Anderson S. Sant'Ana

Growth potential of Salmonella spp. and Listeria monocytogenes in nine types of ready-to-eat vegetables stored at variable temperature conditions during shelf-life.

Growth potential (δ) is defined as the difference between the population of a microorganism at the end of shelf-life of specific food and its initial population. The determination of δ of Salmonella and Listeria monocytogenes in RTE vegetables can be very useful to determine likely threats to food safety. However, little is known on the behavior of these microorganisms in several RTE vegetables. Therefore, the aim of this study was to determine the δ of both pathogens in nine different types of RTE vegetables (escarole, collard green, spinach, watercress, arugula, grated carrot, green salad, and mix for yakisoba) stored at refrigeration (7°C) and abuse temperature (15°C). The population of aerobic microorganisms and lactic acid bacteria, including those showing antimicrobial activity has been also determined. Results indicated that L. monocytogenes was able to grow (δ≥0.5 log(10)) in more storage conditions and vegetables than Salmonella. Both microorganisms were inhibited in carrots, although a more pronounced effect has been observed against L. monocytogenes. The highest δ values were obtained when the RTE vegetables were stored 15°C/6days in collard greens (δ=3.3) and arugula (δ=3.2) (L. monocytogenes) and arugula (δ=4.1) and escarole (δ=2.8) (Salmonella). In most vegetables and storage conditions studied, the counts of total aerobic microorganisms raised significantly independent of the temperature of storage (p<0.05). Counts of lactic acid bacteria were higher in vegetables partially or fully stored at abuse temperature with recovery of isolates showing antimicrobial activity. In conclusion, the results of this study show that Salmonella and L. monocytogenes may grow and reach high populations in RTE vegetables depending on storage conditions and the definition of effective intervention strategies are needed to control their growth in these products.

Prevalence, populations and pheno- and genotypic characteristics of Listeria monocytogenes isolated from ready-to-eat vegetables marketed in Sao Paulo, Brazil

Listeria monocytogenes is a foodborne pathogen of great concern due to the high fatality rates of listeriosis. The consumption of RTE vegetables has increased in Brazil over the last two decades, but little is known about the risks associated to the consumption of these products. This study evaluated the prevalence and counts of L. monocytogenes in 512 packages of ready-to-eat vegetables marketed in São Paulo. The isolates were characterized for their serotypes, ribotypes, positivity for virulence genes inlA, inlC and inlJ, resistance to chlorine, growth rate variability and capability to form biofilm on stainless steel (AISI 304, #4) coupons. L. monocytogenes was detected in 3.1% of the samples. Only five samples presented countable levels, with counts between 1.0×10(1) and 2.6×10(2)CFU/g. Isolates belonged to serotypes 1/2b or 4b and most were positive for genes inlC and inlJ. Ribotypable isolates were grouped into four groups: 1038 (69.4%), 19175 (11.3%), 19191 (17.7%) and 18604 (one isolate). Most isolates survived to exposure to 125 ppm of a chlorine-based disinfectant for 3 min. All isolates were capable to attach to the coupons, reaching counts above 4 log(10) CFU/cm(2) and the growth rate (μ) at 25°C of the majority of the isolates varied between 0.1 and 0.2 log OD/h, but for few strains the μ was as high as 0.26 log OD/h. Results of this survey indicate that RTE vegetables may be vehicles of L. monocytogenes strains with limited variation in serotype, ribotype and virulence factors but varying significantly in resistance to chlorine disinfectants, capability of forming biofilm and growth rate. Data obtained is of foremost importance to serve as baseline for the development of scientific-based policies to control the incidence of L. monocytogenes in RTE vegetables in Brazil.

Modelling the growth of Listeria monocytogenes in fresh green coconut (Cocos nucifera L.) water.

The behaviour of Listeria monocytogenes in the fresh coconut water stored at 4 degrees C, 10 degrees C and 35 degrees C was studied. The coconut water was aseptically extracted from green coconuts (Cocos nucifera L.) and samples were inoculated in triplicate with a mixture of 5 strains of L. monocytogenes with a mean population of approximately 3 log(10) CFU/mL. The kinetic parameters of the bacteria were estimated from the Baranyi model, and compared with predictions of the Pathogen Modelling Program so as to predict its behaviour in the beverage. The results demonstrated that fresh green coconut water was a beverage propitious for the survival and growth of L. monocytogenes and that refrigeration at 10 degrees C or 4 degrees C retarded, but did not inhibit, growth of this bacterium. Temperature abuse at 35 degrees C considerably reduced the lagtimes. The study shows that L. monocytogenes growth in fresh green coconut water is controlled for several days by storage at low temperature, mainly at 4 degrees C. Thus, for risk population this product should only be drunk directly from the coconut or despite the sensorial alterations should be consumed pasteurized.

Modelling the lag time and growth rate of Aspergillus section Nigri IOC 4573 in mango nectar as a function of temperature and pH

Aims: To assess the behaviour of Aspergillus section Nigri IOC 4573 in mango nectar as affected by temperature and pH.

Use of a multivariate approach to assess the incidence of Alicyclobacillus spp. in concentrate fruit juices marketed in Argentina: Results of a 14-year survey

The purpose of this study was to determine the incidence of Alicyclobacillus spp. in fruit/vegetable juices (concentrated pulps and clarified and non-clarified juices) marketed in Argentina between 1996 and 2009. The presence of Alicyclobacillus was determined in a total of 8556 samples of fruit and vegetable juices (apple, pear, grape, peach, blend of juices, tangerine, pineapple, orange, mango, plum, guava, apricot, lemon, banana, kiwi, carrot, strawberry, grapefruit, and beetroot) collected in seven Argentinean provinces. Multiple factor analysis (MFA) was carried out on a data matrix that contained the percentage of positive samples, type of juice, raw material and production year. Except for kiwi and orange, Alicyclobacillus was found in juices from all the evaluated raw materials. The highest percentage of positive samples was found for beetroot, strawberry, banana, peach, mango, carrot and plum juices. The percentage of positive samples for these juices ranged from 100% to 24%. Furthermore, the application of multivariate techniques provided an insight on the relationship between the incidence of Alicyclobacillus and production variables. This approach enabled the identification of the most relevant variables that increased the percentage of positive samples among the juices, which could help in developing strategies to avoid the incidence of this bacterium. By means of hierarchical cluster analysis seven groups (clusters) of juices which showed different percentages of positive samples for Alicyclobacillus spp. were identified. This analysis showed that pineapple, peach, strawberry, mango and beetroot juices had higher rates of positivity for Alicyclobacillus than the rest of the evaluated juices. MFA analysis also showed that some clear relationships could be highlighted between the percentage of samples positive for Alicyclobacillus and five types of fruit juices (strawberry, beetroot, grapefruit, pineapple and mango). It was observed that a large proportion of juices produced in 2000, 2005 and 2008 were located in clusters with higher incidence of Alicyclobacillus spp., whereas a larger proportion of clarified concentrate juice and concentrate pulp samples showed higher probability of incidence of Alicyclobacillus in these products. Data presented in this study brings a contribution to the ecology of Alicyclobacillus in fruit/vegetable juices marketed in Argentina. This information would be useful to enhance the microbiological stability of fruit juices regarding the presence of Alicyclobacillus spp.

Involvement of Clostridium gasigenes and C. algidicarnis in ‘blown pack' spoilage of Brazilian vacuum-packed beef

The objectives of this study were to isolate psychrotrophic clostridia from Brazilian vacuum-packed beef cuts (spoiled or not) and to identify the isolates by using 16S rRNA gene sequencing. Anaerobic psychrotrophic microorganisms were also enumerated and samples were collected to verify the incidence of psychrotrophic clostridia in the abattoir environment. Vacuum-packed beef cuts (n=8 grossly distended and n=5 non-spoiled) and environmental samples were obtained from a beef packing plant located in the state of São Paulo, Brazil. Each sample was divided in three subsamples (exudate, beef surface and beef core) that were analyzed for vegetative forms, total spore-forming, and sulfide reducing spore-forming, both activated by alcohol and heat. Biochemical profiles of the isolates were obtained using API20A, with further identification using 16S rRNA gene sequencing. The growth temperature and the pH range were also assessed. Populations of psychrotrophic anaerobic vegetative microorganisms of up to 10(10)CFU/(g, mL or 100 cm(2)) were found in blown pack samples, while in non-spoiled samples populations of 10(5)CFU/(g, CFU/mL or CFU/100 cm(2)) was found. Overall, a higher population of total spores and sulfide reducing spores activated by heat in spoiled samples was found. Clostridium gasigenes (n=10) and C. algidicarnis (n=2) were identified using 16S rRNA gene sequencing. Among the ten C. gasigenes isolates, six were from spoiled samples (C1, C2 and C9), two were isolated from non-spoiled samples (C4 and C5) and two were isolated from the hide and the abattoir corridor/beef cut conveyor belt. C. algidicarnis was recovered from spoiled beef packs (C2). Although some samples (C3, C7, C10 and C14) presented signs of blown pack spoilage, Clostridium was not recovered. C. algidicarnis (n=1) and C. gasigenes (n=9) isolates have shown a psychrotrophic behavior, grew in the range 6.2-8.2. This is the first report on the isolation of psychrotrophic Clostridium (C. gasigenes and C. algidicarnis) in Brazil. This study shows that psychrotrophic Clostridium may pose a risk for the stability of vacuum-packed beef produced in tropical countries during shelf-life and highlights the need of adopting control measures to reduce their incidence in abattoir and the occurrence of blown pack spoilage.

Influence of the hot-fill water-spray-cooling process after continuous pasteurization on the number of decimal reductions and on Alicyclobacillus acidoterrestris CRA 7152 growth in orange juice stored at 35 °C

In this study, the influence of the hot-fill water-spray-cooling process after continuous pasteurization on the number of decimal reductions (gamma) and growth parameters (lag time; lambda, ratio N(f)/N(o); kappa, maximum growth rate; mu) of Alicyclobacillus acidoterrestris CRA 7152 in orange juice stored at 35 degrees C were investigated. Two different inoculum levels of A. acidoterrestris CRA 7152 (10(2) and 10(3) spores/mL) in orange juice (11(0)Brix, pH 3.7) and a Microthermics UHT-HTST pilot plant were used to simulate industrial conditions. Results have shown that regardless of the inoculum level (10(2) or 10(3) spores/mL), the pasteurization processes were unable to cause even 1 gamma. Predictive modeling using the Baranyi model showed that only kappa and time to reach 10(4)spores/mL (t10(4) - time to juice spoilage) were affected by the spore inoculum used (p<0.05). It has been concluded that A. acidoterrestris was able to survive the hot-fill process and to grow and spoil orange juice in 5-6 days when the final storage temperature was 35 degrees C.

High Incidence of Enterotoxin D Producing Staphylococcus spp. in Brazilian Cow's Raw Milk and Its Relation with Coagulase and Thermonuclease Enzymes

In this study, the enterotoxigenic potential of Staphylococcus strains (n = 574) isolated from raw milk samples (n = 140) was determined for their capacity to produce staphylococcal enterotoxins. In addition, the relationship between the presence of enterotoxins, coagulase, and thermonuclease (Tnase) was assessed. The results showed that 19% of Staphylococcus was enterotoxigenic, being able to produce at least one of the staphylococcal enterotoxins (A, B, C, and D). Most of the strains were able to produce enterotoxin D (68.8%), whereas 12.8% of the Staphylococcus strains were able to produce staphylococcal enterotoxin A. Besides, the production of more than one type of enterotoxins by the same strain was observed. Tnase was considered the best marker for enterotoxigenic potential of isolates, although some of them were negative for coagulase and Tnase but positive for enterotoxin production. Therefore, either the use of Tnase to assess Staphylococcus enterotoxigenic potential or the use of simple and easy screening tests for enterotoxin production should receive more attention when evaluating the pathogenic potential of foodborne Staphylococcus strains. Due to the association of both coagulase positive Staphylococcus and coagulase negative Staphylococcus with foodborne disease outbreaks, regulators and industries should pay more attention to enterotoxigenic Staphylococcus rather than focusing only on S. aureus or coagulase positive Staphylococcus. Finally, data found here suggest a high risk of staphylococcal intoxication with the consumption of raw milk or dairy products made from raw milk.

Influence of different shrinking temperatures and vacuum conditions on the ability of psychrotrophic Clostridium to cause 'blown pack' spoilage in chilled vacuum-packaged beef.

This study determined the ability of psychrotrophic Clostridium strains isolated from vacuum-packaged beefs and abattoir environments to cause blown-pack spoilage of vacuum-packaged beef stored at 2 and 15 °C. The influence of shrinking temperatures (83, 84 and 87 °C) and vacuum pressure (6 and 9 mbar) on the occurrence of such spoilage as well as the effects of simulated transportation (500 km) on the integrity of packages was determined. At 15 °C and 2 °C, twelve and six strains caused blown-pack spoilage, respectively. The combination of vacuum pressure (9 mbar) combined with shrinking temperature (87 °C) retarded the occurrence of spoilage. The simulated transportation under the experimental conditions did not affect the integrity of packages. More studies that assess the factors that may contribute for the occurrence of blown-pack spoilage should be performed to avoid the occurrence of such spoilage during its shelf-life.

Behavior and enterotoxin production by coagulase negative Staphylococcus in cooked ham, reconstituted skimmed milk, and confectionery cream.

In this study, the behavior and enterotoxin production by 10 different coagulase negative Staphylococcus (CNS) strains inoculated in cooked ham, reconstituted skimmed milk, and confectionery cream in the presence or absence of background microbiota have been investigated. After inoculation (103 CFU/g), foods were incubated at 25, 30, and 37 °C and aerobic mesophilic and CNS counts were carried out at 12, 24, 48, and 72 h. Staphylococcal enterotoxins (SE) detection was performed by SET-RPLA (Oxoid, Basingstoke, U.K.) and mini-Vidas® (bioMérieux, La Balme les Grottes, France). CNS counts increased during incubation and approached 10⁶ to 10⁷ CFU/g after 12 h at 37 °C in the 3 foods studied. At 25 °C, counts reached 10⁶ to 10⁷ CFU/g only after 24 to 48 h. The interference of background microbiota on CNS behavior was only observed when they grew in sliced cooked ham, which presented a high initial total count (10⁵ CFU/g). Significantly higher counts of CNS isolated from raw cows milk in comparison with food handlers isolates were found in reconstituted milk and confectionery cream. Although CNS strains were able to produce SEA, SEB, and SED in culture media, in foods, in the presence or absence of background microbiota S. chromogenes LE0598 was the only strain able to produce SEs. Despite the scarcity of reports on CNS involvement with foodborne disease outbreaks, the results found here support the CNS growth and SE production in foods even in the presence of background microbiota and may affect food safety.