Inactivation of Salmonella, Listeria monocytogenes and Enterococcus faecium NRRL B-2354 in a selection of low moisture foods.

The aims of this study were to obtain data on survival and heat resistance of cocktails of Salmonella, Listeria monocytogenes and the surrogate Enterococcus faecium (NRRL B-2354) in four low moisture foods (confectionery formulation, chicken meat powder, pet food and savoury seasoning) during storage before processing. Inoculated samples were stored at 16°C and cell viability examined at day 0, 3, 7 and 21. At each time point, the heat resistance at 80°C was determined. The purpose was to determine a suitable storage time of inoculated foods that can be applied in heat resistance studies or process validations with similar cell viability and heat resistance characteristics. The main inactivation study was carried out within 7days after inoculation, the heat resistance of each bacterial cocktail was evaluated in each low moisture food heated in thermal cells exposed to temperatures between 70 and 140°C. The Weibull model and the first order kinetics (D-value) were used to express inactivation data and calculate the heating time to achieve 5 log reduction at each temperature. Results showed that the pathogens Salmonella and L. monocytogenes and the surrogate E. faecium NRRL B-2354, can survive well (maximum reduction <0.8 log) in low moisture foods maintained at 16°C, as simulation of warehouse raw material storage in winter and before processing. The D80 value of the pathogens and surrogate did not significantly change during the 21day storage (p>0.05). The inactivation kinetics of the pathogens and surrogate at temperatures between 70 and 140°C, were different between each organism and product. E. faecium NRRL B-2354 was a suitable Salmonella surrogate for three of the low moisture foods studied, but not for the sugar-containing confectionery formulation. Heating low moisture food in moisture-tight environments (thermal cells) to 111.2, 105.3 or 111.8°C can inactivate 5 log of Salmonella, L. monocytogenes or E. faecium NRRL B-2354 respectively.

Application of an Impedimetric Technique for the Detection of Lytic Infection of Salmonella spp. by Specific Phages.

This study was performed to evaluate the adaption of the impedimetric method to detect the lytic infection by Salmonella-specific bacteriophages and to provide a higher selectivity to this rapid method in detecting Salmonella spp. by using specific agents. Three bacteriophages and twelve strains of Salmonella spp. were tested. Each of the twelve strains was used separately to inoculate TSB together with each one of the phages. The inoculum concentration was between 10(6) and 10(7) cfu/mL, at a cell: phage ratio of 1 : 100. From the sample analysis, based on conductance (G) measurements (37 degrees C), the infection could be detected, by observation of both detection-time delay and distinct curve trends. The main conclusions were that kinetic detection by impedance microbiology with phage typing constitutes a method of determining whether a test microorganism is sensitive to the bacteriophage and a method to evaluate whether a lytic bacteriophage is present in a sample, by affecting bacterial growth rate/metabolic change.

Characterization of anti-Listeria bacteriocins isolated from shellfish: potential antimicrobials to control non-fermented seafood.

This work had as main objectives to characterize two bacteriocins produced by lactic acid bacteria (LAB) previously isolated from non-fermented seafood, in order to evaluate their potential as new food protective agents. The two bacteriocinogenic isolates were identified by Polymerase Chain Reaction (PCR) using genus- and species-specific primers, and confirmed by 16S rDNA sequencing, as Enterococcus faecium and Pediococcus pentosaceus. The antimicrobial spectrum of each strain included several indicator microorganisms, some of them also isolated from seafood. Growth of Listeria innocua, L. monocytogenes, Staphylococcus aureus, Bacillus cereus and other LAB species were inhibited, although no inhibition of Gram-negative microorganisms was observed. Proteolytic, but not lipolytic or glycolytic enzymes, completely inactivated the antimicrobial effect of both cell-free supernatants confirming the proteinaceous nature of the inhibitors. The antimicrobial activity was maintained after treatment with NaCl, SDS, Triton X-100, Tween 20, Tween 80 and EDTA after 2 h or 5 h of exposure and both bacteriocins were stable over a wide range of pH and temperatures. Production of bacteriocin by E. faecium (bacALP7) was detected initially at exponential phase and reached a maximum activity of 25,600 AU/ml in the early stationary phase, whereas bacteriocin production by P. pentosaceus ALP57 (bacALP57) reached the maximum at exponential phase with 12,800 AU/ml. The bacteriocins did not kill L. monocytogenes ESB54 nor L. innocua 2030c however, cellular growth was reduced. The partially purified bacteriocins, bacALP7 and bacALP57, were below 6.5 kDa in size as determined by Tricine-SDS gel electrophoresis. E. faecium and P. pentosaceus contained DNA fragments corresponding in size to those recorded for enterocin B and pediocin PA-1, respectively. Sequencing of the fragments from both bacteriocins confirmed the homology. To our knowledge, for the first time two LAB producing bacteriocins similar to pediocin PA-1 and enterocin B, were isolated from non-fermented shellfish. The adaptation of the cultures to seafood matrices may be advantageous in terms of application as a biopreservation strategy for reduction of L. monocytogenes levels in seafood products.

Growth control of Listeria innocua 2030c on vacuum-packaged cold-smoked salmon by lactic acid bacteria.

Five bacteriocin-producing lactic acid bacteria (LAB): Enterococcus faecium ET05, Lactobacillus curvatus ET06, L. curvatus ET30, L. deldrueckii ET32 and Pediococcus acidilactici ET34, selected by their capacity for growth and producing inhibition in vitro at high salt-on-water content, low temperature and anaerobic atmosphere, conditions simulating cold-smoked fish, were inoculated onto salmon fillets, in co-culture with Listeria innocua 2030c, and cold-smoked processed (dry salted for 6 h; drying for 6 h; smoke for 2 h). The finished product was then packed under vacuum and stored at 5 degrees C. Enumeration of LAB and L. innocua was performed during storage. Results showed that strain E. faecium ET05 was the best biopreservative candidate for controlling L. innocua growth in vacuum-packaged cold-smoked salmon (CSS) processed under the salting/drying/smoking parameters referred above. L. curvatus ET30 and L. delbrueckii ET32 also showed a good biopreservation potential for CSS although they were less effective than the former. L. curvatus ET06 and P. acidilactici ET34 showed a bacteriostatic mode of action against the target bacteria in vitro as well as when inoculated into the salmon fillets. This study describes a potential application of five different LAB in the biopreservation of Listeria in CSS.

Could modifications of processing parameters enhance the growth and selection of lactic acid bacteria in cold-smoked salmon to improve preservation by natural means?

Several smoking conditions were examined with the objective of enhancing the numbers of lactic acid bacteria (LAB) by natural means in vacuum-packaged cold-smoked salmon during 21 days of storage at 5 degrees C. Three combinations of salting, drying, and smoking were used: (i) dry salting x time of salting (2 or 6 h); (ii) wet salting (6 h) x dry salting (6 h) x with or without sugar; and (iii) wet salting (6 h) x dry salting (6 h) x different times of smoking (2 or 6 h of drying and 2 or 6 h of smoking). Two batches were processed for each set of conditions. Determinations of pH and salt content in the water phase were carried out for products in each treatment. Microbiological analyses (total viable count, total LAB, Lactobacillus spp., and Enterobacteriaceae) also were conducted at the beginning of storage (t0) and after 21 days of refrigerated storage (tl). There were differential increases in total LAB and lactobacilli during the storage period according to the treatment performed. The most effective treatment to enhance LAB growth was 6 h of dry salting with sugar, 6 h of drying, and 2 h of smoking. These salting-drying-smoking conditions also selected the LAB as the dominant flora at the end of the storage period. The LAB promoted by these processing parameters seem to be potentially useful protective cultures because of their anti-Listeria activity. From the results of this research, we conclude that it is possible to enhance the growth of LAB in general and that of inhibitory strains in particular by suitable choices of processing parameters.

Antimicrobial effects of a microemulsion and a nanoemulsion on enteric and other pathogens and biofilms.

Some microemulsions and nanoemulsions may have antimicrobial properties and be effective anti-biofilm agents. We examined the abilities of two fine emulsions, designated BCTP and TEOP, to inactivate suspensions of vegetative cells of Salmonella spp. Escherichia coli 0157:H7 (VT-), Pseudomonas aeruginosa, Staphylococcus aureus and Listeria monocytogenes. BCTP is an O/W nanoemulsion of soybean oil and tri-n-butyl phosphate emulsified with Triton X-100, while TEOP is an O/W microemulsion of ethyl oleate with Tween 80 as emulsifier and n-pentanol as a co-emulsifier. BCTP was effective in reducing the cell numbers of L. monocytogenes, while TEOP was effective against all five organisms investigated. The abilities of these emulsions to reduce preformed biofilms of the five bacteria were also investigated. With the exception of the biofilm formed by L. monocytogenes, which surprisingly was not significantly affected by BCTP, all biofilms were inhibited by both BCTP and TEOP.

Listeriosis in Portugal: an existing but under reported infection.

BACKGROUND: Listeriosis is a rare disease caused by the bacterium Listeria monocytogenes, the normal vehicle of which is food. The disease, which is largely confined to its risk groups of pregnant women, the elderly and immunocompromised individuals, has increased in incidence in recent years. In Portugal, listeriosis is not a notifiable infection and available data are scarce. The objective of this work was to collate the available information concerning listeriosis in Portugal by compiling a retrospective study of cases recorded over a decade. METHODS: Requests for case data on clinically confirmed listeriosis, recorded over the previous decade, were replied to by 23 hospitals and a National Institute of Health delegation. RESULTS: 35 cases of listeriosis were identified for the period between 1994 and 2003 inclusive, the mortality rate being greater than 17%. According to the data collected in this study for the year 2003, the incidence of this disease in Portugal was at least 1.4 cases per million inhabitants in that year. CONCLUSION: The study demonstrates, for the first time in the widely available literature, that despite their being no cases of listeriosis in Portugal recorded in official reports, the threat of L. monocytogenes to public health is of a similar dimension to that in other countries.

Anti-listerial inhibitory lactic acid bacteria isolated from commercial cold smoked salmon.

The natural microflora of cold-smoked fish at the end of shelf-life are lactic acid bacteria (LAB). Some of these display a capacity to inhibit spoilage as well as several strains of pathogenic micro-organisms, e.g. Listeria monocytogenes which is isolated frequently from cold-smoked salmon (CSS). Eight batches of sliced vacuum-packed CSS from Norway, Scotland and Spain were collected at retail. Packs were stored at 5 degrees C and examined for chemical and microbiological characteristics, at purchase date and at expiration date. pH, water activity and salt content were similar to available data on lightly preserved fish products. There was a consistent pattern in the development of the microflora on CSS; the initial level of LAB was low on freshly produced CSS (10(2) cfu g(-1)); however, storage in vacuum packaging at refrigeration temperature was elective for LAB. At the end of the stated shelf-life these micro-organisms, represented mainly by Lactobacillus spp., attained ca.10(7) cfu g(-1) while Enterobacteriaceae counts were consistently lower (10(5) cfu g(-1)), which indicates the ability of LAB to grow and compete with few carbohydrates available and in the presence of moderate salt concentrations. L. monocytogenes was not found in any sample. Forty-one percent of LAB strains isolated exhibited inhibitory capacity against Listeria innocua, in a plate assay. A majority of the inhibitory effects were non-bacteriocinogenic, but nevertheless were very competitive cultures which may provide an additional hurdle for improved preservation by natural means.

Survival of Lactobacillus sakei during heating, drying and storage in the dried state when growth has occurred in the presence of sucrose or monosodium glutamate.

Spray-dried cells of Lactobacillus sakei CTC 494 survived ca. 60% longer in the spray dried state when cells were grown in the presence of 20 g sucrose l(-1) or 12.5 g monosodium glutamate l(-1). No significant differences were observed in viability during storage in the freeze dried state with the addition of these compounds to the growth medium, nor in survival during a heat treatment (55 degrees C). Both sucrose and glutamate in the growth medium suppressed intracellular accumulation of total amino acids and changed the overall pattern of the individual amino acids. Glutamate in the growth medium enhanced intracellular glutamate by ca. 38%.

Modified Pseudomonas agar: new differential medium for the detection/enumeration of Pseudomonas aeruginosa in mineral water.

Pseudomonas aeruginosa has been implicated as a foodborne and waterborne pathogen and is now considered a primary infectious agent. In the present study, the survival of P. aeruginosa inoculated in mineral water was evaluated by drop counts on Pseudomonas Agar Base (PAB), PAB with CN supplement X107, PAB with cetrimide, PAB with nalidixic acid, and these media with added FeSO(4). Initial counts, before starvation, were the same in all media tested. Following this period, P. aeruginosa became sensitive to PAB with added cetrimide. The addition of FeSO(4) did not improve the recovery of stressed P. aeruginosa but gave colonies a typical dark brown colour being easily differentiated from other species that can grow at 42 degrees C. The modified Pseudomonas agar medium was also tested with several P. aeruginosa strains, other species of Pseudomonas, and other genera. Only P. aeruginosa strains (pyocyanin positive) produced the typical colonies. Our results demonstrate that Pseudomonas agar with ferrous sulphate, used for the differentiation of P. aeruginosa colonies, and nalidixic acid, used as an inhibitor of Gram-positive bacteria, might be a useful medium for the detection of injured P. aeruginosa in mineral water.