Tracking microbial quality, safety and environmental contamination sources in artisanal goat cheesemaking factories.

A harmonised microbiological survey was performed in two artisanal factories of raw goat milk cheeses (A and B) located in the Andalusian region (Spain). A total of 165 different control points or samples (raw materials, final products, food-contact surfaces [FCS], and air) were examined as microbial and pathogen sources of contamination of artisanal goat raw milk cheeses. For raw milk samples analysed from both producers, the concentrations of aerobic-mesophilic bacteria (AMB), total coliforms, coagulase-positive Staphylococcus spp. (CPS), lactic-acid bacteria (LAB) and moulds and yeasts ranged between 3.48 and 8.59, 2.45-5.48, 3.42-4.81, 4.99-8.59 and 3.35-6.85 log cfu/mL respectively. For the same microbial groups, the analysis of raw milk cheeses revealed concentrations ranging from 7.82 to 8.88, 2.00-6.82, 2.00-5.28, 8.11-9.57 and 2.00-5.76 log cfu/g, respectively. Although the raw material analysed from producer A presented higher microbial loads and between-batch variability, it was B the producer with the most loaded final products. Regarding the microbial air quality, the fermentation area, storage room, milk reception and packaging room were the most AMB loaded places, while the ripening chamber was the area with higher fungal loads in bioareosol from both producers. Conveyor belts, cutting machine, storage boxes and brine tank were highlighted as the most contaminated FCS evaluated. Staphylococcus aureus was the only pathogen detected within the set of 51 isolates from samples as revealed by MALDI-TOF and molecular PCR, with a prevalence of 12.5% for samples from the producer B. The public health risk attributed to the consumption of artisanal goat cheese should not be neglected, and may consider the whole cheesemaking processing chain, from microbiological quality of raw milk to the ready-to-eat final product, especially concerning the presence of S. aureus.

Exploring communication signals inside the microbial community of a Listeria monocytogenes-carrying biofilm contamination site.

In nature, bacterial pathogens like L. monocytogenes, live in nature associated with other microbial species in spatially-structured communities called biofilms. In the food industry, biofilms contribute to the survival and persistence of L. monocytogenes within processing facilities, thereby enhancing its risk of cross-contaminating food products. The challenge of combating biofilms has triggered the search for new antibiofilm strategies including devising ways to interfere with cell communication mechanisms (quorum-sensing) that are known to be involved with biofilm development regulation. The aim of this study was to explore cell communication signals in a L. monocytogenes-carrying microbial community isolated from a meat processing plant (location No. 96) in order to elucidate the ecological interactions that could serve as a starting point for the development of new antibiofilm strategies. Quorum quenching (QQ) and quorum sensing (QS) activities were screened among 31 bacterial strains isolated from location No. 96. Whereas no QQ activity was detected against short-chain lactone N-hexanoyl-DL-homoserine lactone (C6-HSL), it was detected against N-dodecanoyl-DL-homoserine lactone (C12-HSL) in 7 isolates (23%), particularly in Pseudomonas monteilli, Rhodococcus sp. and Rhodococcus erythropolis. QS activity assays detected HC4, C4, C6, OC6, HC10 and C16 in all the extracts, being C4, C6 and OC6 with predominantly produced by Pseudomonas monteilli, Pseudomonas gesardii, Psychrobacter maritimus and Paracoccus sp. High production levels of C16-HSL by Paracoccus sp. and the role of this long-chain lactone as a self-inhibitor of cell aggregation led us to carry out further studies focused on the effects of a Paracoccus lactone extract (PLE) against the biofilm formation by L. monocytogenes. A quantitative microscopic analysis demonstrated a significant decrease (p < 0.05) in the area occupied by biofilms formed on stainless steel (SS) coupons by different strains of L. monocytogenes in the presence of PLEs. Conversely, no significant differences were observed in the total number of viable adhered cells on SS coupons with or without PLE. The observed effect was partially reproduced by the addition of pure C16-HSL to 24 h-biofilms of L. monocytogenes L1.96. These results demonstrate that the observed effects can be attributed, at least partially, to the HSLs contained in the PLE. Overall, the present results highlight how interspecies communication within a biofilm can open up new insights for the development of new ways to combat biofilm.

Optimization of E. coli Inactivation by Benzalkonium Chloride Reveals the Importance of Quantifying the Inoculum Effect on Chemical Disinfection.

Optimal disinfection protocols are fundamental to minimize bacterial resistance to the compound applied, or cross-resistance to other antimicrobials such as antibiotics. The objective is twofold: guarantee safe levels of pathogens and minimize the excess of disinfectant after a treatment. In this work, the disinfectant dose is optimized based on a mathematical model. The model explains and predicts the interplay between disinfectant and pathogen at different initial microbial densities (inocula) and dose concentrations. The study focuses on the disinfection of Escherichia coli with benzalkonium chloride, the most common quaternary ammonium compound. Interestingly, the specific benzalkonium chloride uptake (mean uptake per cell) decreases exponentially when the inoculum concentration increases. As a consequence, the optimal disinfectant dose increases exponentially with the initial bacterial concentration.

Modeling Reveals the Role of Aging and Glucose Uptake Impairment in L1A1 Listeria monocytogenes Biofilm Life Cycle.

Listeria monocytogenes is a food-borne pathogen that can persist in food processing plants by forming biofilms on abiotic surfaces. The benefits that bacteria can gain from living in a biofilm, i.e., protection from environmental factors and tolerance to biocides, have been linked to the biofilm structure. Different L. monocytogenes strains build biofilms with diverse structures, and the underlying mechanisms for that diversity are not yet fully known. This work combines quantitative image analysis, cell counts, nutrient uptake data and mathematical modeling to provide a mechanistic insight into the dynamics of the structure of biofilms formed by L. monocytogenes L1A1 (serotype 1/2a) strain. Confocal laser scanning microscopy (CLSM) and quantitative image analysis were used to characterize the structure of L1A1 biofilms throughout time. L1A1 forms flat, thick structures; damaged or dead cells start appearing early in deep layers of the biofilm and rapidly and massively loss biomass after 4 days. We proposed several reaction-diffusion models to explain the system dynamics. Model candidates describe biomass and nutrients evolution including mechanisms of growth and cell spreading, nutrients diffusion and uptake and biofilm decay. Data fitting was used to estimate unknown model parameters and to choose the most appropriate candidate model. Remarkably, standard reaction-diffusion models could not describe the biofilm dynamics. The selected model reveals that biofilm aging and glucose impaired uptake play a critical role in L1A1 L. monocytogenes biofilm life cycle.

Quantifying the combined effects of pronase and benzalkonium chloride in removing late-stage Listeria monocytogenes-Escherichia coli dual-species biofilms.

This work presents the assessment of the effectivity of a pronase (PRN)-benzalkonium chloride (BAC) sequential treatment in removing Listeria monocytogenes-Escherichia coli dual-species biofilms grown on stainless steel (SS) using fluorescence microscopy and plate count assays. The effects of PRN-BAC on the occupied area (OA) by undamaged cells in 168 h dual-species samples were determined using a first-order factorial design. Empirical equations significantly (r2 = 0.927) described a negative individual effect of BAC and a negative interactive effect of PRN-BAC achieving OA reductions up to 46%. After treatment, high numbers of remaining attached and released viable and cultivable E. coli cells were detected in PRN-BAC combinations when low BAC concentrations were used. Therefore, at appropriate BAC doses, in addition to biofilm removal, sequential application of PRN and BAC represents an appealing strategy for pathogen control on SS surfaces while hindering the dispersion of live cells into the environment.

Removal of Listeria monocytogenes dual-species biofilms using combined enzyme-benzalkonium chloride treatments.

The effects of pronase (PRN), cellulase (CEL) or DNaseI alone or combined with benzalkonium chloride (BAC) against Listeria monocytogenes-carrying biofilms were assayed. The best removal activity against L. monocytogenes-Escherichia coli biofilms was obtained using DNaseI followed by PRN and CEL. Subsequently, a modified logistic model was used to quantify the combined effects of PRN or DNaseI with BAC. A better BAC performance after PRN compared to DNaseI eradicating L. monocytogenes was observed. In E. coli the effects were the opposite. Finally, effects of DNaseI and DNaseI-BAC treatments were compared against two different L. monocytogenes-carrying biofilms. DNaseI-BAC was more effective against L. monocytogenes when co-cultured with E. coli. Nonetheless, comparing the removal effects after BAC addition, these were higher in mixed-biofilms with Pseudomonas fluorescens. However, a high number of released viable cells was observed after combined treatments. These results open new perspectives of enzymes as an anti-biofilm strategy for environmental pathogen control.

Antimicrobial activity of essential oils against Staphylococcus aureus biofilms.

The present study was aimed to evaluate the potential of essential oils to remove the foodborne pathogen Staphylococcus aureus from food-processing facilities. The effectiveness of 19 essential oils against planktonic cells of S. aureus was firstly assessed by minimal inhibitory concentration. Planktonic cells showed a wide variability in resistance to essential oils, with thyme oil as the most effective, followed by lemongrass oil and then vetiver oil. The eight essential oils most effective against planktonic cells were subsequently tested against 48-h-old biofilms formed on stainless steel. All essential oils reduced significantly (p < 0.01) the number of viable biofilm cells, but none of them could remove biofilms completely. Thyme and patchouli oils were the most effective, but high concentrations were needed to achieve logarithmic reductions over 4 log CFU/cm(2) after 30 min exposure. Alternatively, the use of sub-lethal doses of thyme oil allowed to slow down biofilm formation and to enhance the efficiency of thyme oil and benzalkonium chloride against biofilms. However, some cellular adaptation to thyme oil was detected. Therefore, essential oil-based treatments should be based on the rotation and combination of different essential oils or with other biocides to prevent the emergence of antimicrobial-resistant strains.

A new and efficient method to obtain benzalkonium chloride adapted cells of Listeria monocytogenes.

A new method to obtain benzalkonium chloride (BAC) adapted L. monocytogenes cells was developed. A factorial design was used to assess the effects of the inoculum size and BAC concentration on the adaptation (measured in terms of lethal dose 50 -LD50-) of 6 strains of Listeria monocytogenes after only one exposure. The proposed method could be applied successfully in the L. monocytogenes strains with higher adaptive capacity to BAC. In those cases, a significant empirical equation was obtained showing a positive effect of the inoculum size and a positive interaction between the effects of BAC and inoculum size on the level of adaptation achieved. However, a slight negative effect of BAC, due to the biocide, was also significant. The proposed method improves the classical method based on successive stationary phase cultures in sublethal BAC concentrations because it is less time-consuming and more effective. For the laboratory strain L. monocytogenes 5873, by applying the new procedure it was possible to increase BAC-adaptation 3.69-fold in only 33 h, whereas using the classical procedure 2.61-fold of increase was reached after 5 days. Moreover, with the new method, the maximum level of adaptation was determined for all the strains reaching surprisingly almost the same concentration of BAC (mg/l) for 5 out 6 strains. Thus, a good reference for establishing the effective concentrations of biocides to ensure the maximum level of adaptation was also determined.

The use of water and ice with bactericide to prevent onboard and onshore spoilage of refrigerated megrim (Lepidorhombus whiffiagonis).

This study investigates the effectiveness of ozonated water and flake ice (combined Petfrost system) to increase the quality and stability of fresh megrim on fishing boats. The captured fish were washed, placed in plastic boxes, covered with flake ice and refrigerated at 2°C for up to 2-weeks onboard and, thereafter, for 11 days onshore. The experiments employed sterile, filtered and ozonated water at a concentration of 2ppm for washing the fish and making the flake ice. The results are compared with samples from a traditional treatment consisting of water and flake ice of marine origin. Fish were caught in four different hauls, which took 14, 12, 8 and 3 days in being landed. Subsequently, fish were stored for 1, 5, 7 and 11 days at 3°C. The different treatments were evaluated using sensory, microbiological and chemical techniques. Fish treated with ozone always showed the best quality. Megrim treated with ozone was still suitable for consumption after 14 days on board, and megrim stored for 12, 8 and 3 days on board could be stored for a further five days in the ice state once landed with an acceptable quality. In contrast, control fish were not suitable for consumption if stored for longer than three days on board.The results indicate that treatment with water and ice flakes made from sterile and ozonated water maintains the quality of fresh megrim onboard fishing boats and upon arrival onshore.

Possible role for cryostabilizers in preventing protein and lipid alterations in frozen-stored minced muscle of Atlantic mackerel.

Adding DE 18 maltodextrin (80 g kg(-1)) to high-fat minced mackerel was highly effective against lipid oxidation and protein and color changes during frozen storage. It increased the temperature of ice-melting onset (Tm') and decreased freeze concentration of solutes in the unfrozen water (UFW) phase, which would have allowed it to effectively slow such perturbations. This maltodextrin showed a higher effectiveness against lipid oxidation, but was slightly less effective in preventing the loss of protein solubility than common cryoprotectants, that is, an equiproportional mixture of sucrose and sorbitol. Such differences in effectiveness were much higher in low-fat minces, in which lipid oxidation proceeded to a much lower extent. Consequently, prior to replacing traditional cryoprotectants with maltodextrins, it should be known which processes limit the shelf life of the food. Decreasing (from 80 to 50 g kg(-1)) the proportion of maltodextrin added to high-fat minced mackerel showed that although it affected only slightly the effectiveness against lipid oxidation, it did notably affect the effectiveness in preventing the loss of protein solubility and color changes. Therefore, such a decrease could be accepted only if lipid oxidation is the most limiting process of shelf life, but does not seem appropriate when protein changes are important.

Modified method for the analysis of free fatty acids in fish.

The objective of this study was the development of a method for the quantification of free fatty acids (FFA) using less aggressive reactants against the handler and the environment than those used in the classic method of Lowry and Tinsley. The modified procedure is a variation of the Lowry and Tinsley method employing cyclohexane in place of benzene. The use of benzene is prohibited in certain work processes and laboratories, and the competent authority in each country is actively promoting research into harmless or less harmful products that could replace benzene. A comparison with the traditional AOCS titration method for oil analysis was performed. FFA content in mackerel frozen at -10 degrees C was measured according to the three methods over a 12 month period. The results showed similar values, and good correlations were obtained.