Investigating the influence of Food Safety Management Systems (FSMS) on microbial diversity of Canastra cheeses and their processing environments.

Canastra Cheese is one of the most commercialized artisanal cheeses in Brazil and intrinsic characteristics of its production, such as the use of raw milk and natural whey starter cultures as well short ripening time on wooden shelves, offer risk of contamination by a plethora of microorganisms. Here, we used 16 S rRNA gene amplicon sequencing approach to characterize the bacterial communities from Canastra cheese processing environments and final products, accessing cheesemaking facilities with distinct profiles of Food Safety Management Systems (FSMS), in order to estimate whether differences in microbial composition and diversity could also be observed between the two sampled groups of facilities. Our results revealed that the diversity of bacterial communities in the processing environments was much higher than that observed for cheeses, with greater discrepancy for facilities with inadequate FSMS. Additionally, in facilities with inadequate FSMS the bacterial communities from environments, especially hand surfaces and ripening wooden shelves, were similar to those during processing and finished cheese. These evidences highlight the importance of implementing and maintaining FSMS in the facilities, in order to assure quality and safety of Canastra cheese, but also the stability and economic viability of the Canastra cheese production chain.

Assessment of Consumer Exposure to Salmonella spp., Campylobacter spp., and Shiga Toxin-Producing Escherichia coli in Meat Products at Retail in the City of Sao Paulo, Brazil.

Meat products may be vehicles of bacterial pathogens to humans, and Salmonella spp., Campylobacter spp., and Shiga toxin-producing Escherichia coli (STEC) are the most relevant. The aim of this study was to generate data on prevalence of these three pathogens in 552 samples of meat products (hot dogs, pork sausages, raw ground beef, and raw chicken legs) sold at retail in the city of Sao Paulo, Brazil. Salmonella spp. was detected in 5.8% (32/552) of samples, comprising pork sausages 62.5% (20/32) and chicken legs 37.5% (12/32). The counts of Salmonella spp. were low, ranging from < 0.3 to 9.3 × 10 most probable number per gram and the most frequent serovars were Salmonella Typhimurium (28.1%), Salmonella I 4,[5],12:i:- (15.6%), Salmonella Enteritidis (12.5%), Salmonella Derby, and Salmonella Brandenburg (9.4%). Campylobacter spp. was detected in 33 samples (6.0%), comprising chicken legs (82%) and ground beef (18%). All samples were negative for STEC. These results suggest that meat products when subjected to inadequate cooking and/or cross-contamination with other products ready for consumption can lead to occurrence of outbreaks, highlighting the risks associated with them.

Improving safety of salami by application of bacteriocins produced by an autochthonous Lactobacillus curvatus isolate.

The aims of this study were to isolate LAB with anti-Listeria activity from salami samples, characterize the bacteriocin/s produced by selected isolates, semi-purify them and evaluate their effectiveness for the control of Listeria monocytogenes during manufacturing of salami in a pilot scale. Two isolates (differentiated by RAPD-PCR) presented activity against 22 out of 23 L. monocytogenes strains for bacteriocin MBSa2, while the bacteriocin MBSa3 inhibited all 23 strains in addition to several other Gram-positive bacteria for both antimicrobials and were identified as Lactobacillus curvatus based on 16S rRNA sequencing. A three-step purification procedure indicated that both strains produced the same two active peptides (4457.9 Da and 4360.1 Da), homlogous to sakacins P and X, respectively. Addition of the semi-purified bacteriocins produced by Lb. curvatus MBSa2 to the batter for production of salami, experimentally contaminated with L. monocytogenes (10(4)-10(5) CFU/g), caused 2 log and 1.5 log reductions in the counts of the pathogen in the product after 10 and 20 days respectively, highlighting the interest for application of these bacteriocins to improve safety of salami during its manufacture.

High Level of Interaction between Phages and Bacteria in an Artisanal Raw Milk Cheese Microbial Community.

Microbial starter cultures are used in the production of many cheeses around the world, such as Parmigiano-Reggiano, in Italy, Époisses, in France, and Canastra, in Brazil, providing many of the unique features of these cheeses. Bacteriophages (phages) are ubiquitous and well known to modulate the structure of bacterial communities, and recent data indicate that cheeses contain a high abundance of naturally occurring phages. Here, we analyze the viral and bacterial metagenomes of Canastra cheese: a traditional artisanal Brazilian cheese produced using an endogenous starter culture and raw milk. Over 1,200 viral operational taxonomic units were recovered using both isolated viral-like particles and complete metagenomic DNA. Common viral families identified included Siphoviridae and Myoviridae, with 40% of putative phage genomes unidentified at the family level of classification. We observed very high phage diversity, which varied greatly across different cheese producers, with 28% of phage genomes detected in only one producer. Several metagenome-assembled genomes were recovered for lactic acid-producing bacteria, as well as nonstarter bacterial species, and we identified several phage-bacterium interactions, at the strain level of resolution, varying across distinct cheese producers. We postulate that at least one bacterial strain detected could be endogenous and unique to the Canastra cheese-producing region in Brazil and that its growth seems to be modulated by autochthonous phages present in this artisanal production system. This phage-host relationship is likely to influence the fermentation dynamics and ultimately the sensorial profile of these cheeses, with implications for other similar cheese production systems around the world. IMPORTANCE Our work demonstrated a dynamic yet stable microbial ecosystem during cheese production using an endogenous starter culture. This was observed across several distinct producers and was marked by genomic evidence of continued phage-bacterium interactions, such as the presence of bacterial defense mechanisms. Furthermore, we provide evidence of unique microbial signatures for each individual cheese producer studied in the region, a fact that may have profound consequences on product traceability. This was the first effort to describe and understand the bacteriophage composition and ecological dynamics within the Brazilian Canastra cheese production system. The study of this prototypical backslopping production system provides a solid background for further mechanistic studies of the production of many cheeses around the world.

Genomic and functional characterization of bacteriocinogenic lactic acid bacteria isolated from Boza, a traditional cereal-based beverage.

Boza is a traditional low-alcohol fermented beverage from the Balkan Peninsula, frequently explored as a functional food product. The product is rich in Lactic Acid Bacteria (LAB) and some of them can produce bacteriocins. In this study, a sample of Boza from Belogratchik, Bulgaria, was analyzed for the presence of bacteriocinogenic LAB, and after analyses by RAPD-PCR, three representative isolates were characterized by genomic analyses, using whole genome sequencing. Isolates identified as Pediococcus pentosaceus ST75BZ and Pediococcus pentosaceus ST87BZ contained operons encoding for bacteriocins pediocin PA-1 and penocin A, while isolate identified as Pediococcus acidilactici ST31BZ contained only the operon for pediocin PA-1 and a CRISPR/Cas system for protection against bacteriophage infection. The antimicrobial activity of bacteriocins produced by the three isolates was inhibited by treatment of the cell-free supernatants with proteolytic enzymes. The produced bacteriocins inhibited the growth of Listeria monocytogenes, Enterococcus spp. and some Lactobacillus spp., among other tested species. The levels of bacteriocin production varied from 3200 to 12,800 AU/ml recorded against L. monocytogenes 104, 637 and 711, measured at 24 h of incubation at 37 °C. All bacteriocins remained active after incubation at pH 2.0-10.0. The activity mode of the studied bacteriocins was bactericidal, as determined against L. monocytogenes 104, 637 and 711. In addition, bactericidal activity was demonstrated using a cell leakage ß-galactosidase assay, indicating a pore formation mechanism as a mode of action. The present study highlights the importance of combining genomic analyses and traditional microbiological approaches as way of characterizing microbial interactions in fermented foods.

Fermentation of Gluten by Lactococcus lactis LLGKC18 Reduces its Antigenicity and Allergenicity.

Wheat is a worldwide staple food, yet some people suffer from strong immunological reactions after ingesting wheat-based products. Lactic acid bacteria (LAB) constitute a promising approach to reduce wheat allergenicity because of their proteolytic system. In this study, 172 LAB strains were screened for their proteolytic activity on gluten proteins and α-amylase inhibitors (ATIs) by SDS-PAGE and RP-HPLC. Gliadins, glutenins, and ATI antigenicity and allergenicity were assessed by Western blot/Dot blot and by degranulation assay using RBL-SX38 cells. The screening resulted in selecting 9 high gluten proteolytic strains belonging to two species: Enterococcus faecalis and Lactococcus lactis. Proteomic analysis showed that one of selected strains, Lc. lactis LLGKC18, caused degradation of the main gluten allergenic proteins. A significant decrease of the gliadins, glutenins, and ATI antigenicity was observed after fermentation of gluten by Lc. lactis LLGKC18, regardless the antibody used in the tests. Also, the allergenicity as measured by the RBL-SX38 cell degranulation test was significantly reduced. These results indicate that Lc. lactis LLGKC18 gluten fermentation can be deeply explored for its capability to hydrolyze the epitopes responsible for wheat allergy.

Pediocin PA-1 production by Pediococcus pentosaceus ET34 using non-detoxified hemicellulose hydrolysate obtained from hydrothermal pretreatment of sugarcane bagasse.

Listeria monocytogenes is one of the foodborne pathogens of most concern for food safety. To limit its presence in foods, bacteriocins have been proposed as natural bio-preservatives. Herein, a bacteriocin was produced on hemicellulose hydrolysate of sugarcane bagasse by Pediococcus pentosaceous ET34, whose genome sequencing revealed an operon with 100% similarity to that of pediocin PA-1. ET34 grown on hydrolysate-containing medium led to an increase in the expression of PA-1 genes and a non-optimized purification step sequence resulted in a yield of 0.8 mg·L-1 of pure pediocin (purity > 95%). Culture conditions were optimized according to a central composite design using temperature and hydrolysate % as independent variables and validated in 3-L Erlenmeyers. Finally, a process for scaled-up implementation by sugar-ethanol industry was proposed, considering green chemistry and biorefinery concepts. This work stands up as an approach addressing a future proper sugarcane bagasse valorisation for pediocin production.

Exploring Beneficial/Virulence Properties of Two Dairy-Related Strains of Streptococcus infantarius subsp. infantarius.

The genus Streptococcus includes various species, remarkably different in their behavior, applications, virulence, and safety. Taxonomically Streptococcus infantarius subsp. infantarius belonging to the Streptococcus bovis group, which includes several pathogen species, however, has been found as predominant species in some African dairy products that are widely consumed and considered to be safe. Streptococcus infantarius subsp. infantarius' safety may be questioned due to the association of this species with clinical cases. In this study, isolates from dairy origin were selected based on their bacteriocinogenic potential and differentiated by their RAPD-PCR profiles. Two strains were identified by 16S rRNA sequencing as St. infantarius subsp. infantarius and investigated regarding their potential beneficial properties and factors related to virulence and safety. A series of in vitro tests included properties related to survival in the gastrointestinal tract and beneficial intestinal activities. Production of bacteriocin/s, detection of related genes, and partial characterization of expressed antimicrobial protein were evaluated. Genes related to folate biosynthesis were detected in both studied strains. Evaluation of physiological tests related to strains virulence, adhesion, and resistance to antibiotics and detections of virulence and biogenic amines production-related genes were also investigated. Taking in consideration all the aspects of the specific nature of St. infantarius subsp. infantarius K1-4 and K5-1 (beneficial properties and virulence characteristics), both strains cannot be considered safe for human or other animals application, even though they have been isolated from dairy products. This study is highlighting the importance of evaluation for presence of potential virulence factors in newly characterized strains in order to be confident in their safety.

Nisin Production by Enterococcus hirae DF105Mi Isolated from Brazilian Goat Milk.

The purpose of this study was to select the promising biopreservation bacteriocin producer strain from goat milk and characterize the expressed bacteriocin, related to its physiological and biochemical properties and specificity of operon encoding production and expression of antimicrobial peptide. Brazilian goat milk was used as the source for the selection of bacteriocin-producing lactic acid bacteria. One strain (DF105Mi) stood out for its strong activity against several Listeria monocytogenes strains. Selected strain was identified based on the biochemical and physiological characteristics and 16s rRNA analysis. The bacteriocin production and inhibitory spectrum of strain DF105Mi were studied, together with the evaluation of the effect of temperature, pH, and chemicals on bacteriocin stability and production, activity, and adsorption to target cells as well as to the cell surface of bacteriocin producers. Physiological and bio-molecular analyses based on targeting of different genes, parts of nisin operon were performed in order to investigate the hypothesis that the studied strain can produce and express nisin. Based on biochemical, physiological, and 16s rRNA analysis, the strain DF105Mi was classified as Enterococcus hirae. The selected strain produces a bacteriocin which is stable in a wide range of pH (2.0-12.0), temperature (up to 120 °C), presence of selected chemicals and presents adsorption affinity to different test organisms, process influenced by environmental conditions. Higher bacteriocin production by Ent. hirae DF105Mi was recorded during stationary growth phase, but only when the strain was cultured at 37 °C. The strain's genetic analysis indicated presence of the genes coding for the production of the bacteriocin nisin. This result was confirmed by cross-checking the sensitivity of the produced strain to commercial nisin A. The strong anti-Listeria activity, bacteriocin adsorption, and stability of produced bacteriocin indicate that Ent. hirae DF105Mi presents a differentiated potential application for biopreservation of fermented dairy products.

Behaviour of L. monocytogenes in sliced, vacuum-packed mortadella.

This study evaluated the growth of naturally occurring L. monocytogenes in sliced, vacuum-packed mortadella samples during storage at 5°C until the expiration date. Tukey's test indicated that counts of L. monocytogenes on 0, 10, 20, 30 and 40 days of storage were significantly different (p<0.05), indicating growth during shelf life. In three trials, the mean increase was 1.72 log cycles. Vacuum packing and storage under refrigeration were not effective in controlling the growth of L. monocytogenes in sliced mortadella, indicating that good manufacturing practices and implemented HACCP programs are essential to assure safety of this product.

Occurrence and phenotypic and molecular characterization of Listeriamonocytogenes and Salmonella spp. in slaughterhouses in southern Brazil.

This study addressed the occurrence of Listeriamonocytogenes and Salmonella spp. in bovine carcasses at two slaughterhouses in southern Brazil. Then, the antimicrobial susceptibility profile and the virulence potential of the isolates were evaluated. Two hundred carcasses were sampled at four steps of the slaughter process, with L. monocytogenes being isolated in 12 and Salmonella spp. in 17 carcasses. All L. monocytogenes isolates carried the hlyA, prfA, plcA, plcB, actA, iap, mpl, inlA, inlB, inlC, and inlJ genes, while Salmonella spp. carried invA and hilA. Among the L. monocytogenes isolates, all of them presented virulence determinants and one showed multi-drug resistance. In relationship to Salmonella spp. isolates, many serogroups frequently related to outbreaks of foodborne diseases were identified and four isolates showed resistance to more than one antimicrobial agent. This data highlights the importance of a rigid hygienic-sanitary control during the slaughter process to reduce the risk of cross-contamination and lower the consumer exposure to L. monocytogenes and Salmonella spp. infections.