Pseudomonas carnis isolated from blue discolored fresh cheese and insights into the phylogeny.

Most Pseudomonas spp. are responsible for spoilage in refrigerated foods such as alteration in flavor, texture and appearance. Samples of Minas Frescal cheese with blue discoloration were analysed and contained a high Pseudomonas concentration (7.72 ± 0.36 log CFU/g). Out of the 26 Pseudomonas isolates that were analyzed in our study, 19 demonstrated the capability of producing a diffusible dark pigment. Thus, a pigment-producing isolate (C020) was selected by rep-PCR fingerprinting and subsequently subjected to whole-genome sequencing. The draft genome assembled comprises 42 contigs totaling 6,366,75 bp with an average G + C content of 59.97%, and the species prediction performed by TYGS server, based on the draft genome sequence, identified the C020 as Pseudomonas carnis. In order to investigate the phylogenetic relationships of this isolate with strains already identified of this species, we performed an analysis based on whole-genomic sequences. First, an analysis of all P. carnis genomes deposited in GenBank to date shows that 11% (4/37) are misidentified, and belong to the Pseudomonas paracarnis species. A comparative analysis based on phylogenomic analysis has showed that there is no evolutionary relationship between P. carnis strains carrying second copies of trp genes related to blue discoloration (trpABCDF). This finding reinforces the assertion that these genes are contained in a mobile genetic element. However, it is worth noting that all strains carrying these secondary gene copies have exclusively been isolated from food sources. This observation provides valuable insights into the potential origins and dispersion dynamics of this genetic trait within the species.

Phylogenetic characterization and biodiversity of spore-forming bacteria isolated from Brazilian UHT milk.

Among the milk contaminating microorganisms, those which are able to form heat-resistant spores are concerning, especially for dairy companies that use ultra-high temperature (UHT) technology. These spores, throughout storage, can germinate and produce hydrolytic enzymes that compromise the quality of the final product. This study evaluated 184 UHT milk samples from different batches collected from seven Brazilian dairy companies with a possible microbial contamination problem. The bacteria were isolated, phenotypically characterized, clustered by REP-PCR, and identified through 16S rDNA sequencing. The presence of Bacillus sporothermodurans was verified using biochemical tests (Gram staining, catalase and oxidase test, glucose fermentation, esculin hydrolysis, nitrate reduction, and urease test). According to these tests, none of the isolates presented typical characteristics of B. sporothermodurans. In sequence, the isolates, that presented rod-shapes, were submitted to molecular analyses in order to determine the microbial biodiversity existing among them. The isolates obtained were grouped into 16 clusters, four of which were composed of only one individual. A phylogenetic tree was constructed using the sequences obtained from the 16S rDNA sequencing and some reference strains of species close to those found using BLAST search in the NCBI nucleotide database. Through this tree, it was possible to verify the division of the isolates into two large groups, the Bacillus subtilis and the Bacillus cereus groups. Furthermore, most isolates are phylogenetically closely related, which makes it even more difficult to identify them at the species level. In conclusion, it was possible to assess, in general, the groups of sporulated contaminants in Brazilian UHT milk produced in the regions evaluated. In addition, it was also possible to determine the biodiversity of spore-forming bacteria found in UHT milk samples, thus opening up a range of possible research topics regarding the effects of the presence of these microorganisms on milk quality.

Ozonation for Pseudomonas paracarnis control: biofilm removal and preservation of chicken meat during refrigerated storage.

Ozone has been studied to control microorganisms in food, as well as to control biofilm. In this context, the goals of this work were to determine the effect of ozonated water in the removal of Pseudomonas paracarnis biofilm and the effect of ozone gas and ozonated water on inactivating P. paracarnis in deboned chicken breast meat and its effect on product color. AISI 304 coupons were used as a surface for biofilm formation. The coupons were immerged into minimal medium for Pseudomonas inoculated with the P. paracarnis overnight culture (1% w/v) followed by incubation at 25 °C for 7 days. To obtain ozonized water, two different systems were used: system with microbubble generator (MB) and system with porous stone diffuser (PSD). The inlet ozone concentration was 19 mg/L and flow rate of 1 L/min. The coupons were subjected to ozonized water for 10 and 20 min. The chicken breast meat was exposed to gaseous ozone and ozonized water for 40 min. After the ozonation process, chicken meat samples were stored at 8 °C, for 5 days. More expressive removals of biofilm were obtained when using ozonized water obtained in the system with microbubble generator (MB for 20 min-reduction of 2.3 log cycles) and system with porous stone diffuser (PSD for 10 min-reduction of 2.7 log cycles; PSD for 20 min-reduction of 2.6 log cycles). The treatment of chicken meat with ozone gas resulted in lower counting of Pseudomonas, when compared with the control treatments and with ozonized water, both immediately after ozonation (day 1) and after 5 days of storage. The luminosity in the chicken meat samples treated with ozonized water was higher than that verified in the control treatments and with ozone gas, immediately after ozonation (day 1). A similar trend was observed in hue angle and color difference, in which the highest values were obtained for treatment with ozonized water. Based on the results obtained in this study, it was concluded that ozonated water can be used to remove P. paracarnis biofilm from stainless steel under static conditions and gaseous ozone is more efficient in the inactivation of P. paracarnis from chicken breast meat, when compared to ozonated water.

Diversity of Filamentous Fungi Associated with Dairy Processing Environments and Spoiled Products in Brazil.

Few studies have investigated the diversity of spoilage fungi from the dairy production chain in Brazil, despite their importance as spoilage microorganisms. In the present study, 109 filamentous fungi were isolated from various spoiled dairy products and dairy production environments. The isolates were identified through sequencing of the internal transcribed spacer (ITS) region. In spoiled products, Penicillium and Cladosporium were the most frequent genera of filamentous fungi and were also present in the dairy environment, indicating that they may represent a primary source of contamination. For dairy production environments, the most frequent genera were Cladosporium, Penicillium, Aspergillus, and Nigrospora. Four species (Hypoxylon griseobrunneum, Rhinocladiella similis, Coniochaeta rosae, and Paecilomyces maximus) were identified for the first time in dairy products or in dairy production environment. Phytopathogenic genera were also detected, such as Montagnula, Clonostachys, and Riopa. One species isolated from the dairy production environment is classified as the pathogenic fungi, R. similis. Regarding the phylogeny, 14 different families were observed and most of the fungi belong to the Ascomycota phylum. The understanding of fungal biodiversity in dairy products and environment can support the development of conservation strategies to control food spoilage. This includes the suitable use of preservatives in dairy products, as well as the application of specific cleaning and sanitizing protocols designed for a specific group of target microorganisms.

Identification and characterization of a heat-resistant protease from Serratia liquefaciens isolated from Brazilian cold raw milk.

The cold storage of raw milk before heat treatment in dairy industry promotes the growth of psychrotrophic microorganisms, which are known for their ability to produce heat-resistant proteolytic enzymes. Although Pseudomonas is described as the main causative genus for high proteolytic spoilage potential in dairy products, Serratia liquefaciens secretes proteases and may be found in raw milk samples as well. However, at the present there is no information about the proteolytic spoilage potential of S. liquefaciens in milk after heat-treatment. The main aim of this research was to assess the proteolytic spoilage potential of S. liquefaciens isolated from Brazilian raw milk and to characterize the involved protease. S. liquefaciens was shown to secrete one heat-resistant spoilage metalloprotease of, approximately, 52 kDa encoded by the ser2 gene. The heat-resistance of Ser2 was similar to the aprX encoded metalloprotease produced by Pseudomonas. Although the ser2 gene was detected in all S. liquefaciens isolates tested in this study, the proteolytic activity of the isolates in milk was highly heterogeneous. Since nucleotide and deduced amino acid sequences of ser2 of all tested isolates are identical, this heterogeneity may be attributed to differences in enzyme expression levels or post-translational modifications.

Performance of two alternative methods for Listeria detection throughout Serro Minas cheese ripening.

The ability of pathogens to survive cheese ripening is a food-security concern. Therefore, this study aimed to evaluate the performance of two alternative methods of analysis of Listeria during the ripening of artisanal Minas cheese. These methods were tested and compared with the conventional method: Lateral Flow System™, in cheeses produced on laboratory scale using raw milk collected from different farms and inoculated with Listeria innocua; and VIDAS(®)-LMO, in cheese samples collected from different manufacturers in Serro, Minas Gerais, Brazil. These samples were also characterized in terms of lactic acid bacteria, coliforms and physical-chemical analysis. In the inoculated samples, L. innocua was detected by Lateral Flow System™ method with 33% false-negative and 68% accuracy results. L. innocua was only detected in the inoculated samples by the conventional method at 60-days of cheese ripening. L. monocytogenes was not detected by the conventional and the VIDAS(®)-LMO methods in cheese samples collected from different manufacturers, which impairs evaluating the performance of this alternative method. We concluded that the conventional method provided a better recovery of L. innocua throughout cheese ripening, being able to detect L. innocua at 60-day, aging period which is required by the current legislation.

Isolamento e classificação de micro-organismos psicrotróficos proteolíticos presentes em queijo Minas Frescal / Isolation and classification of psychrotrophic and proteolytic microorganisms in “Minas Frescal” cheese

Algumas condições da qualidade de leite e derivados estão conexas à microbiota contaminante deterioradora que é, sobretudo, representada por bactérias psicrotróficas proteolíticas. Portanto, o objetivo desse trabalho foi isolar micro-organismos psicrotróficos proteolíticos de queijo Minas Frescal comercializados em Salinas -MG. Para tanto foi realizado o isolamento de micro-organismos psicrotróficos proteolíticos presentes em queijos Minas Frescal. Logo após, foi realizada a caracterização dos isolados quanto à coloração de Gram, forma e atividade de oxidase. Os resultados obtidos evidenciaram a predominância de micro-organismos psicrotróficos Gram-positivos na microbiota dos queijos comercializados em Salinas-MG.

Performance of two alternative methods for Listeria detection throughout Serro Minas cheese ripening

ABSTRACT The ability of pathogens to survive cheese ripening is a food-security concern. Therefore, this study aimed to evaluate the performance of two alternative methods of analysis of Listeria during the ripening of artisanal Minas cheese. These methods were tested and compared with the conventional method: Lateral Flow System™, in cheeses produced on laboratory scale using raw milk collected from different farms and inoculated with Listeria innocua; and VIDAS®-LMO, in cheese samples collected from different manufacturers in Serro, Minas Gerais, Brazil. These samples were also characterized in terms of lactic acid bacteria, coliforms and physical-chemical analysis. In the inoculated samples, L. innocua was detected by Lateral Flow System™ method with 33% false-negative and 68% accuracy results. L. innocua was only detected in the inoculated samples by the conventional method at 60-days of cheese ripening. L. monocytogenes was not detected by the conventional and the VIDAS®-LMO methods in cheese samples collected from different manufacturers, which impairs evaluating the performance of this alternative method. We concluded that the conventional method provided a better recovery of L. innocua throughout cheese ripening, being able to detect L. innocua at 60-day, aging period which is required by the current legislation.