Cm0733

To validate primer specificity, a selection of Lactobacillus and Bifidobacterium strains were cultivated on MRS (de Man, Rogosa and Sharpe, [18 (link)]) or BSM (Sigma, 88,517) agar at 37 °C under anaerobic conditions for 72 h (Additional file 1: Table S2). Genomic DNA was extracted (PowerLyzer® PowerSoil® DNA isolation kit, MoBio 12,855–100) from colony material according to the protocol provided by the manufacturer and diluted to 1 ng/μl and used for input in the PCR. For spiking fecal samples with known quantities of the strains LGG® and BB-12®, single colonies of these were propagated in 10 ml MRS medium under anaerobic conditions for 48 h at 37 °C, centrifuged at 10,000×g for 5 min at 4 °C, re-suspended in 1 ml maximum recovery diluent (MRD, Oxoid CM0733), and 10-fold serially diluted in MRD. One hundred μl of each dilution (10⁰–10⁻⁷) was spiked into 100 mg LGG®/BB-12® negative baseline fecal samples (based on specific qPCR of the probiotic strains) and also plated in duplicates (dilutions 10⁻⁴–10⁻⁹) on MRS plates and incubated anaerobically at 37 °C for 24 h before CFU counting. Community DNA was extracted (PowerLyzer® PowerSoil® DNA isolation kit, MoBio 12,855–100) from each spiked samples plus none-spiked controls.

Campylobacters were enumerated as previously described [24 (link)]. Briefly, serial 10-fold dilutions were made in MRD (CM0733; Oxoid) and enumerated in triplicate on mCCDA (CM0739; Oxoid) agar with additional agar (L13; Oxoid) to a total of 2% (w/v) added to reduce swarming. Plates were incubated under microaerobic conditions at 42°C for 48 h before typical Campylobacter colonies were counted. Bacteriophages were enumerated using the soft agar overlay method as previously described [30 (link)]. Briefly, serial 10-fold dilutions of phage suspensions were applied as 10 µl droplets in triplicate to the surface of prepared host bacterial lawns and allowed to dry. Plates were then incubated under microaerobic conditions at 42°C for 48 h before the plaques were counted.

Electrosprayed samples containing Bifidobacterium animalis were stored in a desiccant cabinet, (NALGENE^® 5317-0120, Thermo Fisher Scientific™, Rochester, NY, USA), containing a saturated solution of potassium carbonate at a relative humidity (RH) of 40% at 30 °C. The viability of encapsulated and non-encapsulated Bifido cells was determined by colony-forming unit (CFU) analysis. The samples were diluted 1:100 with Maximum Recovery Diluent (MRD, (Oxoid CM0733, Hampshire, UK)) at 37 °C.
The suspension was homogenized by stomaching (Smasher, AES Laboratoire, bioMérieux, Hazelwood, MO, USA) at intermediate speed for 2 min, and further dilutions were performed in MRD. Appropriate volumes of appropriate dilutions were plated in de Man Rogosa Sharpe (MRS) agar supplemented with 0.05 w/v % L-cysteine hydrochloride monohydrate by pour plating, aiming for colony counts between 30–300 CFU/plate. Plates were incubated at 37 °C under anaerobic conditions for 3 days, and the results were calculated as the average of 2 plates. Cell viability was measured at the time points of 0, 2, and 4 weeks.
The encapsulation efficiency (EE) of the microcapsules was calculated by determining the number of viable cells inside the capsules (N) divided by the number of viable cells in the initial solution (N0), as expressed in (1) [2 (link)]: $$\mathrm{EE}(\%)=\frac{\mathrm{Log}\mathrm{N}}{\mathrm{Log}\mathrm{N}0}\times 100$$