Diversity and composition of lactic acid bacteria and Bifidobacterium in breast milk form mothers living in Inner Mongolia

Human breast milk contains commensal lactic acid bacteria (LAB) and Bifidobacterium . Several reports have suggested that human milk is a direct source of commensal bacteria found in the infant gut, and some commensal LAB and Bifidobacterium in human milk may bring positive effects to the health of breast-fed babies by shaping their gut microbiota. Therefore, researching on LAB and Bifidobacterium in breast milk is important. A total of 25 human breast milk samples were collected in Huhhot city of Inner Mongolia. The bacterial DNA of all samples was extracted, and the target sequences of LAB and Bifidobacterium were amplified using specific primers. The PCR products were sequenced by Pacific Biosciences (PacBio) single-molecule, real-time sequencing technology (SMRT). Then the diversity and composition of LAB and Bifidobacterium were analyzed at species level using bioinformatic methods. We found 120 species of LAB including Lactobacillus delbrueckii , Lactobacillus plantarum , Lactobacillus gallinarum , Lactobacillus fermentum , Staphylococcus epidermidis , Streptococcus parauberis , Streptococcus mitis , Streptococcus salivarius and so on, as well as 11 species of Bifidobacterium including Bifidobacterium catenulatum , Bifidobacterium longum , Bifidobacterium stercoris and Bifidobacterium bifidum and so on. Among them, the species Lactobacillus delbrueckii , Lactobacillus plantarum , Lactobacillus gallinarum , Lactobacillus fermentum , Lactobacillus rhamnosus and Streptococcus salivarius were detected in all samples. Additionally, almost all of the detected Bifidobacterium species were present in all samples, except Bifidobacterium pseudolongum and Bifidobacterium mongoliense and. The lactation stage had a significant effect on the diversity and composition of LAB but not Bifidobacterium . Results from PERMANOVA based on Bray-Curtis distances further confirmed the significant effect of lactation stage on LAB structure in human milk ( P 0.05). To compare the structure of the LAB community among three milk groups (i.e. colostrum, less than 5 d after delivery; transitional milk, 5–14 d after delivery; mature milk, more than 15 d after delivery), we performed Bray-Curtis PCoA based on OTU distribution and abundance. The PCoA results suggested apparent LAB structural differences among the three groups, as the symbols representing the individual groups were separated on the score plot with only minor overlap. In addition, the relative abundances of Lactobacillus delbrueckii ( P 0.01) and Lactobacillus plantarum ( P 0.05) in colostrum and mature milk were significantly higher than that of the transitional milk. Significantly more Lactobacillus rhamnosus was found in colostrum than in transition milk and mature milk ( P 0.05), whereas, the Lactobacillus helveticus in transition milk was remarkably higher than another two stages human milk ( P 0.05). However, there was no difference in the distribution of Bifidobacterium species among human milk of three lactation stages. The breast milk from the sampled mothers in Inner Mongolia could be divided into three subtypes according to the LAB composition: the first subtype contained high proportion of Lactobacillus delbrueckii (>50%); the second subtype had less than 50% of Lactobacillus delbrueckii together with more Lactobacillus plantarum and other unclassified LAB species; the third subtype contained more than 5% of Lactobacillus gallinarum . Interestingly, the three subtypes were largely consistent with the grouping by lactation stage except for the colostrum. Nevertheless, most colostrum samples belonged to the first and second subtypes. We also found significant correlations between some LAB and Bifidobacterium . In recent years, we have started isolating probiotics from human breast milk in order to identify suitable probiotic bacteria in breast milk of the Chinese population. This study has established a new method to assess the diversity of LAB and Bifidobacterium , which will serve as reference and guiding protocol for screening probiotics in human breast milk.