Meat-based Medium Research Articles

Production of Aflatoxin B1 by Aspergillus parasiticus Grown on a Novel Meat-Based Media.

The aim of the present study was to develop meat-based media with compositions similar to those of dry-fermented meat products and to evaluate their use in studying the growth of Aspergillus parasiticus and the kinetics of aflatoxin B1 (AFB1) production. In our previous experiments, we found that the strain A. parasiticus ŽMJ7 produced a high amount of AFB1. Cooked meat agar (CMA2) was used as a novel complex meat-based medium with four variations: CMA2G (CMA2 supplemented with 1% glucose), CMA2YE (CMA2 supplemented with 0.2% yeast extract), and CMA2GYE (CMA2 supplemented with 1% glucose and 0.2% yeast extract). Media were inoculated with an A. parasiticus spore suspension (105 spores/mL) and incubated at 25 °C for up to 15 days. The A. parasiticus lag phase lasted less than 1 day, irrespective of the growth medium, with the exception of control medium CMA1 (cooked meat agar) as an already known meat-based medium. The highest mean colony growth rate was observed on CMA2 and CMA2G. Reversed-phase UPLC-MS/MS analysis was performed to determine the AFB1 concentration in combination with solid phase extraction (SPE). The highest AFB1 concentration in meat-based media was detected in CMA2GYE after 15 days of incubation (13,502 ± 2367 ng/mL media). The results showed that for studying AFB1 production in dry-fermented meat products, novel suitable media such as CMA2-based media are required. This finding could represent a potential concern with regard to the production of dry-fermented meat products.

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.

Differential Proteomic Analysis of Lactic Acid Bacteria-Escherichia coli O157:H7 Interaction and Its Contribution to Bioprotection Strategies in Meat.

Human infection by Enterohemorrhagic Escherichia (E.) coli (EHEC) occurs through the ingestion of contaminated foods such as milk, vegetable products, water-based drinks, and particularly minced meats. Indeed EHEC is a pathogen that threatens public health and meat industry. The potential of different Lactic Acid Bacteria (LAB) strains to control EHEC in a meat-based medium was evaluated by using a simple and rapid method and by analyzing the growth kinetics of co-cultures (LAB-EHEC) in a meat-based medium. The activity of LAB toward EHEC in co-cultures showed variable inhibitory effect. Although, LAB were able to control EHEC, neither the produced acid nor bacteriocins were responsible of the inhibition. The bacteriocinogenic Enteroccus (Ent.) mundtii CRL35 presented one of the highest inhibition activities. A proteomic approach was used to evaluate bacterial interaction and antagonistic mechanisms between Ent. mundtii and EHEC. Physiological observations, such as growth kinetics, acidification ability and EHEC inhibitory potential were supported by the proteomic results, demonstrating significant differences in protein expression in LAB: (i) due to the presence of the pathogen and (ii) according to the growth phase analyzed. Most of the identified proteins belonged to carbohydrate/amino acid metabolism, energy production, transcription/translation, and cell division. These results contribute to the knowledge of competition strategies used by Ent. mundtii during its co-culture with EHEC setting new perspectives for the use of LAB to control this pathogen in meat.

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.

Effect of dry-sausage starter culture and endogenous yeasts on Aspergillus westerdijkiae and Penicillium nordicum growth and OTA production

Processed meat products frequently suffer from fungal and mycotoxin contamination, mostly ochratoxin A (OTA). Penicillium nordicum is considered responsible for this contamination, but Aspergillus westerdijkiae has recently been associated with high levels of OTA in meat products.Several biocontrol agents have been tested against P. nordicum growth and OTA production in meat products, but A. westerdijkiae has not been considered. The aim of this work was to evaluate in vitro the effect of a commercial starter culture used in sausage fermentation and of sausage-native yeasts on OTA production by A. westerdijkiae, as compared with the highly studied P. nordicum, in meat-based culture media.Four representative yeasts isolated from dry-cured sausage and a commercial starter culture were co-inoculated with both fungi in different meat-based media, under varying conditions. Fungal growth was determined by measuring colony diameter, and OTA production was quantified by HPLC-FLD.A. westerdijkiae was significantly stimulated to produce OTA under all tested conditions, and, in ham, OTA production by P. nordicum was stimulated by co-culture with the starter culture.In conclusion, endogenous or added microorganisms enrolled in fermentation or in biocontrol in meat products seem to exert varying responses on different ochratoxigenic fungi, thus leading to unforeseen safety problems.

Comparison of microbial transfer rates from Salmonella spp. biofilm growth on stainless steel to selected processed and raw meat

Salmonella biofilm cells can serve as a serious source of cross-contamination, both in the home and at food production sites. The objectives of this study were to determine the transfer rates (RTs) of Salmonella biofilm cells and to model the transfer process of biofilm cells from stainless steel surfaces to raw meat. The results showed that the RTs were significantly influenced by the types of meat products, with bacon and emulsified sausage showing higher RTs and roast pork showing lower RTs. Higher RTs of biofilm grown in a meat-based medium, Meat Thawing-Loss Broth (MTLB), were observed as compared to that in a standard growth medium (TSB). The logistic, exponential and multi-roots models could be used to satisfactorily describe the transfer of biofilm cells as demonstrated by use of MSE, F-test and R2. There was no difference in attachment strength (reflected by the coefficients of transfer models) of biofilm grown in TSB or MTLB, as shown by the coefficients of r, D and B in three models. Compared with the exponential and the multi-roots models, the logistic model was able to more accurately fit the whole transfer process of biofilm cells. Our findings highlight the occurrence of cross-contamination with biofilm cells, and the models may provide useful tools in quantitative microbiological risk assessment of meat products.

Lack of induction of direct protection or cross-protection in Staphylococcus aureus by sublethal concentrations of Origanum vulgare L. essential oil and carvacrol in a meat-based medium

The capacity of Origanum vulgare L. essential oil (OVEO) and its majority compound, carvacrol (CAR), to induce direct tolerance and cross-tolerance in Staphylococcus aureus against high temperature (45 °C), lactic acid (pH 5.2) and NaCl (10 g/100 mL) was assessed. Overnight exposure of S. aureus to sublethal concentrations (1/2 MIC, 1/4 MIC) of either OVEO or CAR in meat broth revealed no induction of direct protection. S. aureus cells pre-adapted to OVEO or CAR showed no induction of cross-protection to high temperature, lactic acid or NaCl. Cells subjected to 24 h cycles of adaptation in increasing amounts (1/2 MIC to 2 × MIC) of OVEO or CAR showed no increase in direct tolerance. These results revealed a lack of induction of direct protection or cross-protection in S. aureus exposed to sublethal amounts of OVEO or CAR in meat-based broth, as determined by monitoring cell survival and growth behavior.

Bacteriocins produced by wild Lactococcus lactis strains isolated from traditional, starter-free cheeses made of raw milk

Sixty bacterial strains were encountered by random amplification of polymorphic DNA (RAPD) and repetitive extragenic palindromic (REP) typing in a series of 306 Lactococcus lactis isolates collected during the manufacturing and ripening stages of five traditional, starter-free cheeses made from raw milk. Among the 60 strains, 17 were shown to produce bacteriocin-like compounds in both solid and liquid media. At a genotypic level, 16 of the strains were identified by molecular methods as belonging to L. lactis subsp. lactis and one to L. lactis subsp. cremoris. Among the L. lactis subsp. lactis strains, phenotypic and genetic data determined that eleven produced either nisin A (nine strains) or nisin Z (two strains), and that five produced lactococcin 972. Variable levels of the two bacteriocins were produced by different strains. In addition, nisin was shown to be produced in inexpensive, dairy- and meat-based media, which will allow the practical application of its producing strains in industrial processes. Specific PCR and nucleotide and deduced amino acid sequence analysis identified the inhibitor produced by the single L. lactis subsp. cremoris isolate as a lactococcin G-like bacteriocin. Beyond the use of bacteriocins as functional ingredients for the biopreservation of foods, the newly identified bacteriocin-producing L. lactis strains from traditional cheeses may also be useful for designing starter cultures with protective properties and/or adjunct cultures for accelerating cheese ripening.

Optimization of casein-based semisynthetic medium for growing of toxigenicCorinebacterium diphtheriaein a fermenter

Diphtheria toxin is produced by growing Corinebacterium diphtheriae either in a semisynthetic casein-based medium or in the Pope-Lingood meat extract based medium. The World Health Organization advises the use of the semisynthetic one, as it has important advantages. Data on the composition of casein-based media and their ability to support high toxin production are not freely available. Important factors affecting toxin production during C. diphtheriae cultivation are the pH of the culture medium and the concentration of casein hydrolysate and of Fe2+. We established that the optimal pH for toxin production is 7.2. The highest yield of toxin was obtained using a casein hydrolysate concentration of 35.0 g/L and a Fe2+ concentration of 0.05-0.41 microg/mL. Under these conditions, diphtheria toxin with higher purity and yield compared with the batches obtained using the meat-based medium of Pope-Lingood was produced.

Molecular composition of diphtheria toxoid produced using semi-synthetic and meat extract-based broths

The use of semi-synthetic broths for cultivation of Corynebacterium diphtheriae instead of a meat extract-based broth avoids the presence of highly undesirable bovine meat antigens in the diphtheria toxoid. As information on the composition of casein digest-based broths used for the production of diphtheria toxoid is scarce, we have now developed one. The composition of a casein-based medium that supports vigorous bacterial growth as well as high toxin production is described below. The comparative analysis of the toxoids, produced using the meat-based Pope–Lingood and the casein digest-based broths, showed considerable differences in their molecular composition. The variance of weight distribution of toxoid-containing molecular complexes was smaller when the semi-synthetic broth was used. Normal human therapeutic IgG recognizes some of the proteins in the meat-based medium but does not react with any components of the semi-synthetic medium. While precipitation at the isoelectric point of the diphtheria toxoid produced by culturing the C. diphtheriae strain in the semi-synthetic medium resulted in a preparation meeting the requirement for purity (more than 1500 limit floculation Lf/mg protein nitrogen PN), the toxoid produced in the Pope–Lingood broth failed to meet this requirement in some cases, even after a second purification step using ultrafiltration.

Effects of Substrate, Water Activity, and Temperature on Growth and Verrucosidin Production by Penicillium polonicum Isolated from Dry-Cured Ham

Penicillium polonicum, a common mold on dry-cured meat products, is able to produce verrucosidin, a potent neurotoxin. The ability of P. polonicum isolated from dry-cured ham to grow and produce verrucosidin from 4 to 40°C at water activities (aw) of 0.99, 0.97, and 0.95 on malt extract agar (MEA) and a medium made up with meat extract, peptone, and agar (MPA) was evaluated. Verrucosidin was quantified by high-pressure liquid chromatography and mass spectrometry. P. polonicum was able to grow on MEA and MPA at all the aw values tested from 4 to 37°C but not at 40°C. The optimal environmental conditions for growth were 20°C, 0.99 aw on MEA and 20 to 25°C, 0.97 aw on MPA, but the highest amount of verrucosidin was obtained at 25°C, 0.99 aw in both media. No direct correlation between extension of mold growth and verrucosidin production was found. Temperature appears to be the most important factor ruling mycelial growth, whereas verrucosidin accumulation is mostly influenced by aw. However, analysis of variance of the data showed that there was a complex interaction among all the environmental factors (medium, temperature, and aw) that significantly (P < 0.0001) affected growth and verrucosidin production. The reduction of aw to intermediates values of 0.95 has a stronger effect on growth on MEA than on MPA. Given that the meat-based medium proved to be an appropriate substrate for the biosynthesis of verrucosidin by P. polonicum, the ability of this mold to produce the toxin on meat products should be established.