*3.1. Olives' Fermentation*

Initial brine pH was 8.21, after 5 days it showed a pH of 7.22, representing an optimal condition for the proliferation of initial lactic bacteria but also enterobacteria. The pH decreasing was due to lactic bacteria, which ferment a wide variety of watersoluble carbohydrates and produce lactic acid [20,29,30]. In this phase the olives lost their

*Fermentation* **2021**, *7*, x FOR PEER REVIEW 6 of 12

In fact, enterobacteria counts showed a rapid increase within the first 10 days, followed by a sharp decline thereafter. No viable counts were enumerated after 25 days of fermentation, according with Sanchez et al. [28] (Figure 1). classic hardness: this indicates the presence of yeasts and molds that degrade pectin and cellulosic substances, important for the compactness of the olives [31,32].

**Figure 1.** Lactic bacteria (square), yeast and molds (triangle), enterobacteria (circle) concentration, reported as CFU per mL. **Figure 1.** Lactic bacteria (square), yeast and molds (triangle), enterobacteria (circle) concentration, reported as CFU per mL.

Lactic acid bacteria (LAB) and yeasts increased steadily and became the dominant members of the microflora during fermentation [33]. Lactic acid bacteria reached their maxima within the first 30 days (1.6× 108 CFU mL−1), and their enumeration was stable until the end of the fermentation process. Yeasts coexisted with lactic acid bacteria The pH decreasing was due to lactic bacteria, which ferment a wide variety of watersoluble carbohydrates and produce lactic acid [20,29,30]. In this phase the olives lost their classic hardness: this indicates the presence of yeasts and molds that degrade pectin and cellulosic substances, important for the compactness of the olives [31,32].

throughout the whole fermentation period. Their counts were lower than those of the LAB by approximately 3–4.5 log cycles. They grew in similar populations with lactic acid bacteria during the first week, and their presence was stable until the end of fermentation reaching 1.7× 104 CFU mL−1 (Figure 1). Subsequently, the pH of the brine after 25 days was 5.87. In this phase, no viable counts of enterobacteria were enumerated. At the end of the fermentation process, after 60 days, the olives were very soft and the epicarp was easily detached from the pulp, and the pH was 4.52. Lactic acid bacteria (LAB) and yeasts increased steadily and became the dominant members of the microflora during fermentation [33]. Lactic acid bacteria reached their maxima within the first 30 days (1.6 <sup>×</sup> <sup>10</sup><sup>8</sup> CFU mL−<sup>1</sup> ), and their enumeration was stable until the end of the fermentation process. Yeasts coexisted with lactic acid bacteria throughout the whole fermentation period. Their counts were lower than those of the LAB by approximately 3–4.5 log cycles. They grew in similar populations with lactic acid bacteria during the first week, and their presence was stable until the end of fermentation reaching 1.7 <sup>×</sup> <sup>10</sup><sup>4</sup> CFU mL−<sup>1</sup> (Figure 1).

The pH, throughout the fermentation phase, decreased slowly compared to the common fermentation processes of table olives (Spanish and Greek method) [19,34]; moreo-Subsequently, the pH of the brine after 25 days was 5.87. In this phase, no viable counts of enterobacteria were enumerated.

ver, anomalous phenomena such as the loss of hardness of the olives were observed [21,35,36]. At the end of the fermentation process, after 60 days, the olives were very soft and the epicarp was easily detached from the pulp, and the pH was 4.52.

*3.2. Oil Analysis*  The comparison of the values of acidity and peroxides of the NEFO and EVO oils are The pH, throughout the fermentation phase, decreased slowly compared to the common fermentation processes of table olives (Spanish and Greek method) [19,34]; moreover, anomalous phenomena such as the loss of hardness of the olives were observed [21,35,36].

#### shown in Table 1. NEFO oil showed a low number of peroxides (produced by oxidative *3.2. Oil Analysis*

ural fermentation [38,39]

rancidity processes) but a high amount of free fatty acids (produced by hydrolytic rancidity processes) [37]. The comparison of the results obtained is interesting; the acidity of NEFO oil is 255 times higher than the EVO oil, while the quantity of peroxides of the first oil is only 4.7 times higher than the second. The comparison of the values of acidity and peroxides of the NEFO and EVO oils are shown in Table 1. NEFO oil showed a low number of peroxides (produced by oxidative rancidity processes) but a high amount of free fatty acids (produced by hydrolytic rancidity processes) [37].

The high acidity of NEFO oil is caused by the lipases present in the pulp of the drupes but also by lipolytic enzymes produced by bacteria, yeasts, and molds developed in nat-

The comparison of the results obtained is interesting; the acidity of NEFO oil is 255 times higher than the EVO oil, while the quantity of peroxides of the first oil is only 4.7 times higher than the second.

The high acidity of NEFO oil is caused by the lipases present in the pulp of the drupes but also by lipolytic enzymes produced by bacteria, yeasts, and molds developed in natural fermentation [38,39]

The oxidative rancidity, which forms peroxides, was probably limited by the brine because it blocked the direct contact of the drupes with oxygen. Furthermore, some species of lactobacilli degrade hydrogen peroxides and prevent the formation of peroxide radicals and hydroperoxide radicals, thus limiting the formation of peroxides and stabilizing the drupes in brine [38,40,41].

Natural fermentation with aerobic conditions determines the high AV and low PV of non-edible olive oil, these results are in agreement with the results of Girgis A. Y. and Alajtal A. I. et al. [40,41].
