growth models for food pathogens are developed based on experiments in broth media, resulting in models which do not incorporate the influence of food microstructure. The use of model systems with various microstructures is a promising concept to get more insight into the influence of food microstructure on microbial dynamics. By means of minimal variation of compositional and physicochemical factors, these model systems can be used to study the isolated effect of certain microstructural aspects on microbial growth, survival and inactivation. In this study, the isolated effect on microbial growth dynamics of Listeria monocytogenes of two food microstructural aspects and one aspect influenced by food microstructure were investigated, i.e., the nature of the food matrix, the presence of fat droplets, and microorganism growth morphology, respectively. To this extent, fish-based model systems with various microstructures were used, i.e., a liquid, a second more viscous liquid system containing xanthan gum, an emulsion, an aqueous gel, and a gelled emulsion. Growth experiments were conducted at 4 and 10 °C, both using homogeneous and surface inoculation (only for the gelled systems). Results regarding the influence of the growth morphology indicated that the lag phase of planktonic cells in the liquid system was similar to the lag phase of submerged colonies in the xanthan system. The lag phase of submerged colonies in each gelled system was considerably longer than the lag phase of surface colonies on these respective systems. The maximum specific growth rate of planktonic cells in the liquid system was significantly lower than for submerged colonies in the xanthan system at 10 °C, while no significant differences were observed at 4 °C. The maximum cell density was higher for submerged colonies than for surface colonies. The nature of the food matrix only exerted an influence on the maximum specific growth rate, which was significantly higher in the viscous systems than in the gelled systems. The presence of a small amount of fat droplets improved the growth of L. monocytogenes at 4 °C, resulting in a shorter lag phase and a higher maximum specific growth rate. The obtained results could be useful in the determination of a set of suitable microstructural parameters for future predictive models that incorporate the influence of food microstructure on microbial dynamics.

Development of fish-based model systems with various microstructures.

The effectiveness of predictive microbiology is limited by the lack of knowledge concerning the influence of food microstructure on microbial dynamics. Therefore, future modelling attempts should be based on experiments in structured food model systems as well as liquid systems. In this study, fish-based model systems with various microstructures were developed, i.e., two liquid systems (with and without xanthan gum), an emulsion, an aqueous gel, and a gelled emulsion. The microstructural effect was isolated by minimising compositional and physico-chemical changes among the different model systems. The systems were suitable for common growth and mild thermal inactivation experiments involving both homogeneous and surface inoculation. Average pH of the model systems was 6.36±0.03 and average aw was 0.988±0.002. The liquid system without xanthan gum behaved like a Newtonian fluid, while the emulsion and the liquid containing xanthan gum exhibited (non-Newtonian) pseudo-plastic behaviour. Both the aqueous gel and gelled emulsion were classified as strong gels, with a hardness of 1.35±0.07N and 1.25±0.05N, respectively. Fat droplet size of the emulsion and gelled emulsion model systems was evenly distributed around 1µm. In general, the set of model systems was proven to be suitable to study the influence of important aspects of food microstructure on microbial dynamics.

Salmonella Typhimurium and Staphylococcus aureus dynamics in/on variable (micro)structures of fish-based model systems at suboptimal temperatures.

The limited knowledge concerning the influence of food (micro)structure on microbial dynamics decreases the accuracy of the developed predictive models, as most studies have mainly been based on experimental data obtained in liquid microbiological media or in/on real foods. The use of model systems has a great potential when studying this complex factor. Apart from the variability in (micro)structural properties, model systems vary in compositional aspects, as a consequence of their (micro)structural variation. In this study, different experimental food model systems, with compositional and physicochemical properties similar to fish patés, are developed to study the influence of food (micro)structure on microbial dynamics. The microbiological safety of fish products is of major importance given the numerous cases of salmonellosis and infections attributed to staphylococcus toxins. The model systems understudy represent food (micro)structures of liquids, aqueous gels, emulsions and gelled emulsions. The growth/inactivation dynamics and a modelling approach of combined growth and inactivation of Salmonella Typhimurium and Staphylococcus aureus, related to fish products, are investigated in/on these model systems at temperatures relevant to fish products' common storage (4°C) and to abuse storage temperatures (8 and 12°C). ComBase (http://www.combase.cc/) predictions compared with the maximum specific growth rate (µmax) values estimated by the Baranyi and Roberts model in the current study indicated that the (micro)structure influences the microbial dynamics. Overall, ComBase overestimated microbial growth at the same pH, aw and storage temperature. Finally, the storage temperature had also an influence on how much each model system affected the microbial dynamics.