Cordless motor

Thirty-six Asian seabass at the age of three months (body weight: ~50 g) originally maintained in a 1,000 L tank containing 500 L of freshwater were divided equally into two tanks containing 3,00 L of freshwater. Eighteen fishes, used as a control, were fed twice daily with pelleted feed (Biomar, Nersac, FR, France), and the other eighteen fishes in the test tank were not given access to feed before sampling. Six fishes from each group were sacrificed at three, six and twelve days post fasting, respectively. Before experiments, the pestles and dissecting tools were first soaked in 5% concentration of Clorox bleach (The Clorox Company, Oakland, USA) for ~15 minutes, and autoclaved for 20 minutes after washing and rinsing in ddH2O. The working surface area and the deceased fish were decontaminated with 70% ethanol. Small sections (~1 cm of length) from the middle part of the intestine samples were taken and homogenized in 1 ml of Trizol reagent (Invitrogen, Carlsbad, USA) with sterile KONTES® pellet pestle driven by a cordless motor (Fisher Scientific, New Hampshire, USA). RNA isolation was conducted using the Trizol reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions. The remaining intestine samples from each fish was taken for DNA isolation using the QIAamp DNA Stool Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions.

For the determination of cultivable spray-dried bacteria, cells were previously released from the microparticles by disintegration using a pellet pestle mixer (Cordless^® Motor; Fisher Scientific, Hampton, NH, USA) for 30–60 s in PBS 1×. This treatment was previously tested and was shown not to affect the viability of A. muciniphila cells (unpublished results). From here, both cell suspensions (homogenized spray dried and non-encapsulated A. muciniphila cells) were treated equally: aliquots were serial diluted with PBS; 10 µL of each dilution were spotted, in triplicates, on PYGM agar plates (PYGM broth supplemented with 1.5% (m/v) agar (Liofilchem, Roseto degli Abruzzi, Italy)). The plates were incubated for 5–7 days at 37 °C under anaerobic conditions and results were expressed in CFU/mL, in the case of free cells for the initial suspensions controls, or CFU/g, for the spray dried cells.
Akkermansia muciniphila viability was also assessed, using the same methodology, throughout aerobic storage at 4 °C and 22 °C, after 7, 14, 21, and 28 days of storage.