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.

Effect of temperature and water activity on growth and aflatoxin production by Aspergillus flavus and Aspergillus parasiticus on cured meat model systems.

Dry-cured hams may be colonised by aflatoxin-producing Aspergillus flavus and Aspergillus parasiticus during the ripening process. The objective of this study was to evaluate the interaction between non-ionic water stress and temperatures may have on lag phases prior to growth, growth rates and aflatoxin production by two strains of each A. parasiticus and A. flavus on meat matrices over a period of 12days. Results showed that A. flavus CBS 573.65 had shorter lag phases than A. parasiticus CECT 2688, however the growth rates were quite similar. For both species, no growth occurred at 10°C and all aw tested and optimum growth happened at 25°C and 0.95 aw. Similar aflatoxin B1 production profiles between both species were found, however A. flavus produced much higher concentration of such toxin than A. parasiticus. Both species produced aflatoxins when the temperature and the aw were ≥15°C and ≥0.90.

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.

Characterisation and detection of spoilage mould responsible for black spot in dry-cured fermented sausages.

Moulds responsible for black spot spoilage of dry-cured fermented sausages were characterised. For this purpose, samples were taken from those dry-cured fermented sausages which showed black spot alteration. Most of the mould strains were first tentatively identified as Penicillium spp. due to their morphological characteristics in different culture conditions, with one strain as Cladosporium sp. The Cladosporium strain was the only one which provoked blackening in culture media. This strain was further characterised by sequencing of ITS1-5.8S-ITS2 rRNA and ß-tubulin genes. This mould strain was able to reproduce black spot formation in dry-cured fermented sausage 'salchichón' throughout the ripening process. In addition, a specific and sensitive real-time PCR method was also developed to detect Cladosporium oxysporum responsible for the black spot formation in sausages. This method could be of great interest for the meat industry to detect samples contaminated with this mould before spoilage of product avoiding economic losses for this sector.

Influence of temperature and substrate conditions on the omt-1 gene expression of Aspergillus parasiticus in relation to its aflatoxin production.

Most strains of Aspergillus parasiticus are able to produce high concentrations of aflatoxin (AF) B1 and G1 which are among the most potent mutagenic, teratogenic, and carcinogenic mycotoxins. Molecular studies in relation to activity of secondary metabolite gene clusters are crucial to improving food safety. In the present work, reverse transcriptase quantitative PCR (RT-qPCR) was used to monitor the influence of temperature, substrates containing nitrogen and incubation time on the omt-1 gene expression in A. parasiticus. Phenotypic AFB1 and G1 production was also evaluated by high-performance liquid chromatography-mass spectrometry (HPLC-MS). The results demonstrated that temperature (25°C and 30°C) influenced relative expression of omt-1 gene throughout the time (maximum at 25°C) while substrate composition was not affected by it. However, when effect of temperature and substrate was analyzed at each incubation time, significant effects were found. Optimal conditions for biosynthesis of AFB1 and AFG1 were similar, and they were related to changes in temperature and sodium nitrate. The highest AFB1 and G1 production levels were found at 25°C. However, lower AFB1 and G1 values were obtained when A. parasiticus grew on the substrate containing sodium nitrate and there was no production of these AFs at 37°C in any of the conditions tested. In addition, omt-1 gene expression was correlated to AFB1 and G1 syntheses at the different conditions. Use of temperature conditions and sodium nitrate concentrations which limit production of AFs holds potential for preventing AF from entering the food chain.