# Screening of Some Romanian Raw Honeys and Their Probiotic Potential Evaluation
## 3 Results
### Antibacterial Activity for the Identified Probiotic Bacteria  
_Bacillus subtilis_ complex species (_B. subtilis_, _B. amyloliquefaciens_, _B. velezensis_), identified in samples 1, 4, 5, 6, and 8, seems to have antibacterial activity especially against Gram-negative bacteria: _Pseudomonas aeruginosa_, _Escherichia coli_, followed by _Salmonella enteritidis_, but also against Gram-positive: _Listeria monocytogenes_ and less against _Staphylococcus aureus_. No effect was observed against _Enterococcus faecalis_.  
Antimicrobial activity of _Bacillus spp_ isolates against pathogenic bacteria is presented in Table 6 and Figure 7.  
\begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline
**Antimicrobial Effect** & **1** & **2** & **3** & **4** & **5** & **6** & **7** & **8** \\ \hline _Enterococcus faecalis_ & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 \\ _Staphylococcus aureus_ & 2.10 (0.11) \({}^{\mathrm{c}}\) & 0 & 0 & 1.80 (0.09) \({}^{\mathrm{d}}\) & 1.70 (0.38) \({}^{\mathrm{c}}\) & 1.80 (0.08) \({}^{\mathrm{c}}\) & 2.40 (0.12) \({}^{\mathrm{a}}\) & 1.40 (0.07) \({}^{\mathrm{c}}\) \\ _Listeria monocytogenes_ & 3.4 (0.17) \({}^{\mathrm{b}}\) & 1.8 (0.09) \({}^{\mathrm{a}}\) & 1.9 (0.09) \({}^{\mathrm{a}}\) & 3.50 (0.18) \({}^{\mathrm{b}}\) & 2.60 (0.13) \({}^{\mathrm{b}}\) & 2.80 (0.14) \({}^{\mathrm{b}}\) & 2.20 (0.10) \({}^{\mathrm{b}}\) & 1.80 (0.08) \({}^{\mathrm{c}}\) \\ _Escherichia coli_ & 3.4 (0.17) \({}^{\mathrm{b}}\) & 0 & 0 & 3.40 (0.16) \({}^{\mathrm{b}}\) & 2.80 (0.13) \({}^{\mathrm{b}}\) & 2.80 (0.15) \({}^{\mathrm{b}}\) & 0 & 2.40 (0.12) \({}^{\mathrm{b}}\) \\ _Salmonella enteritidis_ & 3.2 (0.15) \({}^{\mathrm{b}}\) & 0 & 0 & 2.99 (0.15) \({}^{\mathrm{c}}\) & 2.80 (0.14) \({}^{\mathrm{b}}\) & 2.80 (0.15) \({}^{\mathrm{b}}\) & 0 & 2.60 (0.13) \({}^{\mathrm{b}}\) \\ _Pseudomonas aeruginosa_ & 5.2 (0.26) \({}^{\mathrm{a}}\) & 0 & 0 & 4.90 (0.23) \({}^{\mathrm{a}}\) & 4.50 (0.21) \({}^{\mathrm{a}}\) & 4.40 (0.23) \({}^{\mathrm{a}}\) & 0 & 6.40 (0.31) \({}^{\mathrm{a}}\) \\ \hline \hline \end{tabular}  
* The values are mean of three samples (n = 3, standard deviation in parentheses), analyzed individualy in triplicate. Different letters within a column denote significant differences (\(p<0.05\)). 1 = B. subtilis, 2 = B. thuringiensis 1, 3 = B. mycoides, 4 = B. amyloliquefaciens 5 = B. velezensis 1, 6 = B. velezensis 2, 7 = B. thuringiensis 2, 8 = B. velezensis 3, N = negative control.  
\end{table}
Table 6: Antimicrobial effect of Bacillus spp. isolates from the raw honey samples (zone of inhibition, mm).  
\begin{table}
\begin{tabular}{c c} \hline \hline
**Raw Honey Samples** & **NTG CFU/mL** & **Identifified Species** \\ \hline P1 & \(1\times 10^{2}\) & _Bacillus mycoides_ \\ P2 & 0 \\ P3 & \(100\times 10^{6}\) & _Ewingella americana_, _Staphylococcus hominitis_ ssp. _homitis_ \\ P4 & \(51\times 10^{4}\) & _Staphylococcus epidermidis_, _Bacillus thuringiensis_, _Aerococcus viridans_ \\ P5 & \(150\times 10^{4}\) & _Bacillus amyloliquefaciens_ * \\ P6 & \(25\times 10^{4}\) & _Staphylococcus hominitis_ ssp. _homitis_, _Candidia glabrata_ \\ P7 & 0 \\ P8 & \(1\times 10^{2}\) & _Leuconostoc mesenteroides_ ssp. _remoris_ \\ P9 & \(20\times 10^{2}\) & _Bacillus subtilis_, _Bacillus velezensis_ * \\ P10 & \(28\times 10^{3}\) & _Corynebacterium amycolatum_, _Staphylococcus haemolyticus_, _Staphylococcus warneri_, _B. thuringiensis_ *, _Bacillus velezensis_ *, _Candidia glabrata_ \\ \hline \hline \end{tabular}  
* Identified by BLASTn analysis of Bacteria 165 sequence.  
\end{table}
Table 5: NTG quantification, probiotic, and other species identification in the raw honey samples analyzed.