# Use of Autochthonous Lactobacillusli to Increase the Safety of Zgougou
## 3 Discussion  
Seeds of _Pinus halepensis_ are used for preparing zgougou, a spontaneously fermented matrix giving juice and seeds debris, consumed in many Arabian countries, including Tunisia [1; 2; 4]. Similarly, to all the food processes based on spontaneous fermentation, zgougou hides health risks due to eventual pathogenic microorganisms and derived toxins [19]. In this study, two lactobacilli, previously isolated from zgougou [18], were used as combined starters for driving fermentation, aiming especially to standardize the process and to inhibit the growth of undesired bacteria. The effect of these starters on microbiota of zgougou was evaluated by comparing the results of culture-dependent and -independent analyses, performed on juice and seeds obtained after lactobacilli-driven or spontaneous fermentation. Culture-dependent analyses after 1 h of fermentation showed that the basic bacterial biota of zgougou was mainly represented by LAB, regardless of the use of starter lactobacilli. Spontaneously fermented fruits and vegetables normally harbor LAB [20; 21]. As expected, the addition of autochthonous strains belonging to the _L. plantarum_ group in our zgougou samples favored mesophilic lactobacilli over the other LAB groups. During fermentation, coccus-shaped LAB and coliforms increased their number. However, the highest increases were found in spontaneously fermented zgougou samples (J LAB- and S LAB-). In addition, in the latter samples enterococci, microococci and staphylococci increased during fermentation. On the opposite, these bacterial groups remained constant or decreased after 24 h of fermentation with autochthonous lactobacilli (J LAB+ and S LAB+), suggesting that the starter lactobacilli exerted a target-dependent inhibitory effect towards undesired bacterial groups. The results of culture-dependent analyses indicated that the decrease of pH observed in the spontaneously fermented zgougou was due to the acidifying activity of LAB, including enterococci, naturally present in the Aleppo pine's seeds. As expected, the acidification of zgougou was higher when autochthonous lactobacilli were used as starters. In agreement with our results, an inhibitory effect of autochthonous LAB strains (belonging to _L. plantarum_, _Leuconostoc mesenteroides_, and _Pedicoccus pentosaceus_) towards coliforms was found during fermentation of carrots and French beans [22]. Figure 4: Sensory analysis of pudding prepared using juice from spontaneously fermented (P LAB–, ———) or fermented with autochthonous lactobacilli (P LAB+, ——) zgougou. Results from 16S metagenomic analysis of bacterial biota through Illumina MiSeq partially confirmed the results of culture-dependent analyses. In detail, _Enterobacter_ sp., belonging to the group of coliforms, and _Lc. raffinolactis_ were dominant in spontaneously fermented zygougou samples. On the contrary, the use of autochthonous lactobacilli, possibly in combination with chemical sterilization of raw matter (seeds), inhibited growth of _Lc. raffinolactis_ and/or inactivated this LAB during fermentation. Similarly, to the results of this study, the use of protective lactobacilli strains (belonging to _L. plantarum_ and _Lactobacillus rhamnus_) inhibited coccus-shaped LAB (lactococci and streptococci) during cheese-making [23]. _Lc. raffinolactis_ has been isolated especially from dairy environment (raw milk and ripening cheese), but rarely from vegetables [24]. The role of this LAB in food has not been elucidated so far. Inactivation/inhibition of _Enterobacter_ sp. occurred in juice obtained from zgougou fermentation with autochthonous lactobacilli and, at a much lesser extent, in seeds. _Enterobacter_ sp. has been reported as producer of biogenic amines [25]. In addition, it may occasionally cause food-borne infections [26]. _L. plantarum_ dominated the bacterial biota of fermented juices, especially, as expected, when autochthonous lactobacilli were used as starters.