Bacillus cereus cell and spore properties as influenced by the micro-structure of the medium.

AIM: To investigate the effect of different growth conditions on Bacillus cereus cell and spore properties. METHODS AND RESULTS: Bacillus cereus was grown on agar plates with different surface water conditions (wet and dry) or viscosity. Cell populations displayed different types of behaviour, and heterogeneity was manifested in cell motility and dimension. Spore populations were heterogeneous regarding their properties, namely size and thermal resistance. The smallest spores were produced from flagellated cells, which also displayed jet-motility, growing on the wettest agar. Cytometric analysis also revealed within the smallest spores a sub-population labelled by propidium iodide (PI), indicating that spore populations were partly damaged. Nonmotile cells grown on diffusion-limiting media were elongated and produced the least thermal-resistant spores. CONCLUSIONS: The micro-structural properties of the media were found to influence cell and spore properties. Abundant surface water enabled flagellar motility and resulted in a heterogeneous cell and spore population, the latter including small and damaged spores. High viscosity gave rise to filamentous cells and more heat-sensitive spores. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides useful information on conditions resulting in heterogeneous populations of damaged and heat-sensitive spores.

The food safety management system.

A comprehensive food safety strategy involves establishing risk management goals, food safety objectives and, for production systems, performance objectives and performance criteria. The working instructions for each step of the process should be validated for their effect before integration within a specific HACCP plan. The importance of realistic inactivation models to predict the hygienic equivalence of food processing operations is discussed.

Probabilistic representation of the exposure of consumers to Clostridium botulinum neurotoxin in a minimally processed potato product.

We have examined the potential of a well-specified, minimally processed potato product as a vehicle for the exposure of consumers to Clostridium botulinum neurotoxin. The product is a relatively simple combination of raw potato flakes, flour, starch and other minor ingredients and has an extended lifetime under refrigeration conditions. A combination of information and data, from a variety of sources that includes the manufacturer, has shown that the product is particularly safe with respect to non-proteolytic C. botulinum hazards. The model concentrates on a simple end point, the toxicity of an individual retail unit of the product at the point of consumer preparation, which is related to an individual risk. The probabilistic analysis was built using Bayesian Belief Network (BBN) techniques.

Prevalence of Clostridium species and behaviour of Clostridium botulinum in gnocchi, a REPFED of italian origin.

Sales and consumption of refrigerated processed foods of extended durability (REPFEDs) have increased many-fold in Europe over the last 10 years. The safety and quality of these convenient ready-to-eat foods relies on a combination of mild heat treatment and refrigerated storage, sometimes in combination with other hurdles such as mild preservative factors. The major hazard to the microbiological safety of these foods is Clostridium botulinum. This paper reports on the prevalence and behaviour of proteolytic C. botulinum and non-proteolytic C. botulinum in gnocchi, a potato-based REPFED of Italian origin. Attempts to isolate proteolytic C. botulinum and non-proteolytic C. botulinum from gnocchi and its ingredients were unsuccessful. Based on assessment of the adequacy of the methods used, it was estimated that for proteolytic C. botulinum there was < 25 spores/kg of gnocchi and < 70 spores/kg of ingredients. The total anaerobic microbial load of gnocchi and its ingredients was low, with an estimated 1 MPN/g in processed gnocchi. Most of the anaerobic flora was facultatively anaerobic. A few obligately anaerobic bacteria were isolated from gnocchi and its ingredients and belonged to different Clostridium species. The protection factor, number of decimal reductions in the probability of toxigenesis from a single spore, was determined for eight different gnocchi formulations by challenge test studies. For all gnocchi stored at 8 degrees C (as recommended by the manufacturer) or 12 degrees C (mild temperature abuse), growth and toxin production were not detected in 75 days. The protection factor was >4.2 for proteolytic C. botulinum, and >6.2 for non-proteolytic C. botulinum. When inoculated packs were stored at 20 degrees C (severe temperature abuse), toxin production in 75 days was prevented by the inclusion of 0.09% (w/w) sorbic acid (protection factors as above), however in the absence of sorbic acid the packs became toxic before the end of the intended shelf-life and the protection factors were lower. Providing sorbic acid (0.09% w/w) is included in the gnocchi, the safety margin would seem to be very large with respect to the foodborne botulism hazard.

Determination of peroxy radical-scavenging of lactic acid bacteria.

Responses of lactic acid bacteria (LAB) to peroxy radicals generated via thermal (40 degrees C) decomposition of the diazocompound 2,2,-azo-bis (2-amidinopropane) dihydrochloride (ABAP), were studied. In general, LAB displayed survival curves with shoulders and tails indicative of 'multihit' killing by exposure to peroxy radicals. One strain, Lactococcus lactis subsp. lactis DIP15, producing a slope of 0.0105 in the kinetic analysis when exposed to 4 mM ABAP, exhibited a measurable antioxidant capacity. The other LAB failed to show any significant antioxidant capacity. The antioxidant capacity of strain DIP15 remained constant after cells have been heat-treated, suggesting that compounds bearing free radical scavenging capacity are rather stable.

Influence of agar content on the growth parameters of Bacillus cereus.

Factors governing Bacillus cereus colony growth on agar media as modified by the agar content (1-7%, w/v) were studied. Agar had a significant effect on the radial growth rate which diminished as the agar content increased. Cell density in colonies (colony density) was found to decrease during the incubation time, with lower values occurring in the presence of 1% agar. Size and DNA content of the cells grown on 1 and 7% agar were similar. An increasing proportion of cell population growing on 7% agar produced spores during the 24-h incubation period. It was shown that 'water condition' on the agar surface could be associated with colony density, with 7% agar media presenting the thinnest nominal thickness of the liquid film. On the other hand, the partial drying phenomena of the agar media which occur during preparation and incubation, could not account for the observed differences in colony growth.

Prevalence and behaviour of Bacillus cereus in a REPFED of Italian origin.

This paper reports on the prevalence and behaviour of Bacillus cereus in gnocchi, a REPFED of Italian origin. A survey of gnocchi under varying storage conditions, revealed that, although B. cereus was found in 33% of the samples, the contamination level was lower than 10(2) CFU/g for the unstored and the refrigerated (8 degrees C) samples. Counts increased with increasing storage temperatures and prolonged storage times in samples prepared without sorbic acid. The effect of different formulations (sorbic, citric and lactic acid) and storage conditions (8, 12 and 20 degrees C) have been evaluated in a challenge testing with spores of B. cereus. Results indicate that the use of sorbic acid in association with citric or lactic acid to pH 5.0 is effective in inhibiting growth of B. cereus and the anticipated shelf life of the product is safe even if temperature abuse occurs. If sorbic acid is omitted, lactic acid can inhibit B. cereus growth during storage at 8 degrees C. On the contrary, when temperature abuse occurs (12 and 20 degrees C), lactic or citric acid are not able to prevent growth of B. cereus.

Growth of Bacillus cereus on solid media as affected by agar, sodium chloride, and potassium sorbate.

The effect of two independent variables: microstructure, as modified by the agar content (1.0, 4.0, 7.0%), and water activity (a(w)), as modified by the NaCl content (0.5, 2.5, 4.5%), in the absence or in the presence of potassium sorbate (0.0; 2,000 ppm) on Bacillus cereus growth on solid media was studied. The time to visible growth (TVG) and the radial growth rate (RGR) of colonies were evaluated. TVG was not affected by microstructure and K-sorbate, although when a(w) was reduced, TVG tended to increase. RGR depended on linear effects of microstructure and a(w) variables and their interaction. When K-sorbate was added to cultural media, RGR was reduced significantly. However, in the presence of K-sorbate, RGR was found to change only when a(w) vas varied.

Investigation of the ability of proteolytic Clostridium botulinum to multiply and produce toxin in fresh Italian pasta.

The ability of proteolytic Clostridium botulinum (types A, B, and F) to produce toxin in filled fresh Italian pasta (tortelli) packed under a modified atmosphere was investigated. Four types of tortelli (filled with artichoke, meat, ricotta-spinach, or salmon) were inoculated with a suspension of heat-shocked spores to give an initial concentration of approximately 10(3) spores per piece. Samples were incubated at both 12 and 20 degrees C for up to 50 days and examined at selected time intervals for the presence of toxin by an ELISA and the mouse test. Toxin was not detected in any tortelli stored at 12 degrees C. When storage was at 20 degrees C, toxin was detected in the salmon-filled tortelli at day 30, in the meat and ricotta-spinach tortelli at day 50, but not in the artichoke-filled tortelli at day 50.

Influence of structural properties and kinetic constraints on Bacillus cereus growth.

The influence of structural properties and kinetic constraints on the behavior of Bacillus cereus was investigated on agar media. Dimensional criteria were used to study the growth in bacterial colonies. The architecture of the agar gel as modified by the agar content was found to influence the colony size, and smaller colonies were observed on media containing 50 to 70 g of agar liter-1. Except at low nutrient levels, colonies responded to nutrient gradients by decreasing in size the farther away they were from the nutrient source, and the decrease in colony size was influenced by the agar content. The diffusivities of glucose and a protein (insulin-like growth factor) were not affected by the gel architecture, suggesting that other factors, such as mechanical factors, could influence microbial growth in the agar systems used. Increasing the viscosity of the liquid phase of the agar media by adding polyvinylpyrrolidone resulted in a reduction in colony size. When the agar concentration was increased, the colony areas were not influenced by the viscosity of the system.

Behavior of Listeria monocytogenes in Mozzarella cheese in presence of Lactococcus lactis.

The behavior of Listeria monocytogenes (Scott A) on fully processed Italian Mozzarella cheese was examined in presence and in absence of bacteriocins produced by Lactococcus lactis ssp. lactis strains (DIP 15 and DIP 16). These strains, isolated from raw milk, produced heat stable bacteriocins that were inactivated by pronase, alpha- chymotrypsin and proteinase K, but not by pepsin, trypsin and catalase. The addition of crude bacteriocins to the growing culture of Listeria monocytogenes resulted in a significant reduction in cell number at 5 degrees C, but not at 30 degrees C. Mozzarella cheese was inoculated with the Listeria culture to obtain an initial level of approximately 30 CFU/cm2 surface of Mozzarella and approximately 10(3) CFU/ml of the surrounding fluid and then packaged in bags containing the heat-treated neutralized-cultures of Lactococcus lactis ssp. lactis in skim milk (in Italy, Mozzarella is sold in small size pieces, individually packaged in bags containing some fluid). Bags were stored at 5 degrees C up to 21 days. The presence of bacteriocins resulted in apparent death of Listeria monocytogenes after 24 h storage. After 7 days of storage, a revival of Listeria monocytogenes was observed, followed by an increase in number. However, for a storage period of 2-3 weeks the number of Listeria monocytogenes remained significantly below the number observed for Mozzarella cheese packaged in absence of the heat-treated cultures of Lactococcus lactis.

Thermal Inactivation of Aeromonas hydrophila As Affected by Sodium Chloride and Ascorbic Acid.

The combined effects of sodium chloride (0, 1.0, 1.5, and 3.0%) and ascorbic acid (0, 1.0, and 2.0 mmol/liter) with mild heat (46 degrees C) on the survival of Aeromonas hydrophila were evaluated. Because of the nonlinear nature of the survivor curves obtained, several equations yielding an R (coefficient of multiple determination) of congruent with 1 were tested. The equation that most closely fit the curvature of the observed data set was a hyperbolic function. Equation coefficients were combined to obtain a so-called death value. This value (46.67% explained variance) was calculated by extracting the larger eigenvalue and the relative eigenvector from the correlation matrix of the coefficients. the effects of the experimental factors on the death value were described by a quadratic response surface model. Results revealed that the death value was not influenced by the presence of ascorbic acid. However, increased mortality resulted from the interaction between sodium chloride and ascorbic acid.

Tertiary myotubes in postnatal growing pig muscle detected by their myosin isoform composition.

The postnatal development of skeletal muscles was studied in growing pigs from 8 to 210 d of age. Indirect immunoperoxidase staining of frozen sections of porcine semimembranosus muscle and longissimus muscle revealed a distinct population of small fibers (tertiary myotubes) that were stained specifically by an antibody (anti-NE) selective for the developmental (embryonic and neonatal) isoforms of muscle myosin. At 8 d of age the other larger fibers were already anti-NE negative and differentiated into Types I and II. A gradual decrease in the number of anti-NE positive fibers together with a gradual increase in area of the remaining positive fibers was observed throughout the pigs' growth. These results may indicate that hyperplastic growth does not cease at birth. Possible mechanisms to explain the origin of these tertiary myotubes containing developmental isoforms of myosin are suggested.

Antibiotic and disinfectant susceptibility in Enterobacteriaceae isolated from minced meat.

The resistance of Enterobacteriaceae isolated from minced meat to antibiotics and to an amphoteric surfactant, was examined. Despite the wide range of antibiotic resistance occurring in the strains tested, resistance to the disinfectant was not observed.

The influence of Lactobacillus plantarum culture inoculation on the fate of Staphylococcus aureus and Salmonella typhimurium in Montasio cheese.

The growth and survival of Staphylococcus aureus and Salmonella typhimurium were investigated during the manufacturing and ripening of raw milk Montasio cheese. Initial inoculated populations in the cheese milk were about 10(5) cfu/ml for S. aureus and 10(6) cfu/ml for S. typhimurium. Samples of curds and cheeses were taken during manufacturing and storage and analysed for pH and microbial populations. S. aureus increased slightly in number during the early period of ripening and attained a population of about 10(6) cfu/ml during the remaining period of storage. S. typhimurium decreased during cheesemaking and storage but persisted through 90 days. The addition of Lactobacillus plantarum culture (0.2% v/v) produced a marked reduction in populations of the test strains in 10 days of storage. Enterotoxin A was not detected in Montasio cheese even with a S. aureus population of 1.1 X 10(7) cfu/ml. L. plantarum strains were also tested by the spot method and the associative growth approach for their antagonistic activity against S. aureus and S. typhimurium. The compound excreted by L. plantarum was active only toward S. aureus. Furthermore, its activity was destroyed by protease treatment. These results indicated that while the growth of S. typhimurium is reduced by the acid production, S. aureus inhibition can be ascribed to bacteriocin production.

Influence of breeding systems on pH and histochemical properties of muscle fibres in porcine M. semimembranosus.

The purpose of this study was to investigate pH values, fibre type frequency and histological features of Semimembranosus muscle of 15 pigs subjected to three breeding systems. The A group was raised in a large paddock; the B group animals were raised in a collective pen until they reached a live weight of 120 kg and then in the same paddock as the A group; the C group was raised in a collective pen. Breeding systems had no significant effect on pH values and fibre type frequency. Histopathological changes in muscle fibres and connective tissue were observed in 12 of the animals studied regardless of the group. However, analysis of the incidence of alterations showed a higher frequency of lesions in the B group, followed by the C group. Except for one case of DFD meat, muscle alterations were not associated with an adverse pH. In addition, regenerating fibres, with strong reactivity for neonatal serum, were observed both in apparently healthy and in pathological muscle samples.