## 3 Results  
Breast milk was collected from 100 healthy women in Greece, aged from 20 to 47 years. Twenty-six (26) samples were colostrum and seventy-four (74) mature breast milk. The probiotic bacteria _Bifidobacterium_ were detected in 61.5% samples of colostrum, and 37.8% of mature breast milk. The presence of the genus _Lactobacillus_ was identified in the 46.2% of colostrum and 24.3% of mature breast milk. The statistical analysis indicated significance differences in the two types of breast milk (\(p<0.05\)). In 18% of the samples the presence of both genera was detected. The colostrum milk samples were found to have higher percentages of either _Bifidobacterium_ or _Lactobacillus_ when compared to the mature milk samples (76.9% and 48.6%, respectively) (\(p<0.05\)) (Table 2). The samples positive for _Bifidobacterium_ were further tested in order to determine the species. From the 44 positive samples for _Bifidobacterium_ spp. tested, 20 (45.5%) were found to be _Bifidobacterium longum_, 14 samples (31.8%) were _Bifidobacterium bifidum_, and 10 (22.7%) were identified as other _Bifidobacterium_ species. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Target Microorganism** & **Primer** & **Sequence (\(\mathbf{5^{\prime}}\) to \(\mathbf{3^{\prime}}\))** & **Reference** \\ \hline _Bifidobacterium_ & Bifid-F & CTC CTG GAA ACG GGT GG & [PERSON] et al., 2002 \\ _Lactobacillus_ & Bifid-R & GGT CTT CTCC GAT ATC TAC A & [PERSON] et al., 2002 \\ _Lactobacillus_ & Lab 159 & GGA AAC AGT TGC TAA TAC CG & [PERSON] et al., 2014 \\ _Bifidobacterium bifidum_ & Lab 677 & CACCG TAC ACA TGG AG & [PERSON] et al., 2014 \\ _Bifidobacterium bifidum_ & B.Bifidum FWD & CCA CAT GAT CGC ATG TGA TTG & [PERSON] et al., 2005 \\ _Bifidobacterium bifidum_ & B.Iopum FWD & TTC CAG TTG ATC GCA TGG TC & [PERSON] et al., 2005 \\ _Bifidobacterium longum_ & B. longum Rev & GGG AAG CCG TAT CTC TAC GA & [PERSON] et al., 2005 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Primers used for the detection of genera _Bifidobacterium_ spp., _Lactobacillus_ spp., and _Bifidobacterium_ _bifidum_ and _Bifidobacterium_ _longum_. As age progresses, this incidence declines. In almost all women (97.1%) from smaller communities (villages), probiotic bacteria _Bifidobacterium_ and _Lactobacillus_ were detected (and this was statistically significant) more than in the milk of women from urban areas/major cities (33.8%) (\(p<0.05\)). In the majority of women with a BMI less than 25, milk bacteria were detected, while in the other BMI categories the incidence decreased substantially. The consumption of probiotics did not affect the composition of the breast milk microbiota (\(p>0.05\)). Women consuming yogurt regularly in their diet, had in their majority (64.3%) milk bacteria detected, and this was statistically significant more than that in the non-yogurt eaters (\(p<0.05\)) (Table 4). \begin{table}
\begin{tabular}{c c c} \hline \hline
**Data** & **Number of Women** & **Total Positive Samples** \\ \hline Age & & \\
18–24 & 7 & 7 (100\%) \\
25–29 & 15 & 15 (100\%) \\
30–34 & 35 & 23 (65.7\%) \\
35–39 & 23 & 6 (26.1\%) \\
40–44 & 15 & 5 (33.3\%) \\ \(\geq\)45 & 5 & 0 \\ BMI & & \\ \(<\)20 & 15 & 13 (86.7\%) \\
20–25 & 40 & 32 (80\%) \\
25–30 & 35 & 8 (22.9\%) \\ \(>\)30 & 10 & 3 (30\%) \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Correlation of maternal demographics, anthropometric characteristics, and nutritional habits with positive samples in _Bifidobacterium_ and/or _Lactobacillus_. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Type of Breast Milk** & **Number of Samples** & **Positive for** & **Positive for** & **Positive Samples for** \\  & **Analyzed (\(n\))** & **Bifidobacterium** & **Lactobacillus** & **either Bifidobacterium** \\  & & & & **or** _Lactobacillus_ \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Realtime PCR detection of probiotic bacteria _Bifidobacterium_ and _Lactobacillus_ in human breast milk.