Samples

Pistachio samples (Pistachia vera L. cv Kerman) were provided by Pisté S.R.L., an industrial factory located in Carpintería, Pocito district, from the province of San Juan, Argentina (2014 crop year). Samples were obtained at both riverbanks of San Juan River (lat. 31° S, long. 69° W, altitude 650–750 m.a.s.l.).

Sample treatment

Pistachio seeds dried at 40 °C (4 h) up to 3 % moisture content in a grain drier (Mega S.A., Argentina), were named natural pistachios (NP). A sub-sample of these NP were roasted (RP) using a rotating bakery oven (Argental, Argentina) at 120 °C during 90 min. Salted and roasted pistachios (SRP) were prepared by immersion of NP in a brine solution (NaCl 10 % w/v) during 1 min, then slurred and toasted (Penci et al. 2013). All samples were stored under vacuum, in individual bags (500 g each) at 5 °C until theirs use.

Pistachio seed composition

Samples of NP, RP and SRP were analysed according to standards AACC (AACC 2003) and American Oil Chemist’s Society (AOCS) (AOCS 2009) for total oil content, fatty acid profile, total protein, total carbohydrates and ash.

Screw press extraction

To optimize oil extraction by screw-press operations, pistachio seeds were conditioned to adjust moisture content to 10 % (w/w) level. Moisture conditioning was achieved by instant water sprinkling according to Singh and Bargale (2000). The water sprinkled samples were then packed in air-tight metal containers and stored for about 48 h for equilibration. The containers were shaken at regular intervals to distribute moisture uniformly throughout the sample. Extractions were carried out in a single step, with a Komet screw-press (Model CA 59 G, IBG Monforts, Monchengladbach, Germany) at a pilot plant scale (5 mm restriction die, 20 rpm screw speed). The screw press was firstly run for 15 min without material, but by heating via, an electrical resistance-heating ring attached around the press barrel, to raise the screw-press barrel temperature to the desired temperature (25 °C). Running temperature was checked with a digital thermometer inserted into the restriction die. After each run, all press devices were cleaned and dried (Martínez et al. 2008).

Oil yield

The oil yield was calculated considering the initial oil content in the incoming material, and the residual oil content in the cake. Oil yield was expressed as g extracted oil per 100 g of total oil present in the incoming material (g/100 g oil).

Fines amount in oil

Press-extracted oils were centrifuged at 11,000 x g during 30 min. The solid sediment was recovered and its content was calculated as g per 100 g of the total extracted oil (Martínez et al. 2008).

Oil analysis

Free fatty acid content (AV), peroxide value (PV), and specific extinction coefficients (K₂₃₂ and K₂₇₀) were determined according to standard methods of AOCS (AOCS 2009). The fatty acid profiles were analysed by gas chromatography (GC) according to Martínez et al. (2006). Carotenoids and chlorophylls were measured according to Minguez-Mosquera et al. (1991). The antiradical activity (AA) was analyzed by means of spectrophotometric determinations (Shimadzu Corporation, Kyoto, Japan MultiSpec-1501, equipped with a holder for multiple cells and temperature control) according to Martínez and Maestri (2008).

Pistachio flours

From each treatment (NP, RP, SRP), three independent samples of pistachio flours were collected immediately after pressing pistachio seeds. Flour samples were lyophilized and stored under vacuum in individual bags (50 g each) at 5 °C until theirs further use. Samples were named as follow: natural pistachio flour (NPF), roasted pistachio flour (RPF) and salted roasted pistachio flour (SRPF).

NPF, RPF and SRPF samples were homogenized, weighted (200 mg) and extracted by sonication (40 kHz, 30 min, 25 °C, ultrasound bath model TB02TACA, TESTLAB S.R.L, Buenos Aires, Argentina) using acidified methanol (0.1 % HCl, v/v) (MeOH-H⁺), according to Fabani et al. (2013). The homogenates was then centrifuged at 10,000 x g during 10 min using a Biofuge® 28RS Heraeus Sepatech Centrifuge (Heraeus Instruments, Hanau, Germany). The supernatant was separated, filtered (0.45 μm) and used for further analyses.

Determination of total phenolic (TP) and flavonoids (FT) content

The total phenolic (TP) and flavonoids (FT) content of acidified methanolic extracts (NPF, RPF and SRPF) were determined according Folin-Ciocalteu method and a colorimetric method with AlCl₃ respectively. TP were determined by linear regression from a calibration plot constructed using gallic acid (0–250 μg/mL), and expressed as mg of gallic acid equivalents (GAE) per 100 g of pistachio flour (PF) on a dry weight (d.w.) (mg GAE/100 g PF d.w.). The values of FT were expressed as mg of quercetin equivalents (QE) per 100 g of pistachio flour (PF) on a d.w. basis (mg QE/100 PF g d.w.). For both, the values from triplicates were reported as mean ± SD.

Identification and quantification of phenolic compounds by HPLC-ESI-MS/MS

The phenolic profile was performed on an Agilent Series 1200 LC System (Agilent, Santa Clara, CA, USA) coupled in tandem to a PDA detector (Agilent Series 1200) and a MicrQTOF Q II (Bruker Daltonics, Billerica, MA, USA) high resolution mass spectrometer (MS and MS/MS) equipped with an ESI source. The HPLC system was equipped with a binary gradient pump, solvent degasser, and autosampler (Agilent Series 1200 L).

HPLC analyses were performed on a thermostatized (40 °C) Luna C18 250 × 4.6 mm (5 μm) column (Phenomenex, Torrance, CA, USA), at 0.4 mL/min flow rate, using 0.5 % (v/v) formic acid-water (solvent A) and 0.5 % (v/v) formic acid-methanol (solvent B). HPLC runs were performed using the following gradient: starting with 20 % B, changing to 50 % B along 3 min, kept for 5 min, followed by a second ramp to 80 % B during 5 min, maintained for 17 min, returning to 20 % B in 1 min, remaining at this last condition for 10 min before the next run. The injection volume was 40 μL. ESI-MS and MS/MS detection was performed in successive runs using both negative and positive ionization mode, with mass acquisition between 100 and 1500 Da. Nitrogen was used as drying and nebulizer gas (7 L/min and 3.5 Bar, respectively), and 180 °C for drying temperature. For MS/MS experiments fragmentation was achieved by using the auto MS² option of the equipment. UV-Vis analyses were carried out in the range between 200 and 700 nm (PDA).

The identification of pistachios flours constituents was achieved by comparison of the spectral properties (UV, ESI-MS and MS/MS) of eluted compounds with those of reference samples, when available, and comparison with literature data. The standards gallic acid, naringenin, apigenin, quercetin, isoquercitrin, (+)-catechin, (−)-epicatechin and myricetin, were prepared at a stock concentration of 1000 mg/L. Calibration standard samples were prepared by appropriate dilutions with methanol from the stock solutions and filtered on Millipore filters (0.45 μm) before used. MS analysis was used for compounds quantification with the specific calibration plot. When reference compounds were not available, the calibration plots from structurally related compounds were used. The compounds concentrations were measured in triplicate, reporting the mean value and the standard deviation in each case.

Antioxidant activity

Ferric-reducing antioxidant power assay (FRAP)

FRAP assay, measures the reducing capability of the samples, evaluating the conversion of a Fe^3+/ferricyanide complex to Fe²⁺. The iron-reducing power of the samples was tested using the assay reported by Oyaizu (1986). The absorbance was read at 700 nm in a Multiskan FC microplate photometer (Thermo Scientific, USA). Quercetin was used as a reference compound. Analyses were performed in triplicate; values were reported as mean ± SD.

Total phenolic (TP), flavonoid (FT) content and antioxidant activity in pistachio flour

The acidified methanolic extraction yields from pistachio defatted flour were studied. Significant differences (P < 0.05) among samples of different heat treatments were observed. The results showed that SRPF with 28.5 ± 0.9 % was the best in the extraction, followed by RPF (20.0 ± 0.2 %) and NPF (18.6 ± 0.9 %), respectively.

Folin-Ciocalteau assay was used as rapid methods to evaluate total phenolic (TF) compounds in the pistachio flours samples (Fig. ​Fig.22). The TP concentration of acidified methanolic extracts was similar between NPF (11.4 ± 0.2 mg GAE/g PF d.w.) and RPF (11.1 ± 0.6 mg GAE/g PF d.w.). However, a slight and significant decrease was observed in SRPF (10.4 ± 0.6 mg GAE/g PF d.w.) (Fig. ​(Fig.2).2). The results obtained were lower when compared with pistachio green hull extract from Fandoghi and Ahmadaghaei variety, 32.8 and 28 mg/g sample, respectively (Goli et al. 2005; Rajaei et al. 2010). Concerning flavonoids (FL) content, pistachio flours varied from 0.48 ± 0.05 to 0.7 ± 0.1 mg QE/g PF d.w. (Fig. ​(Fig.2).2). The SRPF presented the minor FT content, which was significantly different in relation to the others pistachio flours analyzed.

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Fig. 2

Total phenolic (TP) (grey bars) and flavonoids (FT) (dot) content of acidified methanolic pistachios flours extracts (NPF: natural pistachio flour, RPF: roasted pistachio flour, SRPF: salted roasted pistachio flour). Results are expressed as mean ± SD (standard deviation). Different letters indicate significant difference among treatments, Duncan (P < 0.05)

Otherwise, the scavenging effect on DPPH radicals assay showed concentration-dependent activity, and similar trends were observed for NPF and RPF extracts (Fig. ​(Fig.3a).3a). The EC₅₀ values in NPF extract had the lowest value (2.2 mg/mL), followed by RPF (2.7 mg/mL) and SRPF (4.1 mg/mL). The correlation between antioxidant activity and TP content was evaluated applying simple correlation analysis. A positive significant Pearson’s correlations was found between DPPH activity and TP (r = 0.69, P < 0.01). Also, a major statistically significant correlation was observed between FT content and DPPH antioxidant capacity (r = 0.78, P < 0.01). The Fig. ​Fig.3b3b shows that the reducing antioxidant power (FRAP assay) of pistachio flour extracts have a direct relation with extracts concentration being NPF the one that have the highest reducing power. TP and FL content are also directly correlated with FRAP assay results (r = 0.68 and r = 0.72, respectively, at P < 0.01). The roasting process not diminish total phenolic (TP) and flavonoids (FL) content significantly compared to natural pistachio flour (NPF), even so reduced the DPPH antioxidant capacity (approximately 20 %) and antioxidant power in the roasted pistachio flour (RPF). Furthermore, salted roasted pistachio flour (SRPF) showed a slight and significant decrease on TP and FL content in relation to the others samples.

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Fig. 3

Scavenging activity of acidified methanolic pistachio flour extracts (NPF: natural pistachio flour, RPF: roasted pistachio flour, SRPF: salted roasted pistachio flour) on 2,2-Diphenyl-1-picrylhydrazyl (DPPH) (3-A) and ferric reducing power (FRAP) (3-B)

Identification and quantification of pistachio flour phenolics

The results of the HPLC-PDA-ESI-MS and MS/MS assays of pistachio flours extract were summarized in Table ​Table3.3. In all samples, a total of 13 compounds belonging to the family of phenolic acids and flavonoids, and also 2 anthocyanins were identified and quantified. The total amount of polyphenolic was a little higher in NPF (173 ± 18 μg/g PF d.w.) compared with SRPF (155 ± 15 μg/g PF d.w.) and RPF (129 ± 11 μg/g PF d.w.) (Table ​(Table3).3). Results of the Folin-Ciocalteu were compared with HPLC findings and no relation was found among the results obtained. It is well know that the Folin-Ciocalteu reagent is not specific and detects all phenolic groups found in extracts. However, this assay still provides a very useful index for phenolic content, but it would not be expected to correlate with the weight of phenolics quantified by HPLC.

The major polyphenol identified in pistachio flours was (+)-catechin (38–65.5 μg/g PF d.w.), followed by gallic acid (23–36 μg/g PF d.w.), procyanidin dimer (10–15 μg/g PF d.w.) and eriodictyol (9–13 μg/g PF d.w.). Conversely, Mandalari et al. (2013) reported gallic acid as the phenolic present in major quantities in natural and roasted salted pistachio kernels (Pistachia vera L.) from California (USA).

In the present study, the treatments have different effects on the phenolics constituents of pistachio flour. According to Xu and Chang (2008) the thermal treatment applied to foods of plant origin by roasting causes evaporation of intracellular water, resulting in a greater availability of plant phenolic compounds in the matrix. Roasting caused a significant reduction of some phenolics in pistachio flour, gallic acid and (+)-catechin, and increased others, (naringenin and luteolin) (Table ​(Table3).3). On the other hand, a hypothesis that may explain the increase of the level of some phenolics compounds in salted-roasted pistachio flour is that during salting the integument (the outer skin) is in contact with a brine solution (NaCl 10 % w/v). Then, during moisture conditioning the proteins could be partly solubilized, resulting in increased levels of free proteins and phenolic compounds (gallic acid and naringenin) on the SRPF. The anthocyanins identified in PF extracts were cyanidin-3-O-galactoside and cyanidin-3-O-glucoside and content ranged from 21 to 23 to 0.81–1.1 μg/g PF d.w., respectively. In the current study roasting at 120 °C during 90 min did not influence significantly in the degradation of anthocyanins (Table ​(Table3).3). Mandalari et al. (2013) reported similar behaviour for certain compounds in sated roasted pistachios from Californian and Bonilla-Lemos et al. (2012) in Brazilian baru nuts roasted at 150 °C for 45 min.

Pistachio nuts analysed here present higher amounts of polyphenolic compounds in comparison with almonds, hazelnuts, peanuts, macadamia and pistachio nuts from other origins (Yang et al. 2009). Polyhydric phenols with high number of OH-groups, such as several compounds identified in pistachio methanolic extracts (Fig. ​(Fig.3),3), have been also recognized for their antioxidant activity in lipid peroxidation reactions owing to their capacity of hydrogen-atom transfer to lipid alkyl radicals. However, such effect must be interpreted with caution because such polyphenols are rather polar and hydrophilic substances and could have low solubility in oil. In summary, considering the whole set of compounds with potential antioxidant capacities, it is possible that tocopherols and carotenoids could be the main contributors to the radical scavenging activity observed in pistachio oil, with a minor contribution of polyphenolic compounds. Conversely, (+)-catechin and gallic acid could be the responsible of the bioactivity in pistachio flour extracts. These well known for their antioxidant activity in different trials. (+)-catechin is a flavonoid that has been indicated as a factor that reduce cardiovascular risk by lowering serum cholesterol levels, diminishing platelet aggregation and reducing blood pressure (Marinou et al. 2010). On the other hand, procyanidins are reported to be potent antioxidants. Human studies show that a diet rich in procyanidins decreases/inhibits lipid peroxidation of LDL cholesterol and increases free radical scavenging capacity (Natella et al. 2002).

Authors are grateful to CICITCA, Universidad Nacional de San Juan and Universidad Nacional de Córdoba, SECITI Gobierno de la Provincia de San Juan (IDeA Exp N° 1400-0107-2012), Argentina for the financial support. G.E.F., M.P.F., M.V.B., M.L.M., D.M.M. and D.A.W. are researchers from CONICET, Argentina. R.N.M.H. is fellow CONICET. We would like to express our gratitude to Piste´ S.R.L. for providing pistachio samples.