The Influence of Debaryomyces hansenii, Candida deformans and Candida zeylanoides on the aroma formation of dry-cured “lacón”

The volatile profile of dry-cured “lacón” that has been inoculated with three different yeasts were determined and compared with a non-inoculated dry-cured “lacón”. Yeasts (Debaryomyces hansenii, Candida deformans and Candida zeylanoides) that were used as starter cultures in the present study were selected among yeasts that were isolated from native dry-cured “lacón” at different stages of ripening process. These starters were spread on dry-cured “lacón” surface in order to test their capacity to contribute on the generation of volatile compounds. A total of forty two volatile compounds were detected by dynamic headspace sampling followed by gas chromatography–mass spectrometry analysis. Significant differences (P < 0.001) on the volatile profiles of different batches were found in comparison with non-inoculated samples, showing the highest total area values for the inoculated ones. Esters were the most abundant chemical family in all batches studied except for C. zeylanoides batch, which showed greater amount of hydrocarbons than esters. The second more abundant family was hydrocarbons for control and C. deformans batches (147.6 and 445.24 × 10⁶ area units, respectively), alcohols for D. hansenii (363.77 × 10⁶ area units) and esters for C. zeylanoides (248.33 × 10⁶ area units). However, the aldehyde compound group in control batch samples was found to be significantly higher than in the inoculated ones (P < 0.001). Among inoculated batches, D. hansenii batch showed the lowest hexanal content (14.42 × 10⁶ area units) in comparison with non-inoculated batch (105.99 × 10⁶ area units). Among all batches studied, D. hansenii batch presented the highest area values for esters, alcohols, linear hydrocarbons, ketones, acids and furans; control batch for aldehydes and C. zeylanoides batch for branched hydrocarbons. Therefore, the study showed that every yeast strain produced a specific volatile profile which was also different from that of the control dry-cured “lacón”.     

Heat stress adaptation induces cross-protection against lethal acid stress conditions in Arcobacter butzleri but not in Campylobacter jejuni

The ability of many bacteria to adapt to stressful conditions may later protect them against the same type of stress (specific adaptive response) or different types of stresses (multiple adaptive response, also termed cross-protection). Arcobacter butzleri and Campylobacter jejuni are close phylogenetic relatives that occur in many foods of animal origin and have been linked with human illness (mainly diarrhoea). In the present study, sublethal stress adaptation temperatures (48 °C and 10 °C) and mild and lethal acid conditions (pH 5.0 and pH 4.0) were determined for A. butzleri and C. jejuni. In addition, it was evaluated whether these sublethal stress adaptations cause specific adaptive responses or cross-protection against subsequent mild or lethal acid stresses in these bacteria. The studies were conducted in broth adjusted to the different conditions and the results were determined by the dilution series plating method. It was shown that heat stress adapted A. butzleri (incubated for 2 h at 48 °C) were significantly more resistant to subsequent lethal acid stress (pH 4.0) than non-adapted cells at the 1 h time-point (p < 0.01 in Wilcoxon rank sum test). No specific adaptive responses against the stresses in A. butzleri or C. jejuni and no cross-protection in C. jejuni were found. The ability of heat stressed A. butzleri to tolerate later lethal acid conditions should be taken into account when designing new food decontamination and processing strategies.     

Barcode High Resolution Melting (Bar-HRM) analysis for detection and quantification of PDO “Fava Santorinis” (Lathyrus clymenum) adulterants

Legumes considered as one of the most important crops worldwide. Due to high price as a PDO product, commercial products of “Fava Santorinis” are often subjected to adulterations from other legume products coming from other Lathyrus or Vicia and Pisum species. Using plant DNA barcoding regions (trnL and rpoC) coupled with High Resolution Melting (Bar-HRM) we have developed a method allowing us to detect and authenticate PDO “Fava Santorinis”. Bar-HRM proved to be a very sensitive tool able to genotype Lathyrus and its closed relative species and to detect admixtures, being sensitive enough to as low as 1:100 of non-“Fava Santorinis” in “Fava Santorinis” commercial products. In conclusion, Bar-HRM analysis can be a faster, with higher resolution and cost effectiveness alternative method to authenticate PDO “Fava Santorinis” and to quantitatively detect adulterations in “Fava Santorinis” with other relative commercial “Fava” food products.     

“Framboisé” spoilage in French ciders: Zymomonas mobilis implication and characterization

Biodiversity of 17 Zymomonas mobilis strains isolated from French “framboisé” ciders from 7 different geographical areas of France was analysed using random amplified polymorphic DNA (RAPD) and 6 different strain fingerprints were observed. One of the fingerprints was identical for 10 of the 17 cases. No correlation between geographical origin and strain fingerprint was observed. In parallel, the cider-making process was followed from the orchard to the final product for Z. mobilis presence. Detection of the bacterium only occurred during the fermentation process. The French isolate, strain AN0101, recently proposed as a novel subspecies (Z. mobilis subsp. francensis) was characterized in a synthetic medium using various growth conditions that can be encountered during cider-making (T (°C), pH, ethanol, SO₂, polyphenols) to determine growth limits. Results clearly showed that cider is in a risk zone for Z. mobilis contamination as this bacterium was able to grow at temperatures as low as 4 °C, in pH range from 3.5 to 6.0, in 0-8 ml/100 ml ethanol, at the legal limit of 200 mg/l SO₂ and in the presence of a cider marc polyphenol extract up to 3 g/l. These results should allow for a better understanding of Z. mobilis conditions of contamination in cider and lead to possible preventive measures in the future.     

Survival of poultry-derived Campylobacter jejuni of multilocus sequence type clonal complexes 21 and 45 under freeze,chill, oxidative,acid and heat stresses

The application of multilocus sequence typing (MLST) for studying Campylobacter jejuni diversity reveals that MLST clonal complex (CC) 21 and CC-45 occupies significant proportion in the diverse population of C. jejuni. These two complexes are ecologically abundant and represent an interesting subpopulation for studying C. jejuni survival under different stress conditions. In the present study we characterize and compare 19 C. jejuni strains assigned to CC-21 and CC-45, isolated from chicken meat, based on laboratory stress models maintained in Muller-Hinton broth. Model conditions were mimicking freeze, chill, oxidative, acid and heat stresses. Results show that survival patterns varied between the strains. C. jejuni strains of CC-21 survived significantly better than C. jejuni strains of CC-45 under heat (P value = 0.022) and chill (P value = 0.001) stress models. On the other hand, C. jejuni strains of CC-45 showed significantly better survival compared to C. jejuni strains of CC-21 in response to oxidative (P value = 0.003) and freeze (P value = 0.021) stress models. C. jejuni strains assigned to the founder ST-45 showed significantly better survival (P value = 0.017) under heat stress model compared to their ancestral sequence types. However, an association between survival fitness and the diversification of a clonal group cannot be demonstrated directly from the obtained results. In conclusion, findings of the present study show that genotypic variations of C. jejuni might play a role in enabling certain lineages to be selected when encountering adverse and stressful environments. In future stress response studies, it is recommended to consider the effect of genotypic diversity among C. jejuni strains as that might bias the experimental findings.     

Quantitative analysis of viable,stressed and dead cells of Campylobacter jejuni strain 81-176

Campylobacter jejuni is an important foodborne gastrointestinal pathogen and highly sensitive to environmental stresses. Research has shown that changes in culturability, cell morphology, and viability occur when C. jejuni cells are subjected to stresses. In this study, real-time PCR, ethidium monoazide (EMA) in combination with real-time PCR (EMA-PCR), BacLight bacterial viability staining, and agar plate counting methods were used to quantitatively analyze viable, stressed, and dead C. jejuni strain 81-176. The real-time PCR assay provides highly sensitive and specific quantification of total genome copies of C. jejuni culture in different growth phases. Our results also reveal that real-time PCR can be used for direct quantification of Campylobacter genome release into Phosphate Buffered Saline (PBS) as an indicator of cell lysis. Using EMA-PCR, we obtained a dynamic range of greater than 3 logs for differentiating viable vs. dead cells. The viability and morphological characteristics of the stressed cells after one-week incubation at 25 °C, in air, and under nutrient-poor conditions were investigated. Our results indicated that, over 99% of the stressed cells were converted from the spiral to the coccoid form and became non-culturable. However, more than 96% of the coccoid cells retained their membrane integrity as suggested by both the BacLight staining and EMA-PCR analyses. Thus, to detect C. jejuni under stress conditions, conventional culturing method in conjunction with EMA-PCR or BacLight staining might be a more appropriate approach.     

Removal of polychlorinated dioxins by semi-aerobic fed-batch composting with biostimulation of “Dehalococcoides”

A semi-aerobic, mesophilic, fed-batch composting (FBC) reactor loaded with household garbage was used to remove polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs). The reactor was packed with woodchips as the solid matrix and PCDD/F-contaminated soil or flyash and then operated at a waste-loading rate of 0.5 kg (wet wt) day^- 1. All congeners of PCDD/Fs (initial concentration, 200–830 pmol g^- 1 [dry wt]) were totally reduced during the over period of operation, with a half reduction time of 4 months. Direct cell counting and respiratory quinone profiling showed that the reactors at the fully acclimated stage harbored a high population density of bacteria (10¹¹ g^- 1 [dry wt]) with members of the Actinobacteria predominating. Real-time quantitative PCR showed that the population of “Dehalococcoides” and its phylogenetic relatives of Chloroflexi as the possible dechlorinators varied between at the order of 10⁷ to 10⁸ g^- 1 (dry wt). A “Dehalococcoides”-containing dechlorinating culture from the soil-treating reactor was successfully enriched with a model PCDD/F compound, fthalide. 16S rRNA gene-targeted PCR-denaturated gradient gel electrophoresis and clone library analyses showed that this culture comprised at least three major phylogenetic groups of bacteria, Acidaminobacter, “Dehalococcoides,” and Rhizobium. These results suggest that the semi-aerobic FBC process is applicable for the bioremediation of PCDD/Fs and possibly other haloorganic compounds with the biostimulation of “Dehalococcoides” and its relatives as the potent dechlorinators.     

The biofilm forming potential of bacterial species in the genus Campylobacter

The biofilm forming abilities of 16 strains representative of 14 of the 16 species comprising the genus Campylobacter were determined on glass, stainless steel, and polystyrene plastic. The formation of biofilms has been suggested as a means by which Campylobacter is able to persist within an inhospitable environment. Of the eight microaerophilic Campylobacter species, including two strains each of Campylobacter jejuni and Campylobacter fetus, only C. jejuni strain 81–176 reliably produced a visible biofilm on multiple surfaces. Alternately, all six strains of the anaerobic Campylobacter species reliably produced visible biofilms on multiple surfaces. Electron micrographs of the individual biofilms showed relatively homogeneous biofilms produced by the anaerobic strains, while the microaerophilic C. jejuni strain 81–176 produced a biofilm containing similar quantities of both the spiral and coccoid forms. This survey suggests a difference in the biofilm forming potentials and the morphologies of the bacteria comprising the biofilms between anaerobic and microaerophilic species of Campylobacter. Additionally, differences observed in the biofilm forming ability of two strains of C. jejuni suggest the need for a further investigation of the biofilm forming potential of this species using a larger number of strains.     

PorA specific primers for the identification of Campylobacter species in food and clinical samples

Campylobacteriosis is a public health problem with considerable socio-economic impact. As the European Food Safety Authority has emphasized the importance of a surveillance programme for campylobacteriosis, the aim of the present study was the optimization of a specific and sensitive PCR protocol able to detect Campylobacter species responsible for gastrointestinal infections. Raw poultry meat samples were analysed for the presence of Campylobacter sp., by plating onto mCCD (Modified Charcoal-Cefoperazone-Deoxycholate) Agar and Campylobacter Selective Preston Agar and using four sets of species-specific primers for Campylobacter jejuni, Campylobacter coli, Campylobacter upsaliensis and Campylobacter lari designed to bridge the porA gene. The resulting primers demonstrated a sensitivity of 0.01 ng/μl for the C. coli-specific, C. lari-specific, and C. upsaliensis-specific primer sets and 0.5 ng/μl for the C. jejuni-specific primer sets using DNA from pure cultures. Non-specific amplification of non-target DNA was not observed indicating excellent specificity. The primers were useful for the analyses of poultry meat samples both for direct plating onto mCCDA, and for DNA extracted directly from the cells grown for 48 h in Preston enrichment broth. The sets of primers were also useful when used for species identification of human isolates.     

Control of foodborne pathogens on fresh-cut fruit by a novel strain of Pseudomonas graminis

The consumption of fresh-cut fruit has substantially risen over the last few years, leading to an increase in the number of outbreaks associated with fruit. Moreover, consumers are currently demanding wholesome, fresh-like, safe foods without added chemicals. As a response, the aim of this study was to determine if the naturally occurring microorganisms on fruit are “competitive with” or “antagonistic to” potentially encountered pathogens. Of the 97 and 107 isolates tested by co-inoculation with Escherichia coli O157:H7, Salmonella and Listeria innocua on fresh-cut apple and peach, respectively, and stored at 20 °C, seven showed a strong antagonistic capacity (more than 1-log unit reduction). One of the isolates, CPA-7, achieved the best reduction values (from 2.8 to 5.9-log units) and was the only isolate able to inhibit E. coli O157:H7 at refrigeration temperatures on both fruits. Therefore, CPA-7 was selected for further assays. Dose-response assays showed that CPA-7 should be present in at least the same amount as the pathogen to adequately reduce the numbers of the pathogen. From the results obtained in in vitro assays, competition seemed to be CPA-7's mode of action against E. coli O157:H7. The CPA-7 strain was identified as Pseudomonas graminis. Thus, the results support the potential use of CPA-7 as a bioprotective agent against foodborne pathogens in minimally processed fruit.     

Development and evaluation of aptamer magnetic capture assay in conjunction with real-time PCR for detection of Campylobacter jejuni

A prototype method for the concentration and detection of Campylobacter jejuni was developed using a previously reported biotinylated DNA aptamer in conjunction with qPCR. The so-called aptamer-based magnetic capture-qPCR (AMC-qPCR) assay was compared to a similar immunomagnetic separation (IMS)-qPCR assay. In small volume experiments (300 μl) applied to serially diluted C. jejuni suspended in buffer containing a mixed culture of other common food borne pathogens, the lower detection limit of the AMC-qPCR method was 1.1 log₁₀/300 μl C. jejuni cells, one log₁₀ better (lower) than that of IMS-qPCR (2.1 log₁₀ CFU/300 μl). AMC-qPCR capture efficiency was 10–13% at assay detection limit. In 10 ml scale-up experiments, the lower detection limit of AMC-qPCR was 2.0 log₁₀ CFU/10 ml with corresponding capture efficiency of 4–7%. Nucleic acid aptamers are promising alternatives to antibodies for magnetic bead-based capture followed by qPCR detection.     

Glucosinolates profile of Brassica rapa L. subsp. Sylvestris L. Janch. var. esculenta Hort

Glucosinolates in different ecotypes of Brassica rapa L. subsp. sylvestris L. Janch. var. esculenta Hort. widely distributed in Southern Italy and locally known as “friariello” and “cima di rapa”, were characterized and their glucosinolate composition was compared with that of broccoli (Brassica oleracea L. var. italica).     

Acorns (Quercus rotundifolia Lam.) and grass as natural sources of antioxidants and fatty acids in the “montanera” feeding of Iberian pig: Intra- and inter-annual variations

In two consecutive years (2006/2007 and 2007/2008), fortnightly samples were taken to characterise the antioxidant composition and fatty acid profile of acorns and grass on which Iberian pigs feed during the period of “montanera” (free-range rearing system of the south-western of Iberian Peninsula). The antioxidant parameters analysed were: α- and γ-tocopherol, total phenolics compounds (TPC), lipophilic and hydrophilic antioxidant activities (LAA, HAA) (acorn and grass) and condensed and hydrolysable tannins (CT, HT) and protein precipitating capacity (PP) (acorns). To characterise the fatty acid profiles, the thirteen major fatty acids were assayed. For the acorns, it was found that there was intra-annual variability in all the antioxidants studied except α-tocopherol, and inter-annual variability in all except the protein precipitating capacity. The fatty acid profile also varied depending on the sampling date and the study year, especially in the saturated fatty acids (SFA) and C18:1 n-9 content. For the grass, there was intra- and inter-annual variability in all the antioxidant parameters studied, and in the proportions of the fatty acids C18:0, and C18:1. It could explain variations in the antioxidant and fatty acids composition of Iberian pig tissues from animals raised in different “montanera” seasons and even in the same “montanera” season.     

In vitro activity of methanol extracts of plants used as spices against Mycobacterium tuberculosis and other bacteria

This study determined the phenolic composition of two thyme species (Lamiaceae), Origanum minutiflorum O. Schwarz and P.H. Davis and Thymbra spicata L. var. spicata, and assessed their antibacterial and antimycobacterial activities. “Kekik” is a collective term used in Turkey for plants that smell like thyme. O. minutiflorum, (locally “Sutculer kekigi”, endemic) and T. spicata var. spicata (locally “Karakekik”) are widely used in Turkey and are important export commodities.     

Evaluation of the strain variability of Salmonella enterica acid and heat resistance

The inherent acid and heat resistances of 60 Salmonella enterica strains were assessed in tryptone soy broth without dextrose acidified to pH 3.0 or heated at 57 °C. A total of 360 inactivation curves were generated. Regarding the acid challenge experiments, the inactivation rate (k_acid), estimated using the log–linear model, ranged from 0.47 to 3.25 h⁻¹. A log–linear model with a “survival tail” was used to describe the thermal inactivation of the strains, and the estimated inactivation rate (k_heat) ranged from 0.42 to 1.33 min⁻¹. The strain variability of k_acid was considerably higher than that of k_heat with the coefficient of variation of this kinetic parameter among the tested strains being 39.0% and 18.3%, respectively. No correlation was observed between the estimated k_acid and k_heat values of the 60 S. enterica strains. Furthermore, no trends among the tested strains related to origin, serotype or antibiotic resistance profile were evident. The present study is the first one to comparatively evaluate the inherent acid and heat resistance profiles of multiple S. enterica strains. Beyond their value in strain selection for use in food safety challenge studies, the collected data should be useful in describing and integrating the strain variability of S. enterica acid and heat resistance profiles in quantitative microbial risk assessment.     

Survival of Pseudomonas fluorescens on beef carcass surfaces in a commercial abattoir

The influence of a commercial chilling process (18 h at 10 °C followed by up to 78 h at 2 °C) on Pseudomonas fluorescens inoculated on beef carcass surfaces at four sites, neck (NE), outside round (OR), brisket (BR) and foreshank/brisket (FB) before chilling (“hot inoculated”) or after chilling for 24 h (“cold inoculated”) was investigated. Pseudomonas counts increased significantly at all sites on “hot inoculated” carcasses during storage, but on “cold inoculated” carcasses, counts declined or remained unchanged. On hot and cold inoculated carcasses, differences in Pseudomonas growth or survival were demonstrated between sites. No clear relationships were observed between Pseudomonas growth or survival and chiller relative humidity (RH) or surface water activity (a_w) at the different sites. These results were unexpected, and are discussed in relation to environmental factors that affect the growth/survival of P. fluorescens on carcass surfaces during chilling i.e. temperature, RH, and the relationship of these parameters to surface water activity (a_w).     

Peroxidase-like activity of Ilex paraguariensis

Ilex paraguariensis St. Hil. (Aquifoliaceae) is widely exploited in northeastern Argentina, southern Brasil and eastern Paraguay. The commercial product named “Mate” or “Yerba Mate” is used to prepare a tea like beverage (infusions or decoctions).     

Biofilm formation by Campylobacter jejuni in controlled mixed-microbial populations

This study was to screen the ability of biofilm formation by Campylobacter jejuni strains found in New Zealand, and investigate the biofilm growth of C. jejuni in a controlled mixed-microbial population that includes five different bacteria. The ability of C. jejuni to form a biofilm in monoculture and mixed-microbial populations was measured in a laboratory assay using a microtiter plate screening assay. The optical density of the biofilm and cell growth from mixed-microbial populations was converted to a Biofilm Formation Index (BFI). This index was used to standardize the biofilm formation in the mixed-microbial populations. High BFI was observed for Enterococcus faecalis (2.30) and Staphylococcus simulans (3.75) when they were grown with C. jejuni multilocus sequence type ST-474: a dominant poultry and human-associated type in New Zealand. C. jejuni cells were recovered from most of the biofilms containing E. faecalis and/or S. simulans. These results suggest that E. faecalis and S. simulans may play a role in biofilm formation in the poultry environment as both of these microorganisms are found in poultry processing environments and were able to form a biofilm in association with C. jejuni under microaerobic conditions. Understanding the relationships among C. jejuni, E. faecalis and S. simulans in poultry processing plants and farms may help in the design of strategies to reduce the reservoir of contamination of these bacteria and reduce the incidence of campylobacteriosis.     

Initial evaluation of a field-friendly incubation procedure for the colorimetric assay of cassava linamarase

The Cooke colorimetric assay of cassava linamarase activity is temperature- and time-inflexible, making “real time” monitoring of linamarase activity in remote cassava-processing sites practically impossible. A modified incubation procedure is described, in which the 30 °C linamarase incubation step is terminated through acidification, yielding a stable cyanohydrin solution. Using partially purified linamarase as a “standard extract”, the solution – held for up to 21 days at ambient/refrigeration temperatures before colorimetry – showed reductions of up to 21% compared with the standard Cooke assay. In a separate trial, a strong linear relationship (r² > 0.95) was observed between recorded linamarase activity values and incubation temperature in the 25–40 °C range, indicating that incubation may take place in remote processing sites without a water bath, and resulting data may reliably be adjusted in keeping with the standard 30 °C incubation assay. The novel procedure thus appears to offer a satisfactory “field-friendly” means of assaying linamarase activity.     

Survival of Campylobacter jejuni on beef and pork under vacuum packaged and retail storage conditions: examination of the role of natural meat microflora on C. jejuni survival

The ability of Campylobacter jejuni ATCC 11168 to survive on beef and pork stored under chilled, vacuum packaged and retail display conditions were examined. In addition, the effect of natural microflora on commercial beef and pork on the survival of C. jejuni under these storage conditions was examined. When sterile cores of beef and pork were inoculated with ∼10⁵ to 10⁶ cfu cm⁻²C. jejuni, and were stored under aerobic or vacuum packaged conditions at −1.5 or 4 °C, its numbers dropped significantly and C. jejuni could not be enumerated by direct plating after 21 d of the 6 wks study. In contrast, survival of C. jejuni on commercial vacuum packaged beef and pork was significantly enhanced, resulting in only 1 log cfu cm⁻² reduction at the end of 6 wks. During 7 d of display in a retail case, numbers of C. jejuni dropped quickly, but could be enumerated by direct plating even after the 7 d. The presence of high numbers of inoculated C. jejuni on beef and pork had no significant effect on the natural microflora numbers compared to uninoculated controls when the meat was stored either in vacuum or in a retail display case. These results show that natural microflora on vacuum packaged meat afford enhanced survival of C. jejuni present on the surfaces of both beef and pork when stored at refrigeration temperatures. Hence, strict hygienic practices or the implementation of decontamination technologies are recommended to ensure safety of meat with respect to this pathogen.