Salting of dry-cured meat - A potential cause of contamination with the ochratoxin A-producing species Penicillium nordicum

Penicillium nordicum is a known contaminant of protein-rich foods and is primarily found on dry-cured meat products. It is an important producer of the mycotoxin ochratoxin A, which has nephrotoxic and cancerogenic activities. Recently a high number of P. nordicum strains was isolated from different dry-cured meat products from one of the Slovenian meat-processing plants. Since we have isolated P. nordicum in high counts also from Artic habitats, such as sea water and sea ice and due to its ability to grow well at low temperatures and at increased salinity, sea salt was suspected as the possible source of P. nordicum. In the present study contamination of meat products, air in the meat-processing plant and sea salt used for salting were analysed. When 50 g of salt sample from a sealed package was dissolved in sterile water and filtered, 12 colonies of P. nordicum were obtained on solid medium incubated at 15 °C, while a salt sample from an open vessel in the meat-processing area developed high, uncountable number of colonies. Amplified fragment length polymorphism analyses of P. nordicum isolates from different sources showed that contamination of meat products via salt was possible. Three selected isolates examined for extrolites all produced ochratoxin A. As contamination of dry-cured meat products with P. nordicum represents a potential health risk for consumers and workers in the meat-processing plants, salt should be taken into account as a potential cause of such contaminations.     

The biosynthesis of ochratoxin A by Penicillium as one mechanism for adaptation to NaCl rich foods

Penicillium.nordicum is an ochratoxin A producing filamentous fungus, which is adapted to sodium chloride and protein rich food environments like certain cheeses or dry cured meats. Penicillium.verrucosum usually occurs on cereals but can also be isolated from brined olives. It could be shown that sodium chloride has a profound influence on the regulation of ochratoxin A biosynthesis in both Penicillium species. High amounts of ochratoxin A are produced by P. nordicum over a wide concentration range of NaCl (5-100 g/l) with a weak optimum at about 20 g/l after growth on YES medium. P. verrucosum shifts secondary metabolite biosynthesis after growth on YES medium from citrinin at low to ochratoxin at elevated NaCl concentrations. The ochratoxin A biosynthesis of P. nordicum is accompanied by an induction of the otapksPN gene, the gene of the ochratoxin A polyketide synthase. A mutant strain unable to produce ochratoxin showed a drastic growth reduction under high NaCl conditions. Determination of the dry weight and the chloride content in the mycelium of the P. nordicum wild type strain and a non-ochratoxin A producing mutant strain showed a much higher increase of both parameters in the mutant compared to the wild type. These results suggest, that the constant biosynthesis and excretion of ochratoxin A, which itself contains a chloride atom, ensures a partial chloride homeostasis in the fungal cell. This mechanism may support the adaptation of ochratoxin A producing Penicillia to NaCl rich foods.     

Gene Expression Analysis as a Method to Predict OTA Accumulation in Dry-Cured Meat Products

Dry-cured meat products are frequently colonised by toxigenic Penicillium nordicum and Penicillium verrucosum throughout their ripening that can produce ochratoxin A (OTA). The predictive nature of the molecular analyses can be used to determine the risk of toxigenic species. For this reason, the objective of the present work was to analyse the temporal changes in the expression of the otapks and otanps genes by P. nordicum and P. verrucosum in relation to OTA production on slices of dry-fermented sausage salchichón and dry-cured ham. Gene expression was higher in P. nordicum than in P. verrucosum in both meat matrices. The otapks gene was overexpressed by both Penicillium species, especially in salchichón. OTA was only detected in inoculated salchichón regardless of the ochratoxigenic species used. The high significant correlation found between the early relative expression of the otapks gene and OTA production in salchichón leads to propose the relative gene expression of the otapks gene as a good indicator to predict OTA accumulation throughout the ripening of this product.     

Use of recA gene sequence analysis for the identification of Staphylococcus equorum strains predominant on dry-cured hams

Spanish dry-cured ham is an uncooked meat product highly appreciated due to its characteristics flavour. In this study, we examined the accuracy of biochemical tests and 16S rDNA sequencing in the identification of 56 staphylococcal strains isolated during industrial Spanish dry-cured ham processes. Important differences were observed comparing genotypic and phenotypic data. Staphylococcus xylosus was the prevalent species identified by biochemical methods (87.5%), however, sequencing of the 16S rDNA resulted in an unambiguous identification of Staphylococcus equorum (73.2%) and Staphylococcus vitulinus (8.9%) strains. Reliable identification of meat staphylococci, mainly among S. xylosus and S. equorum strains could be also achieved by means of recA gene sequence comparison. Two degenerate primers previously described for lactic acid bacteria were used to amplify an internal fragment of the recA gene. This fragment was amplified from twelve staphylococcal type strains representing frequent meat species. The results indicated that recA sequencing is an adequate method to discriminate among meat staphylococci. In addition, S. xylosus and S. equorum strains could be more accurately discriminated by recA sequencing than 16S rDNA or sodA sequencing. The S. equorum sequence diversity showed at the intra-species level by recA gene sequencing confirmed the high heterogeneity described among S. equorum strains.     

Presence of ochratoxin A on the surface of dry-cured Iberian ham after initial fungal growth in the drying stage

Accumulation of ochratoxin A (OTA) on the surface and to a 0.5 cm depth of dry-cured Iberian ham after initial fungal growth was investigated. For this, 20 dry-cured Iberian hams from the drying stage showing incipient fungal growth on the surface were analyzed. In addition, the presence of OTA-producing molds was examined on the surface of the hams by real-time quantitative PCR (qPCR) based on the otanpsPN gene. Quantification of specific OTA-producing molds, such as Penicillium nordicum and Penicillium verrucosum was also achieved on the hams by specific qPCR methods. Ten of 20 dry-cured hams showed OTA at higher levels than those established by legal regulation. OTA was even detected in the deep section of hams. OTA-producing molds ranged from 1.5 to 7.3 log cfu/cm². Accumulation of OTA on the hams seems to be related to the presence of OTA-producing molds and especially to P. nordicum.     

Antibiotic susceptibility of enterococci isolated from traditional fermented meat products

Antibiotic susceptibility was evaluated for 182 Enterococcus spp. isolated from Alheira, Chouriça de Vinhais and Salpicão de Vinhais, fermented meat products produced in the North of Portugal. Previously, a choice was made from a group of 1060 isolates, using phenotypic and genotypic tests. From these, 76 were previously identified as Enterococcus faecalis, 44 as Enterococcus faecium, one as Enterococcus casseliflavus and 61 as Enteroccocus spp. In order to encompass several of the known chemical and functional classes of antibiotics, resistance to ampicillin, penicillin G, ciprofloxacin, chloramphenicol, erythromycin, nitrofurantoin, rifampicin, tetracycline and vancomycin was evaluated. All the isolates were sensitive to antibiotics of clinical importance, such as penicillins and vancomycin. Some differences in Minimal Inhibitory Concentrations (MICs) of antibiotics, could be associated with the enterococcal species.     

Campylobacter contamination of broiler caeca and carcasses at the slaughterhouse and correlation with Salmonella contamination

In order to estimate the prevalence of Campylobacter spp. and Salmonella spp. on broiler chicken carcasses and the prevalence of Campylobacter spp. in caeca, 58 French slaughterhouses were investigated in 2008. Enumeration of Campylobacter spp. was also performed in order to study the relation between caeca and carcass contamination. A pool of 10 caeca and one carcass were collected from 425 different batches over a 12-month period in 2008. Salmonella was isolated on 32 carcasses leading to a prevalence of 7.5% ([5.0-10.0]_95%CI). The prevalence of Campylobacter was 77.2% ([73.2-81.2]_95%CI) in caeca and 87.5% ([84.4-90.7]_95%CI) on carcasses. No significant correlation was found between Campylobacter and Salmonella. Positive values of Campylobacter were normally distributed and the average level was 8.05 log₁₀ cfu/g ([7.94-8.16]_95%CI) in caeca and 2.39 cfu/g ([2.30-2.48]_95%CI) on carcasses. A positive correlation (r = 0.59) was found between the mean of Campylobacter in caeca and on carcasses (p < 0.001). Thus, carcasses from batches with Campylobacter-positive caeca had significantly (p < 0.001) higher numbers of Campylobacter per gram than batches with negative caeca. These results show that Campylobacter can be present in both matrices and reduction in caeca could be a possible way to reduce the amount of bacteria on carcasses. Of the 2504 identifications performed, 3 species of Campylobacter (Campylobacter jejuni, Campylobacter coli and Campylobacter lari) were identified. The main species recovered were C. jejuni and C. coli, which were isolated in 55.3% and 44.5% of positive samples, respectively. These two species were equally represented in caeca but C. jejuni was the most frequently isolated on carcasses with 57.1% and 42.5% of positive carcasses for C. jejuni and C. coli, respectively. This study underlines that target a reduction of Campylobacter on final products requires a decrease of contamination in caeca.     

Prevalence and characterization of Escherichia coli O157:H7 isolates from meat and meat products sold in Amathole District,Eastern Cape Province of South Africa

Meat and meat products have been implicated in outbreaks of Escherichia coli O157:H7 in most parts of the world. In the Amathole District Municipality of the Eastern Cape Province of South Africa, a large number of households consume meat and meat products daily, although the microbiological quality of these types of food is questionable. The present study investigated the prevalence of E. coli O157:H7 isolated from selected meat and meat products (45 samples each of biltong, cold meat, mincemeat, and polony) sold in this area. Strains of E. coli O157:H7 were isolated by enrichment culture and confirmed by polymerase chain reaction (PCR). Also investigated were the antibiogram profiles of the E. coli O157:H7 isolates. Five (2.8%) out of 180 meat and meat products examined were positive for E. coli O157:H7 that carried the fliC_H7, rfbE_O157, and eaeA genes. Two of the E. coli O157:H7 isolates were resistant against all the eight antibiotics tested. To prevent E. coli O157:H7 infections, meat and meat products such as biltong, cold meat, mincemeat and polony should be properly handled, and packed in sterile polyvinyl wrappers.     

Detection of cold-tolerant clostridia other than Clostridium estertheticum in raw vacuum-packed chill-stored meat

Samples from raw chill-stored vacuum-packed beef, lamb and venison or the meat processing environment, associated with a spoilage problem, but negative for Clostridium estertheticum using a specific real-time PCR test, were examined for other Clostridium spp. using direct 16S rDNA PCR-RFLP and sequencing. Of 291 samples tested by PCR, presence of clostridia was indicated in 123 and there was sufficient PCR product in 35 to be further investigated. Presence of Clostridium spp. was confirmed by RFLP and sequencing in 25/35 samples (11 of 14 incidents). Species detected in spoiled meat were (incidents): Clostridium tagluense-like (4), Clostridium putrefaciens (2), Clostridium algidicarnis (3), Clostridium frigoris/estertheticum-like (3) and Clostridium. gasigenes (2). More than one species was detected in some incidents. All of the above species have previously been associated with spoiled meat apart from the Cl. tagluense-like species. Clostridia were also confirmed in 4/7 samples from the environment, with two Cl. frigoris/estertheticum-like and two mesophilic species of Clostridium. Our study showed that, cold-tolerant Clostridium species other than Cl. estertheticum are occasionally associated with spoiled vacuum-packed meat, particularly lamb. Further studies are required to confirm the exact identity of the Cl. tagluense-like species and its role in meat spoilage.     

Selection of antifungal protein-producing molds from dry-cured meat products

To control unwanted molds in dry-cured meats it is necessary to allow the fungal development essential for the desired characteristics of the final product. Molds producing antifungal proteins could be useful to prevent hazards due to the growth of mycotoxigenic molds. The objective has been to select Penicillium spp. that produce antifungal proteins against toxigenic molds. To obtain strains adapted to these products, molds were isolated from dry-cured ham. A first screening with 281 isolates by the radial inhibition assay revealed that 166 were active against some of the toxigenic P. echinulatum, P. commune, and Aspergillus niger used as reference molds. The activity of different extracts from cultured medium was evaluated by a microspectroscopic assay. Molds producing active chloroform extracts were eliminated from further consideration. A total of 16 Penicillium isolates were screened for antifungal activity from both cell-free media and the aqueous residues obtained after chloroform extraction. The cell-free media of 10 isolates that produced a strong inhibition of the three reference molds were fractionated by FPLC on a cationic column. For protein purification, the fractions of the three molds that showed high inhibitory activity were further chromatographed on a gel filtration column, and the subfractions containing the highest absorbance peaks were assayed against the most sensitive reference molds. One subfraction each from strains AS51D and RP42C from Penicillium chrysogenum confirmed the inhibitory activity against the reference molds. SDS-PAGE revealed a single band from each subfraction, with estimated molecular masses of 37 kDa for AS51D and 9 kDa for RP42C. Although further characterisation is required, both these proteins and the producing strains can be of interest to control unwanted molds on foods.     

A survey of pre-harvest ear rot diseases of maize and associated mycotoxins in south and central Zambia

Maize ear rots reduce grain yield and quality with implication on food security and health. Some of the pathogenic fungi produce mycotoxins in maize grain posing a health risk to humans and livestock. Unfortunately, the levels of ear rot and mycotoxin infection in grain produced by subsistence farmers in sub-Saharan countries are not known. A survey was thus conducted to determine the prevalence of the ear rot problem and levels of mycotoxins in maize grain. A total of 114 farmsteads were randomly sampled from 11 districts in Lusaka and southern provinces in Zambia during 2006. Ten randomly picked cobs were examined per farmstead and the ear rot disease incidence and severity were estimated on site. This was followed by the standard seed health testing procedures for fungal isolation in the laboratory. Results indicated that the dominant ear rots were caused by Fusarium and Stenocarpella. Incidence of Fusarium verticillioides ranged from 2 to 21%, whereas that of Stenocarpella maydis reached 37% on ear rot diseased maize grain. In addition, 2-7% F. verticillioides, and 3-18% Aspergillusflavus, respectively, were recovered from seemingly healthy maize grain. The mean rank of fungal species, from highest to lowest, was F. verticillioides, S. maydis, A.flavus, Fusarium graminearum, Aspergillus niger, Penicillium spp., Botrydiplodia spp., and Cladosporium spp. The direct competitive ELISA-test indicated higher levels of fumonisins than aflatoxins in pre-harvest maize grain samples. The concentration of fumonisins from six districts, and aflatoxin from two districts, was 10-fold higher than 2 ppm and far higher than 2 ppb maximum daily intake recommended by the FAO/WHO. The study therefore suggested that subsistence farmers and consumers in this part of Zambia, and maybe also in similar environments in sub-Saharan Africa, might be exposed to dangerous levels of mycotoxins due to the high levels of ear rot infections in maize grain.     

Degradation of ascorbic acid and potassium sorbate by different Lactobacillus species isolated from packed green olives

The aim of this research was to ascertain the lactic acid bacteria responsible for the degradation of ascorbic acid and/or potassium sorbate, isolated from packed green olives where these additives had diminished. A total of 14 isolates were recovered from samples of different green olive containers. According to partial sequencing of the 16S rRNA coding gene, Lactobacillus parafarraginis, Lactobacillus rapi, Lactobacillus pentosus, Lactobacillus paracollinoides, and Pediococcus ethanolidurans were identified. With the exception of L. pentosus and L. paracollinoides, the other species had not been mentioned in table olives before this study. Only three of the 14 isolates metabolized ascorbic acid in MRS broth, and the products from ascorbic acid in modified MRS broth without carbon sources were acetic and lactic acids. Except for the two L. rapi and the two P. ethanolidurans strains, the remaining 10 isolates depleted potassium sorbate added into MRS broth to some extent. The product generated by three of these strains was confirmed to be trans-4-hexenoic acid. The degradation of ascorbate or sorbate by lactic acid bacteria should be taken into account when these additives are used in food products where this group of bacteria may be present.     

Diversity of culturable microorganisms and occurrence of Listeria monocytogenes and Salmonella spp. in Tuber aestivum and Tuber melanosporum ascocarps

The aim of this study was to investigate the total mesophilic microorganisms, Pseudomonas genus, Enterobacteriaceae family, mold and yeast counts and the presence of Listeria monocytogenes and Salmonella spp on Tuber aestivum and Tuber melanosporum ascocarps. The results confirmed that the major percentage of the microorganisms, approximately 9.0 log ufc/g, were present in the peridium, the glebas of healthy truffles being practically free of microorganisms. The predominant microbial group was the Pseudomonas averaging 8.3 and 8.4 log cfu/g on T. aestivum and T. melanosporum whole ascocarps, respectively. The Enterobacteriaceae also achieved high populations, especially in T. aestivum truffles, with 6.3 log cfu/g. Molds and yeasts never exceeded 5.0 log cfu/g. The characterization of the isolates revealed that the fluorescens pseudomonads were the most prevalent. Raoultella terrigena and Enterobacter intermedius were the dominant Enterobacteriaceae. The identification of the yeast isolates revealed five species: Debaryomyces hansenii, Issatchenkia scutulata, Rhodotorula aurantiaca, Saccharomyces dairensis and Trichosporon beigelii subspecies A and B. The mold genera detected in both species of truffles were Aspergillus, Cladosporium, Penicillium and Fusarium, Trichoderma being present only in T. aestivum. L. monocytogenes was found in 10% of the samples of T. aestivum analysed but Salmonella spp. was not detected. Knowledge of the microbial population in terms of possible food borne and pathogen microorganisms is very useful for establishing successful disinfection and storage methods to prolong the shelf-life of ascocarps of T. aestivum and T. melanosporum.     

Tracking spore-forming bacterial contaminants in fluid milk-processing systems

The presence of psychrotolerant Bacillus species and related spore formers (e.g., Paenibacillus spp.) in milk has emerged as a key biological obstacle in extending the shelf life of high-temperature, short-time pasteurized fluid milk beyond 14 d. A recently developed rpoB DNA sequence-based subtyping method was applied to characterize spoilage bacteria present in raw milk supplies for 2 processing plants, and to assess transmission of these organisms into pasteurized products. Thirty-nine raw milk samples and 11 pasteurized product samples were collected to represent the processing continuum from incoming truck loads of raw milk to packaged products. Milk samples were held at 6°C for up to 16 d and plated for bacterial enumeration at various times throughout storage. Among the 88 bacterial isolates characterized, a total of 31 rpoB allelic types representing Bacillus and Paenibacillus spp. were identified, including 5 allelic types found in both raw milk and finished product samples. The presence of the same bacterial subtypes in raw and commercially pasteurized milk samples suggests that the raw milk supply represents an important source of these spoilage bacteria. Extension of the shelf life of high-temperature, short-time pasteurized fluid milk products will require elimination of these organisms from milk-processing systems.     

Biodiversity of indigenous staphylococci of naturally fermented dry sausages and manufacturing environments of small-scale processing units

The staphylococcal community of the environments of nine French small-scale processing units and their naturally fermented meat products was identified by analyzing 676 isolates. Fifteen species were accurately identified using validated molecular methods. The three prevalent species were Staphylococcus equorum (58.4%), Staphylococcus saprophyticus (15.7%) and Staphylococcus xylosus (9.3%). S. equorum was isolated in all the processing units in similar proportion in meat and environmental samples. S. saprophyticus was also isolated in all the processing units with a higher percentage in environmental samples. S. xylosus was present sporadically in the processing units and its prevalence was higher in meat samples. The genetic diversity of the strains within the three species isolated from one processing unit was studied by PFGE and revealed a high diversity for S. equorum and S. saprophyticus both in the environment and the meat isolates. The genetic diversity remained high through the manufacturing steps. A small percentage of the strains of the two species share the two ecological niches. These results highlight that some strains, probably introduced by the meat, will persist in the manufacturing environment, while other strains are more adapted to the meat products.     

Evaluation of hygiene practices and microbiological quality of cooked meat products during slicing and handling at retail

Cooked meat ready-to-eat products are recognized to be contaminated during slicing which, in the last years, has been associated with several outbreaks. This work aimed to find out possible relation between the hygiene practice taking place at retail point during slicing of cooked meat products in small and medium-sized establishments (SMEs) and large-sized establishments (LEs) and the microbiological quality of sliced cooked meat products. For that, a checklist was drawn up and filled in based on scoring handling practice during slicing in different establishments in Cordoba (Southern Spain). In addition, sliced cooked meats were analyzed for different microbiological indicators and investigated for the presence of Listeria spp. and Listeria monocytogenes. Results indicated that SMEs showed a more deficient handling practices compared to LEs. In spite of these differences, microbiological counts indicated similar microbiological quality in cooked meat samples for both types of establishments. On the other hand, Listeria monocytogenes and Listeria inocua were isolated from 7.35% (5/68) and 8.82% (6/68) of analyzed samples, respectively. Positive samples for Listeria spp. were found in establishments which showed acceptable hygiene levels, though contamination could be associated to the lack of exclusiveness of slicers at retail points. Moreover, Listeria spp presence could not be statistically linked to any microbiological parameters; however, it was observed that seasonality influenced significantly (P < 0.05) L. monocytogenes presence, being all samples found during warm season (5/5). As a conclusion, results suggested that more effort should be made to adequately educate handlers in food hygiene practices, focused specially on SMEs.     

A survey on distribution and toxigenicity of Aspergillus section Flavi in poultry feeds

Thirty-five samples of poultry feeds and corresponding raw materials (maize, soybean and meat meal) from a processing plant were analyzed to evaluate the distribution and toxigenicity of Aspergillus section Flavi isolates. Mycological analysis of the samples indicated the presence of five fungal genera (Aspergillus, Penicillium, Fusarium, Cladosporium, and Eurotium). Aspergillus flavus was the predominant species being present in 48.5% of the analyzed samples. Ninety-one isolates belonging to Aspergillus section Flavi were isolated; ninety were identified as A. flavus and only one as A. parasiticus. Fifty-seven isolates were capable of producing sclerotia, 41 were identified as L-type strains and 16 as type S. Fifty-seven percent of the isolates produced AFB₁ levels ranging from 0.05 μg/kg to 27.7 μg/kg whereas 86.8% produced CPA from 1.5 μg/kg to 137.8 μg/kg. L-strains produced from 0.05 to 14.8 μg/kg of aflatoxin and type S produced levels from 0.05 to 1.65 μg/kg. No significant differences in CPA production among S- and L-strains were observed. Sclerotial isolates produced AFB₁ levels ranging between 0.05 and 27.7 μg/kg and CPA levels from 3.8 to 47.3 μg/kg. More than half of the A. flavus isolates were able to produce AFB and CPA simultaneously. Twenty percent of the 35 samples were contaminated with aflatoxin B₁ whereas 34.3% were contaminated with CPA. The high rate of CPA producing isolates represents a potential risk of contamination with this toxin in poultry feeds.     

A combined morphologic and molecular approach for characterizing fungal microflora from a traditional Italian cheese (Fossa cheese)

Phenotypic and genotypic methods were used to identify filamentous fungi that characterize traditional Italian Fossa cheese and its ripening environment. After ageing for 60 days at a dairy, it was ripened for an additional three months in a pit. In the fully ripened cheese, moulds ranged from 3 to 3.4 log cfu g^?1 and Penicillium was the prevalent species. Pit environmental fungi ranged from 530 to 750 cfu m^?3 (air) and from 130 to 340 cfu cm^?2 (surfaces). The dominant pit strains were Alternaria spp., Aspergillus spp., Cladosporium spp. and Penicillium spp. Phylogenetic analyses of 18S rRNA gene and ITS1-5.8S-ITS2 regions highlighted Penicillium camemberti, Aspergillus nidulans and Aspergillus versicolor as traceable species occurring in both the cheese and pit environment, suggesting their involvement in the development of typical Fossa cheese characteristics. This approach may be used for the identification of microflora on other cheese varieties to better understand the fungal contribution in cheese ripening.