The environmental conditions reached during the ripening of dry-cured meat products favour the proliferation of moulds on their surface. Some of these moulds are hazardous to consumers because of ...their ability to produce ochratoxin A (OTA). Biocontrol using Debaryomyces hansenii could be a suitable strategy to prevent the growth of ochratoxigenic moulds and OTA accumulation in dry-cured meat products. The aim of this work was to evaluate the ability of two strains of D. hansenii to control the growth and OTA production of Penicillium verrucosum in a meat model under water activities (aw) values commonly reached during the dry-cured meat product ripening. The presence of D. hansenii strains triggered a lengthening of the lag phase and a decrease of the growth rate of P. verrucosum in meat-based media at 0.97 and 0.92 aw. Both D. hansenii strains significantly reduced OTA production (between 85.16 and 92.63%) by P. verrucosum in the meat-based medium at 0.92 aw. Neither absorption nor detoxification of OTA by D. hansenii strains seems to be involved. However, a repression of the expression of the non-ribosomal peptide synthetase (otanpsPN) gene linked to the OTA biosynthetic pathway was observed in the presence of D. hansenii. To confirm the protective role of D. hansenii strains, they were inoculated together with P. verrucosum Pv45 in dry-fermented sausage and dry-cured ham slices. Although P. verrucosum Pv45 counts were not affected by the presence of D. hansenii in both meat matrices, a reduction of OTA amount was observed. Therefore, the effect of D. hansenii strains on OTA accumulation should be attributed to a reduction at transcriptional level. Consequently, native D. hansenii can be useful as biocontrol agent in dry-cured meat products for preventing the hazard associated with the presence of OTA.
•D. hansenii reduced OTA concentration in dry-cured sausages and ham.•D. hansenii repressed the expression of a key gene OTA-related in P. verrucosum.•Neither absorption nor detoxification of OTA by D. hansenii strains was involved.
Lactic-acid bacteria isolated throughout the ripening process from traditional RTE soft cheeses and dry-cured fermented sausages were characterized and selected for their anti-L. monocytogenes ...activity. For this, the LAB isolates were first screened for their activity against this pathogen in agar spot assay and further evaluated in cheese and dry-cured fermented sausage models simulating conditions of ripening of these products. From a total of 371 LAB isolates, 84 showed anti-listerial activity in agar spot assay and only 10 of them were selected for reducing L. monocytogenes counts in food models. The selected LAB strains belonged to the species Enterococcus faecium, Lacticaseibacillus casei, Lacticaseibacillus paracasei, Lactilactobacillus sakei and Lactococcus garvieae. All of them have at least one gene encoding known bacteriocins. The most active strains, Lc. casei 116 and Ll. sakei 205, provoked reductions higher than 2 log cycles of L. monocytogenes levels in food models, respectively. The selected LAB strains may be utilized to control L. monocytogenes throughout the ripening process in ripened soft cheeses and dry-cured fermented sausages. The combination of 16S rRNA sequencing with PFGE analysis with restriction SgsI and NotI enzymes could be useful to control the implantation of these selected strains throughout the ripening process.
Display omitted
•371 lactic acid bacteria were isolated from traditional ripened sausages and cheeses.•The procedure followed allows to select 6 LAB active strains against L. monocytogenes.•Lc. casei 116 and Ll. sakei 205 reduced 2 log of this pathogen in foods.•The optimized PFGE analysis allows to control implantation of selected LAB during ripening.
Listeria monocytogenes is an important foodborne pathogen, causative agent of listeriosis. The epidemiology and persistence of this bacterium in meat processing plants may be related to its serotype, ...so it is of utmost importance to carry out a correct differentiation of L. monocytogenes serotypes. The objective of this study was to develop a unique quadruplex real-time quantitative PCR (qPCR) method able to differentiate the four most predominant and worrying L. monocytogenes serotypes (1/2a, 1/2b, 1/2c and 4b) in isolates from meat processing plants and ready-to-eat (RTE) dry-cured meat products. The design of specific primers and probes was based on the lmo0737, lmo0308, ORFC (locus genomically equivalent to gltA-gltB) and ORF2110 genes. A qPCR based on a fragment of the 16S rRNA gene was used to ensure the amplification of Listeria spp. genomic DNA. The standard curves showed efficiency values ranging between 92.3% and 105.8% and, R2 values > 0.98. The specificity of the method was also confirmed by the comparison of the results with those obtained by a previously reported conventional multiplex PCR. In addition, none of the strains which were not ascribed to L. monocytogenes amplified any of the target genes related to the four major serotypes of this pathogenic species. The qPCR, therefore, provides a sensitive, specific and rapid tool for identifying the L. monocytogenes serotypes 1/2a, 1/2b, 1/2c and 4b. This method could be very useful for identifying sources of L. monocytogenes contamination in the meat industry or for epidemiological monitoring of persistent strains throughout the processing of RTE meat products.
•A multiplex qPCR to differentiate the four main L. monocytogenes serotypes was optimised.•Lmo0308, lmo0737, ORFC and ORF2110 genes were targeted for serotype differentiation.•Serotypes by PCR matched with those obtained by the developed multiplex qPCR.•The designed qPCR is an efficient tool to differentiate L. monocytogenes serotypes.
“Torta del Casar” is a Spanish soft-ripened cheese made with sheep's raw milk and subjected to a short ripening process, which favors the growth of pathogenic microorganisms including Listeria ...monocytogenes. The development of strategies to control pathogens and minimize health risks associated with the presence of L. monocytogenes in these products is of great interest. In this regard, the anti-Listeria activity of a whey protein hydrolysate (ProH) alone or combined with six lactic acid bacteria strains isolated from cheese was evaluated in this study as a biocontrol strategy using a “Torta del Casar” cheese-based medium. The most active combinations of lactic acid bacteria assayed induced a reduction higher than two logarithmic units in the growth of L. monocytogenes (serotype 4b) compared to their respective control when they were co-inoculated in “Torta del Casar” cheese-based medium at 7 °C for 7 days. In addition, the observed downregulation of some key virulence genes of L. monocytogenes suggests that the strain Lactiplantibacillus plantarum B2 alone and combined with the strain Lactiplantibacillus spp. B4 are good candidates to be used as biocontrol agents against L. monocytogenes growth in traditional soft cheeses based on raw milk during their storage at refrigeration temperatures.
•Some of the LAB showed antagonist potential against Listeria monocytogenes in a soft cheese-based model.•The ProH presence repressed L. monocytogenes growth but stimulated its virulence determinants.•The strain B2 alone and combined with B4 controlled L. monocytogenes growth.
Environmental conditions during ripening of dry-cured meat products favour growth of fungal population on their surface. Some of these moulds can produce mycotoxins. Paprika is one of the ingredients ...usually used in the formulation of raw-cured sausages, and its addition could influence the growth and production of mycotoxins of the moulds present in these products. In this work the effect of Spanish smoked paprika “Pimentón de la Vera” on growth of Aspergillus parasiticus and Penicillium nordicum and production of aflatoxins B1 (AFB1), G1 (AFG1) and ochratoxin A (OTA) respectively, was evaluated. Moulds were grown in a culture medium made from lyophilized fresh pork meat added with 4% salt and different concentrations of Spanish smoked paprika (1, 2 and 3%) at several water activity values (0.98, 0.94 and 0.87) and temperature (20–25 °C), to simulate conditions usually found during ripening of dry-cured meat products. Mould growth was evaluated by measuring the diameter of the colony every 24 h, and the production of mycotoxins by UHPLC-MS/MS every 2 days, during 10 days of incubation. Addition of paprika favours growth of the two mould species tested. However, the synthesis of mycotoxins was reduced at 0.94 and 0.98 aw when at least a 2% of paprika was added. Therefore, the addition of Spanish smoked paprika at 2–3% in the formulations may help to minimize AFs and OTA production in dry-cured meat products such as loins or “chorizo” sausages.
•Paprika provokes a reduction of lag phases of A. parasiticus and P. nordicum.•Growth of A. parasiticus and P. nordicum increases when paprika is added.•2% of paprika reduced aflatoxins and OTA concentration at 0.94 and 0.98 aw.•Addition of paprika could help to control the mycotoxins hazard in dry-cured meat products.
The anti-Listeria monocytogenes activity of 30 Debaryomyces hansenii strains was evaluated to be used as protective cultures in sliced dry-cured ham. Some of the yeast strains exhibited inhibitory ...potential against the L. monocytogenes strains S2, S4-2, S12-1 and S7-2 belonging to the serotypes 1/2a, 1/2b, 1/2c and 4b, respectively, when they were co-inoculated in a dry-cured ham-based medium at 0.95 and 0.93 water activity (aw). The antagonistic activity varied with the yeast and bacterium strains and the aw. The most active strain (D. hansenii 258H) was selected for assessing its activity on slices of dry-cured ham. D. hansenii 258H showed a limited action and even a promotion of the L. monocytogenes growth in the slices of dry-cured ham. In addition, the upregulation of some key virulence genes of L. monocytogenes and the unsuitability appearance on the slices of dry-cured ham inoculated with D. hansenii 258H suggest that such yeast is not a good candidate to be used as protective culture against L. monocytogenes in sliced dry-cured ham. Such effect should be considered even if D. hansenii is not used as a protective culture due to its presence as native microbial population in dry-cured ham.
•A minority of yeasts inhibited L. monocytogenes in a dry-cured ham-based medium.•The antagonistic effect of yeasts was L. monocytogenes strain and aw dependent.•D. hansenii 258H affected relative expression of L. monocytogenes virulence genes.•D. hansenii 258H affected the visual acceptance of inoculated dry-cured ham slices.
The Listeriamonocytogenes diversity throughout deboning, compressing and slicing of Spanish dry-cured ham processing among and within 6 production plants after cleaning and disinfection in two ...different seasons (autumn/winter and spring/summer) was evaluated in this work. L. monocytogenes isolates (n = 66), collected from deboning area (72.7%) and compressing and slicing areas (27.3%), were serotyped as 1/2b (65.1%), 1/2a (27.3%), 1/2c (6.1%) and 4b (1.5%) by PCR-based methods. The typing of the 66 isolates of L. monocytogenes by PFGE using the ApaI and SgsI enzymes revealed a high diversity with 52 pulsotypes and showed cross-contamination within each plant. The frequency of isolation of L. monocytogenes was higher in spring/summer than in autumn/winter. Although L. monocytogenes was not detected in any of the analyzed dry-cured ham samples, the presence of some pulsotypes and cross-contamination in the different industries suppose a hazard of contamination by this pathogen during deboning and slicing of dry-cured ham. The use of molecular typing techniques, such as PCR-based methods and PFGE characterization, could be used to trace the sources, distribution and routes of L. monocytogenes contamination throughout deboning and slicing of dry-cured ham.
•L. monocytogenes prevalence in dry-cured ham deboning and slicing areas was 9.16%.•Most of the L. monocytogenes in deboning and slicing areas were serotyped as 1/2b.•The frequency of L. monocytogenes was higher in spring/summer than in autumn/winter.•Cross-contamination between deboning, compressing and slicing areas was detected.
The effect of the dry-cured fermented processing of "salchichón" inoculated with a selected strain of
(205) on the growth and transcriptional response of three virulence genes (
and
) of
was ...evaluated. For this, three different batches of "salchichón" were analyzed: batch B (inoculated only with
), batch L (inoculated only with
), and batch L + B (inoculated with both microorganisms). Sausages were ripened for 90 days according to a traditional industrial process. The processing of "salchichón" provoked a reduction in
counts of around 2 log CFU/g. The downregulation of the expression of the three genes was found at the end of ripening when the water activity (a
) of "salchichón" was <0.85 a
. The combined effect on the reduction in
counts together with the downregulation in the expression of the virulence genes throughout the "salchichón" processing could be of great interest to control the hazard caused by the presence of this pathogenic bacterium.
The combined effect of temperature, water activity (aw) and NaCl content, usually found in dry-cured ham, on the growth and expression of the virulence and stress-related genes of Listeria ...monocytogenes was evaluated in a dry-cured ham model system. The highest growth of this pathogen was observed at 15 °C and, at 0.98 and 0.96 aw values. At 0.94 and 0.92 aw values, moderate NaCl levels stimulated the L. monocytogenes growth and repressed the expression of the four tested genes. At 7 °C, the expression of the plcA gene was favored while at 15 °C the hly and iap genes were activated. Preventive actions based on temperature, aw and salt content should be taken to minimise risks associated with growth and gene expression of L. monocytogenes in dry-cured ham.
PDF
