Combining High-Pressure Processing and Supercritical Carbon Dioxide for Inactivation of Listeria innocua.

The effect of high-pressure treatment with supercritical CO2 on the inactivation of Listeria innocua in a fish soup was investigated. The soup was inoculated with L. innocua, packaged in modified atmosphere with 50:50 or 95:5 CO2:N2, high-pressure processed (300, 350, 400 and 600 MPa, 2 min) under subcritical (T < 304 K) or supercritical conditions (T > 304 K) and stored at 4 °C for up to 53 days. Treatment at 400 and 600 MPa had a significant (p < 0.05) effect on L. innocua under both supercritical and subcritical conditions. In contrast, pressurization at 350 MPa and supercritical conditions were needed to significantly (p < 0.05) inactive L. innocua. Increased levels of CO2 in the headspace significantly (p < 0.05) reduced the bacterial load during processing, and supercritical conditions had a significant (p < 0.01) interaction with both CO2 levels and pressure. Increased storage time gave significantly increased levels of L. innocua at 400 and 600 MPa. In addition, high levels of CO2 significantly decreased (p < 0.001) growth. However, 350 MPa under supercritical conditions seemed to set the L. innocua in a permanent lag phase, with slow and steadily decreasing numbers of bacteria during storage. All the design variables resulted in significant inactivation of L. innocua, and supercritical conditions combined with high levels of CO2 inhibited the recovery of L. innocua to a large degree.

Effect of the Application of Ultrasound to Homogenize Milk and the Subsequent Pasteurization by Pulsed Electric Field, High Hydrostatic Pressure, and Microwaves.

The efficacy of applying ultrasound (US) as a system to homogenize emulsions has been widely demonstrated. However, research has not yet shown whether the effect achieved by homogenizing milk with US is modified by subsequent pasteurization treatments that use new processing technologies such as pulsed electric fields (PEF), microwaves (MW), and high hydrostatic pressure (HPP). The aim of this study was, therefore, to optimize the application of US for milk homogenization and to evaluate the effect of PEF, HPP, and MW pasteurization treatments on the sensorial, rheological, and microbiological properties of milk throughout its shelf life. To homogenize whole milk, a continuous US system (20 kHz, 0.204 kJ/mL, 100%, 40 °C) was used, and different ultrasonic intensities (0.25, 0.5, and 1.0 kJ/mL) were evaluated. The optimal ultrasonic treatment was selected on the basis of fat globule size distribution and pasteurization treatments by MW (5800 W, 1.8 L/min), PEF (120 kJ/kg, 20 kV/cm) and HPP (600 MPa, 2 min, 10 °C) was applied. The ultrasound intensity that achieved the highest reduction in fat globule size (0.22 ± 0.02 µm) and the most homogeneous distribution was 1.0 kJ/mL. Fat globule size was smaller than in commercial milk (82% of volume < 0.5 µm for US milk versus 97% of volume < 1.2 µm for commercial milk). That size was maintained after the application of the different pasteurization treatments, and the resulting milk had better emulsion stability than commercial milk. After 28 days of storage, no differences in viscosity (4.4-4.9 mPa s) were observed. HPP pasteurization had the greatest impact on color, leading to higher yellowness values than commercial milk. Microbial counts did not vary significantly after 28 days of storage, with counts below 102 CFU/mL for samples incubated at 15 °C and at 37 °C. In summary, the homogenization of milk obtained by US was not affected by subsequent pasteurization processes, regardless of the technology applied (MW, PEF, or HPP). Further research is needed to evaluate these procedures' effect on milk's nutritional and functional properties.

Metabolomics workflow for quality control of differently-processed pre-cooked chicken fillets.

The contents and profiles of small molecules in a food can provide information about quality-related properties. Processing methods and deterioration during storage, e.g. from bacterial proliferation and degradation, might also lead to changes in the metabolome, which can be determined by mass spectrometry-based metabolomics. By measuring as many metabolites as possible in differently treated pre-cooked chicken fillets in an untargeted approach, we studied individual and combined effects of vacuum packaging (VP), soluble gas stabilisation (SGS), high pressure processing (HPP), and microwave volumetric heating (MW) on the quality and shelf-life of the finished product. The extensive dataset was processed using an optimised workflow of consecutive software tools with stringent statistical analysis to prevent over-interpretation, which is an inherent risk of metabolomics data. Our results showed the predominant influence of VP on storage quality since SGS, HPP, and MW did not have the potential to extent shelf-life.

The complete genome sequence of Listeria monocytogenes strain S2542 and expression of selected genes under high-pressure processing.

OBJECTIVES: The study aims to generate the whole genome sequence of L. monocytogenes strain S2542 and to compare it to the genomes of strains RO15 and ScottA. In addition, we aimed to compare gene expression profiles of L. monocytogenes strains S2542, ScottA and RO15 after high-pressure processing (HPP) using ddPCR. RESULTS: The whole genome sequence of L. monocytogenes S2542 indicates that this strain belongs to serotype 4b, in contrast to the previously reported serotype 1/2a. Strain S2542 appears to be more susceptible to the treatment at 400 MPa compared to RO15 and ScottA strains. In contrast to RO15 and ScottA strains, viable cell counts of strain S2542 were below the limit of detection after HPP (400 MPa/8 min) when stored at 8 °C for 24 and 48 h. The transcriptional response of all three strains to HPP was not significantly different.

High-pressure processing-induced transcriptome response during recovery of Listeria monocytogenes.

BACKGROUND: High-pressure processing (HPP) is a commonly used technique in the food industry to inactivate pathogens, including L. monocytogenes. It has been shown that L. monocytogenes is able to recover from HPP injuries and can start to grow again during long-term cold storage. To date, the gene expression profiling of L. monocytogenes during HPP damage recovery at cooling temperature has not been studied. In order identify key genes that play a role in recovery of the damage caused by HPP treatment, we performed RNA-sequencing (RNA-seq) for two L. monocytogenes strains (barotolerant RO15 and barosensitive ScottA) at nine selected time points (up to 48 h) after treatment with two pressure levels (200 and 400 MPa). RESULTS: The results showed that a general stress response was activated by SigB after HPP treatment. In addition, the phosphotransferase system (PTS; mostly fructose-, mannose-, galactitol-, cellobiose-, and ascorbate-specific PTS systems), protein folding, and cobalamin biosynthesis were the most upregulated genes during HPP damage recovery. We observed that cell-division-related genes (divIC, dicIVA, ftsE, and ftsX) were downregulated. By contrast, peptidoglycan-synthesis genes (murG, murC, and pbp2A) were upregulated. This indicates that cell-wall repair occurs as a part of HPP damage recovery. We also observed that prophage genes, including anti-CRISPR genes, were induced by HPP. Interestingly, a large amount of RNA-seq data (up to 85%) was mapped to Rli47, which is a non-coding RNA that is upregulated after HPP. Thus, we predicted that Rli47 plays a role in HPP damage recovery in L. monocytogenes. Moreover, gene-deletion experiments showed that amongst peptidoglycan biosynthesis genes, pbp2A mutants are more sensitive to HPP. CONCLUSIONS: We identified several genes and mechanisms that may play a role in recovery from HPP damage of L. monocytogenes. Our study contributes to new information on pathogen inactivation by HPP.

Genomic characterization of the most barotolerant Listeria monocytogenes RO15 strain compared to reference strains used to evaluate food high pressure processing.

BACKGROUND: High pressure processing (HPP; i.e. 100-600 MPa pressure depending on product) is a non-thermal preservation technique adopted by the food industry to decrease significantly foodborne pathogens, including Listeria monocytogenes, from food. However, susceptibility towards pressure differs among diverse strains of L. monocytogenes and it is unclear if this is due to their intrinsic characteristics related to genomic content. Here, we tested the barotolerance of 10 different L. monocytogenes strains, from food and food processing environments and widely used reference strains including clinical isolate, to pressure treatments with 400 and 600 MPa. Genome sequencing and genome comparison of the tested L. monocytogenes strains were performed to investigate the relation between genomic profile and pressure tolerance. RESULTS: None of the tested strains were tolerant to 600 MPa. A reduction of more than 5 log10 was observed for all strains after 1 min 600 MPa pressure treatment. L. monocytogenes strain RO15 showed no significant reduction in viable cell counts after 400 MPa for 1 min and was therefore defined as barotolerant. Genome analysis of so far unsequenced L. monocytogenes strain RO15, 2HF33, MB5, AB199, AB120, C7, and RO4 allowed us to compare the gene content of all strains tested. This revealed that the three most pressure tolerant strains had more than one CRISPR system with self-targeting spacers. Furthermore, several anti-CRISPR genes were detected in these strains. Pan-genome analysis showed that 10 prophage genes were significantly associated with the three most barotolerant strains. CONCLUSIONS: L. monocytogenes strain RO15 was the most pressure tolerant among the selected strains. Genome comparison suggests that there might be a relationship between prophages and pressure tolerance in L. monocytogenes.

Effect of high pressure and thermal processing on shelf life and quality of strawberry purée and juice.

The purpose of the study was to determine the effects of high pressure processing (HPP; 400-600 MPa, 20 °C, 1.5 or 3 min) and heat treatment (HT; 85 °C for 2 min) of strawberry purée and juice made from the same raw material. Microbiological and enzymatic inactivation, Brix, pH, anthocyanins, vitamin C, colour and sensory properties were analysed after processing and cold storage. The microbiological shelf life of the products was at least 49 days when processed at 500 or 600 MPa. Anthocyanins, vitamin C and colour were well preserved after HPP and HT. During storage, anthocyanins, vitamin C and sensory quality were better conserved in HT than in HPP purées, while there were minor differences between HT and HPP juices. This was probably due to higher enzyme activity in HPP purées, and indicates that raw materials with lower initial enzyme activity, like juices, are more suited for HPP than e.g. purées.

Survival of Five Strains of Shiga Toxigenic Escherichia coli in a Sausage Fermentation Model and Subsequent Sensitivity to Stress from Gastric Acid and Intestinal Fluid.

The ability of foodborne pathogens to exhibit adaptive responses to stressful conditions in foods may enhance their survival when passing through the gastrointestinal system. We aimed to determine whether Escherichia coli surviving stresses encountered during a model dry-fermented sausage (DFS) production process exhibit enhanced tolerance and survival in an in vitro gastrointestinal model. Salami sausage batters spiked with five E. coli isolates, including enterohaemorrhagic E. coli strains isolated from different DFS outbreaks, were fermented in a model DFS process (20°C, 21 days). Control batters spiked with the same strains were stored at 4°C for the same period. Samples from matured model sausages and controls were thereafter exposed to an in vitro digestion challenge. Gastric exposure (pH 3) resulted in considerably reduced survival of the E. coli strains that had undergone the model DFS process. This reduction continued after entering intestinal challenge (pH 8), but growth resumed after 120 min. When subjected to gastric challenge for 120 min, E. coli that had undergone the DFS process showed about 2.3 log10⁡ lower survival compared with those kept in sausage batter at 4°C. Our results indicated that E. coli strains surviving a model DFS process exhibited reduced tolerance to subsequent gastric challenge at low pH.

Survival of Shiga toxin-producing Escherichia coli and Stx bacteriophages in moisture enhanced beef.

Moisture enhancement of meat through injection is a technology to improve the sensory properties and the weight of meat. However, the technology may increase the risk of food borne infections. Shiga toxin-producing Escherichia coli (STEC) or bacteriophages carrying cytotoxin genes (Shiga toxin genes, stx), which is normally only present on the surface of intact beef, may be transferred to the inner parts of the muscle during the injection process. Pathogens and bacteriophages surviving the storage period may not be eliminated in the cooking process since many consumers prefer undercooked beef. Measures to increase the microbial food safety of moisture enhanced beef may include sterilization or washing of the outer surface of the meat before injection, avoiding recycling of marinade and addition of antimicrobial agents to the marinade. This paper reviews the literature regarding microbial safety of moisture enhanced beef with special emphasis on STEC and Stx bacteriophages. Also, results from a European Union research project, ProSafeBeef (Food-CT-16 2006-36241) are presented.

Shiga toxigenic Escherichia coli show strain dependent reductions under dry-fermented sausage production and post-processing conditions.

Dry-fermented sausages (DFS) are considered possible risk products regarding Shiga toxigenic Escherichia coli (STEC). We have compared the reduction of 11 E. coli isolates of various serogroups in salami during the sausage production process and during post-process measures including storage, heating and freezing. The 11 E. coli isolates, mainly STEC, included enterohaemorrhagic E. coli (EHEC) outbreak strains linked to DFS along with apathogenic E. coli. During sausage production, there was a statistically significant difference in reduction between the E. coli strains ranging from 1.3 to 2.4 log10 (p<0.001). When sausages were subjected to post-process heat treatment of 43 °C for 24 h, a total reduction of more than 5 log10 was obtained for all E. coli isolates. Freezing and thawing of DFS with subsequent storage for 1 month at 16 or 20 °C generally contributed to large E. coli reductions with the latter conditions giving an average additional 3.9 log10 reduction, with a range from 3.4 to 4.4 log10. The combination of freezing and 1 month of storage gave higher reductions compared with storage for 2 months for all examined temperatures. No systematic differences in survival of E. coli of different serogroups were detected for the different post-process measures. The reductions were also similar to those of apathogenic control isolates. Isolates showing higher survival during the ripening process did not have a lower reduction when exposed to post-process stress like storage, heating and freezing. The ability of the isolates to survive in salami was also compared with their survival at equivalent conditions in a tryptic soy broth (TSB) model. There was a low and not significant correlation (p>0.1) between the reductions of E. coli in salami and in the TSB broth model. Results based on broth models and/or single or surrogate strains must therefore be interpreted with caution. The EHEC reducing post-processing measures tested can easily be implemented in DFS production with marginal influence on the quality of the sausages.

High stability of Stx2 phage in food and under food-processing conditions.

Bacteriophages (phages) carrying Shiga toxin genes constitute a major virulence attribute in enterohemorrhagic Escherichia coli (EHEC). Several EHEC outbreaks have been linked to food. The survival of such strains in different foods has received much attention, while the fate of the mobile Shiga toxin-converting phages (Stx phages) has been less studied. We have investigated the stability of an Stx phage in several food products and examined how storage, food processing, and disinfection influence the infectivity of phage particles. The study involved a recombinant Stx phage (Δstx::cat) of an E. coli O103:H25 strain from a Norwegian outbreak in 2006. Temperature, matrix, and time were factors of major importance for the stability of phage particles. Phages stored at cooling temperatures (4°C) showed a dramatic reduction in stability compared to those stored at room temperature. The importance of the matrix was evident at higher temperatures (60°C). Phages in ground beef were below the detection level when heated to 60°C for more than 10 min, while phages in broth exposed to the same heating conditions showed a 5-log-higher stability. The phages tolerated desiccation poorly but were infective for a substantial period of time in solutions. Under moist conditions, they also had a high ability to tolerate exposure to several disinfectants. In a dry-fermented sausage model, phages were shown to infect E. coli in situ. The results show that Stx phage particles can maintain their infectivity in foods and under food-processing conditions.

Reduction of verotoxigenic Escherichia coli in production of fermented sausages.

After a number of foodborne outbreaks of verotoxigenic Escherichia coli involving fermented sausages, some countries have imposed regulations on sausage production. For example, the US Food Safety and Inspection Service requires a 5 log(10) reduction of E. coli in fermented products. Such regulations have led to a number of studies on the inactivation of E. coli in fermented sausages by changing processing and post-processing conditions. Several factors influence the survival of E. coli such as pre-treatment of the meat, amount of NaCl, nitrite and lactic acid, water activity, pH, choice of starter cultures and addition of antimicrobial compounds. Also process variables like fermentation temperature and storage time play important roles. Though a large variety of different production processes of sausages exist, generally the reduction of E. coli caused by production is in the range 1-2 log(10). In many cases this may not be enough to ensure microbial food safety. By optimising ingredients and process parameters it is possible to increase E. coli reduction to some extent, but in some cases still other post process treatments may be required. Such treatments may be storage at ambient temperatures, specific heat treatments, high pressure processing or irradiation. HACCP analyses have identified the quality of the raw materials, low temperature in the batter when preparing the sausages and a rapid pH drop during fermentation as critical control points in sausage production. This review summarises the literature on the reduction verotoxigenic E. coli in production of fermented sausages.

Responses of Staphylococcus aureus exposed to HCl and organic acid stress.

Staphylococcus aureus is an important food poisoning bacterium. In food preservation, acidification is a well-known method. Permeant weak organic acids, like lactic and acetic acids, are known to be more effective against bacteria than inorganic strong acids (e.g., HCl). Growth experiments and metabolic and transcriptional analyses were used to determine the responses of a food pathogenic S. aureus strain exposed to lactic acid, acetic acid, and HCl at pH 4.5. Lactic and acetic acid stress induced a slower transcriptional response and large variations in growth patterns compared with the responses induced by HCl. In cultures acidified with lactic acid, the pH of the medium gradually increased to 7.5 during growth, while no such increase was observed for bacteria exposed to acetic acid or HCl. Staphylococcus aureus increased the pH in the medium mainly through accumulation of ammonium and the removal of acid groups, resulting in increased production of diacetyl (2,3-butanedione) and pyrazines. The results showed flexible and versatile responses of S. aureus to different types of acid stress. As measured by growth inhibition, permeant organic acid stress introduced severe stress compared with the stress caused by HCl. Cells exposed to lactic acid showed specific mechanisms of action in addition to sharing many of the mechanisms induced by HCl stress.

MALDI-TOF mass spectrometry for quantitative gene expression analysis of acid responses in Staphylococcus aureus.

Microorganisms are constantly exposed to new and altered growth conditions, and respond by changing gene expression patterns. Several methods for studying gene expression exist. During the last decade, the analysis of microarrays has been one of the most common approaches applied for large scale gene expression studies. A relatively new method for gene expression analysis is MassARRAY, which combines real competitive-PCR and MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry. In contrast to microarray methods, MassARRAY technology is suitable for analysing a larger number of samples, though for a smaller set of genes. In this study we compare the results from MassARRAY with microarrays on gene expression responses of Staphylococcus aureus exposed to acid stress at pH 4.5. RNA isolated from the same stress experiments was analysed using both the MassARRAY and the microarray methods. The MassARRAY and microarray methods showed good correlation. Both MassARRAY and microarray estimated somewhat lower fold changes compared with quantitative real-time PCR (qRT-PCR). The results confirmed the up-regulation of the urease genes in acidic environments, and also indicated the importance of metal ion regulation. This study shows that the MassARRAY technology is suitable for gene expression analysis in prokaryotes, and has advantages when a set of genes is being analysed for an organism exposed to many different environmental conditions.

Characterization of crustins from the hemocytes of the spider crab, Hyas araneus, and the red king crab, Paralithodes camtschaticus.

Crustins are distributed across the decapods and are believed to play a significant part in the humoral defense system of their host. In this study, two crustin isoforms from Hyas araneus hemocytes were purified and tested for antimicrobial activity against selected microorganisms. They show both antibacterial and antifungal activity, with highest activity against the Gram-positive bacteria Corynebacterium glutamicum. Sequencing of the transcripts showed them to have a mature peptide of 90 amino acids and differing in three positions in the mature peptide. They were named CruHa1 and CruHa2. Real-time RT-PCR revealed that they mainly are expressed in hemocytes. Screening a cDNA library detected a crustin sequence in Paralithodes camtschaticus hemocytes, coding for a mature peptide of 98 amino acids. It was named CruPc. Based on phylogenetic inference and primary structure, CruHa1 and CruHa2 were placed within the Type I group of crustins, while CruPc belongs to the Type II.

Acid-shock responses in Staphylococcus aureus investigated by global gene expression analysis.

A general overview is presented of the changes in the genetic expression along a time curve through the first 20 min after acidification to pH 4.5 of exponentially growing cultures of the food pathogenic strain Staphylococcus aureus 50583. A newly developed method for statistical significance testing was used to detect significant gene expression responses. Most responses showed an increase or decrease from time zero to 10 min after acidification, and then generally a stabilization in expression level from 10 to 20 min. Increased urease activity appeared to be an important factor in the acid defence, along with proton excretion by NADH dehydrogenase and macromolecule repair mechanisms. Oxidative-stress responses, such as increased expression of thioredoxin genes and upregulation of pentose phosphate pathway genes to generate more reducing power, were also induced. A general reduction in the expression of genes encoding ribosomal proteins and genes involved in nucleotide synthesis, as well as fatty acid and lipoprotein metabolism, reflected the lowered growth rate after acidification. The pH shock did not appear to trigger major virulence responses or biofilm formation. Metal ion regulation and transport were affected by the acid shock, and production of several cofactors such as molybdopterin was increased. Many of the presented observations could be explained, while some represent still-unknown mechanisms. The patterns of regulation were confirmed by quantitative reverse transcriptase PCR (QRT-PCR). Together, these results showed the main responses of S. aureus and will be a good starting point for future, more specific, in-depth studies of specific gene responses that occur in conjunction with the acid-stress defence of S. aureus.

Different patterns of biofilm formation in Staphylococcus aureus under food-related stress conditions.

Staphylococcus aureus and its biofilm formation are recognized as a serious clinical problem. S. aureus is also a food borne pathogen, and little is known regarding biofilm formation of food-related strains. We have studied biofilm formation of both food-related and clinical S. aureus strains grown under different stress conditions (temperature, sodium chloride, glucose and ethanol) relevant for food processing. Strong biofilm formers were identified among food-related S. aureus strains, and biofilm formation was affected by environmental conditions relevant for the food industry. The results showed that temperatures suboptimal for growth increased the production of biofilm. The combined presence of sodium chloride and glucose enhanced the biofilm formation. Both temperature and osmolarity affected the expression of several biofilm associated genes (e.g. icaA and rbf). Variations in gene expression (e.g. icaA, agrA and sigB) between strains were also observed. Our results support the existence of both ica-dependent and ica-independent mechanisms of biofilm production in S. aureus. The phenotypic and genotypic results showed highly diverse and complex patterns of biofilm formation in S. aureus. This clearly demonstrates that caution must be exercised before drawing general conclusions about gene expression in S. aureus in relation to regulation of biofilm formation. The results are relevant for food safety as they indicate that food processing conditions could promote biofilm formation by S. aureus.