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.

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.

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.

Development of a HOG-based real-time PCR method to detect stress response changes in mycotoxigenic moulds.

There is a need to understand the mechanism of adaptation of toxigenic fungal species which are able to colonise highly specialised foods such as cured meats where there is a high osmotic stress due to the presence up to 20-22% NaCl during the ripening process. A new tool able to detect changes in stress related genes would be useful to understand the ecological reasons for the ability of these species to grow in specialised niches. In this work a real-time PCR (qPCR) using SYBR Green was developed. Primers were designed from the Hog1 gene involved in osmo-adaptation in fungi. For this, conserved regions resulting from the alignment of 26 published partial sequences of such gene were used. Specificity of primers HogF2/R2 was demonstrated when amplified, producing a unique 131-bp PCR product with a Tm value of 84 °C. The qPCR method showed an efficiency of 98%, R(2) value > 0.99 and a detection limit of 0.7 log Hog1 gene copies. The qPCR method to measure changes in the Hog1 gene expression in relation to growth in ionic and non-ionic stressed environments (using 10-40% NaCl and sorbitol concentrations) was found to be suitable for two mycotoxigenic species (Penicillium nordicum, P. expansum). This assay will be a valuable tool for generating relevant Hog1 expression data from different mould species in relation to different stresses in food habitats. It will also be a good tool for a better understanding of the ability of xerophilic and xerotolerant species to colonise extreme environments.

Biocontrol of L. monocytogenes with Selected Autochthonous Lactic Acid Bacteria in Raw Milk Soft-Ripened Cheese under Different Water Activity Conditions.

The effect of selected autochthonous Lactic Acid Bacteria (LAB) against Listeria monocytogenes was evaluated in two elaborations of soft-ripened cheese performed under high and low relative humidity (RH) elaborations, to achieve aw ranging from 0.97 to 0.94 in ripened cheeses. Two selected autochthonous strains of Lacticaseibacillus casei 31 and 116 were used. In each elaboration, 8 batches were physicochemically and microbiologically evaluated throughout the ripening process. The aw and pH decreased during ripening to final values ranging from 0.944 to 0.972 aw and 5.0 to 5.3 pH, respectively. LAB was the only microbial group that increased throughout the ripening in high and low RH elaborations. In batches that were uninoculated with LAB strains, L. monocytogenes was either maintained at the initial inoculation level or showed a slight reduction by the end of the ripening process. However, in LAB-inoculated batches in the two elaborations, steady decreases of L. monocytogenes were observed throughout maturation. L. casei 31 alone or in combination with strain 116 provoked reductions of 2 to 4 log CFU/g in L. monocytogenes over 60 days of ripening, which could be enough as a strategy for biocontrol to deal with the usual contamination by L. monocytogenes during cheese processing.

Effect of a Selected Protective Culture of Lactilactobacillus sakei on the Evolution of Volatile Compounds and on the Final Sensorial Characteristics of Traditional Dry-Cured Fermented "Salchichón".

BACKGROUND: In this work, the effect of a selected starter culture of Lactilactobacillus sakei 205 on the evolution of volatile compounds throughout the ripening process and on the final sensorial characteristics of traditional dry-cured fermented "salchichón" was evaluated. METHODS: "Salchichón" sausages were prepared, inoculated with L. sakei 205, and ripened for 90 days. Volatile compounds were analyzed throughout the ripening by GC-MS. In the final product, instrumental texture and color were determined. In addition, sensorial analysis was performed by a semi-trained panel. RESULTS: The inoculation of L. sakei 205 does not influence the texture and color parameters of ripened "salchichón". However, an increase in volatile compounds derived from amino acid catabolism and microbial esterification and a decrease in compounds derived from lipid oxidation, mainly hexanal, were observed throughout the ripening time as a consequence of L. sakei inoculation, which could have a positive effect on the flavor development of the dry-cured fermented "salchichón". CONCLUSIONS: The use of selected strains of lactic acid bacteria (LAB) such as L. sakei 205 as a protective culture could be recommended to improve the quality of traditional "salchichón".

Effect of acidic conditions on the growth and expression of two virulence genes of Listeria monocytogenes serotype 4b.

In this work, the effect of the usual acidic conditions of dry-cured fermented foods (pH values between 4.5 and 6), on the growth and expression of the virulence genes, hly and inlA, of Listeria monocytogenes serotype 4b, was evaluated. To analyse the expression of the inlA gene, a novel real-time PCR (qPCR) method using SYBR® Green methodology was developed. L. monocytogenes levels increased as the pH did and they were kept constant throughout incubation time at pH 4.5. However, a significant increase in the relative expression of the virulence genes was detected in most of the acidic conditions in all the incubation times. The most pronounced upregulation of the relative expression of the virulence genes was found at pH 4.5. The efficient inlA-based qPCR method could be of interest to check changes in the expression of such virulence gene of this pathogenic bacterium in acidic environments.

Prevalence of Listeria monocytogenes in RTE Meat Products of Quevedo (Ecuador).

Listeria monocytogenes is a foodborne pathogen that causes listeriosis and can be a problem in areas where meat products are sold at unregulated storage temperatures. In this work, the prevalence of L. monocytogenes was determined in the five most widely traded meat products in the province of Quevedo (Ecuador): bacon, "chorizo paisa", grilled hamburger meat, mortadella, and salami. A total of 1000 samples of these products were analyzed in two seasons of the year (dry season/rainy season). All L. monocytogenes isolates were confirmed by PCR with primers designed for the iap gene. Furthermore, the positive samples were quantified for L. monocytogenes. Of the 1000 meat products analyzed, 163 were positive for L. monocytogenes (16.3%). The prevalence of L. monocytogenes in the two seasons in different meat products was as follows: 22.5% in mortadella, 19% in hamburger meat, 15% in bacon, 14.5% in chorizo paisa and 10.5% in salami. In addition, the concentration of L. monocytogenes in most of the positive samples was in the range of 4-6 log CFU/g or even higher. The results show the need for improvements in the hygienic measures and meat storage temperatures in Quevedo (Ecuador) to avoid risks of foodborne listeriosis.

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.

Quantitative real-time PCR method with internal amplification control to quantify cyclopiazonic acid producing molds in foods.

A quantitative TaqMan real-time PCR (qPCR) method that includes an internal amplification control (IAC) to quantify cyclopiazonic acid (CPA)-producing molds in foods has been developed. A specific primer pair (dmaTF/dmaTR) and a TaqMan probe (dmaTp) were designed on the basis of dmaT gene which encodes the enzyme dimethylallyl tryptophan synthase involved in the biosynthesis of CPA. The IAC consisted of a 105 bp chimeric DNA fragment containing a region of the hly gene of Listeria monocytogenes. Thirty-two mold reference strains representing CPA producers and non-producers of different mold species were used in this study. All strains were tested for CPA production by high-performance liquid chromatography-mass spectrometry (HPLC-MS). The functionality of the designed qPCR method was demonstrated by the high linear relationship of the standard curves relating to the dmaT gene copy numbers and the Ct values obtained from the different CPA producers tested. The ability of the qPCR protocol to quantify CPA-producing molds was evaluated in different artificially inoculated foods. A good linear correlation was obtained over the range 1-4 log cfu/g in the different food matrices. The detection limit in all inoculated foods ranged from 1 to 2 log cfu/g. This qPCR protocol including an IAC showed good efficiency to quantify CPA-producing molds in naturally contaminated foods avoiding false negative results. This method could be used to monitor the CPA producers in the HACCP programs to prevent the risk of CPA formation throughout the food chain.

Real-time PCR assays for detection and quantification of aflatoxin-producing molds in foods.

Aflatoxins are among the most toxic mycotoxins. Early detection and quantification of aflatoxin-producing species is crucial to improve food safety. In the present work, two protocols of real-time PCR (qPCR) based on SYBR Green and TaqMan were developed, and their sensitivity and specificity were evaluated. Primers and probes were designed from the o-methyltransferase gene (omt-1) involved in aflatoxin biosynthesis. Fifty-three mold strains representing aflatoxin producers and non-producers of different species, usually reported in food products, were used as references. All strains were tested for aflatoxins production by high-performance liquid chromatography-mass spectrometry (HPLC-MS). The functionality of the proposed qPCR method was demonstrated by the strong linear relationship of the standard curves constructed with the omt-1 gene copy number and Ct values for the different aflatoxin producers tested. The ability of the qPCR protocols to quantify aflatoxin-producing molds was evaluated in different artificially inoculated foods. A good linear correlation was obtained over the range 4 to 1 log cfu/g per reaction for all qPCR assays in the different food matrices (peanuts, spices and dry-fermented sausages). The detection limit in all inoculated foods ranged from 1 to 2 log cfu/g for SYBR Green and TaqMan assays. No significant effect was observed due to the different equipment, operator, and qPCR methodology used in the tests of repeatability and reproducibility for different foods. The proposed methods quantified with high efficiency the fungal load in foods. These qPCR protocols are proposed for use to quantify aflatoxin-producing molds in food products.

Strategies for Biocontrol of Listeria monocytogenes Using Lactic Acid Bacteria and Their Metabolites in Ready-to-Eat Meat- and Dairy-Ripened Products.

Listeria monocytogenes is one of the most important foodborne pathogens. This microorganism is a serious concern in the ready-to-eat (RTE) meat and dairy-ripened products industries. The use of lactic acid bacteria (LAB)-producing anti-L. monocytogenes peptides (bacteriocins) and/or lactic acid and/or other antimicrobial system could be a promising tool to control this pathogen in RTE meat and dairy products. This review provides an up to date about the strategies of use of LAB and their metabolites in RTE meat products and dairy foods by selecting the most appropriate strains, by analysing the mechanism by which they inhibit L. monocytogenes and methods of effective application of LAB, and their metabolites in these kinds of products to control this pathogen throughout the processing and storage. The selection of LAB with anti-L. monocytogenes activity allows to dispose of effective strains in meat and dairy-ripened products, achieving reductions form 2-5 logarithmic cycles of this pathogen throughout the ripening process. The combination of selected LAB strains with antimicrobial compounds, such as acid/sodium lactate and other strategies, as the active packaging could be the next future innovation for eliminating risk of L. monocytogenes in meat and dairy-ripened products.

Evolution of Volatile Compounds during Ripening and Final Sensory Changes of Traditional Raw Ewe's Milk Cheese "Torta del Casar" Maturated with Selected Protective Lactic Acid Bacteria.

In traditional soft ripened cheeses made with raw milk, the use of protective cultures is infrequent. In the present work, the effect of selected (for their activity against Listeria monocytogenes) protective cultures of Lactocaseibacillus casei 116 and Lactococcus garvieae 151 was evaluated, on the evolution of volatile compounds throughout the ripening and on the final sensory characteristics of traditional soft ripened "Torta del Casar" cheese. For this, both strains were separately inoculated in raw cheeses and ripened for 90 days. The selected LAB strains did not affect physicochemical parameters, including texture and color of the ripened cheeses. However, they could have a positive effect on the aroma, for the generation of methyl branched acids and for the reduction in compounds derived from ß-oxidation of fatty acids. Thus, these protective cultures, in addition to contributing to their safety, could improve quality of the ripened cheeses.

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.

Development of real-time PCR methods to quantify patulin-producing molds in food products.

Patulin is a mycotoxin produced by different Penicillium and Aspergillus strains isolated from food products. To improve food safety, the presence of patulin-producing molds in foods should be quantified. In the present work, two real-time (RTi) PCR protocols based on SYBR Green and TaqMan were developed. Thirty four patulin producers and 28 non-producers strains belonging to different species usually reported in food products were used. The patulin production was tested by mycellar electrokinetic capillary electrophoresis (MECE) and high-pressure liquid chromatography-mass spectrometry (HPLC-MS). A primer pair F-idhtrb/R-idhtrb and the probe IDHprobe were designed from the isoepoxydon dehydrogenase (idh) gene, involved in patulin biosynthesis. The functionality of the developed method was demonstrated by the high linear relationship of the standard curves constructed with the idh gene copy number and Ct values for the different patulin producers tested. The ability to quantify patulin producers of the developed SYBR Green and TaqMan assays in artificially inoculated food samples was successful, with a minimum threshold of 10 conidia g(-1) per reaction. The developed methods quantified with high efficiency fungal load in foods. These RTi-PCR protocols, are proposed to be used to quantify patulin-producing molds in food products and to prevent patulin from entering the food chain.

Role of an autochthonous starter culture and the protease EPg222 on the sensory and safety properties of a traditional Iberian dry-fermented sausage "salchichón".

The effect of the addition of an autochthonous starter culture and the protease EPg222 on the physico-chemical and sensory characteristics of dry-fermented sausage ''salchichon" was investigated. Sausages were prepared with purified EPg222 and Pediococcus acidilactici MS200 and Staphylococcus vitulus RS34 as starter culture (P200S34), separately and together, ripened for 90 days, and compared with a control batch. Dry-fermented sausages ripened with EPg222 and starter culture showed higher amounts of AN and volatile compounds derived from amino acid catabolism than the control, especially in samples in which was added the association of enzyme and starter culture (P200S34+EPg222). There were clear differences shown by the texture analysis, with the P200S34+EPg222 batch being less hard. Especially important was the result found in biogenic amines, since the association P200S34+EPg222 reduced their accumulation compared to the EPg222 batch. The use of EPg222 may be of great interest to improve the sensory characteristics of dry-fermented sausages, but its association with the selected starter culture with low decarboxylase activity is necessary to guarantee healthiness and homogeneity.

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.

Effect of the Dry-Cured Fermented Sausage "Salchichón" Processing with a Selected Lactobacillus sakei in Listeria monocytogenes and Microbial Population.

In the present work, the effect of processing of dry-cured fermented sausage "salchichón" spiked with the selected Lactobacillus sakei 205 was challenge-tested with low and high levels of L. monocytogenes. The evolution of the natural microbial population throughout the "salchichón" ripening was also evaluated. For this, a total of 150 "salchichón" were elaborated and divided into six equal cases which were inoculated with different levels of L. monocytogenes, and L. sakei 205. Afterwards, sausages were ripened for 90 days according to a typical industrial process. Moisture content (%) and water activity (aw) decreased throughout the ripening up to values around 26% and 0.78, respectively. No differences for moisture content, aw, pH, NaCl and nitrite concentration were observed between the analyzed cases. Lactic acid bacteria counts in the L. sakei 205 inoculated cases were always higher than 6 log CFU g-1 during ripening. Enterobacteriaceae counts were reduced during ripening until non-detectable levels at the end of processing. Reductions in L. monocytogenes counts ranged from 1.6 to 2.2 log CFU g-1; therefore, the processing of "salchichón" itself did not allow the growth of this pathogen. Reduction in L. monocytogenes was significantly higher in the cases inoculated with L. sakei 205.

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.

Relationship between cyclopiazonic acid production and gene expression in Penicillium griseofulvum under dry-cured ham processing environmental conditions.

Cyclopiazonic acid (CPA)-producing Penicillium griseofulvum is usually found on the dry-cured ham surface during its ripening. The objective of this work was to evaluate the effect of temperature and water activity (aw) of dry-cured ham processing on growth, CPA production, and temporal relative expression of genes involved in CPA biosynthesis on dry-cured meat-based media. P. griseofulvum CECT 2919 grew faster than P. griseofulvum IBT 14319 in all conditions tested, although no growth occurred at 0.85 aw. Besides, the dry-cured ham-based medium favoured CPA synthesis for both strains compared to the meat-based medium. For the strain CECT 2919, the expression of the mfs-1 and pks-nrps genes were stimulated at 0.90 and 0.95 aw, respectively, while the dmaT gene expression was inhibited during the incubation time. By contrast, the strain IBT 14319 showed that the dmaT gene expression was stimulated at 0.90 aw, while the pks-nrps and mfs-1 genes were repressed throughout incubation time. In conclusion, it is necessary to reduce aw on the surface of the hams below 0.85 during ripening before to increase temperature to reduce growth of P. griseofulvum and CPA production. This information may be useful to design preventive and corrective actions to minimise risks associated with the presence of CPA in dry-cured ham.