Carvalho, Nelson Mota deOliveira, Diana LuaziSaleh, Mayra Anton DibPintado, ManuelaMadureira, Ana Raquel2021-05-242021-05-242021-032311-5637http://hdl.handle.net/10400.14/33189The use of fecal inoculums for in vitro fermentation models requires a viable gut microbiota, capable of fermenting the unabsorbed nutrients. Fresh samples from human donors are used; however, the availability of fresh fecal inoculum and its inherent variability is often a problem. This study aimed to optimize a method of preserving pooled human fecal samples for in vitro fermentation studies. Different conditions and times of storage at -20 degrees C were tested. In vitro fermentation experiments were carried out for both fresh and frozen inoculums, and the metabolic profile compared. In comparison with the fresh, the inoculum frozen in a PBS and 30% glycerol solution, had a significantly lower (p < 0.05) bacterial count (<1 log CFU/mL). However, no significant differences (p < 0.05) were found between the metabolic profiles after 48 h. Hence, a PBS and 30% glycerol solution can be used to maintain the gut microbiota viability during storage at -20 degrees C for at least 3 months, without interfering with the normal course of colonic fermentation.engGut microbiota viabilityGlycerolHuman in vitro modelsColon fermentationOrganic acidsPreservationjournal article10.3390/fermentation701001485106961632000633057500001