# Use of Autochthonous Lactobacillusli to Increase the Safety of Zgougou
## 3 Discussion
In the seeds, it was sub-dominant, but was harbored at higher abundance in those obtained upon zgougou fermentation with lactobacilli. Among the sub-dominant/minor OTUs in the seeds, _Aeromonas_ sp. and _Acinetobacter_ sp. seemed to be controlled over by the use of autochthonous lactobacilli. _Aeromonas_ sp. includes species causing food-borne infections [27]. _Acinetobacter_ sp. includes food spoiler bacterial species [28], _Acinetobacter baumannii_, responsible for nosocomial infections [29], and other species probably responsible for food-borne diseases [30]. Unexpectedly, _Pantoea_ sp. seemed to be favored, in the seeds' debris, by the use of lactobacilli as starters for fermentation. However, we have to take into account the effect of chemical sterilization, which was carried out exclusively in the seeds destined to lactobacilli-driven fermentation. This treatment probably acted just on the surface of seeds and, therefore, did not inactivate _Pantoea_ sp., which is frequently reported as endophytic bacterial genus [31]. On the contrary, other bacterial species harbored on the surface layers of seeds were inactivated by sterilization. Fermentation of zgougou caused more complex VOC profiles of both juices and seeds, containing ca. 20 compounds, which had not been detected before fermentation. Some compounds, such as ethanol, acetic acid and acetoin, clearly derived from energy metabolism of bacteria. Besides being an end-product of fermentation of carbohydrates, a relevant amount of ethanol can participate in esterification of carboxylic acids, generating ethyl acetate (from acetic acid and ethanol), ethyl ester of hexanoic acid, and ethyl ester of octanoic acid [32]. Esters confer pleasant, sweet, fruity flavors to fermented food [32] and were found in rice fermented with _Aspergillus oryzae_ or _Rhizopus oryzae_[33]. Accordingly, in the current study, some esters (e.g., ethyl acetate in juice, butanoic acid pentyl ester) were detected just after fermentation. Acetic acid, at relatively low concentrations, may act as flavor enhancer in fermented food, such as _obushera_ obtained from spontaneous or starter-driven sorghum fermentation involving LAB and yeasts [34]. Acetoin, a yogurt-like odorant, was found at relatively high concentrations in rice fermented with _Lactobacillus fermentum_[33]. In the current study, zgougou fermentation differently affected concentrations of terpen, which are intrinsic VOC for Aleppo pine's organs (including cones, bringing seeds) and show antioxidant and antimicrobial activity [35]. Some terpen, such as limonene, may be used as flavoring and antioxidant additives in food [36]. In the current study, ethanol, acetoin, phenol,2-methoxy and cryophyllene were present at higher concentrations in juice and seeds obtained upon spontaneous zgougou fermentation, compared to the samples deriving from fermentation with autochthonous lactobacilli. Caryophyllene decreased in fermented dry sausage with increasing doses of immobilized cells of _Lactobacillus casei_[37]. The use of autochthonous lactobacilli for zgougou fermentation caused an increase of acetic acid, decane, 1-nonanol, bornyl acetate and bornyl formate in both juices and seeds. Acetic acid may be produced upon various fermentations, including hetero-lactic fermentation. _L. plantarum_ may operate hetero-lactic fermentation, depending on the presence of pentoses, whereas _Weissella_ sp. performs hetero-lactic fermentation, regardless of the type of carbohydrates in the ecosystem [38]. Fermented juices and seeds from fermented zgogou showed antibacterial activity, probably resulting from a combination of compounds naturally contained in Aleppo pine's seeds and products of LAB metabolism. Essential oil extracted from Aleppo pine's seeds contains several antimicrobial terpen, such as \(\alpha\)-pinene, \(\beta\)-myrrene, and caryophyllene [2], detected also in the zgogou samples analysed in this study. LAB may release a wide array of antimicrobial compounds, including organic acids, diacetyl and bacteriocins [39].