Volatile, Sensory and Functional Properties of HydroSOS Pistachios

Climate change, the increase in world population, and the intensification of urban and industrial activities, will cause a shortage of water for agriculture. This situation requires conscientious studies to manage water deficits without affecting the quality of the crops. In this study, regulated deficit irrigation (RDI) strategies and three rootstocks (P. atlantica, P. integerrima, and P. terebinthus) were applied to pistachio cultivation to study the quality of fruits obtained based on the morphological, functional, aroma, and their sensory properties. The results obtained demonstrated that RDI T1 (during phenological phase II of cultivation the stem water potential was maintained around −1.5 MPa) led to pistachios with same morphological properties, total polyphenol content, antioxidant activity, volatile composition, sensory properties, better profile of fatty acids, and being the favorite ones for international consumers, as compared to pistachios obtained under full irrigation treatments. On the other hand, when P. integerrima was used, pistachios obtained had the highest weight, the lowest content of sucrose and the best functional properties.


Introduction
Mediterranean and South American countries, Southern California, Southern Australia and South Africa are characterized by partially wet springs and autumns, mostly rainy winters and hot dry summers. Water scarcity and water deficits in plants, mainly due to scarce rainfall, must be supplemented with irrigation treatments. In addition, different factors, such as climate change, the increase in world population, and the intensification of urban and industrial activities, will cause a shortage of water for agriculture, and it will become more and more severe in the near future [1]. This situation requires more conscientious studies to manage water deficits without affecting the quality of crops. These studies should focus on crops which are able to withstand deficit irrigation or have low water needs but without drastic impacts on production and fruit quality [2].

Sensory Analysis
The sensory analysis of samples was focused only on the analysis of the pistachios obtained under different irrigation treatments, in order to minimize the number of samples and, thus, maintain the panelists concentration to the maximum. Based on previous results [7], pistachio nuts obtained by P. atlantica rootstock were used on the sensory study.
To obtain information about the consumer opinion on the sensory properties of pistachios, a sensory evaluation with consumer panel was carry out in 3 countries: Mexico, Poland, and Spain. At least 60 consumers were recruited in each country. Consumers had to complete a screening questionnaire stating their age, gender, and allergies or diet restrictions. Consumers were asked about nut consumption frequency and willingness to taste pistachios. Consumers who stated that they were 18-70 years old, had no diet restrictions or allergies, ate any kind of nut at least once per week and were willing to taste pistachios were recruited for testing. In the specific case of Poland, the ballots, screeners and demographic questionnaires were translated from Spanish to Polish, and, then, back to Spanish.
Ten pistachio nuts were served, to each panelist, in odor-free disposable 60 mL covered plastic cups, coded using three-digit numbers, and at room temperature. Unsalted crackers and drinking water were used between samples to clean the panelists' palate. Natural illumination was used during the test, and testing room was at 20 ± 2 • C.
Consumers responded using a 9-point hedonic scale, where 9 = like extremely and 1 = dislike extremely. Consumers were, then, asked to indicate their order of preference for the samples, and mark the reasons of their preference regarding the attributes under study (size, peel, color, pistachio-ID, toasted, sweet, sour, aftertaste, oiliness, hardness, crunchiness, friability and adhesiveness). Then, consumers were asked about their "global" satisfaction degree for the samples under evaluation and for their intent to purchase.

Determination of Sugars and Organic Acids
Sugars and organic acids were identified and quantified according to Hernández [15], with some modifications. Approximately 1 g of sample was diluted in 5 mL of phosphate buffer (pH 7.8), homogenized by Ultra-TurraxTM (IKA L004640, Staufen, Germany) for 1 min, and centrifuged at 15,000× g for 10 min. Finally, samples were filtered through a 0.45 µm Millipore filter. For the determination of the content of sugars and organic acids on samples, an HPLC (high-performance liquid chromatograph) Hewlett-Packard series 1100 (Hewlett-Packard, Wilmington, DE, USA) was used. The elution buffer consisted of 0.1% phosphoric acid with a flow rate of 0.5 mL/min. Sugars and organic acids were isolated using a Supelco column (Supelcogel TM C-610H column 30 cm × 7.8 mm, Supelco, Inc., Bellefonte, PA, USA) and a precolumn Supelguard (5 cm × 4.6 mm; Supelco), and the absorbance was measured at 210 nm using a diode-array detector (DAD). Standards of sugars (glucose, fructose, sucrose, raffinose, maltitol, and sorbitol) and organic acids (oxalic, citric, tartaric, malic, quinic, shikimic, succinic and fumaric) were obtained from Sigma (Poole, UK). Calibration curves were used for the quantification of sugars and organic acids, showing good linearity (R 2 = 0.999). Results for both organic acids and sugars were expressed as concentrations g/L of dry weight (dw).

Total Polyphenol Content and Antioxidant Activity
For the total polyphenol content (TPC) determination and the antioxidant activity of the pistachios affected by rootstock and irrigation treatments, a methanol extract was prepared. Half a gram of pistachios (crushed with a grounder) was introduced in a test tube with 10 mL of MeOH/water (80:20, v/v) in 1% HCl. Then, the mixture was sonicated at 20 • C for 15 min and left at 4 • C for 24 h. After that, the extract was sonicated again for 15 min and centrifuged at 10,000× g for 10 min [16].
TPC was quantified using the Folin-Ciocalteu colorimetric method described by Gao [17], with some modifications. To 0.1 mL of the methanolic extract was added 2 mL of distilled water, 0.2 mL of Folin-Ciocalteu reagent, and was incubated for 3 min at room temperature. After that, 1 mL of 20% sodium carbonate was added and the mixture was incubated again for 1 h [16]. The absorbance was determined by measurement at 765 nm using an UV-visible spectrophotometer (Thermo Fisher Scientific Helios Gamma model, UVG 1002E, Waltham, MA, USA). Quantification was carried out according to the standard curve of gallic acid. The results were expressed as gallic acid equivalents (GAE), mg/kg (dw).
For the analysis of the antioxidant activity, the methods ABTS + [18], FRAP [19], and DPPH • [20] were used. Ten microliters of the supernatant of the methanolic extract was mixed with 990 µL of reagent ABTS + or FRAP. After a reaction time of 10 min, the absorbance was measured at 734 nm for ABTS + and 593 nm in case of FRAP method. For the DPPH method, 10 µL of the supernatant was mixed with 40 µL of MeOH and 950 µL of DPPH • solution. Then, the mixture was shaken, placed under dark conditions (15 min), and its absorbance was determined at 515 nm. The results obtained on the analysis of the antioxidant activity of pistachio samples were expressed as mmol Trolox/kg dw.

Fatty Acids
The determination of fatty acid methyl esters (FAMEs) was carried out according to Noguera-Artiaga [16]. A gas chromatograph Shimadzu GC-17A (Shimadzu Corporation, Kyoto, Japan), coupled with a Shimadzu mass spectrometer detector (GCMS QP-5050A) was used for the analysis of the organic layer of the pistachio extracts. The chromatograph was equipped with a column (polar) Suprawax 280, 100% polyethylene glycol (Teknokroma S. Co. Ltd., Barcelona, Spain; 30 m × 0.25 m × 0.25 µm film thickness). Helium (flow rate of 1.1 mL/min) was used as a carrier gas (split ratio 1:10). The temperature on the injector was 230 • C; on the detector, the temperature was 260 • C. The oven program and the identification of peaks were carry out following the method described by Carbonell-Barrachina [7] and Noguera-Artiaga [16].

Morphological Analysis
Twenty-five pistachio nuts from each treatment were randomly selected and used to determine the size, weight, and color. In addition, pistachios were classified as non-split open, split open, and others (uncommercial: unpeeled, broken shell, dark color, etc.). For the determination of the size, the height, width and length of the edible part from each pistachio were measured using a digital caliper (model 500-197-20 150 mm; Mitutoyo Corp., Aurora, IL, USA). In case of weight, the whole nut, shell and edible part were weighed (model AG204 scale; Mettler Toledo, Barcelona, Spain) with a precision of 0.1 mg. For the color, these 25 pistachios were ground (Taurus Aromatic Ver II; Taurus Group, Barcelona, Spain) and placed in Petri dishes (100 mm × 15 mm). A colorimeter Minolta model CR-300 (Minolta, Osaka, Japan) with an illuminant D65 and an observer of 10 • was used for the measuring of color of samples. Color data were provided as CIEL*a*b* coordinates.

Statistical Analysis
The data presented in this study are the mean values of 3 replicates and was subjected to two-way (irrigation treatment and rootstock) analysis of variance (ANOVA). Then, data were subjected to Tukey's multiple-range test to compare the means. Percentage data were transformed by Arcosen function before statistical analysis. Differences were considered statistically significant at p < 0.05. All statistical analyses were done using XLSTAT software (Addinsoft, version 2014.1, Paris, France).

Volatile Compounds
Thirty-one compounds were identified in the volatile profile of pistachios under study (Table S1) and were characterized according their sensory descriptors. The three most abundant compounds were α-pinene (~35%), limonene (~14%), and β-myrcene (~11%), and it was demonstrated that both irrigation and rootstocks significantly affected the content these compounds (Table 1). These results agreed with those obtained by Hojjati [21] and Penci [22] in previous studies, who also found that the same main compounds predominated in the aromatic profile of pistachios and its essential oils, respectively.
The volatile composition of the T1 pistachios showed no significant differences with that of the control samples (T0). On the other hand, pistachios obtained under RDI T2 had lower amounts of α-pinene, dodecane and tridecane, but higher ones of β-myrcene and limonene than T0. Regarding the effect of the pistachio rootstock, P. integerrima had the highest content of α-pinene (the predominant compound); P. atlantica led to nuts with the highest content of β-myrcene, dodecane and tridecane; and, P. terebinthus had the highest content of limonene.
In previous studies, Carbonell-Barrachina [7] demonstrated that RDI treatments led to pistachio nuts with similar or even higher amounts of the main volatile compounds. These results are in concordance with those obtained in the current study, in the sense that both studies demonstrated that the application of RDI strategies had no negative effects on the volatile composition of pistachio nuts.
Based on the study of the interaction between the two studied factors (irrigation and rootstock), to obtain pistachios with the highest possible content of α-pinene, it is necessary to use the rootstock P. integerrima and the irrigation treatments T0 or T1. On the other hand, if the objective is to obtain pistachios with more citrus aroma (more limonene), the combination of P. terebinthus rootstock and irrigation treatment T1 will be the most successful one.
According to results obtained, the main volatile compounds found in the volatile profile of pistachios α-pinene, limonene, and β-myrcene (compounds sensory related with descriptors of woody, citrus and fruity, respectively) can be used as indicators to evaluate the pistachio aroma quality.

Sensory Analysis
Around 200 consumers from Mexico, Spain and Poland (at least 60 in each country) participated in the pistachio affective sensory analysis. In Mexico, a 68% of panelist were women, 37% in Poland, and 55% in case of Spain. Of the total number of consumers, 35% were between 18 and 25 years old, 32% were between 26 and 35, 15 % between 36 and 45 years old, 17% between 46 and 55 years old, and 2% were older than 55 years old.
The irrigation treatments significantly affected three out of the 13 sensory attributes under analysis ( Table 2): pistachio-ID, oiliness, and overall. Pistachios obtained under RDI T1 obtained higher intensities of pistachio-ID (6.7) than control and T2 (6.4 and 6.4). This result was observed in each of the countries under study ( Table 2).
The oiliness of T1 and T0 samples were slightly but statistically higher, 6.0 than T2 samples, 5.8. Mexican consumers liked the oiliness of the pistachio samples less than Polish and Spanish consumers; a similar trend was observed for hardness and crunchiness. The most consumed dried fruit in Mexico is peanut, so it is possible that consumers in this country are used to high intensities of these attributes and were expecting a little more oiliness, hardness and crunchiness in pistachio samples, hoping to find a texture similar to that of fried peanuts.
In addition, in case of overall liking (the attribute that define the final opinion of consumers about the overall quality of sample), the T1 treatment obtained the highest score (6.7), while T0 got 6.3; T2 was statistically related with both of them (score of 6.5). Regarding the factor country, this same trend was observed in Mexico and Spain. However, there were no statistically significant differences in the satisfaction degree of Polish consumers regarding the three irrigations treatments (Table 2).
When consumers were forced to choose (among the three samples studied) which was their favorite sample, T1 pistachios were the most liked ones in each of the countries (in case of Poland, no statistically significant differences were observed between T1 and T0). On the other hand, the least liked sample in all countries was T2 ( Figure 1). Similar trends were observed when consumers were asked about their willingness to pay for the samples under study. Consumers mentioned that the main reasons for selecting the preferred sample (the most liked one) were: (i) pistachio flavor (~83%), (ii) crunchiness (~65%), (iii) aftertaste (~45%) and (iv) hardness (~30%) (Figure 1). Similar results were reported in previous studies; for instance, Carbonell-Barrachina [7] studied the purchase drivers of international pistachio consumers were pistachio flavor, saltiness, crunchiness and toasted flavor. Although, Noguera-Artiaga [23] concluded that international consumers preferred intense crunchy but low salty nuts. Table 1. Relative content (%) of volatile compounds on pistachios affected by regulated deficit irrigation and rootstocks.

Compound
ANOVA Test and P. terebinthus) were applied to pistachio cultivation to study the quality of fruits obtained based on the morphological, functional, aroma, and their sensory properties. The results obtained demonstrated that RDI T1 (during phenological phase II of cultivation the stem water potential was maintained around −1.5 MPa) led to pistachios with same morphological properties, total polyphenol content, antioxidant activity, volatile composition, sensory properties, better profile of fatty acids, and being the favorite ones for international consumers, as compared to pistachios obtained under full irrigation treatments. On the other hand, when P. integerrima was used, pistachios obtained had the highest weight, the lowest content of sucrose and the best functional properties.

Introduction
NS: not significant at p < 0.05; * and **: significant at p < 0.05 and 0.01, respectively. ‡ Values (mean of 200 replications) followed by the same letter, within the same sensory attribute and factor, were not significantly different (p < 0.05), Tukey's least significant difference test.

Sugars and Organic Acids
Three sugars (maltitol, raffinose and sucrose) and three organic acids (fumaric, oxalic, and shikimic) were identified and quantified in the pistachio samples under study (Table 3). In a previous study, Luh [24], identified four main sugars present in pistachios: sucrose, fructose, glucose and raffinose, being sucrose the main sugar, representing~40% of the total content. T1 samples had lower concentration of fumaric acid (0.287 g/L) than control pistachios (T0 = 0.315 g/L), while T2 nuts (0.287 g/L) were statistically related with both T0 and T1. In the rest of organic acids and sugars, no statistically differences were found among pistachios obtained under different irrigation treatments (Table 3).
Regarding rootstocks, P. integerrima led to pistachios with lower sucrose concentration (19.77 g/L) than P. terebinthus and P. atlantica (22.51 and 24.98 g/L, respectively). In the analysis of organic acids, P. integerrima was the rootstock having the highest concentrations of the three studied acids, while P. terebinthus had the lowest amount of oxalic and fumaric acids (Table 3). Table 3. Sugars (g/L) and organic acids (g/L) on pistachios obtained under regulated deficit irrigation and different rootstocks.

Raffinose Sucrose Maltitol Oxalic Shikimic Fumaric
ANOVA Test study, regulated deficit irrigation (RDI) strategies and three rootstocks (P. atlantica, P. integerrima, and P. terebinthus) were applied to pistachio cultivation to study the quality of fruits obtained based on the morphological, functional, aroma, and their sensory properties. The results obtained demonstrated that RDI T1 (during phenological phase II of cultivation the stem water potential was maintained around −1.5 MPa) led to pistachios with same morphological properties, total polyphenol content, antioxidant activity, volatile composition, sensory properties, better profile of fatty acids, and being the favorite ones for international consumers, as compared to pistachios obtained under full irrigation treatments. On the other hand, when P. integerrima was used, pistachios obtained had the highest weight, the lowest content of sucrose and the best functional properties.

Introduction
NS: not significant at p < 0.05; ***: significant at p < 0.001. ‡ Values (mean of 3 replications) followed by the same letter, within the same column and factor, were not significantly different (p < 0.05), Tukey's least significant difference test.
The application of moderate regulated irrigation treatments (T1) on the cultivation of pistachios had no statistically incidence on the AA and TPC of nuts. On the contrary, when the water restriction was severe (T2), the AA of pistachios was reduced (according to DPPH and FRAP methods). Under situations of moderate water stress, plants redistribute the CO 2 to the formation of secondary metabolites as a physiological response for the removal the free radicals formed; while under high stress, this CO 2 is mainly used by primary metabolism [27,28]. Same results were found, in previous studies under similar conditions, by Noguera-Artiaga [16] who obtained that hydroSOS pistachios had same or even higher TPC than control samples. Similar results of AA and TPC were found in previous studies with pistachios affected by different irrigation treatments [4,7,29].
In case of the study of rootstocks, P. integerrima led to obtain pistachio nuts with the highest concentrations of TPC and AA than rootstocks P. atlantica and P. terebinthus (Table 4). In previous studies, no significant differences were found in the functional composition of pistachios obtained through these same rootstocks.

Introduction
NS: not significant at p < 0.05; * and **, significant at p < 0.05 and 0.01, respectively. ‡ Values (mean of 3 replications) followed by the same letter, within the same column and factor, were not significantly different (p < 0.05), Tukey's least significant difference test.
Pistachios obtained under moderate RDI (T1) had the highest content of oleic acid and the lowest one of α-linolenic as compared to those of control (T0) and T2 treatment (Table 5). In case of rootstocks, no statistically differences were observed on the fatty acid composition except on the content of α-linolenic acid, where P. atlantica had the lowest values (Table 5). Regarding the interaction between the two factors studied, rootstock and irrigation, pistachios obtained by P. integerrima and T1 had the highest content of oleic acid (Table 5).

Morphological Analysis
On the analysis of split-open and non-split open pistachios (Table 6) no statistically significant differences were observed among samples obtained under different irrigation treatments, being the mean values 54% and 42%, respectively. In case of effect of rootstock, P. terebinthus had the highest number of split pistachios (60%) and, consequently, the lowest number of non-split open pistachios (35%); while data on P. atlantica and P. integerrima were statistically equivalent.
The moderated reduction of water during the phenological phase II of pistachios (T1) had no effect on the weight and size of the commercial nuts (Table 6). On the other hand, high reduction of water during this same phase (T2), led to pistachios with the lowest weight of their edible nut (T0 = 0.692 g and T2 = 0.673 g).
Regarding rootstocks, no statistically significant differences were found on the weight of whole nut and shell, and on the length and height of pistachio nuts. However, samples of P. integerrima and P. atlantica had the highest weight of the edible nut ( Table 6).
The color of the samples had statistically significant differences in the parameters L* and a*, in case of irrigation and rootstock. These differences were minimum, and some authors have concluded that differences smaller than two units, as it is the current case (Table 6), are imperceptible for the human eye [31,32].
Similar results were previously reported by Carbonell-Barrachina [7] and Noguera-Artiaga [16], who showed that neither rootstocks nor RDI treatments significantly affected the morphological parameters of pistachios. In previous studies, carried out under similar conditions, Carbonell-Barrachina [7] obtained that moderate RDI increased the content of linoleic acid, while in this study the one that has been increased was that of the oleic acid. Acar [30] reported that main fatty acids found in pistachio were oleic, linoleic and palmitic acids as has been shown in the current study.

Morphological Analysis
On the analysis of split-open and non-split open pistachios (Table 6) no statistically significant differences were observed among samples obtained under different irrigation treatments, being the mean values 54% and 42%, respectively. In case of effect of rootstock, P. terebinthus had the highest number of split pistachios (60%) and, consequently, the lowest number of non-split open pistachios (35%); while data on P. atlantica and P. integerrima were statistically equivalent. conscientious studies to manage water deficits without affecting the quality of the crops. In this study, regulated deficit irrigation (RDI) strategies and three rootstocks (P. atlantica, P. integerrima, and P. terebinthus) were applied to pistachio cultivation to study the quality of fruits obtained based on the morphological, functional, aroma, and their sensory properties. The results obtained demonstrated that RDI T1 (during phenological phase II of cultivation the stem water potential was maintained around −1.5 MPa) led to pistachios with same morphological properties, total polyphenol content, antioxidant activity, volatile composition, sensory properties, better profile of fatty acids, and being the favorite ones for international consumers, as compared to pistachios obtained under full irrigation treatments. On the other hand, when P. integerrima was used, pistachios obtained had the highest weight, the lowest content of sucrose and the best functional properties. The moderated reduction of water during the phenological phase II of pistachios (T1) had no effect on the weight and size of the commercial nuts (Table 6). On the other hand, high reduction of water during this same phase (T2), led to pistachios with the lowest weight of their edible nut (T0 = 0.692 g and T2 = 0.673 g).
Regarding rootstocks, no statistically significant differences were found on the weight of whole nut and shell, and on the length and height of pistachio nuts. However, samples of P. integerrima and P. atlantica had the highest weight of the edible nut ( Table 6).
The color of the samples had statistically significant differences in the parameters L* and a*, in case of irrigation and rootstock. These differences were minimum, and some authors have concluded that differences smaller than two units, as it is the current case (Table 6), are imperceptible for the human eye [31,32].
Similar results were previously reported by Carbonell-Barrachina [7] and Noguera-Artiaga [16], who showed that neither rootstocks nor RDI treatments significantly affected the morphological parameters of pistachios.

Conclusions
The results obtained in this study demonstrated that the application of a moderate deficit irrigation during pistachio cultivation (T1) led to pistachios with same morphological properties, total polyphenol content, antioxidant activity, volatile composition and sensory properties than pistachios obtained using full irrigation (T0). Moreover, T1 led to pistachios with better profile of fatty acids and being the sample preferred by international consumers. On the contrary, when the RDI was severe (T2), pistachio nuts had the lowest antioxidant activity, the lowest total polyphenols content, and the least preferred samples by consumers. In case of pistachios obtained using different rootstocks, P. integerrima led to pistachio nuts with the highest weight, the lowest content of sucrose and better functional properties than P. atlantica and P. terebinthus. These results demonstrated that it is possible to save irrigation water in pistachio farming, with low environmental and economic cost, and leading to pistachio nuts with same or even better quality attributes.