3.1. Total Soluble Solids (TSS) Content, Total Acidity (TA), and pH of Fruits
The chemical analysis results for the apple cultivars investigated are presented in
Table 1. The TSS is a good indicator of sugar content of apples and presumably of sweetness [
26]. The Limoncella cultivar showed higher TSS values (16.07 °Brix) than cultivars Golden Delicious (11.93 °Brix), Annurca (11.73 °Brix) and Red Delicious (10.57°Brix). Titratable acidity (TA), instead, may be an important tool in predicting the taste of apples. This is important during the assessment of fruit quality, since consumers often have distinct preferences for acid or sweet tasting apples [
27].
Also for the titratable acidity, the Limoncella cultivar reported statistically superior values equal to 7.57 g/L, while the Annurca and Golden Delicious cultivars did report value equal to 5.87 and 5.47 g/L respectively; lower acidity was shown in the cultivar Red Delicious with value equal to 3.03 g/L. Similar results have also been observed by Minnocci et al. [
28] who showed that Limoncella cultivar had very high soluble solids (28.2 °Brix) and the highest acidity. This high sugar content is an interesting feature for an apple cultivar, both for its taste and for its effect on storage-life, as sugars are a cryoprotectant. Our results are consistent with those previously reported by Wu et al. [
29] who showed that the summer variety, Delicious, showed relatively low levels of soluble solids, compared to the cultivars harvested in September and October, the level of soluble solids differed considerably. We also analyzed the pH variations in the apple juices, which showed significant differences between Red Delicious (4.07) and Golden Delicious (3.63) and Annurca (3.60); the Limoncella cv with 3.83 pH did not show significant statistical differences with Golden Delicious and Annurca cultivars.
3.2. Antioxidant Activity and Total Polyphenolic Content Measurements
The levels of phenolic compounds and their antioxidant activity are relevant aspects in the analysis of health promoting activity of apple cultivars. The radical scavenging activity was thus determined on apple extracts (peel, flesh, and whole fruit) obtained from not digested samples and on bioaccessible fractions (duodenal and colon bioaccessible fractions) obtained after sequential enzyme in vitro digestion. As the measurement of total antioxidant activity evaluated with different assays is important to get the overall antioxidant potential of any food matrix, in this study we used three well-known spectrophotometric assays to determine ABTS
+ radical scavenging activity, DPPH free radical-scavenging activity, and ferric reducing antioxidant capacity (FRAP). The results are reported in
Table 2 and are expressed as mmol trolox/kg dw.
With regard to the peel, in all the cultivars analyzed there was a higher content of total polyphenols and a higher antioxidant activity compared to the flesh and whole fruit. In agreement with our data, different researches reported that depending on cultivar, apple peel contains about two to nine times more total polyphenolic content than their pulp [
30,
31].
As for the flesh, the cultivar Limoncella was the one with the highest level (2.17 mg/g dw). The cultivar Annurca showed a content of 1.88 mg/g dw while there were no significant differences between the cultivars Red Delicious and Golden Delicious. These results are relatively comparable with those of other papers on apples, although it is unavoidable to have a broad variety of values for apples especially in reference to the cultivar, geographical area of cultivation, and environmental conditions [
12,
32].
Overall, the antioxidant capacity varied significantly (
p < 0.05) among cultivars and was higher in the peel than in the flesh and whole fruit. These results well correlated with the total polyphenolic contents measured by the Folin method. Discrepancies between polyphenolic content and antioxidant activity were noted for the FRAP method, whereas ABTS and DPPH proved to be the most reliable methods [
33,
34]. On the other hand, not completely overlapping results were likely caused by synergistic effects between polyphenols and other chemical constituents such as ascorbic acid and beta-carotene that can contribute to the overall antioxidant activity. Moreover, it is also reported that some polyphenolic compounds show different antioxidant activity depending on the measurement method used [
35].
In the whole apple fruit, the greatest total phenolics content and antioxidant activity was found in Limoncella cultivar, followed by Annurca and Red Delicious, whereas the lowest values were found in Golden Delicious. Higher antioxidant potential and content of total polyphenols in peel and in the whole fruit compared to the flesh fraction was described previously for other apple cultivars [
13]. As shown in
Table 2, whole fruit of Limoncella showed an antioxidant activity and a total phenolic content higher than Annurca which represents one of the traditional varieties from Southern Italy largely appreciated for its special taste and health properties [
36].
These results are in accordance with the previous reports [
36] which have shown that several traditional cultivars, including Limoncella, have a higher polyphenols content and antioxidant activity compared to more common cultivars such as Annurca. As we had previously reported [
20], the total polyphenolic content measured with the Folin assay was slightly higher and not well correlated with the amount of total phenolics provided by UHPLC-HRMS data, for these reasons we used Folin results and UHPLC-HRMS data to evaluate, respectively, quantitative and qualitative cultivar effects.
3.3. Quali-Quantitative Polyphenolic Profile by UHPLC-Q-Orbitrap HRMS
Our results showed that procyanindins, flavanols, and flavonols are present in higher concentrations in peels ranging from 62 to 79% of the total polyphenols, while hydroxycinnamics and dihydrochalcones are present at lower concentration (58–6%). Among procyanidins reported in peels, the most representative were dimers (70–84%), trimers (14–26%), and tetramers (2–4%). The cultivar Limoncella is prominent for the high content of flavanols with levels of 903.13, 860.29, and 915.92 μg/g dw respectively in the flesh, peel, and whole fruit. Panzella et al. [
37] carried out a study on traditional apple cultivars native of Campania Region and reported a higher content of flavanols and hydroxycinnamates for Limoncella than for Annurca [
37].
Table 3 reports the phenolic composition obtained by UHPLC-HRMS analysis in flesh and peel of the four different apple cultivars and
Table 4 shows the quali-quantitative polyphenolic profile of the flesh and whole fruits of the same cultivars.
Single phenolic compounds were quantified using calibration curves built with appropriate reference compounds. Coumaroyl quinic acid and epicatechin trimer and tetramer were quantified using calibration curves of chlorogenic acid and procyanidin b2, respectively. The quantification of polyphenolic compounds was also carried out by HRMS considering that the Folin assay may be affected by interferences such as ascorbic acid and reducing sugars. The investigated compounds were classified into five groups, such as hydroxycinnamic acids (chlorogenic acid, coumaroyl quinic acid, and caffeic acid), flavanols (catechin and epicatechin), flavonols (rutin, hyperoside, kaempferol-3-O-glucoside, apigenin-7-O-glucoside, and isorhamnetin derivatives), procyanidins (procyanidin B1, B2, trimer, and tetramer), and dihydrochalcones (phloretin, phloretin-2-O-xyloglucoside, and phloridzin). As reported by several authors, peel was richer than flesh in total polyphenols as well as the majority of the single polyphenolic compounds, in accordance with its defensive rule against pathogenic pressure which mainly acts on the skin [
14,
38]. The great variability in polyphenol content and profile observed among apple cultivars was in agreement with other studies [
30,
37,
39]. The complex of flavonols was identified as quercetin, kaempferol, apigenin, and isorhamnetin derivatives in all the part of apple with significant predominance in peels (
Table 4;
Table 5). Among the quercetin derivatives, the most representative in peels was the hyperoside (quercetin-3-O-galactoside), for all analyzed cultivars, with Annurca that showed the highest content (468 mg/g dw) followed by Red Delicious (427.82 mg/g dw) and Golden Delicious (401.35 mg/g dw), while Limoncella showed the lowest value (213.77 μg/g dw). Dihydrochalcones also represent a considerable amount of apple polyphenols, especially in the peels, (10–13% of the polyphenolic content) according to previous published results [
13,
40].
The results reported for the flesh and the whole fruits (
Table 4), in accordance with literature data [
12,
13,
15], highlighted that the most representative polyphenols are hydroxycinnamic acids, flavanols, and procyanidins ranging from 85.81 to 93.80% of the total polyphenols, while flavonols represented a very low incidence as well as dihydrochalcones. The most representative compound in the flesh and in the whole fruit was chlorogenic acid which contributes 32–56% of the total polyphenols and even more interestingly with the cultivars Limoncella and Annurca showing the highest values (882.76 and 861.79 μg/g dw for flesh and 841.57 and 820.28 μg/g dw for whole fruits, respectively). As reported for peels, in flesh and whole fruits, catechin, epicatechin, and procyanidins (B1 and B2) were also present at high concentrations, and together accounted for ~32–59% of the whole; lower contributions were observed for flavonols whereas dihydrochalcones it is the less represented class of polyphenols reaching levels between 5–8%.
On the basis of the results obtained, it was therefore possible to define two main groups of apple cultivars. The first group comprises the cultivars with the higher amounts of flavanols and hydroxycinnamic acids in the peel and flesh (Limoncella and Annurca cultivars). The second group comprises the Red Delicious and Golden Delicious cultivars with the greater amounts of flavonols in the fruits (especially peels).
3.4. Bioaccessibility of Apple Polyphenols and Antioxidant Activity upon Digestion
Apples are considered an important source of antioxidant compounds, however, the intake of large quantities of these compounds present in a food matrix does not always produce a significative increase of their concentration in blood and tissues which depends mainly on the digestion and assimilation mechanisms of phytonutrients. Since several studies in the literature have reported the positive effects of apple polyphenols on the colon, in this study, we evaluated the colonic release of totally bioaccessible polyphenols by simulating in vitro digestion through a multistep enzymatic protocol. In particular, in vitro digestion was carried out using the INFOGEST protocol [
24] that simulates gastric and duodenal conditions. Colon bioaccessibility was evaluated using pronase E to simulate the activity of bacterial proteases and a multi-component carbohydrase (Viscozyme L) to hydrolyze plant cell wall polysaccharide [
18].
Table 6 summarizes the results of polyphenols bioaccessibility. Data showed that hydroxycinnamic acids were the most abundant compounds released from flesh and whole fruits in the SDP (soluble duodenal phase) and SCP (soluble colonic phase), the abundance being ~73% of total bioaccessible polyphenols whereas flavonols were the most widely released compounds in both the SDP and the SCP in the case of peels with an abundance of ~68% of total polyphenols. The results obtained show that ~17% of bioaccessible polyphenols from apple (whole and flesh) were flavonols while Annurca and Limoncella flesh showed a release of these compounds equal to ~1% in the SDP and SCP. On the other hand, in vitro digestion of Annurca and Limoncella flesh showed that ~78% of totally bioaccessible polyphenols were hydroxycinnamic acids while dihydrochalcones represented about 17% of totally bioaccessible polyphenols. About 54% of totally bioaccessible polyphenols were released in the SCP, and interestingly, flavanols and procyanidins were entirely released in in this intestinal compartment. Analysis of the colon bioaccessibility under in vitro gastrointestinal digestion (
Table 7) revealed the highest release of polyphenolic compounds for the Limoncella cultivar with a release of 46.10, 67.59, and 71.96% respectively for whole fruit, flesh, and peel. Despite a part of polyphenols ranging between 28.04 and 66.32% were released in the duodenal digestion, a considerable amount was released at the colon level (an average 54% of totally bioaccessible polyphenols). This release is due to the action of enzyme such as Pronase E and Viscozyme L (including polysaccaridases such as arabanase, cellulase, β-glucanase, hemicellulase, and xylanase) on the unsolubilized pellet derived from the previous digestive step. Interestingly, the compounds mainly released in SCP were flavanols and procyanidins accounting for 4.22 and 2.93% of total polyphenols bioaccessible in the SCP.
A previous study had reported no detection of either procyanidin B2 or epicatechin after intestinal digestion leading to hypothesize the degradation to unknown products following the transition from the gastric to the intestinal environment [
41]. It has been calculated that the procyanidin level retrieved only upon colon digestion, is ~0.87% of that present in the undigested apples demonstrating that a large amount of these compounds are unstable under digestion conditions and that the fraction delivered is bound to the apple insoluble fiber. Apples, in fact are a source of both soluble and insoluble fiber. Goristein at al. reported that the content of insoluble dietary fiber in apples is about 50% of the total [
42]. This finding supports the hypothesis that non-extractable procyanidins associated with dietary fibers arrive intact to the colon and undergo the action of cell-wall degrading enzymes on the pellets resulting from the SDP [
43].
Hydroxycinnamic acids were released in the range of 34.86 to 81.84% of totally bioaccessible polyphenols, with Limoncella exhibiting the highest values for whole fruit, flesh, and peel in comparison with other cultivars (
Table 6). In particular, high resolution mass spectrometric analysis carried out on soluble fraction upon duodenal and colonic digestion highlighted a high release of chlorogenic and caffeic acid especially following the viscozyme treatment (
Table 6). Caffeic and chlorogenic acid release could be in part explained by the breakdown of the linkage between these compounds and insoluble dietary fiber following the hydrolitic action of enzyme on cell wall as previously reported for artichoke and oat bran [
44,
45]. On the other hand, chlorogenic acid in vivo can be hydrolyzed by the gut microflora into various aromatic acid metabolites including caffeic acid and quinic acid [
46]. From the health point of view, the potential release of chlorogenic and caffeic acid in the colon is noteworthy because these compounds exert a significant antioxidant activity leading to a decrease of oxidative cell damage in human colon cell lines [
47]. With regard to flavonols and dihydrochalcones it is interesting to underline the highest release (88.30%) in the colon simulated digestion from Limoncella peels, while for other cultivars the release of these compounds in the colon was between 19.91 and 81.08%. The importance of the potential delivery of dihydrochalcones in the colon is widely supported by literature studies that report antioxidative, anti-inflammatory, antiproliferative, and pro-apoptotic properties of these compounds in human colon cancer Caco-2 and HT-29 cell lines [
48,
49]. The release of individual polyphenols after in vitro digestion is reported in the
Supplementary Table S1.
The antioxidant activity of soluble fractions (SDP and SCP) evaluated by the DPPH method after duodenal and colonic digestion is reported in
Table 8. The potential antioxidant activity of polyphenolic compounds from apples along the gastro-intestinal tract is in accordance with previous in vitro and in vivo evidences [
9]. Noteworthy, a first set of experiments to evaluate these activities had also been conducted with the ABTS and FRAP assays although differently from the DPPH assay, these methods resulted to be affected by pH and enzymes interference.
The overall trend of soluble antioxidant capacity from the SDP and SCP followed the concentrations of flavanols, procyandinis and hydroxycinnamic acids under the same digestive steps. Interestingly, on average, 64.2% of the total soluble antioxidant activity was released in the SCP, with Limoncella exhibiting the highest values (82.31, 70.05, and 65.5%, respectively for whole fruit, flesh, and peel). This result suggested that enzymatic treatment with pronase E and viscozyme L responsible for the apple cell-wall breakdown released the so-called non-extractable polyphenols (catechins, procyanidins, chlorogenic, and caffeic acid) mainly bound to the polysaccharides of the dietary fiber matrix with an appreciable antioxidant capacity. Therefore, the antioxidant activity of soluble fractions (SDP and SCP) may play a role in the intestinal tract by maintaining the redox equilibrium against harmful oxidizing agents and preventing intestinal diseases related to the generation of oxygen free radicals during the digestion process.