Recently produced PNEC (5 kg), obtained byfirst cold mechanical pressing of organic certified (OIA, Organización Internacional Agropecuaria) shelled pecan nuts (Nucana, Entre Ríos, Argentina) was processed to obtain a homogeneous sample using a commercial food processor (Universo, Rowenta, Germany, 14 cm blade) and sieved through a 350 μm mesh.

Two different permeability films and two temperatures were studied to store samples of 20–40 g/bag, 1.5 kg per condition) in dark rooms during a one-year period. The packages used were a low permeability (pO₂ = 35 cm³/m² d at 23 ºC and pH₂O = 0.2–0.5 g/m² d) vacuum package BB4L (Cryovac, Buenos Aires, Argentina), and a high permeability (pO₂ = 6000–8000 cm³/m² d at 23 ºC and pH₂O = 14–17 g/m² d) package PD-960 (Cryovac, Buenos Aires, Argentina). Temperatures of 10 ºC and 20 ºC were chosen.

Four treatments were considered, as a combination of package atmosphere and storage temperature. The nomenclature used was L-10 and L-20 for those samples stored under low permeability vacuum package at 10 and 20 ºC, respectively; in the same way, H-10 and H-20 correspond to those stored into the high permeable package at the same temperatures. At different times of storage, samples were taken to analyze possible changes in quality to assure it could be employed in baking or as a food ingredient.

Proximate analysis. The moisture, ash, and protein contents of PNEC were determined in triplicate following standard methods of Analysis of the Association of Official Analytical Chemists (AOAC, 1984). Percent nitrogen was multiplied by 6.25 to obtain percent protein.

Fat content was determined in triplicate by the Soxhlet method, using petroleum ether (Bp: 35–60 ºC) as extraction solvent (Marchetti et al., 2013). Total carbohydrates were determined employing the Anthrone method using glucose as standard (Emelike et al., 2015). Total fiber was determined in duplicate using enzymatic hydrolisis Megazyme kits 991.43 and 958.29 (K-TDRF, Megazyme, Wicklow, Ireland). Water activity was determined in quadruplicate using an Aqualab 4TEV (Decagon Devices INC. MA, USA).

Water and oil absorption. Water and oil absorption capacity (WAC and OAC, respectively) are two important functional properties if the pecan expeller would be used as a component in baking products, thus they were characterized as follows.

Water and oil absorptions were determined (Chakraborty, 1986). Briefly, 1 g of expeller was vortexed with 10 mL of distilled water or oil using a vortex at the highest speed for 2 min. The mixture was allowed to stand at room temperature for 30 min, and then centrifuged at 3000g for 20 min. The supernatant was decanted and the centrifuge tube containing the sediment was weighed.

The results were expressed as WAC = mL/g PNEC and OAC = mL/g PNEC, respectively. Four samples of different packages were analyzed each time.

Microbial quality. Changes in mold and yeast counts in the products were evaluated monthly using the pour plate method. Initial dilution was made by aseptically blending (West Sussex, UK) 20 g of sample with 180 mL of 1 g/L of peptone solution in a Stomacher blender for 1 min. Appropriate serial dilutions were plated with Yeast Extract Glucose Chloramphenicol Agar (YGC agar, Merck KGaA) incubated for 5 d at 30 °C.

At the end of storage, appropriate serial dilutions were plated with Plate Count Agar (PCA, Oxoid, Hampshire, UK) for total mesophilic aerobic count (incubated at 30 °C for 2 d) and total psychrotrophic aerobic count (incubated at 4 °C for 7 d), with Violet Red Bile Glucose Agar (Merck KGaA, Darmstadt, Germany) for Enterobacteriaceae (incubated at 37 °C for 24 h), and with de Man, Rogosa, Sharpe agar (MRS agar, Oxoid) for lactic acid bacteria (incubated at 30 °C for 2 d). Data were expressed as log colony-forming units per gram of sample (logCFU/g). Also, total coliform counts using the most probable number method were made (MPN, 46016) (AOAC 1984).

TBARs assay. Thiobarbituric acid reactive substances (TBARS) were determined in quadruplicate according to Pennisi Forell et al. (2010) to evaluate the lipid oxidation in the PNEC stored under different conditions. Results for malondialdehyde (MDA) were expressed as mg/kg product.

Fatty acids profile determination. Fatty acids of PNECwere analyzed by GC-MS as fatty acid methyl esters (FAME) at initial time and after 6 and 12 months of storage.

Total lipids of PNEC were extracted using a chloroform-methanol mix (2:1, v/v) according to the Folch et al. (1957) procedure. For the derivatization process, 60 mg of the obtained oil were added to a 10 mL Falcon conical tube, followed by the addition of 4 mL n-hexane and 300 µL 2 N methanolic KOH. The solution was mixed vigorously and shaken in a Vortex for 20 min. After that, 0.5 g of sodium sulphate were added and finally, 1 mL of the upper phase was separated to be injected.

Fatty acid methyl ester (FAME) content was assayed by Shimadzu GC-MS model QH-2010 Ultra and Hewlett Packard’s (HP) model HP-6890. An AT-WAX (Alltech Associates, Inc.) capillary column of length 30 m and internal diameter of 0.25 x 10⁻³ m packed with polyethylene glycol (0.25 mm film thickness) was used. In QH-2010 Ultra, hydrogen (> 99.999%) was used as a carrier gas at a constant flow rate of 1.4 mL/min. MS interphase and ion source temperatures were 270 °C and 220 °C, respectively. The temperatures of the injector and the detector were set at 250 °C and 270 °C, respectively. The analysis was performed using full MS scan from m/z 50 to 400. A solvent delay of 3 min was used. Injection volume of 1 µL and a split ratio of 50:1 were used as part of the GC-MS analysis method. FAME profiles were obtained by comparison of retention times with standard FAME (Supelco 37 Component FAME Mix, Cat. No.18919–1 AMP, Sigma Aldrich) previously analyzed in the same conditions.

Methanol (> 99.5% purity), n-hexane and sodium sulphate were purchased from J.T. Baker (Mexico); and potassium hydroxide from Analar (UK).

From obtained FAME profiles iodine values (IV) were calculated using the following equation: IV = (% oleic acid x 0.988) + (% linoleic acid x 1.814) + (% linolenic acid x 2.737) (Martínez and Maestri, 2008).

Antioxidant activity:

Extract preparation. For extract preparation 1g of PNECwas extracted for 60 min in 15 mL of ethanol. Mixtures were centrifuged at 3000 g for 15 min at 4 ºC and supernatants were properly diluted according to the assay.

DPPH^● scavenging capacity of pecan expeller cake extracts. The capacity to scavenge the DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical of PNECextracts was monitored (Barman et al., 2013). Various concentrations of extracts (0.5 mL) were mixed with 2.7 mL of ethanolic solution containing DPPH^● radicals (0.2 mmol/l). The mixture was shaken vigorously and left to stand for 90 min in the dark. The reduction in DPPH^● radical was measured by monitoring absorbance at 517 nm. Radical-scavenging activity was calculated as:

Afterwards, % DPPH radical scavenging activity vs. extract concentration plots were employed to calculate the extract concentration required to scavenge 50% of DPPH in the assay medium, referred to as IC₅₀. Two extracts were prepared for each treatment and storage time and measurements were done in quadruplicate.

ABTS^●+ radical-scavenging activity. The radical-scavenging activity of the extracts was determined also by the ABTS^●+ (2,20-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid)) radical cation discoloration assay (Barman et al., 2013). An ABTS^●+ radical was generated from the reaction of 5 mL of 2,20-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS, 7 mM) in water, with 88 mL of potassium persulphate (2.45 mM). The mixture was left to stand for 12–16 h in the dark at room temperature. The absorbance of the reactant was later adjusted to 0.70 ± 0.02, at room temperature, at 734 nm.

Different concentrations of PNEC extracts (0.5 mL) were mixed with 2 mL of the ABTS^●+ solution and the mixtures were shaken during 7 min. The reduction in ABTS^●+ radical was determined by reading absorbance at 734 nm. The relationship between % ABTS^●+ scavenged as a function of extract concentration was measured and PNEC extracts were assayed within an appropriate range of dilutions.

Finally, the extract concentration required to scavenge 50% of ABTS in the assay medium referred to as IC₅₀ (half-maximal inhibitory concentration) was predicted. Two extracts were prepared for each treatment and storage time; measurements were done in quadruplicate.

Analysis of variance (ANOVA, SYSTAT, Inc., Evanston, IL, USA) was carried out to test the significance of independent variables. Experimental data were reported as mean values ± the corresponding standard error of the mean (SEM) when appropriate. For simultaneous pairwise comparisons, the least significance differences (LSD) test was chosen. Differences in means and F-tests were considered significant when P < 0.05.

Table 1 shows the chemical composition of PNEC. Lipid content resulted remarkably high (51 g/100g), indicating the possibility to enhance the oil extraction process or enrich the FA profile of other foods by PNEC incorporation. In addition, an important amount of dietary fiber was found, making the expeller cake also a useful component to enhance the nutritional quality of food formulations. PNEC exhibited a similar composition to almond flour (De Pilli et al., 2008), which has important ash (4.67 g/100g) and protein contents (22.24 g/100g). This indicates that PNEC could be a good substitute for this most commonly used nut-flour. It is interesting to note that in the case of almond flour production, one nut results in one product, but in the case of the pecan nut, one nut results in two products, oil, and cake that is very similar to almond flour (regarding proximate analysis) and could replace it.

Fiber, proteins and carbohydrates are main components of PNEC with the possibility of establishing hydrophilic interactions since these constituents contain hydrophilic parts, such as polar or charged side chains (Jitngarmkusol et al., 2008). In the present work WAC was 1.83 ± 0.06 g H₂O/g and OAC = 2.61 ± 0.09 g/g. Both parameter values obtained for PNEC were within the range for defatted and full fat pecan flours reported by Joshi et al. (2015). As the results show, PENC-lipid interactions were stronger than interactions with water (OAC > WAC). Pecan nut proteins may play a key role in this effect since a very low pecan protein solubility compared to several other nuts has been reported (Sathe et al., 2009), indicating weak protein-water interaction. Thus, PNEC exhibited a high oil retention leading to a great number of hydrophobic interactions between the protein and lipid phase. The high water and oil absorption capacity of expeller cake makes it a potential ingredient for the baking industry, were protein and fiber can bridge fat and water in bakery products.

Microbial quality. Microbial counts are very important to assure a proper shelf life. high levels of B₁ aflatoxins have been reported in Brazil nuts, which exceed the maximum residue limit (De Mello and Scussel, 2007). Therefore, it is important to establish storage conditions with low microbial levels. Changes in mold and yeast counts in products packaged and stored for one year under different conditions are presented in Figure 1.

PNEC stored in diverse conditions showed no significant differences until day 120. Beyond this time, mold and yeast counts quickly rose for PNEC in high permeability film and 20 ºC and after 220 days samples had to be discarded because of high development.

In addition, extremely low mold and yeast levels were found in PNEC in low permeability vacuum package, regardless of storage temperature (10ºC or 20ºC). Thus, this seems to be the main control parameter for microbial spoilage. When a high permeable film was employed microbial levels exhibited significant differences between temperatures.

PNEC stored in high permeability film (H-10) and 10 ºC showed similar microbial quality to low permeability vacuum package at both temperatures during the first 9 months, being significantly different from day 300 on. However, three treatments showed very low log CFU/g values at the end of experiment.

At the end of storage, treatments with low mold and yeast counts (L-10, L-20, and H-10) were tested for other microorganisms. Nevertheless, total aerobic mesophilic and psychrotrophic, Enterobacteriaceae, lactic acid bacteria, and total coliforms were below detection limits, probably due to the low water activity(a_w) of PNEC (0.59 ± 0.05).

TBARs and fatty acid profile. The FA profile obtained for PNEC at t = 0 days (Table 2) was found to be similar to those reported by Fernandes et al. (2017) for pecan oil. The amounts of monounsaturated FA (MUFA) and polyunsaturated FA (PUFA) were 63.27 and 29.93 g/100g, respectively, with lower saturated FA(SFA) levels (6.09 g/100g). The main PNEC unsaturated FA were oleic and linoleic acids, with 65.1 and 28.6 g/100 g, respectively, followed by linolenic acid.

In recent years a variety of studies have demonstrated that dietary polyunsaturated fatty acids have protective effects on metabolic syndrome diseases such as type 2 diabetes, hyperlipidemia, or cardiovascular disease (Koba and Yanagita, 2014). Equally, promising results are now available regarding their positive effects on inflammatory diseases, cancer, and osteoporosis (Moon et al., 2012). The use of PNEC in cereal mixes, cereal bars, or bakery products would be a good alternative to provide high contents of oleic and linoleic acids, and also linolenic acid, and thus enhance the nutritional quality of foods.

As the TBARs methodology is commonly employed for monitoring secondary oxidation products, i.e., aldehydes or carbonyls, which may contribute to the off-flavor of oxidized oils (Haq et al., 2013), TBARS evolution was employed to follow PNEC lipid oxidation in this work. As can be seen in Figure 2, oxygen restriction had a significant effect on lipid stability. PNEC in a low permeability vacuum package was stable, regardless of the temperature, showing a slight TBARs number increase in MDA from 0.05 to 0.2 mg/kg PNEC at the end of storage, which demonstrates an excellent oxidative stability.

On the other hand, the use of a high permeability film facilitated lipid oxidation. In PNEC stored without O₂ restriction and maintained at 10 ºC (H-10) TBARs remained constant up to day 225, when a sharp increase (> 1 mg/kg PNEC) was observed, indicating an oxidative spoilage which could be potentially harmful (Kofakowska, 2010). As expected, PNEC kept at 20 ºC in high permeability film (H-20) exhibited a significant TBARs increase sooner (day 180), resulting in mold and yeast growth and determining the end of the shelf life.

These results showed that both temperatures (10 and 20 ºC) combined with vacuum package in a low permeability film could preserve PNEC during one year. Other authors had suggested a temperature of 10 ºC and oxygen restriction as optimal conditions for a 12 month-shelf life for shelled walnuts, similar to our results for PNEC, as secondary oxidation and rancidity flavor development were critical parameters to control (Jensen et al., 2003).

From the FA profile analyses (Table 2), it can be seen that L-10, H-10 and L-20 treatments could protect unsaturated fatty acids for up to 6 months, since only minor changes were observed. H-20 profiles exhibited a significant decrease in C18:3, indicating an oxidation of this FA. After 12 months of storage, L-10 showed a better performance than the other two treatments (H-10 and L-20). The level of C18:3 remained unchanged and C18:2 showed a slight decrease.

SFA are the most stable fatty acids and it is not expected to detect a change in their total content. Nevertheless, the observed C16:0 proportional increment is a consequence of MUFA and PUFA percentage reduction through peroxidation.

The FA profiles and observed changes at the end of vacuum-packaged refrigerated storage are in agreement with results obtained in TBARs assay, where an O₂ restrictive barrier resulted in an effective method to preserve PNEC unsaturated FA. In addition, lowering storage temperature delayed lipid oxidation. Estimated iodine values (Table 2) are in agreement with the above mentioned results. A significant decrease in IV was observed at 6 months of storage for all the different conditions, indicating an oxidation process that progressed in L-20 and H-10 lowering IV until the end of storage, but was inhibited in L-10 as no further changes were observed.

Antioxidant capacity. Figure 3 and Figure 4 show the results of antioxidant capacity in terms of DPPH^● scavenging activity and ABTS^●+ radical, respectively. As can be seen in Figure 3 the antioxidant capacities of ethanolic extracts (t = 0 d) of PNEC were high and presented similar or higher values than those reported for hazelnuts and pecan nut kernels IC₅₀ (Salvador et al., 2016). This suggests that cold pressed extraction did not negatively affect antioxidant capacity, and that the pecan nut’s main antioxidants were not extracted in the oil showing a predominantly hydrophilic nature of the compounds. There are precedents that support this conclusion. Gentile et al. (2007) reported that the antioxidant activity of a lipophilic extract was much lower than that of the hydrophilic one in pistachio kernels. Also Wu et al. (2004) informed that water-soluble components contribute > 90% to the total antioxidant potential of most foods.

Both antioxidant mechanisms studied (DDPH^● and ABTS^●+) reflected similar tendencies during storage. It is noteworthy that only samples maintained at 10 ºC with O₂ restriction presented similar DPPH^● values within 365 days of storage and a slightly increase in ABTS^●+ IC₅₀. This suggests that under these conditions the lipid oxidation process is clearly inhibited because the antioxidant potential of the sample remained almost unchanged.

On the other hand, 10 ºC with normal atmosphere (H-10), and 20 ºC with O₂ restriction (L-20) are conditions that can prevent or delay lipid oxidation, as seen previously in Figure 2, but antioxidant potential is reduced by 50%. This indicates that antioxidants in the sample are reduced through an incipient oxidation or other process.

It has been reported that pecan nuts have high levels of tocopherols (> 200 mg/kg, Fernandes et al., 2017), with a high proportion of γ-tocopherol as the isomer with the highest in-vitro antioxidant capacity. This may lead to an important antioxidant capacity of PNEC (Miraliakbari and Shahidi, 2008).

Although both methods measure antioxidant capacity, their mechanisms are different. The ABTS^●+ assay measures antioxidant capacity based on the ability of the antioxidant to scavenge the blue–green colored ABTS radical cation by electron donation, while the DPPH^● method determines the hydrogen donating capacity of a molecule and does not produce oxidative chain reactions or react with free radical intermediates. In spite of these differences there was a significant correlation (P < 0.05) between both IC₅₀ values.

Miraliakbari and Shahidi (2008) affirmed that besides tocopherols, other antioxidative minor components of tree nut oils include phospholipids, phytosterols, and phytosterol conjugates, and possibly non-tocopherol phenolics, among others. Synergistic antioxidant activities have been reported between tocopherols and nitrogen-containing phospholipids such as phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine, all of which are present in tree nut oils and their minor component extracts and may help explain the high antioxidant activities observed for these products. Antioxidant capacity has been previously used as an indicator of the overall quality and oxidative stability of oils with good accuracy (Castelo-Branco et al., 2016). In the present paper we found similar trends in TBARS and antioxidants assays.

Considering the four treatments employed and the different shelf life aspects studied, we can conclude that H-20 treatment was a proper storage condition only for short periods of time (4–5 months) because of microbial growth and the consequent risk of aflatoxin development. Lowering the temperature (H-10) would control the microbial growth but at eighth months lipid peroxidation became a problem and product quality could not be assured. Finally, employing a more effective barrier (L-20 and L-10) would solve these issues, nevertheless L-20 seemed to have suffered some antioxidant loss, and had a less protective effect over FA than L-10, which resulted in the best treatment for assuring long shelf life.