Antilisterial activity of cinnamon essential oil, pomegranate extract, or strawberry tree extract against Listeria monocytogenes in slices of dry-cured ham and pork loin.

Owing to concerns about the antimicrobial resistance of agents that can prevent the growth of Listeria monocytogenes in meat, researchers have investigated natural preservatives with antilisterial effects. However, in vivo application of essential oils and plant extracts usually results in reduced antimicrobial activity in meat products when compared to in vitro studies. This study aimed to evaluate the in vivo antimicrobial activity of cinnamon essential oil, pomegranate, and strawberry tree extracts in slices of dry-cured ham and pork loin against L. monocytogenes. Fragments of sterile dry-cured ham were inoculated with 100 µL cinnamon oil 0.5%, pomegranate, or strawberry crude extract. After 10 min, 100 µL of L. monocytogenes serotype 4b (104 colony-forming unit [CFU]/mL) was inoculated, and samples were incubated at 7 °C for 7 d to simulate the processing and storage temperature conditions of dry-cured meat products. L. monocytogenes was detected and quantified. Only strawberry extract presented significant differences (P < 0.05) from the control; thus, it was selected for the assay with 2% and 4% salt-treated pork loin. The strawberry tree extract significantly (P < 0.05) reduced the growth of L. monocytogenes in dry-cured ham. However, it could not reduce L. monocytogenes growth in pork loin, regardless of the salt concentration. This is the first report on the antimicrobial effect of strawberry tree leaf extract against L. monocytogenes in dry-cured ham.

Antimicrobial activity of essential oils and natural plant extracts against Listeria monocytogenes in a dry-cured ham-based model.

BACKGROUND: Listeria monocytogenes is a widespread common contaminant in food production facilities during preparation, storage, and distribution, and minimally processed ready-to-eat products are considered at high risk of contamination by this bacterium. Increased antibiotic resistance has led researchers to search for plant-based natural alternatives to control pathogenic microorganisms. Among these products, essential oils and plant extracts have previously shown antimicrobial activity and are possible alternatives to manage food pathogens. In this study, commercial essential oils (cinnamon, clove, oregano, ginger, and thyme) and plant extracts (pomegranate, acorn, olive, strawberry tree, and dog rose) were tested against L. monocytogenes in a dry-cured ham-based model. RESULTS: Essential oils and plant extracts were screened by agar diffusion and minimum inhibitory concentration for anti-L. monocytogenes activity. Cinnamon, pomegranate, and strawberry trees returned the strongest results and were therefore evaluated in a dry-cured ham-based medium assay with water activity of 0.93 or 0.95. The 10% essential oil of cinnamon was capable of completely inhibiting bacterial growth, while strawberry tree and pomegranate extract also showed antilisterial activity (P > 0.05). Water activity influenced the bacterial count of L. monocytogenes in a dry-cured ham-based medium. CONCLUSIONS: There was a reduction in L. monocytogenes with the application of cinnamon essential oil but, because of the negative sensory impact of this particular compound in meat products, we suggest the use of pomegranate or strawberry tree for the biocontrol of Listeria in ready-to-eat products. © 2021 Society of Chemical Industry.

Control of Listeria monocytogenes growth and virulence in a traditional soft cheese model system based on lactic acid bacteria and a whey protein hydrolysate with antimicrobial activity.

"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.

Growth and Expression of Virulence Genes of Listeria monocytogenes during the Processing of Dry-Cured Fermented "Salchichón" Manufactured with a Selected Lactilactobacillus sakei.

The effect of the dry-cured fermented processing of "salchichón" inoculated with a selected strain of Lactilactobacillus sakei (205) on the growth and transcriptional response of three virulence genes (plcA, hly, and iap) of Listeria monocytogenes was evaluated. For this, three different batches of "salchichón" were analyzed: batch B (inoculated only with L. sakei), batch L (inoculated only with L. monocytogenes), 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 L. monocytogenes 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 (aw) of "salchichón" was <0.85 aw. The combined effect on the reduction in L. monocytogenes 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.

Impact of Water Activity on the Inactivation and Gene Expression of Listeria monocytogenes during Refrigerated Storage of Pressurized Dry-Cured Ham.

Listeria monocytogenes population and the expression patterns of three virulence (plcA, hly, and iap) and one stress-related (sigB) genes in dry-cured ham with different water activity (aw) values (0.92, 0.88, and 0.84) and treated with high pressure processing (HPP, 450 MPa/10 min and 600 MPa/5 min) were monitored throughout 30 days (d) at 4 °C. The antimicrobial effect of HPP at 600 MPa against L. monocytogenes S4-2 (serotype 1/2b) and S12-1 (serotype 1/2c) was greater in dry-cured ham with aw values of 0.92, with reductions of 2.5 and 2.8 log units, respectively. The efficacy of HPP treatments decreased at lower aw values. Regarding gene expression, L. monocytogenes strains responded differently to HPP. For strain S4-2, the four target genes were generally overexpressed in dry-cured ham immediately after HPP treatments at the three aw values investigated, although the extent of this induction was lower in the samples pressurized at 600 MPa and with aw values of 0.84. For strain S12-1, the expression of all target genes was repressed at the three aw values investigated. The antimicrobial efficacy of HPP against L. monocytogenes could be compromised by low aw values in food products. However, no growth of HPP-survival cells was observed during refrigerated storage in low-aw dry-cured ham, and the overexpression of virulence and stress-related genes decreased.

Development of a multiplex real-time PCR to differentiate the four major Listeria monocytogenes serotypes in isolates from meat processing plants.

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.

Effect of Spanish smoked paprika "Pimentón de La Vera" on control of ochratoxin A and aflatoxins production on a dry-cured meat model system.

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.

Combined effect of temperature, water activity and salt content on the growth and gene expression of Listeria monocytogenes in a dry-cured ham model system.

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.

Potential of yeasts isolated from dry-cured ham to control ochratoxin A production in meat models.

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.

Selection of reference genes to quantify relative expression of ochratoxin A-related genes by Penicillium nordicum in dry-cured ham.

Penicillium nordicum is an important and consistent producer of ochratoxin A (OTA) in NaCl-rich foods such as dry-cured ham. OTA is a toxic secondary metabolite which provokes negative effects on consumer health. Once OTA is produced in ham, this mycotoxin is difficult to remove. Since gene expression always precedes OTA production, analysis of expression of OTA-related genes by reverse transcription real-time PCR (RT-qPCR) could be a valuable tool to predict OTA contamination in ham. However scarce RT-qPCR protocols are properly validated leading to inconsistent data analyses. The objective of this study was to examine reference genes suitable for normalisation in designing and developing new RT-qPCR methods for quantifying the relative expression of genes involved in OTA biosynthesis (otapks and otanps) by P. nordicum on a dry-cured ham model system after 7 days of incubation. Firstly, primers based on three housekeeping genes commonly found in moulds, ß-tubulin, COI and ITS, and on the otapks gene were designed. The primer pair F/R-npstr previously developed on the otanps gene was also used. Although most of the designed primers met the requirements needed to be used in qPCR assays, the primer pairs ß-tubF1/R1, COI-F1/R1, ITSF2/R2 and otapksF3/R3 for the ß-tubulin, COI, ITS and otapks genes, respectively, were selected due to their lowest Cq value. Next, the two assumptions of the 2-ΔΔCT method to evaluate the relative expression of the otapks and otanps genes were fulfilled for two of the three endogenous genes tested, ß-tubulin and COI. However, ß-tubulin was considered more proper as reference gene than COI under the environmental conditions assayed since its expression values by day 7 were more related to OTA production. Therefore, the two RT-qPCR methods for the analysis of the relative expression of the otapks and otanps genes have been properly validated and can be used as control tools to avoid or minimise the presence of OTA in ham.

Identification and control of moulds responsible for black spot spoilage in dry-cured ham.

The aims of this work were to identify moulds responsible for black spot spoilage in the drying and cellar stages of dry-cured ham processing and evaluate the effectiveness of preventive actions for controlling this alteration. Four mould strains isolated from spoiled hams were identified by morphological characteristics and the ITS and ß-tubulin sequencing. Two of them were Cladosporium oxysporum, one was C. cladosporioides and the remaining one was C. herbarum. These spoiling strains reproduced the black spots on dry-cured ham-based media and ham slices. Additionally, the effect of water activity (aw) conditions reached throughout dry-cured ham ripening and the activity of the protective culture Penicillium chrysogenum CECT 20922 against the spoiling moulds were evaluated. In the dry-cured ham model system the growth of the Cladosporium strains was minimised when the aw approaches 0.84 or in P. chrysogenum CECT 20922 inoculated dry-cured ham slices. Therefore such combination could be used to avoid the black spot formation in dry-cured ham.