The most consumed cheese in Brazil, Minas Frescal cheese (MFC) is highly susceptible to microbial contamination and clandestine production and commercialization can pose a risk to consumer health. The storage of this fresh product under refrigeration, although more appropriate, may favor the growth of spoilage psychrotrophic bacteria. The objective of this study was to quantify and compare Pseudomonas spp. and other psychrotrophic bacteria in inspected and non-inspected MFC samples, evaluate their lipolytic and proteolytic activities and their metalloprotease production potentials. Twenty MFC samples were evaluated: 10 inspected and 10 non-inspected. Counts of psychrotrophic bacteria and Pseudomonas spp., evaluation of the proteolytic and lipolytic potential of the isolates, and identification of potential producers of alkaline metalloprotease (AprX) were assessed. The mean total psychrotrophic counts were 1.07 (±2.18) × 109CFU/g in the inspected samples and 4.5 (±5.86) × 108CFU/g in the non-inspected, with no significant difference (p=0.37). The average score of Pseudomonas spp. was 6.86 (±18.6) × 105 and 2.08 (±3.65) × 106 CFU/g for the inspected and non-inspected MFC samples, respectively, with no significant difference (p=0.1). Pseudomonas spp. represented 0.06% and 0.004% of psychrotrophic bacteria found in inspected and non-inspected MFC samples, respectively. Collectively, 694 psychrotrophic strains and 47Pseudomonas spp. were isolated, of which 59.9% and 68.1% were simultaneously proteolytic and lipolytic, respectively. Of the 470 psychrotrophs isolated from inspected and 224 from non-inspected cheese samples, 5.74% and 2.23% contained aprX, respectively, while 100 and 86.96% of the Pseudomonas spp. isolates in inspected and non-inspected cheese samples contained the gene. The production potential of AprX did not, however, determine the proteolytic activity on plates of these isolates under the conditions evaluated in this study. Of total, 65.63% of the psychrotrophs that contained aprX gene were confirmed as Pseudomonas spp., using genus-specific PCR. Phylogenetic analysis of the 16S rRNA gene of the other psychrotrophs that were potential producers of AprX identified them as Serratia spp. (n=7), Raoultella ornithinolytica (n=1), and Acinetobacter schindleri (n=1) in the inspected samples and Psychrobacter sanguinis (n=1) and Leuconostoc mesenteroides (n=1) in the non-inspected samples. The production conditions of Brazilian MFC of these samples, while meeting the legal determinations, are not sufficient to control Pseudomonas and other spoilage-related psychrotrophs. Thus, stricter hygienic measures are required during the formal production of this type of cheese.
The aim of this study was to quantify, identify, evaluate antimicrobial resistance, and characterize the virulence factors of enteropathogenic (EPEC), Shigatoxigenic (STEC), and enterohemorrhagic (EHEC) Escherichia coli in raw milk (RM) and legal (LMFC) and illegal (IMFC) Minas Frescal cheeses in southern and northeast Brazil. Illegal cheeses are those made without official inspection service or sanitary surveillance. We evaluated samples of RM produced in Paraná (southern) and Maranhão (northeast) States, LMFC produced using pasteurized milk in inspected industries, and IMFC potentially produced with raw milk. Mean total coliform counts were 8.4 × 10 4 cfu/mL for RM, 1.4 × 10 7 cfu/mL for LMFC, and 2.9 × 10 7 cfu/ mL for IMFC. Mean E. coli counts were 2.4 × 10 3 cfu/ mL for RM, 1.9 × 10 2 cfu/mL for LMFC, and 1.1 × 10 5 cfu/mL for IMFC. Among the 205 E. coli isolates from RM, 9.75% were identified as EPEC, mainly (90%) in samples from Paraná. Of the total isolates from the cheese samples, 97.4% (n = 111) came from IMFC, of which 1.8 and 2.7% were identified as EPEC and STEC, respectively; no EHEC was detected. The phylogenetic group A (60%) and typical EPEC (68%) predominated, which confirms the possible human origin of pathogenic isolates in RM and IMFC. Of these, 50% were resistant to at least one antibiotic, and streptomycin was the antimicrobial with the highest number (8) of EPEC and STEC resistant isolates. This study reports the first isolation of serogroup O28ac in Brazilian milk. We found no predominance of a specific serogroup of EPEC or STEC in milk or cheese or clonal isolates in the same sample, indicating different origins of the contamination in these products, presumably mostly related to poor hygienic handling.
The consumption of goat's milk has increased significantly in Brazil. In 2014, its production was 153 thousand tons, and the Paraná state is responsible for the production of 380 thousand liters. Although the government regulates goat milk, this raw material is not widely studied in Brazil. The objective of this work was to study its microbiological and physicochemical characteristics. A total of 32 samples from 8 Paraná State farms were analyzed. The microbial indicators of hygiene, psychrotrophic bacteria, L. monocytogenes and Salmonella spp. were evaluated. We used physicochemical tests of goat’s milk that were established by the Brazilian legislation. In addition, phosphatase activity, pH, conductivity, boiling test, and urea content were measured. The average concentrations of mesophilic aerobes, coliforms, E. coli, coagulase positive Staphylococcus and psychrotrophic microorganisms were 4.10, 2.38, 0.65, 2.06 and 4.02 log CFU mL-1, respectively. The samples did not present L. monocytogenes and Salmonella spp. In the physicochemical analysis, 90.63% of the samples presented at least one parameter outside the legal standards. In the somatic cell count, 73.33% of the samples had counts higher than 1 x 106 cells mL-1. Raw goat's milk produced in Paraná has high coliform and psychrotrophic counts, indicating poor hygiene during milking. The fluctuating values of the physicochemical data indicate that additional studies are required to determine the parameters that reflect the Brazilian conditions of goat milk production.
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