# The Influence of Processing on the Bioactive Compounds of Small Berries
## 3 Comparative Analysis of Extraction and Analytical Methods for Determining Antioxidant Activity
### Extraction Methods
The extraction yield of the bioactive compounds improves with an increase in the extraction time, at temperatures above 50 \({}^{\circ}\)C, so as to avoid possible degradation of the thermal-sensitive compounds, and at an ultrasound power of 40 kHz [53]. Ultrasound can be used to complement other conventional [12, 13, 14, 15] or unconventional extraction techniques, such as supercritical fluid and microwave extraction [29], allowing the extraction yield to be improved. With respect to conventional methods, the main advantages of ultrasound-assisted extraction are the short extraction time and high yield, while the most important disadvantage is the low reproducibility [7, 8]. Microwave is an electromagnetic force-based method employed to assist the extraction of bioactive compounds. The method involves a large frequency spectrum between 300 MHz and 300 GHz, in which the extraction of antioxidant compounds occurs upon the electromagnetic energy to heat conversion, as a result of the dipole rotation and ion conduction mechanisms [28, 54]. Due to the heating effect, the solvent penetrates the food matrix, produces the disruption of hydrogen bonds, and leads to the extraction of antioxidant compounds. The extraction yield depends on the microwave power, which ranges from 100 to 900 W [8]. The most suitable solvents used for microwave-assisted extraction are ethanol solutions of different concentrations and water [8, 23], but methanol, acetone, and acetonitrile can also be used for plant phenolic compound extraction [54]. The supercritical fluid extraction (SC-CO\({}_{2}\)) method involves the use of CO\({}_{2}\) as a solvent [8, 41, 42]. The main factors influencing the extraction efficiency are the particle size of the material, flow rate, and type of co-solvent. The material should be ground prior to extraction in such a manner to avoid the formation of too-small particles, which are prone to agglomeration in the channels of the equipment, and too-large particles, which decrease mass transfer. The flow rate is usually adjusted so as to maximize the solubility of the bioactive compounds and the mass transfer. The use of a co-solvent, such as water or ethanol, is required to improve the extraction yield of bioactive compounds [42]. The supercritical fluid extraction method is suitable for polyphenol, flavonoid, and carotenoid extraction [41, 42, 44]. The pressurized liquid extraction (PLE) method involves combining high pressures (4-12 MPa) and moderate or high temperatures (50-300 \({}^{\circ}\)C) with the use of organic solvents above their boiling point [8]. The most important parameters influencing the efficiency of the method are pressure, temperature, extraction time, and type of solvent [8, 42, 43, 44]. The use of high pressures and temperatures allows the solubility of antioxidant compounds and the mass transfer properties to be improved. The high-hydrostatic-pressure extraction (HPPE) method involves mixing pulverized dry material with a solvent of appropriate polarity in a sealed polyethylene bag, followed by treatment at very high pressures (100 to 1000 MPa) in a pressure vessel [8, 45]. The main operating parameters influencing the extraction of bioactive compounds are the type of solvent (pure or mixture of solvents; most often, aqueous ethanol is used), pressure, and extraction time. The high-hydrostatic-pressure extraction method was successfully used for polyphenol, anthocyanin, and flavonoid extraction from blueberries and strawberries [45, 46]. The pressure and extraction time increase ensured higher antioxidant activity of the extracts from blueberries and strawberries (Table 1), as a result of the better extraction efficiency of bioactive compounds [45, 46]. Pulsed electric field (PEF) extraction is a non-thermal method based on the electroporation of cell membranes, upon matrix exposure to an electrical potential [8, 55]. High-voltage electric current (10-80 kV/cm) treatment is usually applied as nano- to microseconds-long pulses [55].