Phenotypic and genotypic characterization of antimicrobial resistances reveals the effect of the production chain in reducing resistant lactic acid bacteria in an artisanal raw ewe milk PDO cheese.

Antimicrobial resistance (AMR) is a significant public health threat, with the food production chain, and, specifically, fermented products, as a potential vehicle for dissemination. However, information about dairy products, especially raw ewe milk cheeses, is limited. The present study analysed, for the first time, the occurrence of AMRs related to lactic acid bacteria (LAB) along a raw ewe milk cheese production chain for the most common antimicrobial agents used on farms (dihydrostreptomycin, benzylpenicillin, amoxicillin and polymyxin B). More than 200 LAB isolates were obtained and identified by Sanger sequencing (V1-V3 16S rRNA regions); these isolates included 8 LAB genera and 21 species. Significant differences in LAB composition were observed throughout the production chain (P ≤ 0.001), with Enterococcus (e.g., E. hirae and E. faecalis) and Bacillus (e.g., B. thuringiensis and B. cereus) predominating in ovine faeces and raw ewe milk, respectively, along with Lactococcus (L. lactis) in whey and fresh cheeses, while Lactobacillus and Lacticaseibacillus species (e.g., Lactobacillus sp. and L. paracasei) prevailed in ripened cheeses. Phenotypically, by broth microdilution, Lactococcus, Enterococcus and Bacillus species presented the greatest resistance rates (on average, 78.2 %, 56.8 % and 53.4 %, respectively), specifically against polymyxin B, and were more susceptible to dihydrostreptomycin. Conversely, Lacticaseibacillus and Lactobacillus were more susceptible to all antimicrobials tested (31.4 % and 39.1 %, respectively). Thus, resistance patterns and multidrug resistance were reduced along the production chain (P ≤ 0.05). Genotypically, through HT-qPCR, 31 antimicrobial resistance genes (ARGs) and 6 mobile genetic elements (MGEs) were detected, predominating Str, StrB and aadA-01, related to aminoglycoside resistance, and the transposons tnpA-02 and tnpA-01. In general, a significant reduction in ARGs and MGEs abundances was also observed throughout the production chain (P ≤ 0.001). The current findings indicate that LAB dynamics throughout the raw ewe milk cheese production chain facilitated a reduction in AMRs, which has not been reported to date.

The effects of storage and hop extract on aroma and flavour compounds in Balkan-style sausages packed under a CO2-containing anaerobic atmosphere.

Changes in the levels of pH, lactic acid, acetic acid, headspace volatile compounds, and spoilage odour intensity during a 35 day refrigerated storage period in two sets of a Balkan-style fresh sausage, a control sausage (CON) and a sausage containing an aqueous hop extract (HOP), packaged under a 20% CO2 and 80% N2 atmosphere were evaluated. Storage resulted in progressive sausage acidification and increased the levels of acetic acid, 1-methylbutanol, ethyl acetate, ethyl hexanoate, and 2-ethylhexanol; all of which are associated with anaerobic microbial metabolism under sugar restricted conditions. Storage decreased the levels of hexanal, heptanal, pentanol, and garlic-derived organosulfur compounds. Hop extract showed oxygen scavenging abilities, and decreased the levels of the volatile compounds derived from lipid auto-oxidation while contributing to the presence of specific terpene compounds. The use of hop extract did not improve the shelf life of sausages packed under anaerobic atmosphere. The spoilage odour appeared in both types of sausages on the 14th day, and it was considered strong from day 21 onwards.