Pablo S. Fernández

Research on factors allowing a risk assessment of spore-forming pathogenic bacteria in cooked chilled foods containing vegetables: a FAIR collaborative project

Vegetables are frequent ingredients of cooked chilled foods and are frequently contaminated with spore-forming bacteria (SFB). Therefore, risk assessment studies have been carried out, including the following: hazard identification and characterisation--from an extensive literature review and expertise of the participants, B. cereus and C. botulinum were identified as the main hazards; exposure assessment--consisting of determination of the prevalence of hazardous SFB in cooked chilled foods containing vegetables and in unprocessed vegetables, and identification of SFB representative of the bacterial community in cooked chilled foods containing vegetables, determination of heat-resistance parameters and factors affecting heat resistance of SFB, determination of the growth kinetics of SFB in vegetable substrate and of the influence of controlling factors, validation of previous work in complex food systems and by challenge testing and information about process and storage conditions of cooked chilled foods containing vegetables. The paper illustrates some original results obtained in the course of the project. The results and information collected from scientific literature or from the expertise of the participants are integrated into the microbial risk assessment, using both a Bayesian belief network approach and a process risk model approach, previously applied to other foodborne hazards.

Combined Effect of Nisin, Carvacrol and Thymol on the Viability of Bacillus Cereus Heat-Treated Vegetative Cells

The influence of mild heat pretreatment on the bactericidal action of nisin, carvacrol and thymol on stationary and exponential phase cells of two Bacillus cereus strains was studied. Carvacrol or thymol concentrations of 0.3 mmol/L had no bactericidal effect on unheated, and a minor effect on heated B. cereus cells either in stationary or exponential growth phases. Nisin (0.15 mg/mL) alone decreased the viable counts in all cases (unheated and heated cells in stationary and exponential phase) although the two strains tested showed different sensitivity to this natural antimicrobial between them. When carvacrol or thymol were combined with nisin, it resulted in a greater loss of viability of heated cells than when nisin was applied alone. There was a synergistic effect of nisin and both essential oils with a mild heat pretreatment on the viability of B. cereus cells. This study pointed out the potential use of nisin alone and in combination with carvacrol or thymol applied after a mild heat pretreatment for preservation of minimally processed foods.

Influence of pH and temperature on growth of Bacillus cereus in vegetable substrates

Bacillus cereus is a food-borne pathogen which most often contaminates foods of plant origin. Spores of psychrotrophic strains have the ability to germinate and grow at refrigeration temperatures in different vegetable substrates, such as carrot broth, zucchini broth, and cooked carrot purée. In some circumstances, factors such as pH, heat treatment, and storage temperature play a fundamental role in controlling the growth of these psychrotrophic strains and in extending the shelf life of refrigerated, minimally processed vegetable-based products in relation to pathogenic spore-forming bacteria. The combination of mild acidification (pH 5.0) and refrigeration (</=8 degrees C) inhibited B. cereus growth for at least 60 days in vegetable substrates similar to those mentioned above. This protection was maintained even when the temperature to which the food was exposed reached 12 degrees C. Psychrotrophic strains of B. cereus were inhibited in carrot broth by heating at 90 degrees C for 7.5 min, if the broth was refrigerated at a temperature of 8 degrees C or lower. If the vegetable product was exposed to temperatures of mild abuse (12 degrees C), it was necessary to implement a more drastic heat treatment (90 degrees C for 30 min).

Predictive Model Describing the Effect of Prolonged Heating at 70 to 80°C and Incubation at Refrigeration Temperatures on Growth and Toxigenesis by Nonproteolylic Clostridium botulinum

There is growing interest in the food industry in the use of long heat treatments in the range of 70 to 90°C to produce minimally processed foods that have an extended shelf life at refrigeration temperatures. The risk of growth and toxin production by nonproteolytic Clostridium botulinum in these foods is of concern. The effect of heat treatments at 70, 75, 80, 85, and 90°C combined with refrigerated storage for 90 days on growth from 106 spores of nonproteolytic C. botulinum (types B, E, and F) in an anaerobic meat medium was studied. The following heat treatments prevented growth and toxin production during 90 days provided that the storage temperature was no higher than l2°C: 75°C for ≥1,072 min, 80°C for ≥230 min, 85°C for ≥36 min, and 90°C for ≥10 min. Following heating at 70°C for 2,545 min and storage at 12°C, growth was first observed after 22 days. A factorial experimental design allowed a predictive model to be developed that described the incubation time required before the first sample showed growth as a function of heating temperature (70 to 80°C), period of heat treatment (up to 2,545 min), and incubation temperature (5 to 25°C). Predictions from the model provided a valid description of the data used to generate the model, and agreed with observations made previously.

Use of carvacrol and cymene to control growth and viability of Listeria monocytogenes cells and predictions of survivors using frequency distribution functions

The antibacterial action of carvacrol and cymene on two Listeria monocytogenes strains (STCC4031 and NCTN4032) was studied. Carvacrol or cymene showed inhibitory effect on the growth of L. monocytogenes during lag and exponential growth phases and was more evident with increasing concentrations in brain heart infusion broth at 30 degrees C. Carvacrol or cymene also decreased the survival of mid-exponential-growth-phase L. monocytogenes STCC4031 cells in potassium-N-2-hydroxy-ethylpiperazine-N-ethanesulfo nic acid, at 30 degrees C. The combination of carvacrol and cymene resulted in an increased antibacterial effect on the growth and a synergistic effect on the viability of L. monocytogenes compared with the natural compounds applied separately. The analysis of survival curves by the Weibull frequency distribution function allowed an accurate prediction of the level of inactivation achieved. Interestingly, an important bactericidal effect (4.7-log reduction) of low concentrations of both antimicrobials combined (0.75 mM) was observed on L. monocytogenes in carrot juice. This study indicates the potential use of carvacrol and cymene applied simultaneously for preservation of minimally processed foods.

Irradiation of spores of Bacillus cereus and Bacillus subtilis with electron beams

The effect of irradiation with accelerated electrons on Bacillus cereus and Bacillus subtilis spore counts and spore heat resistance was investigated. Spore counts reduced approximately two log cycles for B. cereus and up to five log cycles for B. subtilis after a radiation dose of 7.6 kGy, with D values ranging from 1.5to 3.8 kGy. Radiation had an important heat-sensitising 10 effect on bacterial spores. After a 3.3 kGy treatment D values were reduced more than three times for both B. cereus and B. T subtilis spores. This study indicates the potential use of accelerated electrons, alone or followed by a heat treatment, for food preservation.

Thermal resistance of Bacillus stearothermophilus spores heated in acidified mushroom extract

The thermal resistance of Bacillus stearothermophilus spores was studied in bidistilled water as the reference medium, mushroom extract and acidified mushroom extract. Citric acid and glucono-δ-lactone were used as acidulants. Results indicated that mushroom extract affects the heat resistance of spores; D values were lower than for those in bidistilled water. The pH effect was lower with higher treatment temperatures. Acidification reduced the thermal resistance of spores, the reduction being similar for both types of acidulants, and in general it also had the effect of increasing the z values. It has been confirmed that acidification of the canned mushrooms could actually help to control the thermophilic spoilage. This acidification could also be obtained by the use of glucono-δ-lactone, which has been shown to be as effective as citric acid in reducing the heat resistance of spores.

Control of Enterobacter aerogenes by high-intensity, pulsed electric fields in horchata, a Spanish low-acid vegetable beverage

High-intensity pulsed electric fields (HIPEF) is a non-thermal technology used in the processing of liquid foods. Inactivation of foodborne micro-organisms by this novel technology is an important alternative to traditional thermal methods with a great potential for new liquid products. However, the existence of a resistant proportion of microbial population presents a problem to establish the parameters that can guarantee the safety during the shelf-life of each food. The aim of this study was to investigate the influence of field strength, substrate inoculum size (initial cell concentration) and storage conditions after HIPEF treatment to evaluate the potential growth and subsequent spoilage by Enterobacter aerogenes in horchata, a Spanish low-acid vegetable beverage. Although no more than 1.1-log reductions were obtained by any of the HIPEF conditions applied, E. aerogenes growth in both substrates (horchata and a standard broth) at 10°C, 12°C and 16°C was affected by HIPEF treatments compared to untreated samples. The specific growth rate of the bacteria after HIPEF treatment was not modified, but the lag period was increased. A synergistic effect of HIPEF treatment, low temperature (10°C) and low inoculum size on the delay in lag phase was observed.

Predictive model to describe the combined effect of pH and NaCl on apparent heat resistance of Bacillus stearothermophilus

The combined effect of pH and NaCl on the apparent thermal resistance of Bacillus stearothermophilus ATCC 12980 spores was studied. Spores were heated at different temperatures (115-125 degrees C) in mushroom substrate, acidified using glucono-delta-lactone to different pH levels (from 5.75 to 6.7), which contained concentrations of NaCl that ranged from 0.5 to 3% (w/v). The recovery medium was acidified to the same pH level and contained the same NaCl concentration as the heating menstruum. A factorial experimental design allowed a predictive model to be developed, which described the combined effect of heating temperature, pH and NaCl on the thermal resistance of B. stearothermophilus spores. Predictions from the model provided a valid description of the data used to generate the model, and agreed with observations from the literature and from an independent experiment performed using asparagus and bean substrates.

Modeling the Lag Period and Exponential Growth of Listeria monocytogenes under Conditions of Fluctuating Temperature and Water Activity Values

ABSTRACT The dynamic model for the growth of a bacterial population described by Baranyi and Roberts (J. Baranyi and T. A. Roberts, Int. J. Food Microbiol. 23:277-294, 1994) was applied to model the lag period and exponential growth of Listeria monocytogenes under conditions of fluctuating temperature and water activity (aw) values. To model the duration of the lag phase, the dependence of the parameter h0, which quantifies the amount of work done during the lag period, on the previous and current environmental conditions was determined experimentally. This parameter depended not only on the magnitude of the change between the previous and current environmental conditions but also on the current growth conditions. In an exponentially growing population, any change in the environment requiring a certain amount of work to adapt to the new conditions initiated a lag period that lasted until that work was finished. Observations for several scenarios in which exponential growth was halted by a sudden change in the temperature and/or aw were in good agreement with predictions. When a population already in a lag period was subjected to environmental fluctuations, the system was reset with a new lag phase. The work to be done during the new lag phase was estimated to be the workload due to the environmental change plus the unfinished workload from the uncompleted previous lag phase.