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.

Change in the color of heat-treated, vacuum-packed broccoli stems and florets during storage: effects of process conditions and modeling by an artificial neural network.

BACKGROUND: Vacuum-packed broccoli stems and florets were subjected to heat treatment (60-99 °C) for various time intervals. The activity of peroxidase was measured after processing. Thermally processed samples were then stored at 4 °C for 35 days, and the color of the samples was measured every 7 days. Effects of parameters (heating temperature and duration, storage time) on the color of broccoli were modeled and simulated by an artificial neural network (ANN). RESULTS: Simulations confirmed that stems were predicted to be more prone to changes than florets. More color loss was observed with longer processing or storage combinations. The simulations also confirmed that higher temperatures during heat processing could retard color changes during storage. For stems treated at 80 °C for short durations, color loss was more predominant than both 65 and 99 °C, probably due to the incomplete inactivation of enzymes besides more tissue damage, with increased enzyme access to the substrate. CONCLUSION: The greenness of both stems and florets during storage can be better preserved at higher temperatures (99 °C) and short times. The simulation results revealed that the ANN method could be used as an effective tool for predicting and analyzing the color values of heat-treated broccoli. © 2018 Society of Chemical Industry.

A novel model to assess the efficacy of steam surface pasteurization of cooked surimi gels inoculated with realistic levels of Listeria innocua.

Steam surface pasteurization is a promising decontamination technology for reducing pathogenic bacteria in different stages of food production. The effect of the artificial inoculation type and initial microbial load, however, has not been thoroughly assessed in the context of inactivation studies. In order to optimize the efficacy of the technology, the aim of this study was to design and validate a model system for steam surface pasteurization, assessing different inoculation methods and realistic microbial levels. More specifically, the response of Listeria innocua, a surrogate organism of Listeria monocytogenes, on a model fish product, and the effect of different inoculation levels following treatments with a steam surface pasteurization system was investigated. The variation in the resulting inoculation level on the samples was too large (77%) for the contact inoculation procedure to be further considered. In contrast, the variation of a drop inoculation procedure was 17%. Inoculation with high levels showed a rapid 1-2 log decrease after 3-5 s, and then no further inactivation beyond 20 s. A low level inoculation study was performed by analysing the treated samples using a novel contact plating approach, which can be performed without sample homogenization and dilution. Using logistic regression, results from this method were used to model the binary responses of Listeria on surfaces with realistic inoculation levels. According to this model, a treatment time of 23 s will result in a 1 log reduction (for P = 0.1).

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).

Effects of capture-related stress and pre-freezing holding in refrigerated sea water (RSW) on the muscle quality and storage stability of Atlantic mackerel (Scomber scombrus) during subsequent frozen storage.

Crowded (stressed) and unstressed Atlantic mackerel with or without pre-freezing holding in refrigerated sea water (RSW) were stored at -19 °C for ∼12 months and analysed for nucleotide degradation (K value), muscle pH, water holding capacity (WHC), fillet firmness, cathepsin B/L like activity, lipid oxidation and fillet colour. The frozen storage showed the largest and most consistent direct effects on the quality metrics leading to increased lipid oxidation, discolouration (yellowing) and reduction on WHC and cathepsin activity. RSW treatment promoted nucleotide degradation and reduced WHC and fillet firmness in interaction with frozen storage and affected fillet colour lightness and saturation. Although showing only marginal main effects, crowding stress modified WHC, cathepsin activity and fillet firmness and colour through significant interactions with the frozen storage and RSW treatment. Further studies with larger sample sizes would be needed to elucidate their complex effects and interactions on the quality and storage stability of mackerel.

Effect of antioxidants on the sensory quality and physicochemical stability of Atlantic mackerel (Scomber scombrus) fillets during frozen storage.

This study aimed to evaluate the shelf-life of mechanically filleted well-fed Atlantic mackerel during frozen storage at -25 °C and effect of treatment with antioxidants (sodium erythorbate and a polyphosphate mixture) and different antioxidant application methods (dipping, spraying and glazing). Both physicochemical measurements and sensory analysis were applied. Antioxidant treatments prolonged shelf-life of mackerel. Sensory analysis indicated that untreated fillets had a shelf-life of less than 2.5 months, while all antioxidant treated fillets exceeded that. The most effective treatment, dipping fillets into a sodium erythorbate solution, yielding a shelf-life of 15 months. Physicochemical methods used to evaluate degradation of lipids in the fillets were free fatty acids (FFA), lipid hydroperoxides (PV) and thiobarbituric acid reactive substances (TBARS). They did not correlate with sensory results and might therefore be a questionable choice for evaluation of oxidation and development of rancid flavour and odour in complex matrixes such as Atlantic mackerel.

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.

Growth kinetics of listeria isolated from salmon and salmon processing environment: single strains versus cocktails.

The growth dynamics of Listeria monocytogenes strains isolated from salmon or a salmon processing environment and two reference Listeria innocua strains were investigated at refrigerated and close-to-optimal growth temperatures. Estimates for the growth rates and the lag-phase duration at 4, 8, 12, and 30°C were obtained for optical density measurements by using different growth parameter estimation methods, i.e., the serial dilution (SD) method and the relative rate to detection (RRD) method. Both single L. innocua and L. monocytogenes strains and mixtures of L. monocytogenes strains (cocktails) were studied. Both methods show an increase in maximum growth rate (µ(max)) of Listeria with increasing temperatures. Generally, single-strain growth rate estimates were quite similar for both species, although L. monocytogenes showed slightly higher µ(max) estimates at 4°C. The SD method gave the highest estimates for the growth rate, i.e., the estimates from the RRD method were 10 to 20% lower. This should lead to caution when using the latter method for Listeria, particularly at lower temperatures. Overall, the SD method is preferred as this method yields µ(max) estimates close to the biological value and provides estimates for the duration of lag time (λ). For discrimination between different strains, λ appeared to be a more suitable parameter than µ(max). This effect was most prominent for L. innocua. Significant differences were observed between µ(max) and/or λ of L. monocytogenes cocktails and single strains at all temperatures investigated. At 4°C, the average growth rate of cocktails was higher than that of single strains. At 8 and 30°C, this trend was reversed. The average λ of single strains were more than twice as long as those of cocktails at 4°C. At 8 and 30°C, the λ of cocktails were significantly slower than those of single strains, but the variation was considerably less and the differences were less pronounced.

Injection-salting of pre rigor fillets of Atlantic salmon (Salmo salar).

The effects of temperature (-1, 4, and 10 degrees C), brine concentration (12% and 25% NaCl), injection volumes, and needle densities were investigated on fillet weight gain (%), salt content (%), fillet contraction (%), and muscle gaping in pre rigor brine-injected fillets of Atlantic salmon (Salmo salar). Increased brine concentration (12% to 25%) significantly increased the initial (< 5 min after injection) and final contraction (24 h after injection) of pre rigor fillets. Increased brine concentration significantly reduced weight gain and increased salt content but had no significant effect on muscle gaping. The temperatures tested did not significantly affect weight gain, fillet contraction, or gaping score. Significant regressions (P < 0.01) between the injection volume and weight gain (range: 2.5% to 15.5%) and salt content (range: 1.7% to 6.5%) were observed for injections of pre rigor fillets. Double injections significantly increased the weight gain and salt content compared to single injections. Initial fillet contraction measured 30 min after brine injection increased significantly (P < 0.01) with increasing brine injection volume but no significant difference in the fillet contraction was observed 12 h after brine injection (range: 7.9% to 8.9%). Brine-injected post rigor control fillets obtained higher weight gain, higher salt content, more muscle gaping, and significantly lower fillet contraction compared to the pre rigor injected fillets. Injection-salting is an applicable technology as a means to obtain satisfactory salt contents and homogenously distribute the salt into the muscle of pre rigor fillets of Atlantic salmon before further processing steps such as drying and smoking.