## 3 Results and Discussion  
### Olive Oil Bodies Extraction  
The patented protocol, mentioned above, was properly adapted to the OP by applying some modifications described in the following. First, the raw material was stored at \(-20\)\({}^{\circ}\)C rather than at 25 \({}^{\circ}\)C, as suggested in the patent protocol for the oleaginous seeds [24]. This step was necessary to eliminate the issues linked to the seasonality of the product, which could lead to excessive process stress, and to achieve a facile separation of the free lipid fraction, here called oil, of the olive paste and water phases from the solid using cryoconcentration. Before the extraction, we thawed and homogenised the OP (40 g) in 200 mL of the GM. We chose to reduce the wt/vol ratio of the OP/GM at 1:5 instead of at 1:10 [24], because it is more suitable for olive-based raw materials [35]. Applying the protocol stated by [PERSON] et al. [24], consisting of the repeated dilution of the cream collected in the three different liquid buffers (FB\({}_{1}\), FB\({}_{2}\), FB\({}_{3}\)), we obtained no separation of the solid phase from the liquid phase. This behaviour is due to a low difference in density between the liquid solution and solid material and an unsuitable pH. Owing to the low extraction yield obtained, we proceeded to define a new, called Method 1, described in the following. First, we increased the concentration of the solutes in GM, FB\({}_{1}\), FB\({}_{2}\) and FB\({}_{3}\) to improve the density difference between the solid and the liquid phases, as suggested for the separation of liquid and solid phases in the case of olives [35; 36]. Therefore, the compositions of the GM and of the FBs which we used were: GM (1.2 M sucrose), FB\({}_{1}\) (0.8 M sucrose, 1 M NaCl), FB\({}_{2}\) (0.4 M sucrose, 1 M NaCl), FB\({}_{3}\) (3.2 M NaCl). The pH of the suspensions was almost constant at 5.4. After each dispersion step, the suspension was centrifuged at 4 \({}^{\circ}\)C, 3226\(\times g\) for 40 min [24]. The initial composition of the olive paste was 20%. After the first extraction step, the yield was 32%, to follow, in the case of the others extraction steps, the yields were 30%, 25%, and finally a yield of 21.4% was measured at the end of the extraction process. After each centrifugation step, the liquid phase, below the cream containing the OBs, showed a cloudy appearance, highlighting small, suspended solid particles. To eliminate this phenomenon, after each centrifugation step, we introduced a novel step in the extraction protocol reported in the patent [24], called _freezing_. The container with the partially separated suspension was frozen at \(-20\)\({}^{\circ}\)C, thus increasing the collection of OBs by more than two-fold. We recovered the cream and three away the oil (i.e., the free lipid fraction of the olive paste). Each freezing step lasted about 3 h, resulting in an almost total recovery of solid fraction in the container. Figure 1 summarises the main steps of the new extraction, Method 1. Figure 1: Flow chart for OOBC extraction with Method 1. C, centrifugation; F, freezing; R, recovery of OOBC. After the last step of the extraction protocol (see Figure 1), we obtained the cream here called OOBC. As we have stated before, the extraction protocol drives the formation of an upper layer with a creamy texture. Figure 2 illustrates the OOBC extracted. Although the protocol described above yields high extraction efficiencies (47.5%), it is time-consuming (the extraction time was at least 1 week) and is therefore not ideal for industrial applications. Considering these limitations, efforts were expended to determine a new, easy, and quick extraction method which we called Method 2. The outstanding novelty was that we exclusively used one buffer solution in the whole extraction process, whereas Method 1 needs GM, FB1, FB2 and FB3. This buffer solution had suitable chemical-physical characteristics such as reagents used, pH and density. Due to them, we were able to use it in the grinding and in the subsequent centrifugation steps.