*3.1. Pecan Nut Kernels*

### 3.1.1. Moisture and Oil Content

The moisture of dry pecan nuts used as reference was 2.4 ± 0.1 g/100 g db, while control kernels soaked at 3, 20 and 35 min had moisture contents of 13.5 ± 0.6, 21.1 ± 0.9, and 21.7 ± 0.7 g/100 g db, respectively. In PEF-treated kernels moisture contents also increased reaching values of 13.5 ± 1.0, 18.0 ± 0.1 and 19.3 ± 1.1 g/100 g db at 0.8, 7.8 and 15.0 kJ/kg pretreatments, respectively. However, the moisture of kernels processed at 7.8 and 15.0 kJ/kg was significantly lower than their respective control kernels (*p* = 0.004 and *p* = 0.015, respectively). Regarding the oil content, reference kernels contained 69.4 ± 0.7 g/ 100 g db, decreasing to 62.7 ± 0.0 g/100 g db in control kernels and 62.7 ± 0.7 g/100 g db in kernels pretreated by PEF, containing 9.7% less oil than reference kernels. Likewise, the oSW was 5.7 ± 0.0 g for both control and PEF-treated kernels.

### 3.1.2. OEY, OEYTOTAL and Microstructure

OEY and OEYTOTAL of dry pecan nuts are displayed in Figure 1. The highest OEY was observed for reference kernels, being 88.7 ± 3.0%. Control kernels yielded 71.3 ± 1.0, 65.9 ± 3.1 and 72.3 ± 0.7% at 3, 20 and 35 min of soaking, respectively, while OEY of PEF-treated kernels was 74.3 ± 1.1, 69.1 ± 2.1 and 70.6 ± 3.2% at 0.8, 7.8 and 15.0 kJ/kg pretreatments, respectively. No statistical differences were observed between OEY of control and PEF-treated kernels submitted to comparable soaking times.

After considering the oSW, OEYTOTAL of control kernels increased up to 82.0 ± 1.0, 76.5 ± 3.1 and 83.0 ± 0.7% for 3, 20 and 35 min of soaking, respectively, and OEYTOTAL of PEF-treated kernels rose to 85.0 ± 1.1, 79.8 ± 2.1 and 81.3 ± 3.2% for 0.8, 7.8 and 15.0 kJ/kg pretreatments, respectively (Figure 1). No statistical differences were observed between control and PEF-treated kernels. Kernels processed at 0.8 kJ/kg were selected for the microstructural analysis.

**Figure 1.** Oil extraction yields and total oil extraction yields (OEYTOTAL) of dry pecan nuts. Reference kernels were not soaked nor PEF processed. Control kernels ( ) were placed in tap water for 3, 20 and 35 min corresponding to PEF-treated kernels ( ) processed at 0.8, 7.8 and 15.0 kJ/kg, respectively. OEYTOTAL is the oil extraction yield estimated with the oSW.

In order to compare the microstructure of PEF-treated and control kernels, a micrograph of the transversal section of dry kernels cotyledon tissue reported by Wakeling et al. was employed (Figure 2) [30]. In this micrograph, cells delimited by the cell wall containing intracellular oleosomes stained with toluidine blue were showed.

**Figure 2.** Light microscopy micrograph of cotyledon tissue of dry pecan nuts reported by Wakeling et al. [30] employed to compare the microstructure of control and PEF-treated kernels.

In the light microscopy of control kernels (Figure 3a), the cotyledon tissue exhibited similarities to the micrograph of Wakeling et al. [30] displaying delimited cells and oleosomes within the intracellular space stained with Richardson blue. However, at higher magnification, it appeared that the cotyledon tissue was composed of both intact and damaged cells. Additionally, oleosomes seemed to change their shape and aggregate within the intracellular space (Figure 3b). Concerning kernels pretreated by PEF, micrographs showed compaction of cells in testa and cotyledon tissues with a loss of delimited inclusions in the intracellular space (Figure 3c). It seems that PEF processing, rather than inducing the cell rupture, produced the rupture of intracellular inclusions. In Figure 3d, the higher magnification of PEF-treated kernels cotyledon tissue showed no difference between intact and damaged cells due to oleosomes fusion in the periphery of the cell.

**Figure 3.** Light microscopy micrographs of testa and cotyledon tissue of dry pecan nuts. Control kernels (**<sup>a</sup>**,**b**) were placed in tap water for 3 min while PEF-treated kernels (**<sup>c</sup>**,**d**) were processed at 0.8 kJ/kg. Ts, testa; Ct, cotyledon tissue; C, cell; Cd, damaged cell; Cw, cell wall; Ol, oleosomes.

### *3.2. Pecan Nut Oil Stability*

The acidity of oils extracted from dry pecan nuts ranged from 19.0 ± 0.9 to 21.8 ± 0.9 mg KOH/100 g (Table 1). Only the oil extracted from kernels processed at 15.0 kJ/kg was significantly different, being 14.2% higher than its control. Concerning AC of extracted oils, it varied from 45.7 ± 2.2 to 49.0 ± 1.7 mg Trolox/100 g with no significant differences between experimental groups (Table 1).

Based on acidity and AC results, OSI, phytosterols concentration and LOX activity were determined for kernels pretreated at 0.8 kJ/kg, their control kernels, and reference kernels. The OSI values were 10.4 ± 0.4, 10.9 ± 0.3 and 10.6 ± 0.5 h for reference, control and PEF-treated oils, respectively. Neither soaking nor PEF processing significantly changed the stability index of pecan nut oil. Regarding the phytosterols content, oil from reference, control and PEF-treated kernels contained a β-sitosterol concentration of 929.0 ± 89.3, 858.2 ± 62.6 and 910.5 ± 132.2 mg/kg, respectively, while stigmasterol ranged from 324.4 ± 48.5 to 501.5 ± 79.7 mg/kg (Table 2). Campesterol was not detected despite that it has been reported in low concentration in pecan nuts [31,32]. Neither β-Sitosterol nor stigmasterol significantly changed among extracted oils.



*W*, specific energy input. Reference kernels were not soaked nor PEF processed. Control kernels were placed in tap water for 3, 20 and 35 min corresponding to PEF-treated kernels processed at 0.8, 7.8 and 15.0 kJ/kg, respectively. Oil acidity and AC were expressed per 100 g of pecan nut oil. Means with an asterisk indicated a significant difference from the control (α = 0.05).



*W*, specific energy input. Reference kernels were not soaked nor PEF processed. Control kernels were placed in tap water for 3 min corresponding to PEF-treated kernels processed at 0.8 kJ/kg. Concentrations were expressed per kg of pecan nut oil. Means were not significantly different from the control (α = 0.05).

The LOX activity of reference, control and PEF-treated kernels was 4.52 ± 0.14, 4.83 ± 0.10 and 4.41 ± 0.20 μmol/L·s, respectively. Reference and PEF-treated kernels showed LOX activity values significantly lower than control kernels (*p* = 0.006 and *p* = 0.001, respectively). It seemed that the application of PEF at 0.8 kJ/kg did not promote lipid oxidation of pecan nut oil. These results agreed with acidity, AC, and OSI values of the oil extracted from kernels processed at 0.8 kJ/kg. However, it should not be discarded lipid oxidation via enzyme activation since the acidity of pecan nut oil increased at higher *W*.
