# Spontaneous and Controlled Fermentation Tests in Industrial Table Olives Production
## 2 Materials and Methods  
### Experimental Trial  
Olives of cv. Leccino (caliber 17+) were supplied by the company Ficacci Olive Co. (Castel Madama RM, Italy) as part of a batch that fitted several hundred barrels. The company allowed us to use five barrels, each containing about 140 kg of olives and 80 kg of brine. The barrels were randomly selected from the entire batch on 6 November 2021. The initial brine inside the barrels was 13% NaCl in non-sterilized potable water. These barrels were named N1, N2, G1, G2, and FUO, as listed in Table 1. N1, N2, G1, and G2 were placed inside in a company shed, while the barrel named FUO followed all the other barrels of the production batch, and was stored outdoors in a yard exposed to bad weather and sudden changes in temperature (Figure 1). We then left barrels N1 and N2 to spontaneous fermentation due to the natural epiphytic microflora, while barrels G1 and G2 were inoculated with a mix of _Lactiplantibacillus plantarum_ (LAB) B1 strain derived from the CREA-IT Pescara microorganisms collection and _Saccharomyces cerevisiae_ strain CS24 from the CREA-IT Turin collection. The inoculum \begin{table}
\begin{tabular}{l l} \hline \hline
**Code** & **Description** \\ \hline N1 & Spontaneous Fermentation (replica 1) \\ N2 & Spontaneous Fermentation (replica 2) \\ G1 & Guided Fermentation (replica 1) \\ G2 & Guided Fermentation (replica 2) \\ FUO & Spontaneous Fermentation (sealed barrel) \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Barrel codes list. load was 1 \(\times\) 10\({}^{8}\) CFU/mL. The choice of the B1 strain for LAB was settled after a deep screening of the various strains present in the collection [11]. The choice to add a yeast to the inoculum, and especially _S. cerevisiae_ SC24, was due to the known potential role of this yeast to protect the fermentation from spoilage thanks to their killer activity [14; 17; 18]. The FUO barrel was chosen randomly from hundreds of barrels, and it was identical to N1 and N2, except for two differences:  
1. it was kept sealed until the end of the fermentation process;
2. its position, because it was placed in an open space together with all the other barrels. Barrels N1, N2, G1, and G2 have been periodically opened to take olives and brine samples to analyse chemical and microbiological parameters, according to the method suggested by [PERSON] et al. [19]. The FUO barrel was kept sealed until the end of the fermentation process (210 days). Once the correct filling of the 4 barrels had been verified, a special internal crate made from polymeric material intended for food was put just below the cap to prevent the olives rising from out of the brine, thus avoiding the formation of a yeast film [20]. Finally, the barrels were closed with the appropriate screw cap. After a conditioning period, set at 23 days, the inoculations were carried out on barrels G1 and G2 with a 1:1 ratio of YPD Broth (Yeast-extract Pettone Dextrose). According to the schedule illustrated in Table 2, periodic visits were made to the facility to obtain samples of brine and olives, always taking care to replace the removed content with fresh brine in order not to leave excessive empty space in the barrel and avoiding the oxidative action of air. Globally, we performed 7 samplings, as shown in Table 2. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Date (dd/mm/yyyy)** & **Days** & **Description** \\ \hline
6 November 2021 & 0 & Barrels preparation \\
2 December 2021 & 23 & Inoculation of barrels G1 and G2 \\
9 December 2021 & 30 & 1 st sampling (s.) \\
16 December 2021 & 37 & 2 nd sampling (s.) \\
13 January 2022 & 65 & 3 rd sampling (s.) \\
3 March 2022 & 114 & 4 th sampling (s.) \\
16 June 2022 & 219 & 5 th sampling/end fermentation (EP) \\
20 September 2022 & 315 & 6 th sampling/shelf-life 3 months (S3) \\
7 December 2022 & 388 & 7 th sampling/shelf-life 6 months (S6) \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Samplings schedule. Figure 1: Test barrels.