Although free radicals and other reactive oxygen species are continuously formed in the human body, an imbalance between oxidants (free radicals) and antioxidants, in favor of oxidants, has been related to oxidative stress, which is implicated in the pathophysiology of several human diseases.
3.2. Phenolic Composition of Almond Skin
In the industry, almond skin is obtained by blanching or roasting. Upon these processes, the entire almond is subjected to high temperatures (around 100 °C for blanching and up to 200 °C for roasting) to obtain skinless almond kernels. Even though the almond skin represents just around 4.0% of the total almond weight, 60.0–80.0% of the almond phenolic compounds are distributed within the almond skin [23
]. However, thermal processes in the food industry may affect the stability of polyphenols and promote the degradation and loss of bioactive compounds [52
]. For instance, albeit subjected to lower temperatures compared to industrial roasting, almond skin polyphenols are easily lost by hot water blanching and demonstrate a lower phenolic content [53
]. Furthermore, results from one study suggested that the majority of the phenolic compounds in the kernel itself does not consist of flavonoids [23
]. Eleven out of 19 phenolic compounds identified in almond kernels, skin, and blanch water (Table 1
) were found only in the skin, whilst 95.0% of the individual flavonoids identified were also present in the skin [23
]. This situation also points out the role of flavonoids as phytoalexins, secondary metabolites that plants synthesize for self-defense, protecting the skin from different stresses.
Sang et al. (2002) isolated nine phenolic compounds that were identified as flavonol glycosides, flavanone glycoside, and flavanol and benzoic acid derivatives (Table 1
]. Upon the determination of the radical scavenging activity using the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) test, the strong radical scavenging activity of catechin, protocatechuic acid, vanillic acid, and p
-hydroxybenzoic acid, as well as of all identified flavonol glycosides, except kaempferol-3-O
-glucopyranoside, which exhibited very weak DPPH radical scavenging activity, was demonstrated. The basis of the DPPH assay is the scavenging of DPPH radicals by antioxidant molecules [59
] since the radical compound is stable and does not need to be generated [60
]. Thus, this is one of the methods more widely used for determining the antioxidant activity of different food matrices, although frequently its results are complemented by those provided by other methods evaluating molecules with different polarity or the reduction power. This approach is justified by the existence of numerous methods to determine the radical scavenging activity, while their use depends on some factors such as the nature of the sample, the mechanism of generation of target molecules, and the end-product determination, among others [61
]. Many studies have been focused on determining the antioxidant activity of bioactive compounds in almond skin by the radical scavenging methods DPPH [28
] and ABTS [56
], the ferric reducing antioxidant power (FRAP) assay [56
], and the oxygen radical absorbance capacity (ORAC) assay [30
]. Most of them provides data supporting almond skin as a matrix containing a phytochemical composition with highly valuable antioxidant activity. The radical scavenging activity of extracts of almond skin is mainly due to its content of phenolic compounds. Once the interest of these bioactive compounds is determined, the polyphenolic extracts of almond skin are further characterized via high-performance liquid chromatography (HPLC) coupled with UV/VIS detection and mass spectrometry. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) was used by Frison-Norrie and Sporns (2002) [58
] to detect and quantify four flavonol glycosides—isorhamnetin rutinoside, isorhamnetin glucoside, kaempferol rutinoside, and kaempferol glucoside (Table 1
)—in the almond skin. In a further study, these authors assessed skins of 16 different almond varieties using the same MALDI-TOF/MS technique as previously [64
], which allowed for the detection of the same four flavonol glycosides, with isorhamnetin rutinoside being the most plentiful one, in all the studied almond varieties. However, MALDI-TOF/MS cannot distinguish between some compounds, such as ishorhamnetin-3-galactoside and isorhamnetin-3-glucoside, as they have identical molecular weights. Furthermore, Arraez-Roman et al. (2010) have recently developed and compared two rapid methods for the separation and characterization of phenolic compounds in almond skin extracts: CE-ESI-TOF-MS and HPLC-ESI-TOF-MS [55
]. Using a 35 min gradient, the method described by Arraez-Roman allowed for the determination of nine (9) compounds under optimum CE-ESI-TOF-MS conditions (Table 1
), while, using the HPLC-ESI-TOF-MS method, a 9 min gradient allowed for the identification of 23 compounds corresponding to phenolic acids and flavonoids (Table 1
). In addition, Bolling et al. (2009) developed a routine, conventional, noncapillary LC-MS method for quantifying polyphenols from almond skin, which provided the tentative identification and quantification of 16 flavonoids and two phenolic acids (Table 1
), and concentrations ranging from 1.4 to 2.2 mg polyphenols/g almond skin were found [57
]. However, as one of the objectives was to reduce the analysis duration, some of the compounds were not properly differentiated, such as isorhamnetin-3-O
-glucoside and isorhamnetin-3-O
-galactoside, and quercetin-3-O
-galactoside and quercetin-3-O
-glucoside, when using this method. Bolling (2017) recently assessed in his review all methods of analysis used for identification and quantification of almond polyphenols, mainly based on the kernel but also on its by-products [65
Almond skin can be removed in the industry by blanching (and then peeling) or roasting, processes that can change the polyphenolic composition of almond skin. Garrido et al. (2008) have concluded, when comparing different industrial processing of removing skin from the kernel, that the most efficient industrial process of removing almond skin, maintaining its phenolic composition almost intact and with high antioxidant activity, is roasting, followed by blanching and oven drying [53
]. In this study, from three different almond varieties, during distinct harvest years and each industrial process, the occurrence of 31 phenolic compounds corresponding to flavanols (in the highest percentage, 33.0–56.0% of the total phenolics), flavonol glycosides, hydroxybenzoic acids and aldehydes, flavonol aglycones, flavanone glycosides, flavanone aglycones, hydroxycinnamic acids, and dihydroflavonol aglycones was observed (Table 1
As blanching is the most commonly used process in the industry for skin removal, Mandalari et al. (2010) analyzed differences between natural almond skin and blanched almond skin regarding their phenolic composition [32
]. Combinations of flavonols, flavanols, hydroxybenzoic acids, and flavanones were identified in almond skin powders and in industrial blanch water. The most representative flavonoids present in almond skin were catechin, epicatechin, kaempferol, and isorhamnetin (as 3-O
-rutinoside or 3-O
-glucoside). In the same study, the total phenolic content in almond skin was 3474.1 ± 239.8 mg GAE/100 g of fresh skin, while for blanched almond skin the total phenolic content was 278.9 ± 12.0 mg GAE/100 g of fresh skin. As expected, natural almond skin showed the highest radical scavenging activity, followed by blanched almond skin and blanch water. However, blanched almond skin presented excellent radical scavenging activity in the DPPH assay, which can be explained by the biodegradation of bioactive compounds at high temperatures, generating other products with antioxidant properties. Similarly, Smeriglio et al. (2016) characterized the phenolic composition and antioxidant activity of industrial by-products of Avola almonds (blanched skin and blanch water), and compared the results with the values retrieved for natural almond skin [56
]. Hence, resorting to RP-HPLC-DAD, 21 flavanons, flavonols, flavanols, and phenolic acids were identified (Table 1
), with naringenin being the most abundant compound, followed by kaempferol-3-O
-glucoside, kaempferol, and eriodictyol-7-O
-glucoside. From this work, it was retrieved that the blanching process causes a significant decrease in the polyphenol content in almond skin, more than 60.0%, as the total phenolic content expressed in mg GAE/100 g of fresh weight was 703.0 ± 15.9 for natural skin, 313.8 ± 2.3 for blanched skin, and 73.9 ± 0.5 for blanch water. It is obvious that the blanching process can lower the phenolic content of almond skin, and that derived from this differential polyphenolic composition, natural almond skin displays a higher antioxidant power. However, technological requirements make the development of this process in the almond industry mandatory. It is required that knowledge of the development of new value-added products for these residues is gained, as this knowledge would contribute to the reduction of their environmental impact and the enhancement in the sustainability and competitiveness of the almond industry.
In addition to the previously described processes, raw almonds are often pasteurized (to prevent Salmonella
poisoning) and can be stored for long periods of time before being sold. Bolling et al. (2010) compared the influence of roasting, pasteurization, and storage on phenolic content and antioxidant activity [63
]. Indeed, initially, it was expected that these processes decrease the phenolic content and radical scavenging activity. Skin from roasted processed almond showed a significant decrease in total phenolic content and antioxidant activity, while phenolic acids remained unchanged. Pasteurization did not have any significant effect on the phenolic content, while long-term storage (15 months) had a positive effect on the concentration of total phenolic, phenolic acids, and radical scavenging activity, indicating that storage is a dynamic process upon which the physical structure of the food matrix considered could be altered, allowing a more efficient diffusion of the phenolic compounds [63
Almond skin polyphenols are present as flavonoids in aglycone and glycoside forms, with aglycones exhibiting a higher efficiency regarding radical scavenging activity than the glycoside species [6
]. Major phenolic compounds found in almond skin are flavanols and flavonol glycosides, followed by non-flavonoids [30
]. Within flavanols, the most abundant compounds identified are catechin and epicatechin, while isorhamnetin-3-O
-rutinoside and kaempferol-3-O
-rutinoside are the major flavonol glycosides present in almond skin [30
]. Milbury et al. (2006) reported that isorhamnetin (in the form 3-O
-glucoside or 3-O
-rutinoside), representing around 70.0% of the total, is the predominant flavonoid in most of the varieties assessed so far, with 97.1 ± 2.3% of compounds originating from skin and 2.9 ± 2.3% from kernel [23
]. However, during the blanching process around 69.0% of isorhamnetin-3-O
-glucoside and isorhamnetin-3-O
-rutinoside are extracted to the blanch water [23
]. Garrido et al. (2008) stated that catechin, which represents 10.0–23.0% of the phenolics identified, and isorhamnetin-3-O
-rutinoside (6.8–17%) are the most abundant phenolics in almond skin in their study [53
]. The presence and the quantity of each phenolic compound in almond skin might depend on factors such as the industrial process to which it was subjected, the storage conditions, and the extraction method, among others. Predominant phenolic acids (non-flavonoids) in many foods of plant origin are derivatives of hydroxybenzoic and hydroxycinnamic acids. Wijeratne et al. (2006) identified derivatives of cinnamic acids in almonds (with, caffeic, ferulic, p
-coumaric, and sinaptic acids as major ones in almond skin) (Table 1
], whilst others identified hydroxybenzoic acids (protocatechuic, p
-hydroxybenzoic, and vanillic acid) [23
] and hydroxycinnamic acids (chlorogenic acid) [30
] as most abundant in almond skin.
3.3. Phenolic Composition of Almond Blanch Water
Blanching is a process consisting in the removal of almond skin by scalding almonds in hot water for a short period of time, in effect removing most flavonoids and other phenols from almond skin. Milbury et al. (2006) noticed that, in the blanching process, kaempferol and quercetin do not leach to the water [23
], while Mandalari et al. (2010) confirmed that quercetin-3-O
-galactoside and isorhamnetin are two additional compounds that are not extracted to water during blanching [32
]. This fact has been attributed to their poor water solubility. In addition, Hughey et al. (2012) did not detect quercetin-3-O
-rutinoside in blanch water [67
], as well as nonpolar aglycones, such as isorhamnetin, quercetin, and kaempferol. To test the hypothesis of the constraint represented by the “limited water solubility” to the leaching of phenolic compounds into the blanching water, blanch water precipitate was analyzed. This approach demonstrated that nonpolar aglycones (isorhamnetin and kaempferol), partially insoluble glycosides (quercetin glycoside, kaempferol glycoside, rutinoside, isorhamnetin glycoside, rutinoside, and naringenin glucoside), and even more water-soluble catechins were present in blanch water precipitate, indicating that these compounds leach from the skin into blanch water, dissolve until their solubility limit, and then precipitate [67
Furthermore, Mandalari et al. (2013) studied the antioxidant effect of blanch water [43
], determining the polyphenolic content of blanch water extracts, the level of proanthocyanidins, and the vanillin index. The total phenolic content, the antioxidant activity, and the radical scavenging activity were evaluated with different in vitro tests (Folin–Ciocalteu method, DPPH radical scavenging activity, β-carotene bleaching test, reducing power test, UV-induced peroxidation in liposomal membranes). Resorting to these determinations, the total phenolic content expressed as mg of gallic acid equivalents (GAE) per g of the blanch water extract was 90.28 ± 5.47, similar to the concentrations reported by Milbury et al. (2006) that described values ranging between 50.3 and 153.9 mg GAE released from blanching 100 g of fresh natural almonds [23
]. The most representative compounds identified in blanch water extracts were naringenin-7-O
-glucoside and kaempferol-7-O
-rutinoside, followed by catechin. The valuable antiradical activity of the extract was demonstrated within the DPPH and reducing power tests.
Kinetics of the almond skin separation via hot water submersion need to be understood to achieve it more efficiently, save energy, and possibly lower production costs [68
]. Fisklements and Barrett (2014) [68
] have reported that skin separation rates at 90 °C and 100 °C were not significantly different, while the rate decreased rapidly below 90 °C. At the same time, the relevance of these findings lies in understanding the distribution of polyphenols in blanch water and almond blanched skin, as well as those factors with a relevant impact on this distribution. On the other hand, according to Hughey et al. (2012), temperature and time increased the amount of phenolic compounds extracted into water [67
]. These authors reported that, during the first 2 min and under a temperature of almost 100 °C (that is, time and temperature used in the industry for blanching), approximately 73.0% of phenolics leach from almond skin into water, rising to 90.0% after 10 min.
The presence of value-added antioxidant compounds in the blanch water extracts reveals the possibility of further utilization and valorization of the blanch water by the nutraceutical and pharmaceutical industries.
3.4. Phenolic Composition of Almond Hulls
Almond hulls are a major by-product in almond production, being mostly used as cattle feed. The phenolic constituents and the radical scavenging activity, as well as the bioactivities of this almond green cover, have been identified and quantified. Sang et al. (2002) isolated protocatechuic acid, catechin, ursolic acid, and prenylated benzoic acid derivatives from almond hulls (Table 1
]. Protocatechuic acid is one of the major benzoic acid derivatives with strong antioxidant power, and catechin is the most widely distributed flavonoid in edible plants, with a high antioxidant activity and an inhibitory effect on numerous enzymes.
Takeoka and Dao (2003) extracted almond hulls using methanol and identified present compounds with reversed phase HPLC with diode array detection [54
]. In the extract, three hydroxycinnamic acids were identified: chlorogenic acid (5-O
-caffeoylquinic acid), cryptoclorogenic acid (4-O
-caffeoylquinic acid), and neochlorogenic acid (3-O
-caffeoylquinic acid) in the ratio 79.5:14.8:5.7 (Table 1
), chlorogenic acid appearing as the most abundant phenolic acid in the almond hull extract. In addition, extracts were tested for their ability to inhibit the oxidation of methyl linoleate at 40 °C, and the result showed that, at the same concentration, almond hull extracts present a higher antioxidant activity than α-tocopherol. At higher concentrations, almond hull extracts presented increased antioxidant activity, with similar values to chlorogenic acid and morin standards at the same concentration, showing that almond hulls are potential sources of these dietary antioxidants.
In addition, Rubilar et al. (2007) analyzed ethanol extracts and fractions (organic/water fraction) of almond hulls and grape pomace for antioxidant power and their phenolic profiles were determined by HPLC-MS [40
]. In the almond hull extracts, chromatographic peaks evidenced the occurrence of benzoic and cinnamic acid derivatives, with a small presence of flavan-3-ols, while an additional presence of epicatechin and glycosilated flavonols have been identified in the fractions. The antiradical activity was assessed using both DPPH and TBARS assays, and results showed a stronger antiradical activity of grape pomace than almond hulls.
Another study on antioxidant properties of almond hulls has been performed by Barreira et al. (2010), evaluating it through a range of chemical and biochemical assays on different almond varieties in Portugal [41
]. Samples were extracted using water, as the aim of the study was to obtain a clean extract that can be used as an additive due to the synergistic effects of the phytochemicals present in fruits and vegetables and the benefits of the complex mixture in whole foods. All assayed by-products (almond hulls, chestnut leaves, and skins) revealed good antioxidant properties with very low EC50 values.
Wijeratne et al. (2006) identified eight phenolic compounds from almond extracts of almond whole seed, brown skin, and hulls [6
]. High-performance liquid chromatography (HPLC) analysis allowed for a description of the presence of flavonols and flavonol glycosides as major flavonoids in all three extracts (Table 1
). In another study, antioxidant and antiradical activity of phenolic extracts of almond hulls and shells, obtained from different genotypes were compared by Sfahlan et al. (2009) [7
]. The authors selected 18 genotypes from diverse Iran provinces and determined total phenolic content using Folin–Ciocalteu method, reducing power, and scavenging capacity for radicals nitrite, hydrogen peroxide, and superoxide. Within various genotypes significant differences were found in the phenolic content of hulls and shells, as well as on the percentage of radical scavenging capacity. However, among different genotypes, the total phenolic content of almond hulls extract was higher than the one of the correspondent shells.