Bagmixer 400 p

A pre-enrichment step was performed by weighing 25 ± 1 g into a stomacher bag using a Dilumat (AES International). The sample was diluted 1 in 10 with Buffered Peptone Water (BPW) ISO (LAB M Ltd., Lancashire, UK), homogenised by stomaching for 1 min (360 cycles/min; speed setting 6) (Interscience BagMixer® 400 P) and incubated at 37 ± 1 °C for 18 ± 2 h. The pre-enriched samples were examined for the presence of ESBL AmpC β-lactamase and carbapenemase-producing E. coli and Salmonella following the laboratory protocol described by the EU Reference Laboratory for antimicrobial resistance [30 ].

To begin the isolation of B. subtilis bacteria,5 g of fresh natto (fermented soybeans) were weighed into a sterile stomacher bag, followed by adding 20 mL of sterile 0.9% w/v NaCl and a 10-min homogenization (BagMixer 400 P, Interscience^®, Puycapel, France). The homogenized solution was diluted 10-fold before being used for inoculation on solid medium containing 5 g/L bacteriological peptone, 2.5 g/L yeast extract, 1 g/L glucose, and 15 g/L microbial agar, pH 7.2. The culture was grown for 72 h at 28 °C. Inoculation was performed in triplicate with five different samples of fermented soybeans. Following 72 h of cultivation, individual bacterial colonies were transferred to sterile cryobanks using a sterile loop (Graso Biotech^®, Starogard Gdański, Poland). The isolated cultures were stored in cryobanks at −20 °C (according to the manufacturer’s instructions) until they were cultured in a liquid medium. All isolated cultures of microorganisms were Gram-stained for pre-selection of Gram-positive bacteria. The isolated microorganisms were Gram-positive B. subtilis, as confirmed using the 16S rRNA nucleotide sequence. The differences between the isolated strains were confirmed by using the multilocus sequence typing (MLST) method to analyze allelic variation in the pta locus (phosphate acetyltransferase) (Genomed S. A., Warsaw, Poland) [18 (link)].

Microbial counts were carried out immediately after transporting meat samples to the laboratory before other analyses. Total visible bacteria counts were carried out according to ISO 4832:2006 [36 ] and ISO 4833-1:2013 [37 ] after incubation at 30 °C for 72 h. Five to six grams of meat samples were weighed in a sterile stomacher bag filter (Interscience, Saint Nom la Brétèche, France) with 0.1% sterile peptone water (about 100 g). The samples were mixed for three minutes using a stomacher (BagMixer 400 P, Interscience, Saint Nom la Brétèche, France). The homogenized solution was the stock solution (dilution 10⁻¹). The first 10⁻¹ dilution (1 mL was transferred to 9 mL of peptone water) and serial decimal dilutions up to 10⁻⁶ were prepared as described by Mohammed et al. [38 (link)]. The total bacterial count (TBC) was determined on plate count agar (PCA, BIOCORP Poland) after incubation at 30 °C for 72 h. The colonies developed on plates were counted using the cellSence Dimension software version 1.15 (EVIDENT (OLYMPUS), Tokyo, Japan). Antibacterial inhibition was determined by the colony counting method, and the obtained results were presented as log₁₀ CFU·g⁻¹. Each experiment was carried out in triplicate and presented as the mean ± standard deviation.