PLE is another technique that, nowadays, is regarded as an advanced extraction technique, due to the advantages that presents over other traditional extraction mechanism. PLE is based on the use of high temperatures and pressures so that the solvent is maintained in the liquid state during the whole extraction procedure. As a result of the application of these particular conditions, faster extraction processes are obtained in which generally the extraction yield is significantly higher than that obtained using traditional extraction techniques, besides, using lower amounts of organic solvents. Moreover, most of the instruments used for PLE are automated, allowing the development of less labor intensive methods and improving reproducibility.

The principles governing this kind of extraction and providing the above men­tioned characteristics are: (a) the mass transfer rate is improved as a result of the increment on the solubility of the compounds as a consequence of the increase of the extraction temperature; (b) under the PLE experimental conditions, the sur­face tension of the solvent is reduced, allowing a better penetration of the solvent into the sample matrix, increasing likewise the mass transfer; (c) the effect of the pressure theoretically could help to matrix disruption, increasing again the mass transfer rate.

Method development in PLE is by far easier than in SFE, since less parameters influencing the extraction should be considered. Once the solvent has been selected according to the nature of the compounds to be extracted, only two parameters are of significant importance: extraction time and extraction temperature. Although the extraction pressure could help to disrupt the matrix enhancing the mass transfer of the analytes contained on it, as it has been already mentioned, in practice, several reports have shown that the influence of this parameter is not significant once the pressure is high enough to maintain the solvent in the liquid state. The extraction temperature has to be optimized always keeping in mind the possible thermal deg­radation effects that might occur over the interesting extracted compounds. Although generally an increase in the temperature produces the subsequent increase in the extraction yield, for bioactive compounds, too high temperatures might lead to the degradation of these compounds. Therefore, this value should be carefully maxi­mized just to the level in which the interesting compounds start to get degraded. On the other hand, the extraction time has to be minimum enough to have an adequate mass transfer. Longer extraction times would result on slower extraction procedures and could also favor the thermal degradation, once the solvent solution is saturated with analytes from the food matrix. Therefore, quite simple experimental designs, such as full factorial designs with two factors and three levels can be useful to opti­mize the bioactives PLE extraction conditions.

Compared to SFE, the possibility of choosing among a high number of solvents causes PLE to be more versatile in terms of polarity of the bioactive compounds to be extracted and thus, the solvent will be selected depending on their nature. However, this technique is considered by far less selective than SFE. Therefore, it is important to keep in mind, that even if the extraction of the bioactives is attained, it would be possible to find other interfering compounds in the obtained extract. To avoid this problem, other steps can be included. For instance, an extraction step using hexane/acetone as solvent was performed before the PLE of phenolic com­pounds from several algae species using 80% methanol in water at 130°C for 20 min (two 10 min cycles) [132]. Ethanol has been selected to extract antioxidants from different species, such as Synechocystis sp. and Himanthalia elongata [143] or anti­microbial compounds from H. pluvialis [165]. Generally, the best extraction condi­tions in these applications were obtained at mild temperatures, around 100°C.

Moreover, PLE can be applied using a wide variety of extraction solvents, although GRAS extraction solvents, like ethanol, are most commonly used. When the extraction solvent is water, this technique is commonly called subcritical water extraction (SWE). The principles of extraction are the same, but in this case, another parameter has critical importance, the dielectric constant of water. This property of water is greatly modified with the increasing temperature when water is maintained in the liquid state. In fact, the value of dielectric constant of water (e) can vary from 80 at room temperature to values around 25 when is submitted to temperatures of ca. 250°C. This value is similar to the one presented by some organic solvents at room temperature, such as ethanol or methanol, and thus, the use of SWE could be an alternative to the use of this type of solvents in some applications. This technique has been already used to explore the possibility of obtaining antioxidants from dif­ferent microalgae species [52, 55]. However, the wide development of novel appli­cations for the extraction of bioactives from algae by using SWE has not been fully explored so far.