Isolation and Identification of Potential Probiotic Bacteria from Human Milk

Breast milk was long considered a sterile environment, but now it is known to harbor many bacteria that will shape the newborn microbiota. The benefits of breastfeeding to newborn health are, on some level, related to the presence of beneficial bacteria in human milk. Therefore, this study aims to investigate and isolate potential probiotics present in human milk that might be associated with improved health in infants, being potential candidates to be used in simulated human milk formula. Milk samples of 24 healthy mothers were collected at three time points: 30 min (colostrum), 5-9 days (transitional milk), and 25-30 days (mature milk) postpartum. Samples were evaluated by culturing, and the isolated bacteria were identified by MALDI-TOF MS and 16S DNA sequencing. In vitro screening for probiotics properties was performed, and the potential probiotics were mono-associated with germ-free mice to evaluate their ability to colonize the gastrointestinal tract. The microorganisms were submitted to the spray-drying process to check their viability for a potential simulated milk formula production. Seventy-seven bacteria were isolated from breast milk pertaining to four bacterial genera (Staphylococcus, Streptococcus, Leuconostoc, and Lacticaseibacillus). Four potential probiotics were selected: Lacticaseibacillus rhamnosus (n = 2) and Leuconostoc mesenteroides (n = 2). Isolates were able to colonize the gastrointestinal tract of germ-free mice and remained viable after the spray-drying process. In conclusion, breast milk harbors a unique microbiota with beneficial microorganisms that will impact the newborn gut colonization, being an essential source of probiotic candidates to be used in a formula of simulated maternal milk.