Nutritional Value and Volatile Compounds of Black Cherry (Prunus serotina) Seeds

Prunus serotina (black cherry), commonly known in Mexico as capulín, is used in Mexican traditional medicine for the treatment of cardiovascular, respiratory, and gastrointestinal diseases. Particularly, P. serotina seeds, consumed in Mexico as snacks, are used for treating cough. In the present study, nutritional and volatile analyses of black cherry seeds were carried out to determine their nutraceutical potential. Proximate analysis indicated that P. serotina raw and toasted seeds contain mostly fat, followed by protein, fiber, carbohydrates, and ash. The potassium content in black cherry raw and toasted seeds is high, and their protein digestibility-corrected amino acid scores suggest that they might represent a complementary source of proteins. Solid phase microextraction and gas chromatography/flame ionization detection/mass spectrometry analysis allowed identification of 59 and 99 volatile compounds in the raw and toasted seeds, respectively. The major volatile compounds identified in raw and toasted seeds were 2,3-butanediol and benzaldehyde, which contribute to the flavor and odor of the toasted seeds. Moreover, it has been previously demonstrated that benzaldehyde possesses a significant vasodilator effect, therefore, the presence of this compound along with oleic, linoleic, and α-eleostearic fatty acids indicate that black cherry seeds consumption might have beneficial effects on the cardiovascular system.

suppressing growth in cancer cells, and it has been proposed as a chemotherapeutic agent against breast cancer [13].
Although black cherry seeds contain cyanogenic glycosides [14], there are no reports of human intoxication related to their intake, since they are consumed toasted, which indicates that the toasting process decreases the content of these compounds [15]. Heat treatment also provokes emission of volatile compounds contained in the seeds, which undoubtedly contribute to their pleasant and characteristic flavor. These volatile components might also possess pharmacological activity [16].
Currently, the nutritional value of black cherry seeds and their volatile components have not been investigated. Therefore, in the present study the proximate composition, vitamin and mineral content of these seeds were determined in order to assess their nutritional value. In addition, the volatile compounds present in the seeds were analyzed by gas chromatography coupled to mass spectrometry.
These findings indicate that P. serotina edible seeds are a good source of unsatured fatty acids with potential health benefits [21]. The mean values of ash and crude fiber of raw and toasted P. serotina seeds, almonds and peanuts did not differ significantly. The crude fiber content was similar to that of other seeds and nuts that represent a complementary source of fiber, like hazelnut (9%-13%) or pumpkin seeds (12.1%) [22,23]. Adequate intake fiber is related to the obesity prevention and better glycemic control in patients with type 2 diabetes mellitus [24,25].

Minerals
The mineral composition (Ca, Fe, Mg, P, K, Zn and Na) of raw and toasted black cherry seeds, almonds and peanuts are presented in Table 2. The Mg content of raw and toasted black cherry seeds was unaffected by heat processing, but the Ca, Fe, P, K, Zn, and Na content diminished after the toasting process. Usually, nuts and seeds are consumed toasted and salted, and toasting might have an important influence on their nutrimental quality. Chitra et al. [26] and Gamel et al. [27] have pointed out that Ca and Fe content significantly reduced after toasting or autoclaving seeds of soybean and pigeon peas. Raw black cherry seeds possess a higher content of Ca, Fe, Mg, K and Na than that found in peanuts. On the other hand, Fe, K, and Na content is greater than that of almonds. Interestingly, the K content in black cherry seeds (873.22 ± 12.64 mg/100 g) is significantly higher than that of almonds and peanuts, suggesting that P. serotina seeds represent a good complementary source of this mineral.

Vitamins
Vitamin analysis showed that raw and toasted P. serotina seeds do not contain vitamins A and C. Vitamin E or α-tocopherol was detected at a concentration of 3.916 mg/100 g in raw seeds, while in toasted seed it was absent. This lost may be associated with vitamin degradation due to temperature exposure [27]. Our results are in agreement with previous studies carried out on other seeds and nuts, such as pumpkin seeds, walnuts and peanuts, which do not contain detectable values of vitamins A and C [28,29]. Vitamin E content of black cherry seeds is similar to that of pine nuts (4.1 mg/100 g) and peanuts (6.1 mg/100 g), however it is lower than that of almonds and hazelnuts (24.2 mg/ 100 g and 31.4 mg/ 100 g, respectively) [28].

Protein Nutritional Quality
Since black cherry seeds contain higher levels of protein (37.95% ± 0.16%) than other seeds, their protein nutritional quality was assessed. Amino acid composition data are presented in Table 3. It is important to mention that in this experiment tryptophan was not determined. Another protein quality measure is digestibility. Differences among the in vitro protein digestibility values of raw and toasted black cherry seeds were not significant (88.12% ± 0.72% and 89.40% ± 1.32%, respectively). These values were similar to that of almonds (90.15% ± 0.85%), however they were lower than those of peanuts (94.06% ± 0.78%) and casein (98.51% ± 0.82%), which was used as control. It has been reported that in vitro protein digestibility values greater than 80% are related to an efficient amino acid bioavailability [27]. Therefore, these results suggest that black cherry seed proteins are highly bioavailable.
Considering these findings, the protein digestibility-corrected amino acid score (PDCAAS) was determined. This method is accepted as the most recognized approach for assessing the protein quality of foods [30]. Every essential amino acid receives a score, but the final PDCAAS value corresponds to that of the limiting amino acid. The highest PDCAAS value for a given protein is 1.0, which indicates that a protein provides adequate amounts of all the essential amino acids [31]. Table 4 shows that lysine is the limiting amino acid in P. serotina seeds, therefore, PDCAAS values for raw and toasted black cherry seeds protein are 0.13 and 0.18, respectively. These values are lower than those reported for almonds (0.23) [17] and peanuts (0.69) [32]. These results suggest that black cherry seeds could be a complementary source of proteins. Notes: * Amino acid score (AAS) = AA content of test protein/reference AA pattern, where reference AA pattern is the amino acid requirement for a preschool child (2-5 years) [30]. ** Limiting amino acid.

Volatile Compounds
Volatile compounds of raw and toasted black cherry seeds were extracted by head space solid phase micro extraction and analyzed by gas chromatography-mass spectrometry, using DB-5 and Wax capillary columns in order to detect a wide range of polar and non-polar compounds. A total of 59 and 99 volatile compounds were identified in the raw and toasted P. serotina seeds, respectively ( Table 5). The identified volatile compounds comprise aldehydes, alcohols, ketones, carboxylic acids, esters, hydrocarbons, and pyrazines.
The predominant aldehyde identified in raw and toasted seeds was benzaldehyde. This compound has a characteristic pleasant almond-taste and aroma [33] and has been identified in bitter and sweet almonds [34], hazelnuts [33], and seeds of other species of the genus Prunus [35,36]. Benzaldehyde is produced by enzymatic degradation of the cyanogenic glycosides amygdalin and prunasin [35,37,38]. Considering on one hand that benzaldehyde content in toasted seeds was higher than that in raw seeds, and on the other, that bitterness attributed to the presence of amygdalin [39,40] diminished in toasted seeds, it is very likely that the toasting process may induce benzaldehyde formation from cyanogenic glycosides. Benzaldehyde might also be generated from phenylalanine during heating processes [41,42] as was demonstrated by Alasalvar et al. [33] in hazelnuts, pumpkin seeds [43], and peanuts [44]. It is worth noting that we have previously identified benzaldehyde as one of the major components in black cherry leaves essential oil, we also proved that this compound induces a significant concentration-dependent vasodilator effect [8]. Therefore, the presence of this compound along with oleic, linoleic, and α-eleostearic fatty acids indicate that black cherry seeds consumption might have beneficial effects on the cardiovascular system.    The most abundant alcohol identified in P. serotina seeds was 2,3-butanediol, which was also the major volatile compound in raw (17.03 ppm) and toasted seeds (17.44 ppm). This alcohol is the major volatile component of sweet kernels and has been associated to their characteristic taste [34]. 2,3-butanediol is also found in cheeses [45] and honey [46]. Recently, it has been reported that this metabolite is abundant in roasted Trichosanthes kirilowii seeds, however it was absent in the raw seeds, which suggests that 2,3-butanediol was generated during the toasting process [47]. In contrast, our results indicated that 2,3-butanediol levels in black cherry seeds were not significantly different before and after toasting. Benzyl alcohol, predominantly found in toasted seeds, imparts a sharp, burning taste and a faint aroma in toasted almonds [48]. This compound was previously detected as one of the major volatiles of black cherry leaves and also displays a vasodilator effect [8]. Regarding 1-pentanol, detected in both raw and toasted seeds, it has been proposed that it derives from the oxidation of linoleic acid, and its concentration increases with storage time in almonds [49].
Acetic acid was the main volatile carboxylic acid found in raw and toasted P serotina seeds. This carboxylic acid can be regarded as a result of long-chain fatty acids degradation and it is often related to a strong sour odor [50]. Esters were detected in lesser quantities, some of them, such as methyl esters of short chain fatty acids, were found in raw seeds, but not in toasted seeds. Hydrocarbons and ketones were minor compounds in both raw and toasted seeds.
Pyrazines and pyrroles were other major volatiles identified exclusively in toasted black cherry seeds. These compounds are produced during the toasting process from free amino acids and monosaccharides by the Maillard reaction through Strecker degradation [33], particularly, ethyl-methyl-, ethyldimethyl-, and diethylmethylpyrazines, are formed in the reaction of glucose and fructose with alanine and glycine [51]. The most abundant pyrazine in black cherry toasted seeds was 2,5-dimethylpyrazine, followed by methylpyrazine, 3-ethyl-2,5-dimethylpyrazine and trimethylpyrazine. The first one has been previously reported as a major pyrazine in roasted hazelnuts [33] and toasted almonds [48]. Pyrazines contribute to the characteristic toasted aroma in chesnuts [52], and peanuts [53], almonds [54], and pyrroles significantly are responsible for the characteristic toasted aroma of different thermal treated foods [44].

Experimental Section
Reagents and standards were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise noted. Solvents were purchased from Baker-Mallincrodt (JT Baker, Mallinckrodt Baker Inc., Phillipsburg, NJ, USA).

Samples
Ripe black cherry fruits were cultivated in Huejotzingo, Puebla (México) and harvested in May of 2011. Subsequently, the seeds were removed from the pulp with plastic knives, washed and allowed to dry at 25 °C during 48 h. Finally, the seeds were stored at −70 °C until analysis.
All the analyses were carried out on raw and toasted seeds. Toasting was performed in a comal (traditional Mexican iron griddle) during 20 min at 125 °C. Then, the seeds were rotated frequently to avoid overheating and to obtain homogenous toasting. Raw and toasted seeds were cracked and opened with pliers to get the kernel.
Taking into account that black cherry toasted seeds are consumed by Mexican people as snacks, almonds (Prunus dulcis) and peanuts (Arachis hypogaea) samples were analyzed for comparative purposes. Both seeds were purchased in a local market in the city of Queretaro, Queretaro (México).

Chemical Proximate Analysis
Chemical proximate analysis was carried out on raw and toasted black cherry seeds, almonds and peanuts by using AOAC methods (AOAC, 2000). Moisture, protein, fat, ash, crude fiber and carbohydrates contents were determined by methods 950.46, 928.08, 960.39, 920.153 and 985.29, respectively. Total carbohydrate content (on dry weight basis) was calculated by difference [100 − (protein + lipids + ash + crude fiber)].

Determination of Vitamins A, C, and E
Vitamins and carotenes were determined by using AOAC methods (2000) for vitamin A (960.46), vitamin C (967. 22), and vitamin E (992.03).

Mineral Content
Sodium, potassium, calcium, magnesium and iron content of samples was determined by method 985.35 described in Association of Official Analytical Chemists (AOAC, 2000). Phosphorous was determined according to method 965.17 (AOAC, 2000).

In Vitro Protein Digestibility
The in vitro protein digestibility of dry samples was estimated by using the methodology of Hsu et al. [55] and applying the equation Y = 234.84 − 22.56X, where Y is the in vitro protein digestibility (%) and X is the pH of the protein sample suspension, after proteolysis with a multienzyme system consisting of porcine pancreatic trypsin type IX, bovine pancreatic chymotrypsin type II, and porcine intestinal peptidase grade III. The average value of three replicates is reported.

Amino Acid Analysis
Proteins were hydrolyzed in hydrochloric acid (JT Baker) and amino acids were analyzed using a HPLC autoanalyzer (Waters 2487, Millipore, MA, USA), according to Bidlingmeyer et al. [56].

Nutritional Quality of Proteins
For predicting dietary protein quality, the in vitro protein digestibility and amino acid composition were used to calculate PDCAAS (protein digestibility corrected amino acid score) according to the FAO/WHO [30] prescribed formula: PDCAAS = Amino acid score (of the most limiting AA) × Digestibility (1)

Volatile Compounds Analysis
Extraction conditions were based on previously published work of Krist et al. [52] and Agila et al. [54] with some modifications. Fresh seeds (15 g) were blended in an electric blender (model O-20, Osterizer TM , Boca Raton, FL, USA) for 15 s. The ground seeds (1 g) were transferred into a 20 mL vial, along with 7 µg of a menthol solution (0.01% w/w) as an internal standard and sealed with a Nickel-aluminum crimp cap provided with a needle-pierceable polytetrafluroethylene/silicone septum. Vials were pre-equilibrated for 60 min at 50 °C. Solid-phase microextraction (SPME) was performed with a 75 mm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber (Supelco Co, Bellefonte, PA, USA). Extractions and injections were performed using a MPS2 autosampler (Gerstel, Linthicum-Baltimore City, MD, USA) fitted with a vial heater. The fiber was exposed for 10 min in the head space of the vial for analytes adsorption. Subsequently, the fiber was removed from the vial and placed into the chromatograph injection port for desorption.
Gas chromatography separation and quantification was carried out using an Agilent GC 7890A series intrumenbt (Agilent Technologies, Inc., Santa Clara, CA, USA) equipped with a flame ionization detector (GC-FID). Injection port and detector temperature was 230 °C. The injector was operated in the splitless mode. The capillary columns used were an HP-5 60 m × 0.32 mm i.d., 0.32 µm film thickness; Agilent Technologies, Inc.) and a DB-Wax (60 m × 0.32 mm i.d., 0.32 µm film thickness; Agilent Technologies, Inc.). Oven temperature was programmed at initial temperature of 40 °C for 5 min, then raised at 5 °C/min to 230 °C and hold for 15 min. Helium was used as the carrier gas at a constant flow rate of 1 mL/min. A semi-quantitative evaluation was achieved by comparing individual peak area from GC-FID response to that of the internal standard. Each tabulated value corresponds to the average of 3 extraction replicates.
Gas Chromatography Mass Spectrometry (GC/MS) analysis was carried out using an Agilent GC 7890A series equipped with an Agilent 5975C Mass Spectrometer in electron impact mode (EI) and a quadrupole analyzer. The temperatures of ion source and quadrupole were 230 and 250 °C respectively. Transfer line was set at 280 °C. Full scan mode was used in a range of 33-300 uma, at a scan rate of 5.2/s, with an ionization voltage of 70 eV. The same chromatographic conditions as for GC-FID were used. MSD ChemStation E.01.00.237 software (Agilent Technologies, Inc.) was employed for data analysis.
Identification of volatile compounds was performed by comparing their mass spectra with those in the NIST Mass Spectral Library (National Institute of Standards and Technology, Gaithersburg, MD, USA). Retention indices of all the volatile compounds were determined by the modified Kovats method reported by Van den Dool and Kratz [57]. MS identification was confirmed by comparing Kovats retention indices (RI) to RI reported in the literature [58].

Statistical Analysis
Results of the experiments are expressed as the mean ± standard deviation (SD) from n = 3 experiments. The data were analyzed by a one-way ANOVA and the Tukey test. Differences between the means were considered to be significant when p ˂ 0.05. Statistical treatment of data was performed with the program Prism 4.0 (GraphPad Software, San Diego, CA, USA).

Conclusions
This study demonstrates that black cherry seeds have a significant content of minerals, particularly potassium, lipids and proteins. Their PDCAAS values suggest that they might be considered as a complementary source of protein. Additionally, the gas chromatography/mass spectrometry analysis allowed identification of 59 and 99 volatile compounds in the raw and toasted seeds, respectively. The major volatile components identified in raw and toasted P. serotina seeds were 2,3-butanediol and benzaldehyde. The results derived from this study indicate that these seeds have nutraceutical properties attributed to their high protein and potassium content and the presence of bioactive compounds such as benzaldehyde.