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Human milk is a source of nutrients for new-born and infants. Its composition is extremely variable, and it contains several factors such as nutrients, antimicrobial agents, oligosaccharides, IgA antibodies or TGF-beta and fat. These last are represented principally by triacylglycerols which provide 40-60% of required energy and essential fatty acids. Additionally, breast milk contains significant numbers of commensal bacteria, including Streptococci, Corynebacteria, Micrococci and Propionibacteria. Moreover, several research groups reported also the isolation of lactic acid bacteria (LAB) and Bifidobacterium spp. from human breast milk. However, very few publications have tried to find a correlation between the human milk microbiota and its composition of milk fatty acids in relation to the woman lactation stage. Thus, in this context, the principal aim of the present research was to characterize the microbiota and the fatty acid profiles of human breast milk in relation to the woman lactation stage. For this, 30 women were enrolled in the study. Human breast milk samples were voluntarily donated by mothers attending the Department of Gynecology and Obstetrics from M. Bufalini Hospital, Cesena, Italy. Mothers declared to be in good health, having had normal and full-term pregnancy without infant or maternal perinatal problems and they were asked to fill a questionnaire on their diet habits. The collected breast milk samples (0, 1, 3, 6 months) were analyzed by DISTAL and analyzed by dependent and independent culture media methods. For dependent ones, the samples were sampled on MRS with cysteine and TPY media added or not of mupirocin (100 mg/L). Plated were incubated at 37 °C for 24-48 h in anaerobiosis and the isolates were subjected to DNA extraction and sequencing. For fatty acid profile analyses, the samples were analyzed by the method reported by Romeu-Nadal et al., 2008 [1]. The results showed that the human milk samples were characterized for the most by commensal species belonging to different genera. However, some samples were characterized by the presence of species belonging to Bifidobacterium longum and Lactobacillus gasseri. Regarding fatty acid profiles, the breast milk sample collected after 1 and 3 months from the partum were principally characterized by the presence of DHA (C22:6 n-3), AA (C20:4 n-6) and EPA (C20:5 n-3), demonstrating an increase of their precursors, C18:2 n-6 (LA) and C18:3 n-3 (ALA), during the lactation time. Differently, the colostrum collected immediately after the partum was characterized by medium chain fatty acids. In conclusion, next trial on Next Generation sequencing (NGS) will permit to find significant correlation on the milk microbiota and the fatty acid profiles in relation to the lactation stage. [1]. Romeu-Nadal, M., Castellote, A. I., Gayà, A., & López-Sabater, M. C. (2008). “Effect of pasteurisation on ascorbic acid, dehydroascorbic acid, tocopherols and fatty acids in pooled mature human milk”. Food chemistry. doi: doi.org/10.1016/j.foodchem.2007.06.060

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