2.1. Flavonoids, Organic Acids and Proximate Constituents
Flavanones and to a lesser extent flavonols are the predominant flavonoids in the genus
Citrus [
23]. The quali-quantitative distribution of these phenols is largely influenced by the specie and/or the variety [
24,
25,
26]. Flavonoids are thus commonly used as chemotaxonomic markers and evaluate the quality and genuineness of citrus juices [
24,
27,
28,
29,
30]. Since flavanones constitute virtually all of the total flavonoids present (e.g., 98% in grapefruit, 90% in lemons, and 96% in limes) [
29], we focused on the major aglycone flavanones with their rutinose or neohesperidose glycosides as markers to differentiate Neapolitan limmo and lemoncetta Locrese from other citrus juices and between the two populations. The same approach was utilized by Mouly et al. [
24] to effectively differentiate lemon and lime, varieties of grapefruits (white, pink, red, and green), and sweet oranges (Valencia, navel, blood, Thomson, and Malta). The flavonoid profile is also a method widely utilized to detect the possible mixture of different juices as for instance the addition of bergamot to lemon juice [
30]. The range of variability of flavonoids and organic acids for citrus juices are reported in the code of practice of the International Federation of Fruit Juice Producers (IFFJP).
We thus compared by HPLC the population of flavonoids in Neapolitan limmo with those of lemoncetta Locrese (
Figure 2 and
Table 1). An identical flavonoid profile unites both analyzed populations (
Figure 2). Neapolitan limmo had flavanone profiles more like lemoncetta Locrese. The insignificant difference (
p < 0.05) observed in flavonoid contents are within the normal limits of environmental variability of these two juices.
Both Neapolitan limmo and lemoncetta Locrese are qualitatively characterized by a common presence of five different flavonoids: the three rutinosidic flavanones, hesperidin, eriocitrin, and narirutin and the two flavones O-glycosides, rutin and diosmin. The identification of these flavonoids is confirmed not only by the retention times but also by spectra analysis compared to their standards (data not shown). The samples do not contain flavanone O-neohesperidose and the non-bitter flavanone neoponcirin.
Of particular interest between the present flavonoids is eriocitrin, a flavonoid that is exclusively characteristic of lemon juice [
27,
29], and is almost absent in orange and grapefruit juice. Eriocitrin in limmo and lemoncetta strengthens the genetic closeness to lemon of these Mediterranean limes, both hybrids of citron (
C. medica L.) being a sour orange × Citron cross [
5].
The flavonoid composition of limmo and lemoncetta was also compared with that of two other limes, both previously characterized by Nogata et al. [
23]. The first is classified under the
Limonoides subset according to the Tanaka’s system [
8], with the common name Sweet lemon and the scientific name
C. limetta or
C. limetta Risso, with slightly acidulous pulp [
9,
12,
13]. The second is classified in the subsection of
Decumanoides (sect.
Citrophorum) [
8], with common name lumie and the scientific name
C. lumia or
C. lumia Risso, with sweet and non-acidic pulp [
4,
13]. This citrus is most diffused in Italy and in some southern regions of France [
31]. A prevalent similarity emerges from the comparison between the flavonoid profiles of
C. limetta and
C. lumia and those of Neapolitan limmo or lemoncetta Locrese (
Table 1): Neapolitan limmo, lemoncetta Locrese and
C. lumia have a significant content of the flavanone
O-rutinosides esperidine and eriocitrin. They also have a reduced content of the other flavanone rutinosides narirutin (
Table 1) and of the flavones rutin and diosmin. Significative is the common absence of the flavanones neoponcirin, naringin, neohesperidin, neoeriocitrin e poncirin in limmo, lemoncetta and
C. lumia but not in
C. limetta.
Unfortunately, no other paper on the traditional Italian sweet lime varieties besides Nogata et al. [
23] report data on flavonoids [
12,
13]. The data are anyway consistent with those recently found by Smeriglio et al. [
32] on
C. Lumia which reported a similar significant presence of hesperidin and eriocitrin.
A further indication that the Neapolitan limmo can be with good reasons classified in the
Citrus lumie group is also offered by two recent papers by two distinct research groups [
6,
7]. These authors demonstrated by independent methodological approaches that in acid-less varieties of citrus, exceptionally low fruit acidity is associated with absence of anthocyanin pigments in leaves and flowers and of proanthocyanidins in seeds and flowers without pigmentation or white, like those of Neapolitan limmo (
Figure 1e).
Next, we extended our investigation to the quali-quantitative distribution of organic acids, the overall acidity, and the pH of the juice. These parameters can give useful indications on the nature of the lime type discriminating between acid ecotypes. Both limmo and lemoncetta have qualitatively a common acidic chromatographic profile characterized by the presence of five organic acids: malic, citric, quinic, tartaric, and fumaric acids (
Figure 3). Similar are also the quantitative data (
Table 2).
Malic acid is the dominant organic acid of these sweet Mediterranean limes with average values of 1.57 ± 0.03 g/L in Neapolitan limmo and slightly higher, 1.88 ± 0.02 g/L, in lemoncetta. It is probably this significant presence in the acidic profile that confers to the juices of these fruits (acid-less sweet tasting) that smooth tartness acidity given by malic acid. This taste is clearly different from the sensorial sour quality given by citric acid in juices when this is dominant [
33].
Both Neapolitan limmo and lemoncetta showed reduced contents of citric acid with average values of about 0.94 g/L in the group of Neapolitan limmo and even lower in lemoncetta Locrese (0.48 g/L), with an average pH > 5.7 and values of total acidity < 1.4 g/L (
Table 2). This is consistent with the phenotypes of the sweet forms of
C. limetta Risso—Mediterranean sweet lime—sweet Roman [
12], Roman [
13,
34], Lima Dulce, or Dulce lime [
14].
Quinic acid is the most expressed acidic compound after malic acid and citric acids. The average levels of malic acid are between 0.10 and 0.43 g/L with higher average values for lemoncetta Locrese compared to Neapolitan limmo (
Table 2). Also, for this acid, the quantitatively expressed levels appear on average higher in the group of lemoncetta Locrese than in those of Neapolitan limmo. The average values are however completely comparable with each other. Finally, fumaric acid is much less expressed and generally does not exceed 0.01 g/L.
This analysis makes us conclude that limmo and lemoncetta are chemically similar although there is an appreciable quantitative difference in some substances likely due to climatic and soil composition and other environmental differences. The values of flavonoids and other metabolites are, for instance, different likely because of the different degree of activivity of phenylalanine ammonium lyase, the enzyme central to the production of the biosyntesis precursor of flavonoids cinnamic acid [
35].
2.2. Chirospecific Analysis
Biological activity is often correlated with chiral properties. In citrus fruits, chiral compounds are widely used as indicators of adulteration or fraud of essential oils by addition of synthetic or natural compounds of different botanical origin. The GC profiles of volatile aromatic compounds of essential oils from Neapolitan limmo and lemoncetta Locrese (
Figure 4a) were initially compared and analyzed by heart-cutting multidimensional GC [
36] to estimate the enantiomeric distribution (ee%) of chiral β-pinene, sabinene, limonene, linalool and linalyl acetate (
Figure 4b).
As for flavonoids and organic acids, an identical metabolic profile of volatile compounds was common to both citrus populations (
Figure 4a). Forty-three volatile aromatic compounds were identified. In both populations, the more expressed were limonene (61.8 ≥ ± 14.4%) ≥ linalyl acetate (9.2 ± 0.5%) ≥ linalool (6.6 ± 0.4%) ≥ β-pinene (4.4 ± 2.9%) ≥ myrcene (1.3 ± 0.6%) ≥ sabinene (0.8 ± 0.4%) ≥ α-terpineol (0.7 ± 0.6%) ≥ α-pinene (0.5 ± 0.4%) ≥ geranial (0.4 ± 0.3%) ≥ neral (0.3 ± 0.1%) ≥ β-bisabolene (0.2 ± 0.1%) ≥ nerol (0.2 ± 0.1%) ≥ terpinene and citronellol ranged from 0.05 to 0.1%. Camphene, octanal, α-phellandrene, terpinolene, and terpinen-4-ol were less than 0.05%.
The data obtained by four heart-cut multidimensional GC are even more interesting; the enantiomers of β-pinene, sabinene, limonene, linalool, and linalyl acetate were all well-separated on a DiActButylsilyl γ-CDX chiral column (
Figure 4b). The dominant enantiomeric form for limonene was (
R)-(+). Both populations showed (
R)-(+) for β-pinene and sabinene (
S)-(−), and (
R)-(−) for linalyl acetate and linalool (
Table 3).
2.3. Volatile Organic Compounds Analysis
Comparison of the total ion chromatograms of the aroma components collected in the juices of Neapolitan limmo and lemoncetta Locrese showed the presence of 8 terpenes, 5 monoterpenoid alcohols, and 3 sesquiterpene hydrocarbons in the juices. Both fruits presented the same volatile compounds (
Figure 5 and
Figure 6).
In the variety lemoncetta Locrese 12 volatile compounds were in concentrations significantly higher than in limmo (
Table 4).
D-limonene was the main flavor compound found in both varieties, but its concentration was significantly higher in Neapolitan limmo compared to lemoncetta Locrese (
p < 0.05). Other major components (except limonene) are β-pinene, β-myrcene, and bergamol (linalyl acetate) for both varieties. The last two compounds are significantly higher in Neapolitan limmo (
p < 0.05). Moreover, other volatile aromas found at small percentage such as α-phellandrene, α-pinene, β-phellandrene, linalool, trans-α-bergamotene, and β-bisabolene were considered to be important compounds influencing the entire aroma [
37]. Their concentrations were significantly higher in lemoncetta (
p < 0.05). Only the volatile compound α-pinene was not significantly different in the two varieties (
p > 0.05).
Twenty-three volatile compounds were identified in the peels: 10 terpenes, 8 monoterpenoid alcohols, and 5 sesquiterpene hydrocarbons (
Table 5). Both varieties exhibited the same volatile compounds with different intensity (
Figure 6). The peel of lemoncetta Locrese presented significantly higher areas than Neapolitan limmo. The main component of the peels is Limonene followed by bergamol, linalool, β-pinene, and β-myrcene with different intensities. Limonene value was not reported because is no longer linear and the areas were off the charts. Other relevant compounds are α-pinene, β-phellandrene, β-pinene, β-myrcene, linalool, and bergamol which are significantly higher in lemoncetta compared to Neapolitan limmo (
p < 0.05). Only two volatile compounds (nerol acetate and geraniol acetate) are significantly higher in Neapolitan limmo than in lemoncetta Locrese (
p < 0.05).
The compounds found in these varieties were also reported in other Citrus varieties. Particularly, in the peels were found 10 additional volatile compounds: cis-β-terpineol, terpinolene, α-terpineol, acetic acid octyl ester, trans-geraniol, α-terpineol acetate, nerol acetate, geraniol acetate, α-bergamotene, and cis-α-bisabolene. The absence of these compounds in the juices is probably due to the juice squeezing. The extraction pressure conditions will determine different aroma components in juices and in peels.
Our results revealed that there are not qualitative differences between the two varieties. The aromatic profiles are identical and there are not specific volatile compounds that could be used to differentiate the varieties. The main differences are connected only to the intensity of the aromatic profile. The use of SPME-GC-MS thus resulted to be a valuable tool to analyze the volatile profile of the two sweet lime juices and peels and obtain a quality characterization of fruits from different varieties.
2.4. Genetic Comparison by DNA-Based Molecular Markers
The genetic similarity of the two Mediterranean sweet lime populations was finally analyzed using RAPD molecular markers [
38]. This technique was preferred to DNA barcoding and phylogenic analysis because DNA barcoding works best if the sequences have sufficiently diverged. We feared that the assay could prove inconclusive in our case given the indications from chemical data that the evolutionary distance between Limmo and lemoncetta is close [
39]. RAPD has instead proven useful in the identification of Citrus cultivars and the assessment of genetic relatedness for neglected or little-known citrus accessions [
40,
41]. Lemon (
C. limon) cultivars of Campania (Italy) were for instance distinguished by their RAPD profiles using five arbitrary primers, confirming that RAPD markers can successfully identify lemon genotypes [
42]. Iannelli et al. [
43] characterized lemons by combining genome size and RAPD markers. In their work, the primer U19 utilized for distinguishing lemon genotypes.
The RAPD-PCR method allowed the genetic analysis of Neapolitan limmo and lemoncetta Locrese by simply comparing the presence/absence of bands in DNA amplification patterns, visible after electrophoresis on agarose gel, as the bands represent the numerous loci detected randomly and dispersed in their respective genomes. We used primers already successfully considered for the variety discrimination of other species [
44,
45] but we obtained more markers as compared to previous work. The primers used showed high reproducibility of amplification products. They also showed the absence of polymorphisms in DNA in the comparison between limmo and lemoncetta and therefore the impossibility to discriminate between the two populations (
Figure 7a,b).
The RAPD profiles obtained with each primer were extremely different from each other, allowing us to explore different regions of the genome. Alleles corresponding to reproducible amplicons, given the dominant genetic nature of RAPD markers, identified a total of 80 markers or loci (
Table 6). The number detected was dependent on the primer used but not on the variety, with an average of 6.7 loci per primer, going from the minimum of four bands for primers G07 and U4 and at most 12 bands for the AX08 primer. Also, the genetic analysis suggested that the Neapolitan limmo and the lemoncetta Locrese are likely synonyms of the same variety since the loci were not polymorphic and did not allow to discriminate between the two local varieties.