Plant material

Seeds were randomly collected from a population of 25 B. alicastrum trees, free of pests and diseases, located in “El Bramador”, Talpa de Allende, Jalisco, Mexico (20° 22’ 50’’ NL and 104° 49’ 19’’ WL), with a semi-warm sub-humid climate with summer rains (^{Ramírez-Sánchez, Meulenert-Peña, & Gómez-Reyna, 2013}). From this population four B. alicastrum trees were selected, from which their seeds were randomly collected and divided into four parts: three for the application of treatments and one that was discarded. The seeds were washed with drinking water and subjected to an aeration process to remove moisture. Each seed lot had an average weight of 200 g.

A completely randomized design was established for the experiment, where the treatments evaluated were: fresh seeds (FS), seeds with medium roasting at 90 °C for 20 min (SMR) and seeds with high roasting at 90 °C for 35 min (SHR) (similar to that applied by the producers). Five replications were performed per treatment. The seeds of all treatments were mechanically pulverized (Ø = 20 Mesh) in a mill (model 4, Thomas Scientific, USA), and stored at room temperature until analysis.

Quantification of minerals

The Fe, Ca, Mg, K and Na contents were determined in a fresh and ground sample with 3N HCl for acid digestion at a temperature of 100 °C for 10 min. The mixture was brought to 25 mL with deionized water, and then the concentration of these elements was determined by atomic absorption spectrophotometry (Analyst 700, PerkinElmer®, USA). Results were expressed in milligrams per 100 g of fresh weight Maya nut sample (mg∙100 g^-1 f.w.).

Proximal analysis

The percentages of moisture, lipids, crude fiber and ash from each ground sample were determined by methods described by the Association of Official Analytical Chemists (^{AOAC, 1990}). The total carbohydrate content was calculated by difference using the following formula: TC = 100 - (P + L + A+ F + M); where TC is the total carbohydrates (%), P is the protein, L is the lipids, A is the ash, F is the fiber and M is the moisture (^{Audu & Aremu, 2011}). Results were expressed as a percentage of fresh weight.

Quantification of soluble phenolic compounds

One g of ground seed sample was macerated in methanol, acetic acid and water (10:1:9, v/v/v) to obtain a concentration of 0.1 g∙mL^-1 and extract the analysis metabolites. Subsequently, the content of total phenolic compounds was determined by the Folin-Ciocalteu method (^{Singleton & Rossi, 1965}); for this, 1 mL of the extract was mixed with 10 mL of water and 1 mL of Folin-Ciocalteu reagent (2 N). The mixture was left to stand for 2 min; later, 4 mL of Na₂CO₃ (7.5 %, w/v) were added and left to stand in darkness at room temperature for 60 min. Finally, the absorbance was read at 765 nm in a spectrophotometer (Genesys 10s, Thermo Scientific, USA). The concentration of the soluble phenolic compounds was determined from a standard curve based on gallic acid (GA). The total soluble phenolic content was expressed in milligrams of gallic acid equivalents per 100 g of fresh weight ground seed sample (mg GAE·100 g^-1 f.w.).

Quantification of flavonoids

One g of ground seed sample was weighed and macerated in 80 % (v/v) methanol to obtain a concentration of 0.1 g·mL^-1. Flavonoid content was quantified according to the Dowd method, adapted by ^{Arvouet-Grand, Vennat, Pourrat, and Legret (1994)}. First, 0.5 mL of AlCl₃ (2 %, w/v) and distilled water were added to 2 mL of the methanolic extract, up to a final volume of 25 mL. The mixture was homogenized in a vortex and incubated in darkness at room temperature for 30 min. Finally, the mixture’s absorbance was measured at a wavelength of 425 nm in a spectrophotometer (Genesys 10s, Thermo Scientific, USA). Flavonoids were quantified from a standard curve based on flavone quercetin (Q). Results were expressed in milligrams of quercetin equivalents per 100 g of fresh weight ground seeds (mg QE·100 g^-1 f.w.).

Quantification of condensed tannins

First, 200 mg of ground seeds were mixed with 10 mL of 1 % HCl in methanol (v/v). The mixture was kept under constant stirring for 20 min, after which 1 mL of the filtered mixture was taken and 4 mL of 8 % HCl in methanol (v/v) and a solution of vanillin in 4% methanol (v/v) were added at a ratio of 1:1. The resulting mixture was kept in a water bath at 30 °C for 20 min. Finally, the mixture´s absorbance was measured at 500 nm. Tannins were quantified using a standard curve based on catechin (C) (^{Cardador-Martínez, Jiménez-Martínez, & Sandoval, 2011}). The content of condensed tannins was expressed as milligrams of catechin equivalents in 100 g of fresh weight ground seeds (mg CE·100 g^-1 f.w.).

Evaluation of antioxidant activity

The ground seed sample was macerated in 80 % (v/v) methanol at a concentration of 0.1 g·mL^-1. Antioxidant activity was quantified from the inhibitory capacity of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) free radicals, obtained following the methodology described by ^{Re et al. (1999)}; For this, a solution of 7 mM of ABTS in distilled water and another of 2.45 mM of potassium persulfate were prepared and then combined at a 2:1 ratio. The mixture was left to stand for 16 h in darkness to allow free radical generation. Subsequently, the mixture was diluted with ethanol to an absorbance of 0.7 ± 0.001 at 734 nm. On the other hand, 1 mL of the solution of the ABTS^·+ radical solution was taken and 10 µL of the methanolic extract were added; the mixture was incubated in a water bath at 30 °C in darkness for 7 min. Finally, the mixture’s absorbance reading was taken at a wavelength of 734 nm. Antioxidant activity was quantified from a standard curve based on trolox (T) (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). Results were expressed in milligrams of trolox equivalents per 100 g of fresh weigh ground seeds (mg TE·100 g^-1 f.w.). The percentage of inhibition of the ABTS^·+ free radical was calculated with the following formula:

where Ai is the initial absorbance of the free radical at 734 nm and Af is the final absorbance of the reaction with the sample.

Statistical analysis

The results obtained were subjected to an analysis of variance and Tukey’s multiple comparison test (P ≤ 0.05) using Statistical Analysis System (^{SAS, 2003}) software. Results were expressed as the mean (( standard deviation) of the five replications. The relationship between the phytochemical variables was determined by Pearson's correlation coefficient.

Mineral content

No significant differences (P ≥ 0.05) were observed between the mineral contents analyzed in fresh and roasted Maya nuts (Table 1). It is important to note that the Fe and Mg contents were higher than those reported in legumes and nuts, as well as the Ca and K contents found in hazelnut, macadamia nut, pecan nut and pistachio nut. In contrast, Maya nuts showed a lower Na concentration than reported in some legumes, pecan nuts and pistachio nuts (Table 2) (^{Bulló, Juanola-Falgarona, Hernández-Alonso, & Salas-Salvadó, 2015}). It is important to mention that the variation in mineral concentration is explained by differences between species, genetic factors and edaphoclimatic conditions (^{Duarte-Martino et al., 2012}; ^{Reynoso-Camacho, Ramos-Gómez, & Loarca-Pina, 2006}; ^{Román-Cortés et al., 2014}).

^{Moreiras, Carbajal, Cabrera, and Cuadrado (2009)} highlight the importance of eating foods rich in minerals, as they are essential for cellular metabolic reactions in the body. Minerals control the composition of extra and intracellular fluids, and are part of enzymes and hormones, which are essential molecules for life. The two most important minerals for human health are Ca and Fe (^{Guzmán-Maldonado & Paredes-López, 1998}). Mg is an activator of many enzymatic systems and maintains the electrical potential in the nervous system (^{Young, 1992}). Ca and K are usually found together in the body and contribute to the formation of blood, in addition to providing support structure to the body (^{Ogunlade, Olaofe, & Fadare, 2005}). Therefore, the analyzed elements are involved in several vital functions of the human body, and Maya nuts outperformed most of the seeds compared in concentration (Table 2).

Proximal analysis

No significant differences (P ≥ 0.05) were found among treatments in terms of proximal analysis, with the exception of lipid content, which was lower in roasted seeds (Table 3), so roasting did not affect the nutritional quality of the seeds. Variation in lipid content due to temperature has also been reported in other seeds. In this regard, ^{Audu and Aremu (2011)} noted the decrease in lipid content when studying various processes (cooking, roasting, fermenting and sprouting) in some legumes (Phaseolus vulgaris) of Nigeria.

There are no reports on the variation of the concentration of these components in Maya nut due to roasting. However, the comparison with other seeds rich in essential nutrients (unsaturated fatty acids, proteins, carbohydrates, vitamins, minerals, antinutritional compounds and oxalates) could be a reference for the attributes of Maya nut (^{Singh & Kaur, 2013}). A very low lipid content was found in Maya nut (2.40 g·100 g^-1 f.w.) compared with that reported in other seeds of higher concentration such as pecan nuts, almonds, pistachio nuts and pine nuts (66.18, 43.36, 45.09 and 61.73 g·100 g^-1 f.w., respectively) (^{Singh & Kaur, 2013}). However, the fatty acid profile was not analyzed in Maya nut as an additional quality attribute.

The carbohydrate content for Maya nut (67.23 g·100 g^-1 f.w.) was higher than reported by ^{Singh and Kaur (2013)} in almonds, pecan nuts and pistachio nuts (21.67, 13.86 and 27.51 g·100 g^-1 f.w., respectively). However, Maya nuts had a higher carbohydrate content compared to some legumes (beans, broad beans and green guaje, 61.60, 55.30 and 61.31 g·100 g^-1 d.w., respectively) (^{Román-Cortés et al., 2014}). The high carbohydrate content is due to the presence of high purity starch (92.57), unconventional and with peculiar physico-chemical and microscopic characteristics, superior to corn starch. These properties are important for their application in food systems that require high processing temperatures and as a potential use in the production of biodegradable materials (^{Pérez-Pacheco et al., 2014}).

This type of resistant starch is one of five different starches that have been reported and is part of the dietary fiber found mainly in cereals such as corn. This starch is characterized by high amylose content compared to some vegetables and is less susceptible to digestion by acid or amylase enzymes, so it is part of the indigestible carbohydrates (ICs), although during processing it is gelatinized, making it easily digestible (^{Ragaee, Gamel, Seethraman, & Abdel-Aal, 2013}). ICs, although fermented during digestion, are associated with a low glycemic index, low cholesterol levels, and decreased risk factors for colon cancer (^{Reynoso-Camacho et al., 2006}). On the other hand, higher carbohydrate contents in Maya nut may favor the formation of melanoidins (antioxidant pigments) and contribute to the dark coloring (^{Somoza, 2005}; ^{Wang, Qian, & Wei-Rong, 2011}) during the roasting process, as reported in coffee roasting (^{Moreira et al., 2017}); however, these pigments were not analyzed in the present study.

The protein content (9.94 g·100 g^-1 f.w.) in Maya nut exceeded that reported in some seeds such as pecan nuts (8.3 g·100 g^-1 f.w.), but lower than that found in almonds and pistachio nuts (19.48 and 13.08 g·100 g^-1 f.w., respectively) (^{Singh & Kaur, 2013}), and in legumes such as beans (from 20.1 to 23.6 g·100 g^-1 f.w.) (^{Peña-Valdivia, García-Nava, Aguirre, Ybarra-Moncada, & López, 2011}), chickpea (from 14.9 to 29.6 g·100 g^-1 f.w.), pea (from 21.2 to 39.2 g·100 g^-1 f.w.), broad bean (from 22.9 to 38.5 g·100 g^-1 f.w.), soybean (from 32.2 to 45.2 g·100 g^-1 f.w.) and lentil (from 20.4 to 305 g·100 g^-1 f.w.) (^{Phillips, 1993}).

According to ^{Larqué-Saveedra (2014)}, the flour quality of these seeds is high, comparable or better than that of conventional grasses, due to the concentrations of proteins, carbohydrates and fats (approximately 11, 70 and 1.5 %, respectively), and high contents of fiber, vitamins (B1, B2 and folic acid) and minerals (such as calcium, iron and zinc), with a contribution of 318 Kcal·100 g^-1 flour.

Antioxidant compounds

Significant differences (P ≤ 0.05) were found among treatments in the concentrations of phenolic compounds, flavonoids and tannins (Table 4). The content of phenolic compounds and condensed tannins in the seeds with high roasting (SHR) exceeded that of the seeds with medium roasting (SMR) and fresh seeds (FS), while the flavonoid concentration was lower in the SHR treatment. ^{Tiwari, Brunton, and Brennan (2013)} point out that high temperatures can produce isomerization of some flavonoids, and not being free could explain their decrease during the roasting process and the increase in condensed tannins.

Variation in the concentrations of phenolic compounds among treatments may be due to the formation of Maillard reaction products, such as melanoidins (^{Budryn et al., 2009}). These compounds, with antioxidant properties (^{Minatel et al., 2017}), are nitrogenous polymers responsible for the brown color of foods, produced by the interaction of amino groups (free amino acids, peptides and proteins) and carbonyl groups of reducing sugars (fructose and glucose) present in foods (seeds) (^{Somoza, 2005}; ^{Wang et al., 2011}); both groups are substrates of Maillard's reaction due to the effect of temperature. Phenolic compounds also participate in the formation of such pigments, as reported in the early stages of coffee roasting (^{Moreira et al., 2017}; ^{Pastoriza & Rufián-Henares, 2014}; ^{Perrone, Farah, & Donangelo, 2012}); therefore, in the quantification of phenolic compounds, melanoidins may also react with the Folin-Ciocalteu reagent (^{Pastoriza & Rufián-Henares, 2014}; ^{Pérez-Martínez et al., 2010}).

In the only Maya nut study, ^{Ozer (2017)} found 2,467 ± 85 mg GAE·100 g^-1 f.w. of phenolic compounds in B. alicastrum seeds from the United States, a concentration higher than that found in the present work. This difference could be due to the analysis methodology, place of origin and edaphoclimatic conditions. This author also reported the presence of p-hydroxybenzoic acid as the main compound in Maya nut; however, the present work is the first to report the contents of condensed tannins and flavonoids in fresh and processed Maya nut.

The concentration of phenolic compounds in fresh Maya nuts (271.58 mg GAE·100 g^-1 f.w.) exceeded that reported in almonds and pistachio nuts (212.9 and 571.8 mg·100 g^-1 f.w., respectively), but the pecan nut values (1,463.9 mg·100 g^-1 f.w.) (^{Singh & Kaur, 2013}) are similar to those found with the SHR treatment (1,337.19 mg GAE·100 g^-1 f.w.) (Table 4).

Flavonoids are a large and important group of phenolic compounds, one of the most abundant and studied groups of plant origin (^{Drago-Serrano, López-López, & Sainz-Espuñes, 2006}; ^{Lee, Koo, & Min, 2004}). The decrease in the concentration of these metabolites during seed roasting (Table 4) could be due to a degradation caused by temperature (> 80 °C), since these metabolites are more unstable (^{Alvarez-Jubete & Twari, 2013}; ^{Katsube, Keiko, Tsushida, Yamaki, & Kobori, 2003}; ^{Zhang, Chen, Li, Pei, & Liang, 2010}).

Reported flavonoid values are higher in almonds (93.5 mg·100 g^-1 f.w.) and pecan nuts (65.4 mg·100 g^-1 f.w.), but lower in pistachio nuts (55.9 mg·100 g^-1 f.w.) than those found in fresh Maya nuts (62.20 mg·100 g^-1 f.w.) (Table 4). The SHR treatment had the highest concentration of tannins; however, this is lower than that reported in foods with high sorghum levels (2927.0 mg·100 g^-1) (^{United States Department of Agriculture [USDA], 2004}).

Antioxidant activity

Significant differences (P < 0.05) were observed among treatments in antioxidant activity (Table 4), which increased with increasing roasting time. SHR had the highest antioxidant activity, probably due to the increase in phenolic compounds and tannins; however, the possible formation of melanoidins (Maillard reaction) could also contribute to the increase in this activity (^{del Castillo, Ames, & Gordon, 2002}; ^{Votavova et al., 2009}), although the melanoidin content was not evaluated in the present study. Pearson's correlation coefficient (Table 5) allowed associating antioxidant activity with the increase in the concentration of soluble phenolic compounds and condensed tannins, caused by the increase in temperature during the roasting process.