Anaerobic system anaerogen

For the tissue samples collected from all the dogs, bacterial isolation was performed using selective and differential agar (MacConkey agar, Columbia blood agar and Mannitol Salt agar) incubated at 37 °C for 24 h. Moreover, Columbia blood agar plates were anaerobically incubated with the AnaeroGen™ Anaerobic System (Oxoid, Milano, Italy) to isolate anaerobic bacteria.
For the Salmonella spp. culture, pre-enrichment in Buffered Peptone water was performed, followed by two enrichments in Selenite Cystine (SC) and Rappaport-Vassiliadis (RV) broths, and incubated, respectively, at 37 °C and 42 °C for 24 h. The enrichment broths were then plated on Xylose–Lysine Deoxycholate Agar (XLD) and Brilliant Green Agar (BGA).
The identification of the isolated strains was carried out with the biochemical API^® system and Vitek^® 2 system (bioMérieux, Craponne, France). For the Salmonella spp. strains, after identification by API20E^®, serological typing was performed.

The frozen bacterial suspensions with ACE, CAS, or GUA as well as the frozen positive (FOS) and negative (NEC) controls were subjected to freeze-drying at −55 ± 2 °C, with a vacuum pressure of <138 μHG and a freeze-drying rate of 1 mm/h, for approximately 40 h using a benchtop freeze dryer (LIOTOP^®, Model L-101, São Carlos-SP, Brazil). Just after freeze-drying, the viable cells of the tested strains were enumerated. For this, the freeze-dried strains in the different treatments were rehydrated in sterile distilled water (30 ± 0.5 °C) for 15 min. Serial dilutions were subsequently performed using sterile saline solution (NaCl 8.5 g/L) and dispensed onto MRS agar plates (HiMedia, Mumbai, India) using a microdrop inoculation technique [29 (link)]. After an incubation period of 48 h at 37 °C under anaerobic conditions (Anaerobic System Anaerogen, Oxoid), the visible colonies were enumerated and the results expressed as log CFU/g. The detection limit of the assays for viable cell counts was 1 log CFU/g.

The growth kinetics of Lactobacillus strains were assessed in MRS broth and in general edible medium (GEM) broth (40 g/L glucose, 30 g/L soya peptone, 7 g/L yeast extract, and 1 g/L MgSO₄.7H₂O in 0.01 mol/L K-phosphate buffer; pH 6.3 ± 0.2; Saarela et al., 2004 (link)) using two different volume scales, i.e., 200 and 2000 mL. Aliquots of each lactobacilli strain suspension were inoculated (1% v/v; final viable cell count of approximately 7 log CFU/mL) in MRS or GEM broth and incubated aerobically at 37°C under stirring (150 rpm) for 48 h. At different incubation time intervals (16, 24, and 48 h), samples were taken (1 mL) and serially diluted (10⁻¹–10⁻⁵) in sterile peptone (0.1 g/100 mL), spread plated onto MRS agar, and incubated anaerobically (Anaerobic System Anaerogen, Oxoid) at 37°C for 48 h. After the incubation period, the viable cells were counted, and the results were expressed as the log CFU/mL.

The AFM₁ standard was obtained from Sigma Aldrich (St. Louis, MO, USA). High-performance liquid chromatography (HPLC) grade solvents were obtained from Merck (Darmstadt, Germany).
The isolates Lactobacillus plantarum 49, L. fermentum 111, and L. paracasei 108 were examined separately for the removal of AFM₁. These isolates were recovered from fruit processing by-products, identified with a partial 16S rRNA gene sequence analysis and characterized as potential candidates for use as probiotics [17 (link)]. Stocks were stored at −20 °C in de Man, Rogosa, and Sharpe (MRS) broth (HiMedia, Mumbai, India) with glycerol (20 mL/100 mL; Sigma-Aldrich, St. Louis, MO, USA). Working cultures were maintained aerobically on MRS agar (HiMedia, Mumbai, India) at 4 °C and transferred to a new media monthly. Prior to use in assays, each isolate was cultivated anaerobically (Anaerobic System Anaerogen, Oxoid, Hampshire, UK) in MRS broth at 37 °C for 20–24 h (to reach the stationary growth phase), harvested by centrifugation (4500× g, 15 min, 4 °C), washed twice, and resuspended in phosphate buffer solution (PBS; 50 mM K₂HPO₄/KH₂PO₄; pH 6.9) to obtain cell suspensions with an optical density reading at 660 nm (OD₆₆₀) of 0.5. This suspension had viable counts of approximately 1.1 × 10⁹ CFU/mL for each isolate when plated in MRS agar.

The probiotic strains freeze-dried (counts of approximately 7–10 log CFU/g) in the absence (NEC) or presence of the different substrates (FOS, ACE, CAS, and GUA) were stored in sealed vials, which were maintained in desiccators containing silica gel for relative humidity control and stored under refrigerated (4 ± 0.5 °C) or room temperature (25 ± 0.5 °C) conditions for 90 days. At regular storage time intervals of 15 days, the strains freeze-dried without or with substrates were rehydrated in sterile distilled water (1:9 ratio) at room temperature (25 ± 0.5 °C), serially diluted using sterile saline solution (NaCl 8.5 g/L), and inoculated onto MRS agar plates using a microdrop inoculation technique [27 (link)]. After an incubation period of 48 h at 37 °C under anaerobic conditions (Anaerobic System Anaerogen, Oxoid), the visible colonies were enumerated and the results expressed as log CFU/g. The detection limit of the assays for viable cell counts was 1 log CFU/g.

The strains L. fermentum 139, L. fermentum 263, and L. fermentum 296 were kindly provided by the Laboratory of Microbiology, Department of Nutrition, Federal University of Paraíba (João Pessoa, PB, Brazil). Each strain was cultured anaerobically (Anaerobic System Anaerogen, Oxoid, Hampshire, UK) in Mann, Rogosa, and Sharpe (MRS) broth (Himedia, Mumbai, India) at 37 ± 0.5 °C for 20–24 h. To obtain the cell suspension, the cells were collected by centrifugation (8000× g, 10 min, 4 °C), washed twice with sterile PBS solution, resuspended in PBS solution, and homogenized by vortexing (30 s) to obtain standard cell suspensions with optical density (OD) at 625 nm (OD625) of 1.2 and 2.0, corresponding to viable cell counts of approximately 10⁸ colony-forming units per milliliter (CFU/mL) and 10¹⁰ CFU/mL, respectively, when plated on MRS agar (HiMedia, Thane, India). In order to increase the specific strain characteristics and to obtain a multi-strain probiotic, mixed cell suspensions were prepared at a ratio of 1:1:1 (v/v). These doses have been tested to achieve 1 log below and 1 log above a dose widely considered therapeutic (10⁹ CFU).