# Use of Autochthonous Lactobacillusli to Increase the Safety of Zgougou
## 2 Results
### Culture-Independent Analysis of Microbial Community of Fermented Zgougou  
The bacterial diversity of zgougou after 24 h of spontaneous or autochthonous lactobacilli-driven fermentation was described by 16S metagenetic analysis. A total of 195,453 high quality sequences of 16S rRNA gene amplicons were obtained, with an average of about 24,400 sequences per sample and an average sequence length of 537 bp. Higher values of observed species, Chao1 and Shannon indexes were found for seeds, compared to juice, regardless of the use of autochthonous lactobacilli (Table 2). The juice obtained from zgougou fermentation with autochthonous lactobacilli (J LAB+) showed lower values of all the diversity indexes than the juice obtained upon spontaneous fermentation (J LAB-). A similar trend, although with lower differences, was found also when seeds from fermentation with autochthonous lactobacilli (S LAB+) were compared to seeds from spontaneous fermentation (S LAB-).  
J LAB- and S LAB- shared 12 bacterial OTUs (Table 3). _Enterobacter_ sp. was one of the dominant Operational Taxonomic Units (OTUs) in both J LAB- and S LAB-. _L. plantarum_ was the OTU found at the highest relative abundance in J LAB-, whereas it was subdominant in S LAB-. On the opposite, _Lactococcus raffinolactis_, subdominant in J LAB-, co-dominated bacterial community of S LAB-, along with _Enterobacter_ sp. Other sub-dominant OTUs found in zgougou obtained upon spontaneous fermentation were _Aeromonas_ sp. and _Acinetobacter_ sp.  
J LAB+ bacterial community was almost exclusively dominated by _L. plantarum_. Among the OTUs retrieved in S LAB+, _Weissella confusa_, _Pantoea_ sp., and especially _Enterobacter_ sp. showed the highest values of relative abundance. However, the abundance of _Enterobacter_ sp. was lower (\(p<0.05\)) than in S LAB-. An opposite trend was found for _Pantoea_ sp. Among the sub-dominant/minor OTUs, S LAB+ harbored _L. plantarum_, and _Aeromonas_ sp. and _Acinetobacter_ sp. at higher and lower (\(p<0.05\)) abundance, respectively, than S LAB-. Overall, in all the samples, a relative abundance ranging from ca. 13 to 37% could not be attributed to any classified bacterial taxonomic group (Table 3).  
DNA extracted from fermented zgougou was also used as template for describing fungal community, using 18S rRNA high throughput sequencing analysis. However, more than 98% of detected OTUs matched with DNA sequences of vegetable origin, namely _Pinus_ sp. Apart from this, ten fungal OTUs were detected, seven of which were molds (Figure S1). S LAB- was the only sample that harbored all the detected fungal OTUs.  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Sample** & **Observed Species** & **Chao1** & **Shannon** \\ \hline J LAB- & 17.0 \(\pm\) 1.41\({}^{\mathrm{c}}\) & 17.3 \(\pm\) 1.65\({}^{\mathrm{c}}\) & 1.71 \(\pm\) 0.032\({}^{\mathrm{c}}\) \\ S LAB- & 21.5 \(\pm\) 0.71\({}^{\mathrm{a}}\) & 21.5 \(\pm\) 0.71\({}^{\mathrm{a}}\) & 2.06 \(\pm\) 0.004\({}^{\mathrm{a}}\) \\ J LAB+ & 3.0 \(\pm\) 1.41\({}^{\mathrm{d}}\) & 3.0 \(\pm\) 1.41\({}^{\mathrm{d}}\) & 0.44 \(\pm\) 0.021\({}^{\mathrm{d}}\) \\ S LAB+ & 20.0 \(\pm\) 0.00\({}^{\mathrm{b}}\) & 20.0 \(\pm\) 0.00\({}^{\mathrm{b}}\) & 1.91 \(\pm\) 0.030\({}^{\mathrm{b}}\) \\ \hline \hline \end{tabular} \({}^{\mathrm{*}}\) Values (mean of two replicates \(\pm\) standard deviation) in the same column followed by different letters (a–d) are significantly different (\(p<0.05\)).  
\end{table}
Table 2: Alpha diversity indexes\({}^{\mathrm{*}}\) of _Bacteria_ (16S rRNA) found in the juice (J) and seeds (S) obtained after 24 h of zgougou fermentation (spontaneous, “LAB–”, or inoculated with autochthonous lactobacilli, “LAB+”).