## 4 Secondary Metabolites
### Extraction
[67] applied this protocol by blending the apple powder with each solvent for 5 min, using an Ultra-Turrax blender, and the mixture was filtered through a G3 sintered glass filter. Moreover, UAE, ASE and SFE play an important role as real potential sustainable technique for industrial applications for polyphenols extraction. In recent works, ASE conditions have been optimized for the extraction of polyphenols from apple peel and pulp, achieving good recovery and repeatability. ASE allows to reduce the volume of solvent required for the extraction, to shorten the analysis time and the handling  
Figure 3: Main compounds responsible for the taste and flavor of apples. necessary to obtain precise results [69]. As regard to SFE, several works aimed to recover phenolic compounds applying CO\({}_{2}\) in supercritical conditions without and with ethanol (5%) as co-solvent. As CO\({}_{2}\) is non-polar, it is not a favorable solvent for polar polyphenols. However, the addition of organic co-solvents which could improve the solvating power and the yield of the extraction, such as ethanol, methanol and acetone, could be a suitable strategy [70]. Instead, [PERSON] et al. [71] used UAE with a methanol: water mixture for the extraction of polyphenols. Ultrasound can reduce the operating temperature of extraction for thermolabile compounds; moreover, the cavitation process that occurs during sonication causes the rupture of cell walls, thus enhancing solvent contact with available extractable cell material [72]. UAE has also been used for the extraction of organic acids; water or alcoholic mixtures were used as extraction solvents [42; 55; 73; 74]. In regard to carotenoids and vitamins, their extraction from apple skin and pomace is usually carried out with acetone or a solution of acetone/petroleum ether as solvent [3; 53]. Since carotenoids are strongly susceptible to oxidative degradation, due to the high number of conjugated double bonds, extraction must be performed in dark conditions or by adding antioxidant compounds, such as butylated hydroxoluene (BHT), to the extraction solvent, in order to prevent photo-isomerization phenomena [75]. Unlike apple polar triterpenoids, such as betulinic and unsolic acid, whose extraction is carried out by using polar solvents (i.e., methanol and acetone) [76], a separate discussion is needed for the extraction of terpen from apple tissues. As they are non-polar and highly volatile compounds, the better method for their extraction is the solid-phase microextraction (SPME), followed by their analysis by gas chromatographic (GC) methods. With SPME, the analytes are absorbed from the liquid or gaseous sample onto an absorbent coated fused silica fiber, which is part of the syringe needle, for a fixed time. The fiber is then inserted directly into a GC injection port for thermal desorption [77]. Other advantages of this technique are the absence of solvents and the possibility to separate and pre-concentrate the analytes in a single step [78]. Therefore, it is really helpful for the extraction of volatile compounds from apples, due to their low concentration and the complexity of the matrix [79]. As fiber coatings, DVB/CAR/PDMS (Divinylbenzene/Carboxen/Polydimethylsiloxane) and PDMS/DVB have been mainly used because they offer higher extraction efficiency and a clear pattern of volatile compounds [57; 80]. Despite its several advantages, the limited amount of stationary phase on the fused silica fiber used for the SPME often does not ensure enough sensitivity and reproducibility. To overcome this problem, [PERSON] et al. [81] successfully applied stir bar sorption extraction (SBSE), a variation of SPME consisting on the use of magnetized bars covered with an absorbent polymer, for the extraction of apple pomace aroma. SBSE has the same advantages than SPME, but its sensibility increases around 100-fold because it uses a greater amount of stationary phase.