Evaluation of Protein and Antioxidant Content in Apricot Kernels as a Sustainable Additional Source of Nutrition

: Apricot fruits are a favorite for consumption; however, their kernels are a rich source of nutritionally interesting substances, too. Nevertheless, in processing of apricots, the kernels remain often unused. In this study, 32 cultivars of different origin were analyzed for their protein content and content of secondary metabolites (phenolics and ﬂavonoids). The weight and taste of kernels were assessed and these data were summarized for an evaluation of the attractiveness of the studied apricot kernels. Results showed that the protein content of kernels ranged from 14.56% to 28.77% and did not depend on the origin or weight of kernel, or taste. In addition, total phenolic (63.5–1277.3 mg GAE/100 g DW) and total ﬂavonoid (0–153.1 mg CE/100 g DW) contents and antioxidant capacity (483.4–2348.4 mg TE/100 g DW) were measured in kernels. In conclusion, the Czech hybrids LE-5959, LE-5500 and French cultivar Koolgat are prospective for kernel processing and consumption because of their high protein content and sweet taste. Hybrid LI-3-6, originating in China, showed high protein content as well but because of bitter taste could be useful rather in medicine.


Introduction
Apricot is a fruit species grown mainly for fresh fruit, but also for fruit processing (drying, canning, jams or distillation) [1]. However, in recent years, the demand for alternative plant sources of proteins and lipids has been rising. At the same time, there has been emphasis on sustainability of the production of these nutritional resources, suggesting sweet apricot kernels could be a delicacy in the same way as sweet almonds. The developmental anatomy/physiology/biochemistry of apricot kernels (seeds) and those of related species such as almond and cherry has been studied and the reader is referred to Famiani et al. (2020) for details [2].
Nutritional substances of apricot kernel have been studied as well. Turan et al. (2007) [11] reported that the total oil content in apricot kernels ranges from 40.23% to 53.19% and is mostly composed of oleic acid (70.83%). Other substances in apricot kernels are proteins, fibers, phenolic compounds, vitamins and minerals [12][13][14]. Overall, apricot kernels have antioxidant, antibacterial and antiparasitic effects [15,16]. Depending on the variety, they contain toxic cyanogenic glycoside amygdalin [17,18]; the sweet kernels contain ten times less amygdalin than the bitter ones [19]. Thus, it is recommended to avoid consumption of bitter kernels due to possible cyanide poisoning.
Numerous reports mention a substantial amount of dietary protein in apricot kernels [13,[20][21][22][23][24], which ranges from 14.1% to 45.3% [12]. The main proteins are albumin (84.7%), globulin (7.65%), gluteline (3.54%) and prolamin (1.17%). Apricot kernels could represent a good potential source of proteins with the ability of generation of bioactive peptides with angiotensin-converting enzyme (ACE) inhibitory activity. ACE plays a central role in the regulation of blood pressure [25]. The prevalent method used for protein measurement in the food industry is the Kjeldahl method [26], which relies on total nitrogen determination, from which the protein content is estimated [27,28] via the nitrogen-toprotein conversion factor. Some studies have reported a phenolic content of apricot kernels, too [14,29]. Phenolic substances increase antioxidant activity and are required in food. The aim of this study was to determine the total protein content in apricot kernels and to compare measured data with respect to physical properties of kernels and antioxidant content of each cultivar. The secondary metabolites, such as total phenolic content, total flavonoid content and antioxidant capacity, were determined to obtain information about the composition of apricot kernels. Finally, their attractiveness was determined by the taste and size measurements. In addition, modern and newly introduced apricot varieties were studied (Koolgat, Kioto, Samourai, Meligat, Mediabel, Congat and Luizet from France, Bora from Italy, and Flavorcot and Early Blush from the USA).

Site of Planting and Plant Material
In total, 32 apricot cultivars of different origin were analyzed in this study (Table 1): ten cultivars from the Czech Republic, seven from France, four from Ukraine, three from the USA, two from Slovakia, two from China and one each from Moldova, Canada, Italy and Hungary. Trees of these cultivars were grown in the experimental orchard at the Faculty of Horticulture in Lednice, Mendel University in Brno (site coordinates 48.80 • N/16.80 • E, at an altitude of 172 m). Ten fruits from each variety were harvested at their optimum maturity and transported to the laboratory for pomological analyses. The weight of the stone and the kernel was measured and the taste of each kernel was qualified (sweet, bittersweet, bitter).

Determination of Total Protein Content (TPC)
The analysis of total protein content was accomplished in three replications for each variety by the Kjeldahl method [26]. Ten dried kernels were crushed in a mill (Fritsch Pulverisette 2 Mortar Grinder). Approximately 0.3 g of crushed kernels, weighed accurately on a scale, were placed in a 250 mL Erlenmeyer flask, mixed with the catalyst (7 g K 2 SO 4 + 0.4 g CuSO 4 + 0.0035 Se) and mineralized in 10 mL of concentrated H 2 SO 4 when heated. The prepared mix was alkalized with 50 mL of 30% NaOH and distilled into 25 mL of 0.05 M H 2 SO 4 . The final sample was colored by Tashiro indicator and titrated with 0.01 M NaOH to the equivalence point. The final volume of 0.01 M NaOH was used for the calculation of the nitrogen content (1). The whole procedure was performed also without the kernel sample and the volume of NaOH was used as a titration blank (1).
The nitrogen content (x) in% was determined by Equation (1): where V is the volume of NaOH of blank titration, V 1 is the volume of NaOH of sample titration, m is the weight of the sample (g). The TPC (%) was determined by Equation (2): where x is the nitrogen content and f is the nitrogen-to-protein conversion factor (6.25), based on the assumption that proteins consistently contain 16% nitrogen and that all nitrogen is allocated in proteins.

Determination of Total Phenolic (TPHC and Flavonoid (TFC) Contents and Antioxidant Activity
The samples were prepared for analyses by extraction of crushed kernels in 25 mL of 75% methanol for 24 h. The weight of each sample was recorded. Then the extract was filtered through filter paper into a 50 mL measuring flask and methanol was added up to the required volume. Samples were placed into 20 mL plastic bottles and kept at −20 • C until the analyses [30].
The secondary metabolites TPHC and TFC and antioxidant activity were determined according to the methods of Zloch et al. (2004) [31] by using a Specord 50 Plus spectrophotometer (Analytik, Jena, DE). TPHC was determined after reaction of sample methanol extracts with Folin-Ciocalteu reagent at a wavelength of 765 nm. The results were calculated from a calibration curve (y = 0.0009x + 0.0034) and expressed in gallic acid equivalents (GAE) per 100 g of dry extract weight (DW). TFC was determined after reaction of sample methanol extracts with AlCl 3 and NaNO 2 at a wavelength of 510 nm. The results were calculated from a calibration curve (y = 1.3286x + 0.0295) and expressed in catechin equivalents (CE) per 100 g DW. Antioxidant activity was determined after reaction of DPPH (1,1-diphenyl-2-picrylhydrazyl) (Sigma) and methanol extracts at a wavelength of 515 nm. The method is based on the decolorizing property of the hydrogen radical of DPPH with hydrogen donors. The results were calculated from a calibration curve (y = −1.2346x + 0.7143) and expressed in Trolox equivalents (TE) per 100 g DW. Trolox (6-hydroxy-2.5.7.8-tetramethylchroman-2-cyrboxylic acid) was used as a standard.

Statistical Analysis
Statistical analysis was performed with Statistica 12 (TIBCO, Palo Alto, CA, USA) and Microsoft Excel software. Single-factor ANOVA analysis (level of significance α = 0.05) was used for statistical processing, the Fisher LSD test and Tukey HSD test were subsequently used to evaluate the statistical significance of differences between the individually measured content values.

Results
The average weight of a fresh apricot kernel was 1.0 ± 0.2 g; the highest weight was measured in Moldavskij Olimpik (1.7 ± 0.3 g) and the lowest in LI-13-6 (0.6 ± 0.2 g). The ratio of kernel to stone weight was 31 ± 1% on average. The highest ratio value was calculated in Meligat (45 ± 4%) and the lowest in the Bora (19 ± 7%) cultivar. A sweet taste was determined in kernels of 17 cultivars; kernels of 7 cultivars had a bitter taste and 8 cultivars had bittersweet kernels. All data are displayed in the Table 2. Table 2. The physical properties and taste of apricot kernels. The data are displayed as the mean ± standard deviation of ten replications.

Cultivar
Weight of Kernel (g) The average total protein content (TPC) in apricot kernels was 22 ± 4% of the kernel weight. The highest value was measured in the Czech hybrid LE-5959 (28.8 ± 0.2%) and the lowest in the Czech cultivar Marlen (14.6 ± 0.4%). The origin of the cultivar had no significant influence on the TPC of the kernel (data not shown). The average TPC of cultivars from the Czech Republic was 23 ± 4%, from Ukraine 19 ± 4%, France 22 ± 4% and the USA 24 ± 3%. The results are shown in Table 3 and Figure 1.   The values of total phenolic content (TPHC) ranged from 1277.3 to 63.5 mg GAE/100 g DW ( Table 4). The average value of TPHC was 267 ± 156 mg GAE/100 g DW. The highest value was measured in the Italian cultivar Bora, which was as an outlier not included in statistical analysis. The lowest values were measured in the Canadian Harostar and French Congat cultivars. After Bora, the next highest values were measured in LE-5959, Pozdní chrámová and Moldavskij Olimpik.  The values of antioxidant activity ranged from 2348.4 to 483.4 mg TE/100 g DW with the mean value reaching 732 ± 257 mg TE/100 g DW. The cultivar Bora was assessed as an outlier with the highest value (Table 6). Surprisingly, the next highest value was reached by the cultivars Velikyj, Koolgat and LE-5959. The lowest value was measured for Harostar. For final comparison, all results of nutritive substances of sweet, bitter and bittersweet kernels are shown in Figure 2. In our study, the mean value of TPC in sweet kernels was 21 ± 1%, in bitter kernels 24 ± 1% and in bittersweet kernels 24 ± 1%. Interestingly, high values of TPHC were measured in cultivars with sweet kernels (330 ± 174 mg GAE/100 g DW on average). THPC of cultivars with bitter kernels was considerably lower (172 ± 80 mg GAE/100 g DW on average). TPHC of cultivars with bittersweet kernels was between the sweet and the bitter kernel cultivars, resulting in 210 ± 98 mg GAE/100 g DW Sustainability 2021, 13, 4742 7 of 11 on average. The antioxidant activity of sweet kernels, bittersweet kernels and bitter kernels was 753 ± 240, 742 ± 387 and 672 ± 144 mg TE/100 g DW, respectively.
(172 ± 80 mg GAE/100 g DW on average). TPHC of cultivars with bittersweet kernels was between the sweet and the bitter kernel cultivars, resulting in 210 ± 98 mg GAE/100 g DW on average. The antioxidant activity of sweet kernels, bittersweet kernels and bitter kernels was 753 ± 240, 742 ± 387 and 672 ± 144 mg TE/100 g DW, respectively.
Correlation was established between TPHC and TFC, which had the correlation coefficient R = 0.9572 (Figure 3). Cultivars Pastyryk, Beta and Velikyj deviated from this correlation as they had high values of TPHC but low TFC. All of them belong to the bittersweet kernel group. Conversely, cultivars Poljus Južnyj, Orangered and Luizet had higher values of TFC compared to TPHC.

Discussion
High protein and phenolic content in apricot kernels are a positive parameter in terms of their consumption or processing. In our study, the mean value of TPC was 22.  Correlation was established between TPHC and TFC, which had the correlation coefficient R = 0.9572 ( Figure 3). Cultivars Pastyryk, Beta and Velikyj deviated from this correlation as they had high values of TPHC but low TFC. All of them belong to the bittersweet kernel group. Conversely, cultivars Poljus Južnyj, Orangered and Luizet had higher values of TFC compared to TPHC.

Discussion
High protein and phenolic content in apricot kernels are a positive parameter in Figure 3. Correlation between total flavonoid content and total phenolic content of apricot kernels.

Discussion
High protein and phenolic content in apricot kernels are a positive parameter in terms of their consumption or processing. In our study, the mean value of TPC was 22.26% and it corresponded to results from other studies (14.1% to 45.3%) [20][21][22][23][32][33][34]. Lazos et al. (1991) measured 21.2% of TPC in apricot kernels, 26.7% in peach kernels and 25.3% in sweet cherry kernels [35]. Sweet apricot kernels could be used in the food industry similarly to almonds (Prunus amygdalus L.). Barbera et al. (1994) found that almond kernels grown in Spain contained 23.03% to 23.98% TPC, while Calixto et al. (1981) measured 20.51% TPC and Özcan (2000) found only 12.7% to 16.3% TPC in almonds [13,36,37]. With regard to the high amygdalin content in bitter kernels (26 ± 14 mg·g −1 ), only sweet apricot kernels are suitable for consumption as they contain considerably less amygdalin (0.16 ± 0.09 mg·g −1 ) [19]. In our study, the average value of TPC in sweet apricot kernels ranged from 14.56% to 28.77% with a mean value of 21 ± 4%. The highest TPC was in the Czech hybrid LE-5959, which contained the most protein (28.8 ± 0.2%) and its weight was approximately average (1.0 ± 0.1 g). The second highest TPC content was in the French cultivar Koolgat (26.5 ± 0.4%), with weight slightly under the average value (0.79 ± 0.07 g). A similar result was found in the Czech hybrid LE-5500 (0.85 ± 0.06 g) with the third highest TPC (25.9 ± 0.3%). Cultivars with sweet kernels, such as Orangered, Congat and Meligat, had slightly higher weight than average. An interesting cultivar is the Moldavskij Olimpik, which had average TPC values (21.52 ± 0.08%) but the heaviest kernels (1.7 ± 0.1 g). The cultivar Luizet had the second heaviest kernels, but its TPC was much less than average (15.3 ± 0.1%). Processing of bitter apricot kernels is also in the long-term interest. Aside from the use of amygdalin in traditional Chinese medicine [38], the protein concentrate extracted from apricot kernels has been used for preparation of transglutaminase-induced gels, which are used for delivering sensitive compounds into functional foods, as well as dietary supplements and pharmaceutical products [39]. In China, approximately 193 thousand tons of bitter kernels are processed every year [40], which have between 23.6% and 26.2% TPC on average [38]. This value corresponds with our results of the average TPC in bitter kernels (23.7%), but the range was higher (18.6% to 27.7%). Famiani et al. (2020) expressed the hypothesis that proteins in the kernels of stone fruits are accumulated in kernels from the endocarp during the hardening of the stone [2]. The process of translocation of proteins is still unknown and could be an interesting topic in future research. Korekar et al. (2011) analyzed kernels of 14 apricot cultivars for their TPHC which ranged from 92.2 to 162.1 mg GAE/100 g with a mean value of 128.5 mg GAE/100 g DW [41]. Juhaimi et al. (2018) studied phenolic and flavonoid compounds in kernels of four Turkish apricots and their results ranged from 54.41 to 59.61 mg GAE/100 g DW for TPHC and 18.17 to 23.56 mg CE/100 g DW for TFC [14]. In our study, the results for TPHC were much higher (267 ± 156 mg GAE/100 g DW on average). The result for TFC was similar to Juhaimi et al.'s (2018) (22 ± 19 mg CE/100 g DW) [14]. Yıldırım et al.
(2010) measured ten times higher values of TPHC in bitter apricot kernels than in sweet ones [29]. In our study, sweet kernels contained more phenolics than bitter kernels (sweet-331 mg GAE/100 g DW; bitter-172 mg GAE/100 g DW). Just for comparison, 425, 589 and 461 mg GAE/100 g was measured in dry hazelnuts, walnuts and pistachios with seed coats, respectively [42]. In our study, much higher TPHC and TFC were measured in cultivars Bora (1277.3 mg GAE/100 g DW), LE-5959 (647.5 mg GAE/100 g DW) and Moldavskij Olimpik (626.3 mg GAE/100 g DW). These cultivars have sweet tasting kernels and the results of antioxidant activity agreed with the phenolic content of mentioned cultivars. Results of antioxidant activity of Turkish apricot kernels in Juhaimi et al. (2018) ranged from 53.98% to 59.61% DPPH [14]. Yigit et al. (2009) mentioned that higher antioxidant activity was found in sweet kernels than in bitter kernels [43]. These results agreed with our study on average, where the antioxidant activity for sweet kernels was 753 mg TE/100 g DW and for bitter kernels 672 mg TE/100 g DW on average. The cultivar Bora, with bittersweet kernels, showed the highest value of antioxidant activity (2348.4 mg TE/100 g DW) and, surprisingly, a high value of antioxidant activity was measured for Velikyj (1600.1 mg TE/100g DW), which did not correlate to TPHC and TFC. In sweet kernel cultivars, the highest values were measured for LE-5959 (1205.0 mg TE/100g DW) and Koolgat (1427.6 mg TE/100g DW). Thus, these cultivars could be an interesting nutritive source of protein and antioxidants in addition.

Conclusions
At a time when the world is driven by many changes, such as global climate change, rational and sustainable agricultural production, depletion of natural resources and overpopulation, it is important to seek solutions to overcome this critical period in the next years. One of the possibilities is to search for food resources with added value for human health which are not yet widely used in the food industry. Apricot kernels are a nutritionally interesting source of proteins, which are the second most abundant component of their weight. The average value of total protein content of 32 cultivars with different origin was 22.26% and the values ranged from 14.56% to 28.77%. No significant correlation between TPC and origin, weight or taste of kernels was found. The average weight of the kernels was 1.02 g and ranged from 0.57 to 1.7 g. The Czech hybrid LE-5959 could be a prospective cultivar for its sweet kernels and high protein content. Interesting results were measured in the cultivar Moldavskij Olimpik, which had average TPC, however a very high value of kernel weight. The data of protein content were supplemented with total phenolic and total flavonoid contents and antioxidant activity in apricot kernels. On average, 267 ± 156 mg GAE/100 g DW, 22 ± 19 mg CE/100 g DW and 732 ± 257 mg TE/100 g DW were measured in kernels of analyzed cultivars. Despite the knowledge about nutritionally interesting substances in apricot kernels, this study is unique in terms of comparing the content values in the kernels of specific apricot cultivars with various origins. However, it was confirmed that the results correspond to the already measured average values.