Assessing the relationship between organic farming practices and microbiological characteristics of organic lettuce varieties (Lactuca sativa L.) grown in Sao Paulo, Brazil.

AIMS: This study aimed to gather information on farming practices employed in organic lettuce fields in Sao Paulo, Brazil and associate these practices with the microbiological characteristics of the products. METHODS AND RESULTS: Practices were surveyed using a questionnaire applied in ten farms, where 200 heads of lettuce were collected and submitted to enumeration of total coliforms and generic Escherichia coli and tested for Salmonella spp. using culture and molecular (qPCR) methods. Based on the responses, the farms could be clustered in two groups: group 1, comprised by six farms, where chicken manure was used as fertilizer in most of them and the composting process was not performed on site; and group 2, comprised by four farms, where other types of fertilizer were used, and the composting process was performed on site. Generic E. coli was detected in 56 (28%) samples, with an average of 1·1 ± 0·7 log MPN per g. Salmonella DNA was detected in two (1%) samples by qPCR. CONCLUSIONS: The prevalence and bacterial loads of generic E. coli, and the occurrence of Salmonella, even at low populations undetectable by conventional culture methods, highlight the need for control measures during farming practices to reduce microbial contamination and risks of foodborne illnesses. These measures include the use of properly composted manure and appropriate washing procedures for leafy vegetables before consumption. SIGNIFICANCE AND IMPACT OF THE STUDY: The obtained data contribute to a better understanding of the farming practices of organically grown lettuces in Sao Paulo, Brazil.

Bacteriocins of Gram-positive bacteria having activity spectra extending beyond closely-related species.

Bacteriocins are bacterially-produced antimicrobial peptides that have killing activity principally against other relatively closely-related bacteria. Some bacteriocins of the lactic acid bacteria (LAB) have for many years been extensively applied in food biopreservation. However, especially during the last decade, a number of reports have appeared about unanticipated extensions to the generally rather narrow anti-bacterial activity spectrum of some of the LAB bacteriocins and novel applications have been proposed for bacteriocins ranging from controlling the growth of an increasingly-heterogeneous variety of pathogens, including Gram-negative multidrug resistant bacteria, viruses, yeasts, and in particular, difficult to control Mycobacterium spp., to their potential application as anticancer agents. How best can we assess this now rapidly-accumulating stream of reports on potential future applications of bacteriocins? Where is the line between realistic, science-based proposals and highly-speculative fiction and what are the 'critical points' that might help us to draw this line? In this review, we have attempted to analyse a selection of the presently-available data concerning relatively 'unorthodox' (i.e. beyond food preservation) applications of bacteriocins, and, by utilising our set of 'critical points', we endeavour to identify essential or/and missing information that appear crucial for success of the proposed applications.

Brazilian artisanal ripened cheeses as sources of proteolytic lactic acid bacteria capable of reducing cow milk allergy.

AIM: The objective was to obtain lactic acid bacteria (LAB) capable of hydrolysing immunoreactive proteins in milk, to optimize the hydrolysis, to determine the proteolysis kinetics and to test the safety of the best hydrolytic strain. METHODS AND RESULTS: Brazilian cheese was used as source of LAB capable of hydrolysing main milk allergens. Proteolytic isolates were submitted to RAPD-PCR for the characterization of clonal diversity. Optimized hydrolysis was strain and protein fraction dependent. 16S rDNA sequencing identified three proteolytic strains: Enterococcus faecalis VB43, that hydrolysed αS1 -, αS2 - and ß-caseins, α-lactalbumin and ß-lactoglobulin (partial hydrolysis), and Pediococcus acidilactici VB90 and Weissella viridescens VB111, that caused partial hydrolysis of αS1 - and αS2 -caseins. Enterococcus faecalis VB43 tested negative for virulence genes asa1, agg, efaA, hyl, esp, cylLL and cylLS but positive for genes ace and gelE. Ethylenediamine tetra-acetic acid inhibited the proteolysis, indicating that the main proteases of E. faecalis VB43 are metalloproteases. CONCLUSION: Brazilian artisanal cheese is a good source of LAB capable of hydrolysing allergenic proteins in milk. One isolate (E. faecalis VB43) presented outstanding activity against these proteins and lacked most of the tested virulence genes. SIGNIFICANCE AND IMPACT OF THE STUDY: Enterococcus faecalis VB43 presents good potential for the manufacture of hypoallergenic dairy products.

Soymilk fermentation by Enterococcus faecalis VB43 leads to reduction in the immunoreactivity of allergenic proteins ß-conglycinin (7S) and glycinin (11S).

Food allergies represent a serious problem affecting human health and soy proteins rank among the most allergenic proteins from food origin. The proteolytic enzymes produced by lactic acid bacteria (LAB) can hydrolyse the major allergens present in soybean, reducing their immunoreactivity. Many studies have reported the ability of LAB to ferment soy-based products; while the majority of them focus on the improvement of the sensory characteristics and functionality of soy proteins, a lack of information about the role of lactic fermentation in the reduction of immunoreactivity of these proteins exists. The aim of the present study was to evaluate the capability of the proteolytic strain Enterococcus faecalis VB43 to hydrolyse the main allergenic proteins present in soymilk and to determine the immunoreactivity of the obtained hydrolysates. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) results of fermented soymilk demonstrated complete hydrolysis of the ß-subunit from ß-conglycinin and the acidic polypeptide from glycinin. Reversed phase high performance liquid chromatography (RP-HPLC) analysis of the peptides released after hydrolysis revealed the appearance of new peptides and the disappearance of non-hydrolysed proteins, indicating extensive hydrolysis of the substrate. Results from competitive enzyme-linked immunosorbent assay (ELISA) tests clearly indicated a reduction in the immunoreactivity (more than one logarithmic unit) in the fermented sample as compared to the non-fermented control. Our results suggest that the soymilk fermented by E. faecalis VB43 may induce lower allergic responses in sensitive individuals. The strain E. faecalis VB43 may be considered as an excellent candidate to efficiently reduce the immunoreactivity of soymilk proteins.

Proteolytic activity of Enterococcus faecalis VB63F for reduction of allergenicity of bovine milk proteins.

With the aim of screening proteolytic strains of lactic acid bacteria to evaluate their potential for the reduction of allergenicity of the major bovine milk proteins, we isolated a new proteolytic strain of Enterococcus faecalis (Ent. faecalis VB63F) from raw bovine milk. The proteases produced by this strain had strong activity against caseins (αS1-, αS2-, and ß-casein), in both skim milk and sodium caseinate. However, only partial hydrolysis of whey proteins was observed. Proteolysis of Na-caseinate and whey proteins, observed after sodium dodecyl sulfate-PAGE, was confirmed by analysis of peptide profiles by reversed-phase HPLC. Inhibition of proteolysis with EDTA indicated that the proteases produced by Ent. faecalis VB63F belonged to the group of metalloproteases. The optimal conditions for their activity were 42°C and pH 6.5. The majority of assessed virulence genes were absent in Ent. faecalis VB63F. The obtained results suggest that Ent. faecalis VB63F could be efficient in reducing the immunoreactivity of bovine milk proteins.

Evaluation of a cross contamination model describing transfer of Salmonella spp. and Listeria monocytogenes during grinding of pork and beef.

In a previous study, a model was developed to describe the transfer and survival of Salmonella during grinding of pork (Møller, C.O.A., Nauta, M.J., Christensen, B.B., Dalgaard, P., Hansen, T.B., 2012. Modelling transfer of Salmonella typhimurium DT104 during simulation of grinding of pork. Journal of Applied Microbiology 112 (1), 90-98). The robustness of this model is now evaluated by studying its performance for predicting the transfer and survival of Salmonella spp. and Listeria monocytogenes during grinding of different types of meat (pork and beef), using two different grinders, different sizes and different numbers of pieces of meats to be ground. A total of 19 grinding trials were collected. Acceptable Simulation Zone (ASZ), visual inspection of the data, Quantitative Microbiological Risk Assessment (QMRA), as well as the Total Transfer Potential (TTP) were used as approaches to evaluate model performance and to access the quality of the cross contamination model predictions. Using the ASZ approach and considering that 70% of the observed counts have to be inside a defined acceptable zone of ±0.5 log10CFU per portion, it was found that the cross contamination parameters suggested by Møller et al. (2012) were not able to describe all 19 trials. However, for each of the collected grinding trials, the transfer event was well described when fitted to the model structure proposed by Møller et al. (2012). Parameter estimates obtained by fitting observed trials performed at different conditions, such as size and number of pieces of meat to be ground, may not be applied to describe cross contamination of unlike processing. Nevertheless, the risk estimates, as well as the TTP, revealed that the risk of disease may be reduced when the grinding of meat is performed in a grinder made of stainless steel (for all surfaces in contact with the meat), using a well-sharpened knife and holding at room temperatures lower than 4°C.

Assessing the effect of sodium dichloroisocyanurate concentration on transfer of Salmonella enterica serotype Typhimurium in wash water for production of minimally processed iceberg lettuce (Lactuca sativa L.).

UNLABELLED: This study evaluated the impact of sodium dichloroisocyanurate (5, 10, 20, 30, 40, 50 and 250 mg l(-1) ) in wash water on transfer of Salmonella Typhimurium from contaminated lettuce to wash water and then to other noncontaminated lettuces washed sequentially in the same water. Experiments were designed mimicking the conditions commonly seen in minimally processed vegetable (MPV) processing plants in Brazil. The scenarios were as follows: (1) Washing one inoculated lettuce portion in nonchlorinated water, followed by washing 10 noninoculated portions sequentially. (2) Washing one inoculated lettuce portion in chlorinated water followed by washing five noninoculated portions sequentially. (3) Washing five inoculated lettuce portions in chlorinated water sequentially, followed by washing five noninoculated portions sequentially. (4) Washing five noninoculated lettuce portions in chlorinated water sequentially, followed by washing five inoculated portions sequentially and then by washing five noninoculated portions sequentially in the same water. Salm. Typhimurium transfer from inoculated lettuce to wash water and further dissemination to noninoculated lettuces occurred when nonchlorinated water was used (scenario 1). When chlorinated water was used (scenarios 2, 3 and 4), no measurable Salm. Typhimurium transfer occurred if the sanitizer was ≥10 mg l(-1) . Use of sanitizers in correct concentrations is important to minimize the risk of microbial transfer during MPV washing. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, the impact of sodium dichloroisocyanurate in the wash water on transfer of Salmonella Typhimurium from inoculated lettuce to wash water and then to other noninoculated lettuces washed sequentially in the same water was evaluated. The use of chlorinated water, at concentration above 10 mg l(-1) , effectively prevented Salm. Typhimurium transfer under several different washing scenarios. Conversely, when nonchlorinated water was used, Salm. Typhimurium transfer occurred in up to at least 10 noninoculated batches of lettuce washed sequentially in the same water.

In vitro fermentation of prebiotic carbohydrates by intestinal microbiota in the presence of Lactobacillus amylovorus DSM 16998.

The aim of this study was to evaluate the assimilation of the prebiotics fructooligosaccharides (FOS), galactooligosaccharides (GOS), and Konjac glucomannan oligosaccharides (KGMO) by three human (H1, H2 and H3) and pig (P1, P2 and P3) faecal microbiotas in the presence of the potentially probiotic strain Lactobacillus amylovorus DSM 16698, using an in vitro batch fermentation model. Total bacteria and L. amylovorus populations were quantified using qPCR and biochemical features (pH, production of short chain fatty acids (SCFA), lactate, ammonia, and carbohydrate assimilation) were determined. L. amylovorus did not have a competitive advantage under in vitro conditions, reflected by its reduced relative abundance during fermentation despite the carbohydrate sources added. Pig microbiota sustained more stable probiotic counts. Intermittently produced lactate was possibly assimilated by the microbiota and converted to other SCFA as the carbohydrates were assimilated, with H3 probably having a methanogenic metabolism with high lactate and acetate consumption except in the presence of FOS, which assimilation resulted in the highest total SCFA for this volunteer. Addition of FOS also resulted in lower pH and ammonia, which might have been used as nitrogen source by pig microbiota. KGMO needed longer fermentation periods to be completely assimilated by both human and porcine faecal microbiotas. Overall, our results reinforce the notion that care must be taken when generalising the effects claimed for a given probiotic or potentially probiotic strain, including the combination with different prebiotic substrates, since they may vary considerably among individuals, which is important when studying potentially pro- and prebiotic combinations for application as functional foods and feed ingredients.

Purification and characterization of the bacteriocin produced by Lactobacillus sakei MBSa1 isolated from Brazilian salami.

AIMS: The study aimed at determining the biochemical characteristics of the bacteriocin produced by Lactobacillus sakei MBSa1, isolated from salami, correlating the results with the genetic features of the producer strain. METHODS AND RESULTS: Identification of strain MBSa1 was performed by 16S rDNA sequencing. The bacteriocin was tested for spectrum of activity, heat and pH stability, mechanism of action, molecular mass and amino acid sequence when purified by cation-exchange and reversed-phase HPLC. Genomic DNA was tested for bacteriocin genes commonly present in Lact. sakei. Bacteriocin MBSa1 was heat-stable, unaffected by pH 2·0 to 6·0 and active against all tested Listeria monocytogenes strains. Maximal production of bacteriocin MBSa1 (1600 AU ml(-1)) in MRS broth occurred after 20 h at 25°C. The molecular mass of produced bacteriocin was 4303·3 Da, and the molecule contained the SIIGGMISGWAASGLAG sequence, also present in sakacin A. The strain contained the sakacin A and curvacin A genes but was negative for other tested sakacin genes (sakacins T-α, T-ß, X, P, G and Q). CONCLUSIONS: In the studied conditions, Lact. sakei MBSa1 produced sakacin A, a class II bacteriocin, with anti-Listeria activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The study covers the purification and characterization of the bacteriocin produced by a lactic acid bacteria isolated from salami (Lact. sakei MBSa1), linking genetic and expression information. Its heat-resistance, pH stability in acid conditions (pH 2·0-6·0) and activity against L. monocytogenes food isolates bring up a potential technological application to improve food safety.

In vitro study of beneficial properties and safety of lactic acid bacteria isolated from Portuguese fermented meat products.

Many lactic acid bacteria produce bacteriocins with a rather broad spectrum of inhibition, which could offer potential applications in food preservation. Bacteriocin production by starter cultures may bring advantage to these strains in competitive interactions with pathogenic bacteria from the food matrix. The objective of this study was to determine the safety of beneficial strains (Lactobacillus plantarum ST202Ch and ST216Ch, Enterococcus faecium ST211Ch, and Lactobacillus sakei ST22Ch, ST153Ch and ST154Ch) previously isolated from fermented meat products and characterised as bacteriocin producers. Auto-aggregation was strain-specific, and values of 28.97, 27.86 and 28.56% were recorded for L. sakei ST22Ch, ST153Ch and ST154Ch, respectively, 16.95 and 14.58% for L. plantarum ST202Ch and ST216Ch, respectively, and 12.77% for E. faecium ST211Ch. Various degrees of co-aggregation between 28.85 and 44.76% for Listeria monocytogenes 211 and 409, and between 23.60 to 34.96% for E. faecium ATCC 19443 were observed. According to the results of the diffusion method, the studied strains demonstrated susceptibility to penicillin G, ampicillin, amoxicillin, amoxicillin/clavulonic acid, imipenem, linezolid, and tetracycline. In addition, the susceptibility of the six strains to various non-antibiotic commercial drugs was examined. Production of ß-galactosidase by L. sakei ST22Ch, ST153Ch and ST154Ch, L. plantarum ST202Ch and ST216Ch, and E. faecium ST211Ch was confirmed by employing sterile filter paper discs impregnated with o-nitrophenyl-ß-D-galactopyranose. A statistically significant (P<0.001) inhibition of Mycobacterium tuberculosis growth by bacteriocins produced by L. plantarum ST202Ch (38.3%) and ST216Ch (48.6%), L. sakei ST153Ch (16.2%) and ST154Ch (16.1%), and E. faecium ST211Ch (21.7%) was observed. As determined by the polymerase chain reaction, the tested strains showed a low virulence gene profile.

Technological properties of bacteriocin-producing lactic acid bacteria isolated from Pico cheese an artisanal cow's milk cheese.

AIM: Evaluate technologically relevant properties from bacteriocin-producing strains to use as starter/adjunct cultures in cheese making. METHODS AND RESULTS: Eight isolates obtained from Pico cheese produced in Azores (Portugal) were found to produce bacteriocins against Listeria monocytogenes and three isolates against Clostridium perfringens. They were identified as Lactococcus lactis and Enterococcus faecalis and submitted to technological tests: growth at different conditions of temperature and salt, acid production, proteolysis, lipolysis, coexistence, enzymatic profile and autolytic capacity. Safety evaluation was performed by evaluating haemolytic, gelatinase and DNase activity, resistance to antibiotics and the presence of virulence genes. Some isolates presented good technological features such as high autolytic activity, acid and diacetyl production. Lactococcus lactis was negative for all virulence genes tested and inhibit the growth of all Lactic acid bacteria (LAB) isolates. Enterococci were positive for the presence of some virulence genes, but none of the isolates were classified as resistant to important antibiotics. CONCLUSIONS: The bacteriocin-producing Lc. lactis present good potential for application in food as adjunct culture in cheese production. The study also reveals good technological features for some Enterococcus isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriocin-producing strains presented important technological properties to be exploited as new adjunct culture for the dairy industry, influencing flavour development and improve safety.

Isolation and identification of bacteriocinogenic strain of Lactobacillus plantarum with potential beneficial properties from donkey milk.

AIMS: The goal of this study was to isolate and characterize a lactic acid bacteria (LAB) from donkey milk with potential beneficial properties. METHODS AND RESULTS: Lactic acid bacteria were isolated from donkey milk and identified based on physiological, biochemical and molecular methods. The isolate that presented highest bacteriocin potential (Lactobacillus plantarum LP08AD) was evaluated for the production of bacteriocin, including stability in the presence of various enzymes, surfactants, salts, pH and temperatures. Bactericidal effect of bacteriocin LP08AD on Listeria monocytogenes, Enterococcus faecium and Lactobacillus curvatus was shown for actively growing and stationary cells. Similar growth and bacteriocin production were observed when strain LP08AD was cultured in MRS broth at 30°C or 37°C. Bacteriocin LP08AD adhered at low levels on the producer cells (200 AU ml(-1) ). The presence of plantaricin W gene on the genomic DNA was recorded based on PCR. Good growth for strain LP08AD was recorded in MRS broth with pH from 5·0 to 9·0 and LP08AD grew well in the absence of oxbile or concentration below 0·8%. Lact. plantarum LP08AD was applied to the small intestinal epithelial polarized monolayers of H4, PSIc1 and CLAB and demonstrated low attachment ability on all cell lines studied, with values with a similar behaviour for cells from human and pig origin. CONCLUSIONS: Bacteriocin-producing Lact. plantarum LP08AD might be useful in the design of novel functional foods with potential probiotic or biopreservation properties. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first report on detection and characterization of bacteriocinogenic Lact. plantarum from donkey milk. The strain LP08AD shows to have potential beneficial properties, as demonstrated by the use of noncancerogenic cell lines.

Isolation and characterization of a nisin-like bacteriocin produced by a Lactococcus lactis strain isolated from charqui, a Brazilian fermented, salted and dried meat product.

A Lactococcus lactis subsp. lactis strain (L. lactis 69) capable to produce a heat-stable bacteriocin was isolated from charqui, a Brazilian fermented, salted and sun-dried meat product. The bacteriocin inhibited, in vitro, Listeria monocytogenes, Staphylococcus aureus, several lactic acid bacteria isolated from foods and spoilage halotolerant bacteria isolated from charqui. The activity of the bacteriocin was not affected by pH (2.0-10.0), heating (100 °C), and chemical agents (1% w/v). Treatment of growing cells of L. monocytogenes ScottA with the cell-free supernatant of L. lactis 69 resulted in complete cell inactivation. L. lactis 69 harbored the gene for the production of a nisin-like bacteriocin, and the amino acid sequence of the active peptide was identical to sequences previously described for nisin Z. However, differences were observed regarding the leader peptide. Besides, the isolate was able to survive and produce bacteriocins in culture medium with NaCl content up to 20%, evidencing a potential application as an additional hurdle in the preservation of charqui.

Bacteriocinogenic and virulence potential of Enterococcus isolates obtained from raw milk and cheese.

AIMS: To provide molecular and phenotypical characterization of Enterococcus isolates obtained from raw milk and cheese, regarding their bacteriocinogenic and virulence activity. METHODS AND RESULTS: Forty-three bacteriocinogenic enterococci isolates were identified by 16s rDNA, fingerprinted by RAPD-PCR analysis and tested by PCR for the presence of genes for lantibiotics (lanM, lanB and lanC) and enterocins (entA, entB, entP, entL50AB and entAS48) and by phenotypical methods for bacteriocin production and inhibitory spectrum. Also, the virulence of the isolates was evaluated by PCR for genes gelE, hyl, asa1, esp, cylA, efaA, ace, vanA, vanB, hdc1, hdc2, tdc and odc and by phenotypical tests for gelatinase, lipase, DNAse and α- and ß-haemolysis. Most isolates (93·0%) harboured at least one lantibiotic or enterocin gene and were positive for several tested virulence genes, mainly asa1 (100%), gelE (93·0%) and efaA (83.7%). 53.5% of the isolates presented ß-haemolysis [corrected]. CONCLUSIONS: Enterococcus spp. isolates presented an interesting potential application for food preservation because of bacteriocin production; however, virulence-related genes were identified in all RAPD profiles. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrated the contradictory characteristics of the tested Enterococcus isolates: they presented a good potential for application in food biopreservation but contained several virulence factors.

Potential beneficial properties of bacteriocin-producing lactic acid bacteria isolated from smoked salmon.

AIMS: To evaluate the probiotic properties of strains isolated from smoked salmon and previously identified as bacteriocin producers. METHODS AND RESULTS: Strains Lactobacillus curvatus ET06, ET30 and ET31, Lactobacillus fermentum ET35, Lactobacillus delbrueckii ET32, Pediococcus acidilactici ET34 and Enterococcus faecium ET05, ET12 and ET88 survived conditions simulating the gastrointestinal tract (GIT) and produced bacteriocins active against several strains of Listeria monocytogenes, but presented very low activity against other lactic acid bacteria (LAB). Cell-free supernatants containing bacteriocins, added to 3-h-old cultures of L. monocytogenes 603, suppressed growth over 12 h. Auto-aggregation was strain-specific, and values ranged from 7·2% for ET35 to 12·1% for ET05. Various degrees of co-aggregation with L. monocytogenes 603, Lactobacillus sakei ATCC 15521 and Enterococcus faecalis ATCC 19443 were observed. Adherence of the bacteriocinogenic strains to Caco-2 cells was within the range reported for Lactobacillus rhamnosus GG, a well-known probiotic. The highest levels of hydrophobicity were recorded for Lact. curvatus (61·9­64·6%), Lact. fermentum (78·9%), Lact. delbrueckii (43·7%) and Ped. acidilactici (51·3%), which are higher than the one recorded for Lact. rhamnosus GG (53·3%). These strains were highly sensitive to several antibiotics and affected by several drugs from different generic groups in a strain-dependent manner. CONCLUSIONS: Smoked salmon is a rich source of probiotic LAB. All strains survived conditions simulating the GIT and produced bacteriocins active against various pathogens. Adherence to Caco-2 cells was within the range reported for Lact. rhamnosus GG, a well-known probiotic. In addition, the high hydrophobicity readings recorded define the strains as good probiotics.

Epidemiological Survey of Listeria monocytogenes in a gravlax salmon processing line / Epidemiologia de Listeria monocytogenes em uma linha de processamento de salmão gravlax

Listeria monocytogenes is a cause of concern to food industries, mainly for those producing ready-to-eat (RTE) products. This microorganism can survive processing steps such as curing and cold smoking and is capable of growing under refrigeration temperatures. Its presence in RTE fish products with extended shelf life may be a risk to the susceptible population. One example of such a product is gravlax salmon; a refrigerated fish product not exposed to listericidal processes and was the subject of this study. In order to evaluate the incidence and dissemination of L. monocytogenes 415 samples were collected at different steps of a gravlax salmon processing line in São Paulo state, Brazil. L. monocytogenes was confirmed in salmon samples (41 percent), food contact surfaces (32 percent), non-food contact surfaces (43 percent) and of food handlers' samples (34 percent), but could not be detected in any ingredient. 179 L. monocytogenes isolates randomly selected were serogrouped and typed by PFGE. Most of L. monocytogenes strains belonged to serogroup 1 (73 percent). 61 combined pulsotypes were found and a dendrogram identified six clusters: most of the strains (120) belonged to cluster A. It was suggested that strains arriving into the plant via raw material could establish themselves in the processing environment contaminating the final product. The wide dissemination of L. monocytogenes in this plant indicates that a great effort has to be taken to eliminate the microorganism from these premises, even though it was not observed multiplication of the microorganism in the final product stored at 4ºC up to 90 days.

Listeria monocytogenes é um patógenode grande preocupação para as indústrias alimentícias, principalmente aquelas produtoras de alimentos prontos para consumo (RTE). Este microrganismo pode sobreviver às etapas de cura e defumação a frio, além de tolerar temperaturas de refrigeração. A presença de L. monocytogenes em pescados RTE com vida de prateleira longa representa um risco para a população susceptível, sendo o salmão gravlax deste tipo de produto. No presente estudo avaliou-se a incidência e disseminação de L. monocytogenes em 415 amostras de salmão gravlax obtidas de diferentes etapas de processamento de uma indústria localizada no Estado de São Paulo. A presença de L. monocytogenes foi confirmada em amostras de salmão (41 por cento), superfícies de contato (32 por cento) e não contato (43 por cento) e manipuladores (34 por cento), porém não se isolou o microrganismo em nenhum ingrediente. Do total de cepas isoladas, 179 destas foram escolhidas aleatoriamente e submetidas a sorologia e tipagem por PFGE. A maioria dos isolados pertenceu ao sorogrupo 1 (73 por cento), sendo identificados 61 pulsotipos quando se combinou os resultados de sorologia e PFGE e 6 clusters foram distribuídos em um dendrograma. O cluster A agrupou a maioria das cepas (120). Pode-se sugerir que as cepas foram introduzidas na linha de processamento por meio da matéria prima e contaminando o produto final. Estes resultados indicam que a eliminação de L. monocytogenes deste estabelecimento requer um grande esforço, ainda que o microrganismo não se multiplicou no produto final estocado a 4ºC por 90 dias.

Listeria monocytogenes and Salmonella spp. in raw milk produced in Brazil: occurrence and interference of indigenous microbiota in their isolation and development.

This study aimed to verify the occurrence of Listeria monocytogenes and Salmonella spp. in raw milk produced in Brazil. On account of the poor microbiological quality of this product, possible interference from the indigenous microbiota in these pathogens was also evaluated. Two-hundred and ten raw milk samples were collected in four important milk-producing areas in Brazil, tested for L. monocytogenes and Salmonella spp. presence, and for enumeration of indicator microorganisms: mesophilic aerobes, total coliforms and Escherichia coli. The interference of the indigenous microbiota in the isolation procedures was also tested, as well the frequency of naturally occurring raw milk strains with antagonistic activity against both pathogens. The pathogens were not isolated in any raw milk sample, but poor microbiological quality was confirmed by the high levels of indicator microorganisms. When present at high levels, the indigenous microbiota generated an evident interference in the methodologies of L. monocytogenes and Salmonella spp. isolation, mainly when the pathogens appeared at low levels. Three-hundred and sixty raw milk strains were tested for antagonistic activity against both pathogens, and 91 (25.3%) showed inhibitory activity against L. monocytogenes and 33 (9.2%) against Salmonella spp. The majority of the antagonistic strains were identified as Lactic Acid Bacteria species, mainly Lactococcus lactis subsp. lactis and Enterococcus faecium, known by antimicrobial substance production.

Epidemiological Survey of Listeria monocytogenes in a gravlax salmon processing line.

Listeria monocytogenes is a cause of concern to food industries, mainly for those producing ready-to-eat (RTE) products. This microorganism can survive processing steps such as curing and cold smoking and is capable of growing under refrigeration temperatures. Its presence in RTE fish products with extended shelf life may be a risk to the susceptible population. One example of such a product is gravlax salmon; a refrigerated fish product not exposed to listericidal processes and was the subject of this study. In order to evaluate the incidence and dissemination of L. monocytogenes 415 samples were collected at different steps of a gravlax salmon processing line in São Paulo state, Brazil. L. monocytogenes was confirmed in salmon samples (41%), food contact surfaces (32%), non-food contact surfaces (43%) and of food handlers' samples (34%), but could not be detected in any ingredient. 179 L. monocytogenes isolates randomly selected were serogrouped and typed by PFGE. Most of L. monocytogenes strains belonged to serogroup 1 (73%). 61 combined pulsotypes were found and a dendrogram identified six clusters: most of the strains (120) belonged to cluster A. It was suggested that strains arriving into the plant via raw material could establish themselves in the processing environment contaminating the final product. The wide dissemination of L. monocytogenes in this plant indicates that a great effort has to be taken to eliminate the microorganism from these premises, even though it was not observed multiplication of the microorganism in the final product stored at 4°C up to 90 days.

Protective effect of Lactobacillus sakei 2a against experimental challenge with Listeria monocytogenes in gnotobiotic mice.

AIM: Lactobacillus sakei 2a isolated from sausage and presenting an in vitro antagonistic activity against Listeria monocytogenes Scott A was tested for a protective effect in mice experimentally challenged with the enterobacteria. METHODS AND RESULTS: In the experimental group, germ-free mice (n = 24) were inoculated intragastrically with 0.1 ml of a suspension containing 10(8) colony forming units (CFU) of Lact. sakei and 4 days later the animals were challenged intragastrically with 0.1 ml of a suspension containing 10(8) CFU of L. monocytogenes. Control group (n = 24) was only inoculated with the bacterial pathogen. Faecal counts showed that L. monocytogenes reached similar population levels (10(9) CFU g(-1) of contents) in both the groups. Animals in the control group showed lower (P = 0.0004) survival frequency (58.3%) when compared with the experimental one (100%). Anatomopathological examination confirmed the mortality data. CONCLUSIONS: Lactobacillus sakei 2a can survive in the mammal digestive tract where showed a protective effect against L. monocytogenes. This phenomenon was not due to an antagonistic activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Use of Lact. sakei 2a as a meat starter could inhibit not only L. monocytogenes growth in the fermented product but also pathogen virulence in the gastrointestinal tract.

Quantificação de Listeria monocytogenes em salames fatiados embalados a vácuo / Occurrence of Listeria monocytogenes in pre-sliced vacuum-packaged

There is scarce information in Brazil and other South American countries about the occurrence of Listeria monocytogenes in food, mainly refrigerated ready-to-eat products. The consumption of sliced vacuum-packaged meat products has increased in the last few years. Nevertheless, a complete assessment of the risk associated with L. monocytogenes in these products is still necessary. Because of the production and storage characteristics of these products, they can be considered potential vehicles for L. monocytogenes to humans, mainly immunocompromised, elderly, and pregnant women. The objectives of this study was to evaluate the population of L. monocytogenes in salami, a ready-to-eat meat product with extended shelf life, acquired in retail stores in São Paulo-Brazil. The three-tube most probable number technique was used and the methodology was that from Health Canada. Strains were biochemically identified and serotyped. Among the 45 samples, 3 (6.7 per cent) harboured 9.2 MPN/g of L. monocytogenes and the others < 0.3 MPN/g. All the strains belonged to serotypes 1/2a and 1/2b, the most frequent serotypes found in food everywhere. Even being low, the population of L. monocytogenes found in this product could be a cause of concern to public health authorities as it can pose a threat to population at risk. This contamination highlights the importance of implementing systems like HACCP to assure safe products to consumers.