Anette McLeod

Diversity of Lactobacillus sakei strains investigated by phenotypic and genotypic methods

The diversity of 10 strains of Lactobacillus sakei, a commercially important species of lactobacilli, was characterized by studying food isolates. Growth characteristics varied among the strains when examined after growth in a complex medium and a defined medium with either glucose or ribose. A commercial starter culture strain showed the fastest growth rates and high biomass formation on all media, while two of the strains hardly grew on ribose. Based on acidification properties in a meat model, some of the strains had the ability to compete with the indigenous microbiota of the meat batter in addition to being fast acid producers. Carbohydrate-fermentation abilities revealed a relatively wide variation, clustering the strains into two phenotypic groups. The isolates were analyzed using different genetic fingerprinting techniques, demonstrating a distinction between two genetic groups, a grouping consistent with previous studies dealing with L. sakei strains. Comparative genome hybridization (CGH) was introduced for clustering the strains and the same division into two genetic groups was observed. Chromosomal sizes of the strains were estimated by pulsed field gel electrophoresis (PFGE) and were found to vary from 1884 to 2175 kb. The genetic groups did not correlate with the clustering obtained with carbohydrate-fermenting abilities or with chromosomal sizes.

Primary metabolism in Lactobacillus sakei food isolates by proteomic analysis

BackgroundLactobacillus sakei is an important food-associated lactic acid bacterium commonly used as starter culture for industrial meat fermentation, and with great potential as a biopreservative in meat and fish products. Understanding the metabolic mechanisms underlying the growth performance of a strain to be used for food fermentations is important for obtaining high-quality and safe products. Proteomic analysis was used to study the primary metabolism in ten food isolates after growth on glucose and ribose, the main sugars available for L. sakei in meat and fish.ResultsProteins, the expression of which varied depending on the carbon source were identified, such as a ribokinase and a D-ribose pyranase directly involved in ribose catabolism, and enzymes involved in the phosphoketolase and glycolytic pathways. Expression of enzymes involved in pyruvate and glycerol/glycerolipid metabolism were also affected by the change of carbon source. Interestingly, a commercial starter culture and a protective culture strain down-regulated the glycolytic pathway more efficiently than the rest of the strains when grown on ribose. The overall two-dimensional gel electrophoresis (2-DE) protein expression pattern was similar for the different strains, though distinct differences were seen between the two subspecies (sakei and carnosus), and a variation of about 20% in the number of spots in the 2-DE gels was observed between strains. A strain isolated from fermented fish showed a higher expression of stress related proteins growing on both carbon sources.ConclusionsIt is obvious from the data obtained in this study that the proteomic approach efficiently identifies differentially expressed proteins caused by the change of carbon source. Despite the basic similarity in the strains metabolic routes when they ferment glucose and ribose, there were also interesting differences. From the application point of view, an understanding of regulatory mechanisms, actions of catabolic enzymes and proteins, and preference of carbon source is of great importance.

Global transcriptome response in Lactobacillus sakei during growth on ribose.

BackgroundLactobacillus sakei is valuable in the fermentation of meat products and exhibits properties that allow for better preservation of meat and fish. On these substrates, glucose and ribose are the main carbon sources available for growth. We used a whole-genome microarray based on the genome sequence of L. sakei strain 23K to investigate the global transcriptome response of three L. sakei strains when grown on ribose compared with glucose.ResultsThe function of the common regulated genes was mostly related to carbohydrate metabolism and transport. Decreased transcription of genes encoding enzymes involved in glucose metabolism and the L-lactate dehydrogenase was observed, but most of the genes showing differential expression were up-regulated. Especially transcription of genes directly involved in ribose catabolism, the phosphoketolase pathway, and in alternative fates of pyruvate increased. Interestingly, the methylglyoxal synthase gene, which encodes an enzyme unique for L. sakei among lactobacilli, was up-regulated. Ribose catabolism seems closely linked with catabolism of nucleosides. The deoxyribonucleoside synthesis operon transcriptional regulator gene was strongly up-regulated, as well as two gene clusters involved in nucleoside catabolism. One of the clusters included a ribokinase gene. Moreover, hprK encoding the HPr kinase/phosphatase, which plays a major role in the regulation of carbon metabolism and sugar transport, was up-regulated, as were genes encoding the general PTS enzyme I and the mannose-specific enzyme II complex (EIIman). Putative catabolite-responsive element (cre) sites were found in proximity to the promoter of several genes and operons affected by the change of carbon source. This could indicate regulation by a catabolite control protein A (CcpA)-mediated carbon catabolite repression (CCR) mechanism, possibly with the EIIman being indirectly involved.ConclusionsOur data shows that the ribose uptake and catabolic machinery in L. sakei is highly regulated at the transcription level. A global regulation mechanism seems to permit a fine tuning of the expression of enzymes that control efficient exploitation of available carbon sources.

Genome Sequence of Lactobacillus sakei subsp. sakei LS25, a Commercial Starter Culture Strain for Fermented Sausage

ABSTRACT Lactobacillus sakei is a lactic acid bacterium associated primarily with fermented meat and fish. Here, we present the draft genome sequence of L. sakei subsp. sakei strain LS25, a commercial starter culture strain for fermented sausage.

Effect of relevant environmental stresses on survival of enterohemorrhagic Escherichia coli in dry-fermented sausage.

Dry-fermented sausages (DFSs) have been linked to several serious foodborne outbreaks of enterohemorrhagic Escherichia coli (EHEC). The ability of pathogens to utilize adaptive responses to different stressful conditions intended to control their growth in foods, food preparation and production processes may enhance their survival. In certain cases, induced tolerance to one type of stress may lead to enhanced resistance to the applied stress as well as to other stresses. We exposed two EHEC strains, MF3582 of serotype O157:H- and MF5554 of serogroup O145, to different stresses commonly encountered during a production process. The two EHEC strains, previously shown to have different abilities to survive DFS production process conditions, were subjected to low temperatures (4°C and 12°C), 5% NaCl or 1% lactic acid for 6days prior to being added to sausage batters. Survival of EHEC was recorded in salami of two recipes, fermented at two temperatures (20°C and 30°C). The results showed that recipe type had the largest impact on EHEC reductions where Moderate recipe (MR) salami batters containing increased levels of NaCl, glucose and NaNO2 provided enhanced EHEC reductions in salami (2.6 log10) compared to Standard recipe (SR) salami (1.7 log10). Effects of pre-exposure stresses were dependent both on strain and recipe. While acid adaptation of MF5554 provided enhanced log10 reductions from 2.0 to 3.0 in MR sausages, adaptation to a combination of acid and salt stress showed the opposite effect in SR sausages with reductions of only 1.1 log10 as compared to the average of 1.8 log10 for the other SR sausages. Otherwise, the salt and acid adaptation single stresses had relatively small effects on EHEC survival through the DFS production process and subsequent storage and freeze/thaw treatments. Growing cells and cells frozen in batter survived poorly in MR sausages with an average reduction of 3.4 and 3.2 log10, respectively. The reductions of EHEC after storage of DFS increased with higher temperature and storage time. Up to 3.7 log10 additional reduction was obtained when MF3582 was stored for 2months at 20°C. In conclusion, adaptation of EHEC to acid, salt and low temperatures prior to being introduced in a DFS production process has limited, but strain dependent effects on EHEC reductions. Producers should avoid conditions leading to acid and salt adapted cells that can contaminate the sausage batter. Recipe parameters had the largest impact on EHEC reductions while storage at 20°C is effective for enhanced reductions in finished products.

Health and Safety Considerations of Fermented Sausages

Fermented sausages are highly treasured traditional foods. A large number of distinct sausages with different properties are produced using widely different recipes and manufacturing processes. Over the last years, eating fermented sausages has been associated with potential health hazards due to their high contents of saturated fats, high NaCl content, presence of nitrite and its degradation products such as nitrosamines, and use of smoking which can lead to formation of toxic compounds such as polycyclic aromatic hydrocarbons. Here we review the recent literature regarding possible health effects of the ingredients used in fermented sausages. We also go through attempts to improve the sausages by lowering the content of saturated fats by replacing them with unsaturated fats, reducing the NaCl concentration by partly replacing it with KCl, and the use of selected starter cultures with desirable properties. In addition, we review the food pathogenic microorganisms relevant for fermented sausages (Escherichia coli, Salmonella enterica, Staphylococcus aureus, Listeria monocytogenes, Clostridium botulinum, and Toxoplasma gondii) and processing and postprocessing strategies to inhibit their growth and reduce their presence in the products.

Chicken fillets subjected to UV-C and pulsed UV light: Reduction of pathogenic and spoilage bacteria, and changes in sensory quality

Abstract We have compared the efficacy of continuous ultraviolet (UV‐C) (254 nm) and pulsed UV light in reducing the viability of Salmonella Enteritidis, Listeria monocytogenes, Staphylococcus aureus, enterohemorrhagic Escherichia coli, Pseudomonas spp., Brochothrix thermospacta, Carnobacterium divergens, and extended‐spectrum β‐lactamase producing E. coli inoculated on chicken fillet surface. Fluences from 0.05 to 3.0 J/cm2 (10 mW/cm2, from 5 to 300 s) used for UV‐C light resulted in average reductions from 1.1 to 2.8 log cfu/cm2. For pulsed UV light, fluences from 1.25 to 18.0 J/cm2 gave average reductions from 0.9 to 3.0 log cfu/cm2. A small change in the odor characterized as sunburnt and increased concentration of volatile compounds associated with burnt odor posed restrictions on the upper limit of UV treatment, however no sensory changes were observed after cooking the meat. Treatments under modified atmosphere conditions using a UV permeable top film gave similar or slightly lower bacterial reductions. Practical applications Ultraviolet (UV) light may be used for decontaminating the surface of food products and reduce viability of pathogenic and spoilage bacteria. Exposure of raw chicken fillet surface to various doses of continuous UV‐C or pulsed UV light proposed in the present work represent alternatives for microbiological improvement of this product. Chicken fillets can be treated in intact packages covered with UV permeable top film, thus avoiding recontamination of the meat. UV‐C light treatment is a low cost strategy with low maintenance, whereas pulsed UV light involves more elaborate equipment, but treatment times are short and less space is required. Both methods can be helpful for producers to manage the safety and quality of chicken fillets.

Effects of glucose availability in Lactobacillus sakei; metabolic change and regulation of the proteome and transcriptome

Effects of glucose availability were investigated in Lactobacillus sakei strains 23K and LS25 cultivated in anaerobic, glucose-limited chemostats set at high (D = 0.357 h-1) and low (D = 0.045 h-1) dilution rates. We observed for both strains a shift from homolactic towards more mixed acid fermentation when comparing high to low growth rates. However, this change was more pronounced for LS25 than for 23K, where dominating products were lactate>formate>acetate≥ethanol at both conditions. A multivariate approach was used for analyzing proteome and transcriptome data from the bacterial cultures, where the predictive power of the omics data was used for identifying features that can explain the differences in the end-product profiles. We show that the different degree of response to the same energy restriction revealed interesting strain specific regulation. An elevated formate production level during slow growth, more for LS25 than for 23K, was clearly reflected in correlating pyruvate formate lyase expression. With stronger effect for LS25, differential expression of the Rex transcriptional regulator and NADH oxidase, a target of Rex, indicated that maintainance of the cell redox balance, in terms of the NADH/NAD+ ratio, may be a key process during the metabolic change. The results provide a better understanding of different strategies that cells may deploy in response to changes in substrate availability.

Comparison of UV-C and Pulsed UV Light Treatments for Reduction of Salmonella, Listeria monocytogenes, and Enterohemorrhagic Escherichia coli on Eggs

Ten percent of all strong-evidence foodborne outbreaks in the European Union are caused by Salmonella related to eggs and egg products. UV light may be used to decontaminate egg surfaces and reduce the risk of human salmonellosis infections. The efficiency of continuous UV-C (254 nm) and pulsed UV light for reducing the viability of Salmonella Enteritidis, Listeria monocytogenes, and enterohemorrhagic Escherichia coli on eggs was thoroughly compared. Bacterial cells were exposed to UV-C light at fluences from 0.05 to 3.0 J/cm2 (10 mW/cm2, for 5 to 300 s) and pulsed UV light at fluences from 1.25 to 18.0 J/cm2, resulting in reductions ranging from 1.6 to 3.8 log, depending on conditions used. Using UV-C light, it was possible to achieve higher reductions at lower fluences compared with pulsed UV light. When Salmonella was stacked on a small area or shielded in feces, the pulsed UV light seemed to have a higher penetration capacity and gave higher bacterial reductions. Microscopy imaging and attempts to contaminate the interior of the eggs with Salmonella through the eggshell demonstrated that the integrity of the eggshell was maintained after UV light treatments. Only minor sensory changes were reported by panelists when the highest UV doses were used. UV-C and pulsed UV light treatments appear to be useful decontamination technologies that can be implemented in continuous processing.

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.