Marta López Cabo

A method for bacteriocin quantification

M.L. CABO, M.A. MURADO, M.P. GONZÁLEZ and L. PASTORIZA.1999.Different aspects of the most commonly used assay methods in the study of bacteriocins were examined. The conditions under which extraction and incubation (including exposure time) take place were analysed, and several different formal models that are usually employed to calculate ID50 were compared. As an alternative designed to overcome the problems which characterize the response of micro‐organisms that are sensitive to bacteriocins, an operative procedure in a liquid medium and a modified re‐parameterized logistic equation is proposed. When applied to the inhibition of Leuconostoc mesenteroides by nisin, the model allows an optimal experimental procedure to be defined.

Incidence of Staphylococcus aureus and analysis of associated bacterial communities on food industry surfaces.

ABSTRACT Biofilms are a common cause of food contamination with undesirable bacteria, such as pathogenic bacteria. Staphylococcus aureus is one of the major bacteria causing food-borne diseases in humans. A study designed to determine the presence of S. aureus on food contact surfaces in dairy, meat, and seafood environments and to identify coexisting microbiota has therefore been carried out. A total of 442 samples were collected, and the presence of S. aureus was confirmed in 6.1% of samples. Sixty-three S. aureus isolates were recovered and typed by random amplification of polymorphic DNA (RAPD). Profiles were clustered into four groups which were related to specific food environments. All isolates harbored some potential virulence factors such as enterotoxin production genes, biofilm formation-associated genes, antibiotic resistance, or lysogeny. PCR-denaturing gradient gel electrophoresis (PCR-DGGE) fingerprints of bacterial communities coexisting with S. aureus revealed the presence of bacteria either involved in food spoilage or of concern for food safety in all food environments. Food industry surfaces could thus be a reservoir for S. aureus forming complex communities with undesirable bacteria in multispecies biofilms. Uneven microbiological conditions were found in each food sector, which indicates the need to improve hygienic conditions in food processing facilities, particularly the removal of bacterial biofilms, to enhance the safety of food products.

Effect of modified atmosphere packaging on shelf-life of iced fresh hake slices

Shelf-life of hake slices (Merluccius merluccius) stored in the ice state (2 ± 1°C) under modified atmosphere packaging (MAP) conditions was determined by measurements of pH, total volatile bases (TVB) and trimethylamine (TMA) content, mesophilic and psychrophilic bacterial counts, malonaldehyde content, exudation, protein functionality and sensorial analyses (colour and odour). The effect of different gas mixtures were evaluated : (1) 40% CO 2 , 50% N 2 , 10% O 2 ; (2) 60% CO 2 , 30% N 2 , 10% O 2 ; (3) 40% CO 2 , 30% N 2 , 30% O 2 ; (4) 60% CO 2 , 10% N 2 , 30% O 2 and (5) air (control). Important differences were found between MAP-stored and air-stored hake slices. Shelf-life of hake stored under MAP conditions was two-fold extended. Bacterial growth was inhibited, increases of pH, TMA and TVB were reduced, and alterations in protein functionality were delayed, and off-odours were not noted in MAP-stored hake slices after 21 days storage. Significant correlations were found between TMA content and total viable count (TVC), as well as between apparent viscosity and exudation. Hake slices could be stored in the ice state under MAP conditions for about three weeks without an important loss of quality. Fish freshness, handling practices and initial bacterial load have an important influence of shelf-life of hake.

Effect of carbon dioxide atmosphere on microbial growth and quality of salmon slices

Salmon slices (Salmo salar) were packed under CO 2 and stored in a chill room at 2 ± 1°C for 3 weeks. A study was carried out on the effect of CO 2 atmosphere on bacterial growth as well as on chemical deterioration (pH, total volatile bases, (TVB), trimethylamine (TMA), lipid oxidation) and sensory features (exudation, raw fish odour, cooked fish flavour). Results were compared with a control stored in air. Salmon slices stored under CO 2 had a shelf-life of nearly twice as long as those in air, having lower bacterial counts, pH, TMA and TVB, as well as improved sensory features after 18 days at 2 ± 1°C. Exudation was not significantly different between control and CO 2 -stored samples after 10 days ice storage. Exudate values extended from 1 to 2% for CO 2 -stored samples after 10 and 20 days storage, respectively. Salmon slices can be stored under CO 2 at 2 ± 1°C for about 18 days, with no substantial loss of quality. Shelf-life can vary depending upon the state of fish, handling practices, initial bacterial load and storage temperature.

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.

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/cm2 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.

Two mathematical models for the correction of carbohydrate and protein interference in the determination of uronic acids by the m-hydroxydiphenyl method

The most common method in the routine determination of uronic acids, the m‐hydroxydiphenyl reaction, recently adapted to rapid microplate analysis, has as a main inconvenience, in any one of their modalities, interferences due to the frequent presence of proteins and neutral carbohydrates in the samples. Corresponding corrections in the literature are unsatisfactory when applied to complex matrices, and further adaptation to the microplate analysis is not free from additional problems. With particular reference to hyaluronic acid, the interactions between the principal reactants and the interfering materials are studied kinetically under realistic conditions, and simple mathematical models are proposed which satisfactorily describe the experimental results and allow adequate corrections to be made.

Survival of lactic acid bacteria in seawater: a factorial study.

A feasibility study of lactic bacteria as potential probiotics in larval cultures of marine fish was performed by investigating the survival of five strains of lactic bacteria in seawater by readily standardized procedures at different temperatures and salinities. These conditions were chosen in such a way that their combinations define a complete first-order factorial design. Depending on the strain and the ambient conditions, the survival adhered to first-order kinetics in some cases, and to the Gompertz equation in others. The half lives (t0.5) calculated from these models were subsequently introduced as responses to the factorial designs, estimating the coefficients of empirical equations that describe the group effect of temperature and salinity on t0.5. Simply additive effects were found in two cases, a negative first-order interaction in another case, while another two required second-order models.

Numerical spatio-temporal characterization of Listeria monocytogenes biofilms

As the structure of biofilms plays a key role in their resistance and persistence, this work presents for the first time the numerical characterization of the temporal evolution of biofilm structures formed by three Listeria monocytogenes strains on two types of stainless-steel supports, AISI 304 SS No. 2B and AISI 316 SS No. 2R. Counting methods, motility tests, fluorescence microscopy and image analysis were combined to study the dynamic evolution of biofilm formation and structure. Image analysis was performed with several well-known parameters as well as a newly defined parameter to quantify spatio-temporal distribution. The results confirm the interstrain variability of L. monocytogenes species regarding biofilm structure and structure evolution. Two types of biofilm were observed: homogeneous or flat and heterogeneous or clustered. Differences in clusters and in attachment and detachment processes were due mainly to the topography and composition of the two surfaces although an effect due to motility was also found.

A proteomic approach to cold acclimation of Staphylococcus aureus CECT 976 grown at room and human body temperatures

Staphylococcus aureus is an important pathogenic microorganism that has been associated with serious infection problems in different fields, from food to clinic. In the present study, we have taken into account that the main reservoirs of this microorganism are the human body and some parts of food processing plants, which have normal temperatures of around 37 and 25°C, respectively. It can be expected that S. aureus must acclimate its metabolism to colder temperatures before growing in food matrices. Since temperature abuse for foods occurs at approximately 12°C, it is expected that S. aureus must acclimate its metabolism to colder temperatures before growing in food. For this reason, we have performed a proteomic comparison between exponential- and stationary-phase cultures of S. aureus CECT 976 acclimated to 12°C after growing at 25°C or 37°C. The analysis led to the identification of two different protein patterns associated with cold acclimation, denominated pattern A and pattern B. The first was characteristic of cultures at stationary phase of growth, grown at 25°C and acclimated to 12°C. The second appeared in the rest of experimental cases. Pattern A was distinguished by the presence of glycolytic proteins, whereas pattern B was differentiated by the presence of general stress and regulatory proteins. Pattern A was related through physiological experiments with a cross-resistance to acid pH, whereas pattern B conferred resistance to nisin. This prompted us to conclude that both molecular strategies could be valid, in vivo, for the process of acclimation of S. aureus to cold temperatures.