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Foods 2014, 3(1), 128-148; doi:10.3390/foods3010128
Published: 27 January 2014
Abstract: Pollen spectrum, physicochemical parameters and volatile fraction of Corsican “spring” honeys were investigated with the aim of developing a multidisciplinary method for the qualification of honeys in which nectar resources are under-represented in the pollen spectrum. Forty-one Corsican “spring” honeys were certified by melissopalynological analysis using directory and biogeographical origin of 50 representative taxa. Two groups of honeys were distinguished according to the botanical origin of samples: “clementine” honeys characterized by the association of cultivated species from oriental plain and other “spring” honeys dominated by wild herbaceous taxa from the ruderal and/or maquis area. The main compounds of the “spring” honey volatile fraction were phenylacetaldehyde, benzaldehyde and methyl-benzene. The volatile composition of “clementine” honeys was also characterized by three lilac aldehyde isomers. Statistical analysis of melissopalynological, physicochemical and volatile data showed that the presence of Citrus pollen in “clementine” honeys was positively correlated with the amount of linalool derivatives and methyl anthranilate. Otherwise, the other “spring” honeys were characterized by complex nectariferous species associations and the content of phenylacetaldehyde and methyl syringate.
The specificity of Corsican honeys is linked with the environmental characteristics of the island (biodiversity of flora, bioclimatic conditions and topography), the endemic black honeybee and typical hive management. Organoleptic and melissopalynological analysis have permitted Corsican honeys to be classified into six ranges: “spring”, “spring maquis”, “honeydew maquis”, “chestnut grove”, “summer maquis” and “autumn maquis”, according to the harvest season and the geographic location of the apiaries . These honeys have been certified by two official designations of origin: the national Appellation d’Origine Contrôlée (AOC) and the European Protected Designation of Origin (PDO), both marketed as “Miel de Corse-Mele di Corsica” [2,3].
The organoleptic properties of the “spring” honey range are a light color (the lightest among the six ranges) associated with low-to-medium olfactory and aromatic intensities, sometimes with a slight acidity [1,2,3]. These honeys are described in terms such as floral, fresh fruit, or dry vegetal according to the vocabulary of odor and the aroma wheel . Moreover, the physicochemical characteristics of “spring” honeys are low values of coloration and electrical conductivity. Finally, these honeys are harvested from April to May at low altitudes (below 400 m) on the coast, plains or valleys [1,2,3].
The Corsican “spring” honeys can be classified into two categories. First, honeys harvested in the oriental plain of the island. These cultivated zones are dominated by clementine orchards (Citrus sinensis × reticulata) associated with other Citrus species, Actinidia sinensis and various fruit trees. They are always surrounded by maquis; an evergreen scrub of vegetation from Mediterranean area. Second, honeys collected in ruderal and/or littoral maquis areas for their first flowering. Ruderal zones are characterized by herbaceous plants, especially Asphodelus ramosus subsp. ramosus (syn: A. microcarpus Salz et Viv.) associated with various species of Fabaceae, Boraginaceae, wild Brassicaceae, Apiaceae and Asteraceae. The coastal areas also showed a diversity of nectariferous and polleniferous resources .
Unifloral honeys from the Citrus genus, produced principally from oranges or lemons, are often found in the Mediterranean region (Italy, Spain, Greece, France and North Africa), but also in Israel, USA, Brazil and Mexico [6,7]. The nectar of Asphodelus species is frequently found in the composition of honeys from Mediterranean regions (Italy, Sicily, Corsica and Sardinia), but asphodel unifloral honey is produced mainly in Sardinia [8,9]. In Corsica, the Asphodelus genus is represented by three species: A. ramosus subsp. ramosus, A. cerasiferus and A. fistulosus . A. ramosus subsp. ramosus, which flowers from March to May, was the more visited species.
The certification of geographical and botanical origins of Corsican honeys is conventionally based on the melissopalynological analysis of the entire pollen spectrum [5,11]. Furthermore, sensory characteristics and physicochemical parameters are also necessary to specify the botanical origin of honey [5,11,12]. However, this traditional approach is not precise enough to determine the predominant botanical origin exactly, especially when nectar resources are under-represented in the pollen spectrum. For this reason, the chemical composition of honeys has been used to complete the classical approaches of botanical origin determination. Thus, various extraction methods, such as headspace solid-phase microextraction (HS-SPME), simultaneous steam distillation-solvent extraction and ultrasound-assisted extraction associated with gas chromatography (GC) have been developed for the analysis of the volatile fraction of honeys . Some volatile components, including methyl anthranilate, lilac aldehyde and p-menth-1-en-9-al, were therefore suggested as the chemical markers of citrus (species not specified) unifloral honey [13,14,15]. Moreover, Alissandrakis et al.  showed that the volatile fractions of citrus flowers (four species) and the corresponding honeys were dominated by linalool derivatives. The phenolic compound hesperetin was also proposed as a botanical indicator of Spanish citrus honeys for its high levels in nectar and honey . Methyl syringate and/or phenylacetaldehyde were identified as characteristic components of nectar from A. microcarpus Salz et Viv. and corresponding unifloral honeys [18,19].
Several techniques (HS-SPME, infrared spectroscopy and 1H-nuclear magnetic resonance spectroscopy) have been used to distinguish Corsican and non-Corsican honeys, but these studies did not provide results for the differentiation of the botanical origin of different ranges of Corsican honey [20,21,22].
According to the geographical and botanical origins of Corsican “spring” honeys certified by melissopalynological analysis, the chemical composition of volatile fractions of honey samples was established using HS-SPME, GC and GC/mass spectrometry (MS). The aim of the study is to establish for the first time a multidisciplinary method for the qualification of Corsican “spring” honeys, based on relationships between the pollen spectrum, volatile chemical markers and some physicochemical parameters.
2. Experimental Section
2.1. Honey and Flower Sampling
In total, 41 Corsican “spring” honeys (samples 1–41) were selected from our reference bank of honey with AOC and PDO appellations. All these samples were directly packaged in a sealed pot and stored below 14 °C according to the optimal conditions of honey conservation indicated by Gonnet et al. . The honey samples of three years of harvest (2004–2006) collected in April to June were provided from 12 Corsican producers. The apiaries were located from littoral to 400 m (principally under 100 m) in the oriental cultivated plain or in ruderal and/or maquis zone of thermo- and meso-Mediterranean levels. Clementine (Citrus sinensis × reticulate, six samples) and Asphodel (Asphodelus ramosus subsp. ramosus, six sample locations) flower specimens were collected in March–May 2009–2012. The nectar secretion during harvest period was ensured by the observation of foraging nectar by honeybees. Flowers samples were analyzed within 48 h.
2.2. Melissopalynological Analysis
In this study, melissopalynological analysis was performed using the method described by Yang et al. . Identification of pollen in the “spring” honey was based on the comparison with laboratory’s own reference pollen-slides library and also carried out with the palynological expertise practice [5,11] developed for the characterization and the AOC and PDO control of Corsican honeys. Pollen analysis was allowed to establish a total pollen spectrum (qualitative analysis) and pollen density (quantitative analysis) for each honey sample. The identified taxa in the pollen spectrum were expressed in term of relative frequency (RF) and the pollen density was expressed as the absolute number of pollen grain in 10 g of honey (PG/10 g).
2.3. Physicochemical Analysis
According to the description of Corsican honeys [1,5], two physicochemical parameters, coloration and electrical conductivity were chosen to complete the botanical origin characterization of Corsican “spring” honey. The honey coloration was measured using a Lovibond Comparator apparatus . Results were expressed as millimeters (mm) Pfund. Electrical conductivity was measured at 20 °C with a conductivity meter micro CM2210 (CRISON, Spain) following the method described by Bogdanov  and expressed as milliSiemens per centimeter (mS/cm).
2.4. HS-SPME Extraction
Volatile fractions of honey and flower samples were extracted by HS-SPME with a divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS, 30 μm) fiber (Supelco Sigma Aldrich). The optimization of HS-SPME parameters was performed using two honey samples (9 and 24) and two flower samples (clementine and asphodel flowers). These samples and subsequent analyses (all honey and flower samples studies) were performed in triplicate to ensure that the coefficient of variation (CV: ratio of standard deviation to the mean) of the major compounds and the sum of the total peak areas were always <15%. The samples analyzed were placed in a 20 mL vial. The parameter optimization was based on the sum of the total peak areas measured using a gas chromatography-flame ionization detection (GC-FID) system. For each sample (both honeys and flowers): the temperatures (25 °C, 50 °C and 70 °C), the equilibration times (30, 60 and 90 min) and the extraction times (15, 30 and 45 min) were tested in various experiments. The honey concentration in distilled water was optimized after six different experiments (0.5 g/mL, 1 g/mL, 1.5 g/mL and 2 g/mL) with Na2SO4 addition (1 g and 2 g). The maximum sum of the total peak areas was obtained from 4 g of honey sample with 4 mL of water and 2 g of Na2SO4 at a temperature of 70 °C, an equilibrium time of 90 min, and an extraction time of 30 min. The flower weight was optimized after three different experiments (1 g, 3 g and 5 g). For the Asphodel flowers, the maximum sum of the total peak areas was obtained from 3 g of sample at a temperature of 70 °C, an equilibrium time of 90 min, and an extraction time of 30 min. Otherwise, the best sampling conditions of Clementine flowers were 1 g of sample at room temperature (25 °C) with an extraction time of 15 min. Before sampling, the fiber was reconditioned for 5 min in the GC injection port at 280 °C. After sampling, the SPME fiber was consecutively inserted into the GC-FID and GC-MS injection ports for 5 min for desorption of volatile components, both techniques using the splitless injection mode.
2.5. GC-FID and GC-MS Analysis
GC-FID analyses were performed using a PerkinElmer (Waltham, MA, USA) AutoSystem XL GC apparatus equipped with a FID system and a fused-silica capillary column (30 m × 0.25 mm, film thickness 1 μm) coated with Rtx-1 (PDMS). The oven temperature was programmed from 60 to 230 °C at 2 °C/min and then held isothermally at 230 °C for 35 min. The injector and detector temperatures were maintained at 280 °C. The samples were injected with an SPME inlet liner (0.75 mm i.d.; Supelco) using hydrogen as the carrier gas (1 mL/min). The retention indices of the compounds were determined relative to the retention times of a series of n-alkanes (C5–C30) with linear interpolation. The relative concentrations of components were calculated from the GC peak areas without using correction factors. Samples were also analyzed with a PerkinElmer TurboMass detector (quadrupole), coupled to a GC PerkinElmer AutoSystem XL, equipped with a fused-silica Rtx-1 capillary column. The ion source temperature was 150 °C, and the ionization energy was 70 eV. Electronic ionisation (EI) mass spectra were acquired over the mass range of 35–350 Da (scan time 1 s). Other GC conditions were the same as described for the GC-FID analysis. Identification of the components was based on: (1) the comparison of their GC retention indices (RI) on a nonpolar column, determined relative to the retention time of a series of n-alkanes with linear interpolation to the retention times of authentic compounds or data with the laboratory’s library; (2) the comparison of the RI and spectra with commercial mass spectra libraries [27,28].
2.6. Statistical Analysis
The statistical analysis of melissopalynological data was carrying out the methodology previously described by Battesti et al. . In the case of “spring” honey, the inclusion of Citrus and Asphodelus pollen during the nectar foraging is low or very low because of pollen maturity or floral morphology. The “under-representation” of these pollen types and entire pollen spectrum were taken into account for the characterization and comparison of pollen spectrum from “spring” honeys. Principal component analysis (PCA) was carried out using the “PCA” function and canonical correspondence analysis (CCA) was performed with “CCA” function from R software (R Foundation—Institute for Statistics and Mathematics, Austria). CCA is a multidimensional exploratory statistical method in order to demonstrate the correlation between two sets of variables obtained from the same individual.
3. Results and Discussion
3.1. Determination of Geographical and Botanical Origins of Corsican “Spring” Honeys
The analysis of 41 Corsican “spring” honeys allowed the determination of 92 taxa, including 64 nectariferous taxa and 28 only-polleniferous taxa (Table 1). A biogeographical analysis (biogeographical code: BC ) showed the diversity of biogeographical origins of these taxa. Mediterranean species (28 taxa, BC 1–3) associated with Eurasian and Atlantic species (13 taxa, BC 5–6) were well represented in the pollen spectrum. Additionally, cultivated species (four taxa, BC 99) were reported in more than 40% of honey samples. This distribution was consistent with the database of the characterization of the Corsican honey taxa directory [5,11].
To define the most representative taxa of Corsican “spring” honey, the presence ratio (PR) and the relative frequency (RF) distributions (mean, minimum, maximum, standard deviation and coefficient variation) of each taxon were reported. The pollen directory showed that 50 taxa (T1–T50) could be considered as regionally characteristic species of Corsican “spring” honey for their significant PR (>10%) and/or RFmax (>3%). This distribution of taxa was characterized by a wide diversity of nectariferous taxa in variable proportions associated with several only-polleniferous species. Among these taxa, two main only-polleniferous taxa, Quercus sp. T1 (Qeurcus sp. (deciduous), Q. ilex and Q. suber) and Cistus sp. T2 (C. creticus, C. monspeliensis and C. salviifolius), were present in all the samples analyzed, followed by Castanea sativa T3 and Fraxinus ornus T4 (PR > 90%). Additionally, we did not find a common predominant nectariferous taxon, unlike two previous studies [24,29]:“chestnut grove” honey predominated by C. sativa with PR = 100%, FRmax > 80% and FRmean = 92.99% and “spring maquis” honey predominated by the “normal” pollen type of Erica arborea with PR = 100%, FRmax > 45% and FRmean = 47.7%. Quite the contrary, this directly demonstrates a diversity of nectariferous taxa with various pollen representation types: for example, “over-represented” (T7 and T13), “normal” (T5, T6, T8, T9 and T14) and “under-represented” (T17, T18 and T22) pollen types .
|Table 1. Statistical analysis and biogeographical characteristics of Corsican “spring” honeys’ taxa.*|
|No a||Type b||Taxa||PR c||Relative frequency (RF) d||BC g|
|Mean||Min.||Max.||SD e||CV f|
|T3||P||Castanea sativa h||90||10.3||0.3||33.8||8.7||84.2||59|
|T5||N, P||Erica arborea||85||7.8||0.2||35.5||8.7||112.3||21|
|T6||N, P||Genista form i||83||6.0||0.3||31.5||8.0||134.8||14-21-29-51-62|
|T7||N, P||Lotus sp.||76||5.3||0.3||52.8||9.5||178.3||21-51|
|T8||N, P||Salix sp.||73||6.3||0.2||29.9||7.4||117.1||51-52|
|T9||N, P||Trifolium sp.||71||14.2||0.4||53.5||16.8||117.9||21-31-51|
|T10||N, P||Rubus sp.||71||3.6||0.4||11.7||3.4||94.6||31-35|
|T11||N, P||Prunus form j||66||3.0||0.2||24.1||4.7||155.6||99-54|
|T13||N, P||Echium sp.||59||10.5||0.6||71.1||15.6||148.2||31|
|T16||N, P||Brassicaceae others||49||2.8||0.3||14.7||3.3||118.6||nd|
|T17||N, P||Lavandula stoechas||49||1.8||0.4||10.1||2.2||124.1||21|
|T18||N, P||Citrus sp.||44||6.1||0.2||16.1||5.2||86.6||99|
|T19||N, P||Vicia form||44||3.0||0.3||11.8||3.2||107.1||nd|
|T21||N, P||Asteraceae Galactites form||44||1.9||0.2||5.2||1.7||93.6||21|
|T22||N, P||Asphodelus ramosus subsp. ramosus||44||0.7||0.2||2.9||0.7||96.5||21|
|T26||N, P||Viburnum tinus||34||1.9||0.3||16.2||4.2||225.9||21|
|T27||N, P||Asteraceae (fenestrated type)||29||1.1||0.3||3.2||1.0||94.0||21-94|
|T28||N, P||Rosa sp.||27||1.2||0.3||4.5||1.3||108.1||31-51|
|T30||N, P||Fabaceae others/Dorycnopis form||24||0.6||0.3||1.4||0.3||54.5||nd|
|T32||N, P||Asteraceae Achillea form||22||0.8||0.2||2.6||0.7||94.0||21-94|
|T34||N, P||Crataegus monogyna||20||2.1||0.3||7.9||2.7||130.8||51|
|T35||N, P||Jasione montana||17||2.0||0.3||10.0||3.5||176.0||54|
|T36||N, P||Rosaceae others||17||1.2||0.3||3.0||1.0||85.6||nd|
|T37||N, P||Asteraceae Dittrichia form||17||1.0||0.2||1.9||0.8||74.4||21-94|
|T38||N, P||Rhamnus sp.||15||1.0||0.3||3.3||1.1||117.6||21|
|T39||N, P||Psoralea bituminosa||15||0.7||0.3||1.6||0.5||75.6||31|
|T40||N, P||Knautia sp.||15||0.5||0.3||0.9||0.3||52.8||31|
|T41||N, P||Lupinus angustifolius||12||4.8||0.3||18.9||8.0||166.1||21|
|T43||N, P||Hedera helix||12||0.8||0.3||1.3||0.4||46.1||65|
|T44||N, P||Liliaceae others||12||0.4||0.3||0.6||0.2||45.8||nd|
|T45||N, P||Allium sp.||12||0.4||0.3||0.6||0.2||42.9||21-25|
|T46||N, P||Acacia dealbata||12||0.4||0.3||0.4||0||13.3||99|
|T48||N, P||Dorycnium sp.||10||1.5||0.3||3.2||1.2||83.3||35|
|T49||N, P||Rosmarinus officinalis||10||1.7||0.4||3.0||1.3||77.9||21|
a Order of taxa were classified by decreasing presence ratio (PR). b Type of taxa: P, polleniferous taxa; N, nectariferous taxa . c PR: presence ratio, number of honey samples presented/41 samples, expressed as %. d Mean, Min., Max. values expressed as relative frequency RF (number of specify pollen counted/total pollen counted). e SD: standard deviation. f CV: coefficient variation. g Biogeographical Code, according to Battesti : 1—Endemic: 14 Mediterraneo-montane origin; 2—Steno-Mediterranean: 21 Wider stenomedit., 25 Western stenomedit., 29 Western macaronesian stenomedit.; 3—Eury-Mediterranean: 31 Wider eurymedit., 35 Western eurymedit.; 5—Eurasian: 51 Wider eurasian, 52 Eurasian, 54 European-caucasian, 55 European, 58 South east european, 59 Southern European; 6—Atlantic: 62 Subatlantic, 65 Atlantic Mediterranean; 94 sub-Cosmopolitan; 99 Cultivated plants; nd: not defined. h Castanea sativa, taxa of “over-represented” type, could be considered as only-polleniferous taxon according to its RF (<40%) and lower pollen density taking into account its over-represented pollen type [6,24]. i Genista form contained essentially Genista corsica, and also Cytisus villosus, Calicotome spinosa and Calicotome villosa. j Prunus form contained Prunus sp. and other fruit tree. * Forty two other determined taxa (PR < 10%): Populus sp., Boraginaceae others, Rumex sp., Ostrya carpinifolia, Ilex aquifolium, Platanus sp., Silene gallica, Stachys glutinosa, Anthyllis hermanniae, Papaver sp., Urticaceae, Reseda sp., Aesculus hippocastanum, Carpobrotus sp., Cercis siliquastrum, Potentilla form, Ranunculaceae, Corylus avellana, Asteraceae Helichrysum form, Arbutus unedo, Erica others, Cupressaceae, Sambucus ebulus, Anemone hortensis, Smilax aspera, Cynoglossum creticum form, Amaryllidaceae, Cyperaceae, Helleborus lividus subsp. corsicus, Mercurialis annua, Robinia pseudoacacia, Clematis sp., Chenopodiaceae, Caryophyllaceae others, Borago officinalis, Centaurea sp., Verbascum sp., Teucrium sp., Centaurium erythrae, Veronica sp., Asteraceae others, Buxus sempervirens (according to decreasing PR).
According to these considerations, two groups of honeys could therefore be distinguished, based not by their FR distributions, but by characteristic associations of taxa (Table 2, Table S1-supplementary materials). The first group included 18 samples (group I: 1–18) and was characterized by the association of cultivated taxa: Citrus sp. T18 and A. sinensis T15 (PR 100% in 18 samples) followed by Prunus form T11 and Olea sp. T25. Citrus sp. contained essentially C. sinensis × reticulata, which possessed an under-represented pollen type, principally due to nectar secretion of Citrus sp. flowers, often before the maturity of stamens. Citrus pollen varied between 0.2% and 16.1%, with an average of 6.1%. The second group (group II: 23 samples, 19–41) was characterized by the absence of a Citrus sp./A. sinensis association and the significant presence of A. ramosus T22 associated with Pistacia lentiscus T20, Phillyrea sp. T24, Apiaceae T14 and Brassicaceae T16. A. ramosus displayed an extreme “under-represented” pollen type due to the flower form (nectar protected by a large base of long stamens that prevented contact with pollen during bee foraging) and the large pollen size. Asphodelus pollen was present in two samples of group I (0.5%–1.3%) and 16 samples of group II (0.2%–2.9%).
In the case of honey with the “under-represented” pollen type, the contribution of other nectariferous species could not be discounted. It had to note that some honeys samples possessed dominant nectariferous taxa (RF > 45%): Trifolium sp. T9 for sample 2 and 3, Echium sp. T13 for sample 4 and Lotus sp. T7 for sample 38. The nectar contribution of these taxa could not be neglected. Otherwise, several taxa might take part in the honey composition for their high RF in the pollen spectrum: Trifolium sp. T9 and E. arborea T5 were characteristic for both groups (RFmax 53.5% and 35.5% for group I and 44.1% and 29.9% for group II, respectively); Echium sp. T13, Prunus form T11 and Viburnum tinus T26 possessed a higher RFmax in group I (71.1%, 24.1% and 16.2%, respectively) than in group II (30.1%, 3.3% and 3.7%, respectively), while Lotus sp. T7, Genista form T6, Salix sp. T8, Lupinus angustifolius T41 and Apiaceae T14 were higher in group II (FRmax: 52.8%, 31.5%, 29.9%, 18.9% and 17.5%, respectively) than in group I (FRmax: 8.7%, 11.8%, 12.9%, 3.5% and 4.5%, respectively).
A quantitative analysis showed that 32 samples possessed a pollen density between 20 and 100 × 103 PG/10 g, eight samples were between 100 and 300 × 103 PG/10 g and one sample (23) could be distinguished by high pollen density (600 × 103 PG/10 g). Compared with the previous studies of Corsican “chestnut grove” and “Erica arborea spring maquis” honey (636.6 × 103 PG/10 g and 177 × 103 PG/10 g, respectively), the “spring” honey displayed a lower pollen density (90 × 103 PG/10 g) [24,29]. Excluding sample 23, the average pollen density of “clementine” honeys (68 × 103 PG/10 g) was slightly lower than that of other Corsican “spring” honeys (84 × 103 PG/10 g) (Table 2). The decreasing pollen richness was in accordance with the pollen representation type in the spectrum of the predominant nectariferous taxa: “over-represented” (C. sativa), “normal” (E. arborea) and “under-represented” (Citrus sp.) types.
|Table 2. Melissopalynological and physico-chemical characteristics of Corsican “clementine” honeys and other “spring” honeys.|
|Melissopalynological Data||Group I—“Clementine” Honeys 18 Samples (1–18)||Group II—Other “Spring” Honeys 23 Samples (19–41)|
|RF c||RF c|
|No. a||Type b||Taxa||PR||Mean||Min.||Max.||SD||CV||PR||Mean||Min.||Max.||SD||CV|
|Main nectariferous taxa|
|T18||N, P||Citrus sp.||100||6.1||0.2||16.1||5.2||86.6||-||-||-||-||-||-|
|T5||N, P||Erica arborea||89||7.5||0.3||35.5||10.2||135.4||83||8.0||0.2||29.7||7.6||94.9|
|T8||N, P||Salix sp.||78||5.8||0.6||12.9||4.5||77.7||70||6.7||0.2||29.9||9.3||139.4|
|T11||N, P||Prunus form||78||4.8||0.2||24.1||6.1||128.4||57||1.2||0.3||3.3||0.9||75.5|
|T7||N, P||Lotus sp.||72||2.7||0.4||8.7||2.5||91.9||78||7.2||0.3||52.8||12.1||167.5|
|T10||N, P||Rubus sp.||67||2.3||0.4||6.5||1.8||77.9||74||4.5||0.4||11.7||4.0||88.4|
|T9||N, P||Trifolium sp.||67||12.9||0.5||53.5||19.4||150.7||74||15.2||0.4||44.1||15.2||100.2|
|T6||N, P||Genista form||67||3.1||0.3||11.8||3.4||107.1||96||7.5||0.6||31.5||9.4||125.3|
|T13||N, P||Echium sp.||44||19.3||1.3||71.1||23.3||121.0||70||6.1||0.6||30.1||7.5||123.2|
|T26||N, P||Viburnum tinus||22||4.5||0.3||16.2||7.8||174.5||43||0.8||0.3||3.7||1.1||127.7|
|T22||N, P||Asphodelus ramosus subsp. ramosus||11||0.9||0.5||1.3||0.5||57.3||70||0.7||0.2||2.9||0.7||104.1|
|T16||N, P||Brassicaceae others||33||1.2||0.3||2.7||1.0||77.9||61||3.5||0.4||14.7||3.8||108.2|
|T17||N, P||Lavandula stoechas||39||1.7||0.5||4.4||1.4||81.9||57||1.8||0.4||10.1||2.6||142.4|
|T19||N, P||Vicia form||28||2.3||0.7||6.0||2.3||102.4||57||3.3||0.3||11.8||3.6||107.6|
|T41||N, P||Lupinus angustifolius||17||1.5||0.3||3.5||1.8||119.1||9||9.8||0.7||18.9||12.9||131.6|
|Other nectariferous taxa||100||3.3||0.3||11.0||3.0||89.9||100||5.2||1.0||10.0||2.8||53.5|
|Main only-polleniferous taxa|
|Other only-polleniferous taxa||100||1.9||0.3||8.9||2.1||110.0||78||1.7||0.3||8.5||2.0||117.8|
|Pollen density (103 PG/10 g) d||68||20||202||52||77||107||22||603||126||118|
|Physico-chemical data e|
a Taxa number is given in Table 1. b Type of taxa: P, polleniferous taxa; N, nectariferous taxa . c Mean, Min., Max. values expressed as relative frequency RF (number of specify pollen counted/total pollen counted). d Pollen density expressed as the absolute number of pollen grains in 10 g of honey (103 PG/10 g). e Unity of parameters: colour (mm Pfund); electrical conductivity (mS/cm).
3.2. Physicochemical Characteristics of Corsican “Spring” Honeys
Corsican “spring” honeys possessed light to very light colors. The mean value of coloration was 30.0 ± 15.4 mm Pfund, with great variation between 11.0 and 71.0 mm Pfund (Table 2). The two groups exhibited quite similar coloration values: 26.4 ± 13.6 mm Pfund for “clementine” honeys and 33.3 ± 16.3 mm Pfund for the other “spring” honeys. For each group, nine samples possessed a very light coloration value (<20.0 mm Pfund). Only one sample (17) of “clementine” honeys had a coloration value >50.0 mm Pfund while five samples (23, 35, 38, 39 and 41) of other “spring” honeys possessed coloration values between 50.0 and 71.0 mm Pfund.
The average electrical conductivity value of the honey samples was 0.25 ± 0.08 mS/cm with a variation of 0.13–0.45 mS/cm (Table 2). The electrical conductivity of the two groups was also quite similar: 0.25 ± 0.07 mS/cm for “clementine” honeys (range: 0.15–0.42 mS/cm) and 0.24 ± 0.09 mS/cm for other “spring” honeys (range: 0.13–0.45 mS/cm). Only three samples (17, 34 and 41) of these honeys had medium electrical conductivity (>0.4 mS/cm).
3.3. Chemical Variability of Corsican “Spring” Honeys
GC and GC/MS analysis of the headspaces of Corsican “spring” honeys allowed the identification of 43 compounds that accounted for 71.5%–96.8% of the total volatile composition (Table 3, Table S2-supplementary materials). It should be noted that the volatile fraction of “spring” honeys is rich in aldehyde (22.1%–63.1%) and alcohol (2.8%–40.2%) components.
To synthesize the chemical data, PCA was used to examine the relative distribution of the matrix of “spring” honey samples according to their volatile chemical compositions. The analyses included 17 compounds: two hydrocarbons (C2 and C12), eight aldehydes (C5, C9, C14, C25, C27, C28, C31 and C32), two ketones (C23 and C24), two esters (C38 and C41), two oxides (C39 and C42) and one alcohol (C37). As shown in Figure 1a, the principal factorial plane (axes 1 and 2) accounted for 58.91% of the entire variability of the honey samples. Dimension 1 (42.24%) correlated negatively C39 and negatively with other compounds. Dimension 2 (16.67%) correlated negatively with two hydrocarbons (C2 and C12), two aldehydes (C5 and C14) and one oxide C42 and positively with with two ketones (C23 and C24), three aldehydes (C25, C27 and C28), one ester C38 and one oxide other compounds.
The plot established according to the first two principal components suggested the existence of two main groups (Figure 1b). Group I contained 17 samples (1–17), which corresponded to the group of “clementine” honeys (except sample 18). This group I was characterized by the presence of lilac aldehyde isomers (C25, C27 and C28), p-menth-1-en-9-al isomers (C31 and C32) and methyl anthranilate C38, which were absent in the other honey samples (group II). It was rich in furan compounds (group I: 26.2% versus group II: 7.6%), but not in phenolic components (group I: 29.4% versus group II: 40.0%). Aldehyde components were also higher in group I (49.1%) than in group II (39.7%). Group II could be divided into subgroups IIa (five samples: 20, 33, 36, 38 and 39) and IIb (18 samples: 18, 19, 21–32, 34, 37, 40 and 41). These two subgroups were characterized by a greater abundance of phenolic compounds (group IIa: 39.9% and group IIb: 43.0%), but group IIa displayed a higher value for linear compounds (group IIa: 26.2% and group IIb: 19.2%). Additionally, subgroup IIa had a higher amount of aldehyde (group IIa: 40.7% and group IIb: 12.8%) and alcohol (group IIa: 35.6% and group IIb: 9.2%) compounds than subgroup IIb. This latter group displayed a greater abundance of ketones (group IIa: 4.3% and group IIb: 22.5%). Finally, sample 35 was characterized by 32.8% of hydrocarbons, whereas the abundance of hydrocarbons was not >25% in the other honey samples.
|Table 3. Chemical composition of volatile fraction of Corsican “spring” honeys.|
|No a||Components||RI b||Group I “Clementine” Honeys c||Group II “Not-Clementine” Honeys c||Sample 35|
|Mean ± SD d||Min.||Max.||Mean ± SD d||Min.||Max.||Mean ± SD d||Min.||Max.|
|C1||3-Methyl-3-buten-1-ol||704||2.9 ± 2.76||0.3||11.3||1.8 ± 1.19||0.7||3.7||1.8 ± 1.44||0.4||6.0||2.8|
|C2||Methyl-benzene||741||6.5 ± 4.45||1.5||15.6||4.1 ± 2.08||2.4||7.1||6.2 ± 4.08||1.5||17.3||10.4|
|C3||Hexanal||773||1.1 ± 0.46||0.5||2.0||1.6 ± 2.62||0.1||6.3||1.6 ± 1.13||0.3||4.5||1.9|
|C4||Octane||790||1.4 ± 1.08||0.3||4.7||0.9 ± 0.77||0.3||2.2||2.6 ± 1.63||0.7||5.6||1.4|
|C5||3-Furaldehyde||800||2.8 ± 1.56||0.6||5.9||3.5 ± 1.37||2.5||5.9||3.5 ± 1.78||1.9||8.3||18.5|
|C6||2-Methyl butanoic acid||858||0.8 ± 0.91||0.1||3.3||4.6 ± 3.21||1.1||7.6||2.9 ± 4.59||0.1||20.4||2.4|
|C7||2-Methyl octane||873||0.4 ± 0.28||0.1||0.9||0.5 ± 0.44||0.1||1.2||0.7 ± 0.38||0.1||1.7||1.6|
|C8||Nonane||893||0.7 ± 0.65||0.2||2.5||1.2 ± 0.35||0.8||1.7||1.5 ± 0.92||0.2||3.5||3.8|
|C9||Benzaldehyde||924||5.5 ± 3.56||2.4||17.9||10.4 ± 3.85||5.4||14.8||8.8 ± 4.89||2.5||18.4||3.0|
|C10||Hexanoic acid||969||0.7 ± 0.25||0.4||1.4||1.7 ± 1.68||0.3||3.9||1.2 ± 0.75||0.4||3.3||-|
|C11||Octanal||982||1.0 ± 0.47||0.2||2.1||0.6 ± 0.12||0.5||0.7||1.6 ± 1.45||0.5||6.6||1.0|
|C12||2,2,4,6,6-Pentamethylheptane||992||1.1 ± 0.60||0.4||2.4||0.6 ± 0.53||0.1||1.3||2.3 ± 1.83||0.2||5.2||15.6|
|C13||p-Methylanisol||995||0.9 ± 1.13||0.1||4.9||1.1 ± 1.10||0.3||3.0||0.9 ± 1.48||0.1||6.1||-|
|C14||Phenylacetaldehyde||1006||10.1 ± 10.68||0.8||39.1||16.5 ± 6.93||7.2||25.7||21.2 ± 7.83||3.7||36.2||13.0|
|C15||p-Cymene||1008||0.7 ± 0.29||0.1||1.0||0.9 ± 0.42||0.6||1.2||-||-||-||-|
|C16||Acetophenone||1037||0.2 ± 0.08||0.1||0.4||0.4 ± 0.21||0.2||0.5||0.3 ± 0.10||0.2||0.4||-|
|C17||trans-Furanoid-linaloxide||1049||1.5 ± 1.00||0.8||4.0||1.3 ± 1.25||0.5||3.5||2.4 ± 1.20||1.0||6.3||-|
|C18||cis-Furanoid-linaloxide||1064||1.1 ± 0.40||0.7||2.0||1.0 ± 0.29||0.7||1.4||1.1 ± 0.26||0.5||1.6||-|
|C19||β-Phenylethanol||1077||4.2 ± 1.54||2.2||5.8||1.6 ± 0.00||1.6||1.6||3.3 ± 1.67||2.1||5.8||-|
|C20||Nonanal||1079||2.7 ± 1.73||0.9||7.6||1.8 ± 1.28||0.4||3.5||3.0 ± 2.29||0.5||7.2||2.9|
|C21||Linalol||1084||2.4 ± 1.82||0.2||6.6||1.3 ± 1.62||0.3||3.2||12.5 ± 10.42||2.1||32.3||tr|
|C22||Hotrienol||1085||4.1 ± 4.39||0.7||10.5||-||-||-||9.7 ± 0.00||9.7||9.7||-|
|C23||Isophorone||1087||2.8 ± 1.54||0.2||4.9||18.2 ± 8.02||8.8||29.3||3.3 ± 3.5||0.1||9.6||-|
|C24||4-Oxoisophorone||1102||0.9 ± 0.33||0.3||1.4||4.2 ± 1.87||2.3||6.4||1.5 ± 0.99||0.3||5.0||-|
|C25||(2S,2′S,5′S)-Lilac aldehyde||1112||5.4 ± 2.36||1.3||8.9||-||-||-||1.5 ± 0.00||1.5||1.5||-|
|C26||Dihydrolinalool||1116||1.2 ± 0.66||0.5||3.0||-||-||-||1.1 ± 0.00||1.1||1.1||-|
|C27||(2R,2′S,5′S)-Lilac aldehyde||1121||10.5 ± 3.66||4.9||16.5||-||-||-||2.4 ± 0.00||2.4||2.4||-|
|C28||(2R,2′R,5′S)-Lilac aldehyde||1134||4.8 ± 1.85||2.2||8.1||-||-||-||1.1 ± 0.00||1.1||1.1||-|
|C29||Octanoic acid||1167||1.7 ± 1.55||0.3||6.0||0.9 ± 0.38||0.3||1.3||1.6 ± 0.78||0.7||3.8||4.7|
|C30||Decanal||1174||1.2 ± 0.67||0.2||2.8||0.6 ± 0.25||0.2||0.8||1.5 ± 0.54||0.6||2.3||-|
|C31||p-Menth-1-en-9-al (isomer 1)||1184||1.9 ± 0.41||1.2||2.7||-||-||-||-||-||-||-|
|C32||p-Menth-1-en-9-al (isomer 2)||1186||1.7 ± 0.46||0.5||2.5||-||-||-||-||-||-||-|
|C33||p-Anisaldehyde||1208||0.7 ± 1.19||0.1||4.6||0.9 ± 0.37||0.3||1.1||0.4 ± 0.21||0.2||0.8||-|
|C34||2,3,5-Trimethylphenol||1248||0.4 ± 0.30||0.1||1.1||1.0 ± 0.69||0.4||2.0||0.8 ± 0.68||0.1||2.0||-|
|C35||4-n-Propylanisol||1264||1.6 ± 1.78||0.2||5.7||2.4 ± 1.29||0.8||4.3||3.8 ± 2.45||1.4||6.3||-|
|C36||Nonanoic acid||1271||2.7 ± 1.39||0.5||4.9||2.6 ± 0.94||1.4||3.7||3.1 ± 1.24||1.3||6.4||-|
|C37||3,4,5-Trimethylphenol||1290||0.5 ± 0.32||0.2||1.4||5.4 ± 2.71||2.9||9.4||0.5 ± 0.67||0.1||2.0||-|
|C38||Methyl anthranilate||1300||1.4 ± 0.96||0.2||3.5||-||-||-||-||-||-||-|
|C39||cis-p-Mentha-1(7),8-dien-1-hydroperoxide||1348||0.4 ± 0.14||0.2||0.7||-||-||-||-||-||-||-|
|C40||Decanoic acid||1362||1.2 ± 0.45||0.6||2.1||1.3 ± 0.75||0.1||1.9||1.7 ± 1.50||0.6||6.8||3.8|
|C41||Methyl 3,5-dimethoxybenzoate||1494||-||-||-||0.4 ± 0.26||0.2||0.7||0.5 ± 0.19||0.3||0.8||-|
|C42||Methyl syringate||1722||-||-||-||0.5 ± 0.50||0.1||1.4||0.9 ± 1.17||0.1||4.1||-|
|C43||Tricosane||2305||0.3 ± 0.17||0.1||0.5||0.5 ± 0.00||0.5||0.5||0.5 ± 0.22||0.2||0.7||-|
|Total identification (%)||84.2 ± 6.95||71.5||94.5||91.2 ± 5.43||84.2||96.8||86.3 ± 5.49||78.8||96.7||86.8|
|Total peak area(106) e||3.8 ± 1.96||1.3||7.4||2.9 ± 1.27||1.6||4.5||2.4 ± 1.08||0.8||4.4||0.3|
|Hydrocarbons||10.6 ± 5.23||4.7||20.8||7.7 ± 3.59||4.8||13.5||13.3 ± 5.88||5.0||23.9||32.8|
|Oxygenated compounds||73.6 ± 7.23||58.2||82.8||83.6 ± 4.11||79.2||90.3||73.7 ± 7.56||58.1||81.7||54.0|
|Phenolic compounds||29.4 ± 12.3||12.6||59.3||43.0 ± 7.39||34.8||53.0||39.9 ± 11.22||23.2||60.4||26.4|
|Furan compounds||26.2 ± 7.85||12.2||38.6||5.8 ± 2.83||3.9||10.8||7.5 ± 1.99||4.3||11.0||18.5|
|Linear compounds||21.0 ± 7.08||11.3||36.6||19.2 ± 3.88||14.8||23.4||26.2 ± 9.63||11.3||53.4||41.9|
|Terpenic compounds||31.0 ± 9.88||15.4||52.2||3.5 ± 2.64||1.7||8.1||13.6 ± 13.05||1.5||45.8||0|
|Ketones||2.5 ± 2.14||0||6.0||22.5 ± 9.67||11.6||35.7||4.3 ± 3.77||0.8||11.6||0|
|Aldehydes||49.1 ± 8.03||34.4||63.1||35.6 ± 9.73||26.1||47.7||40.7 ± 9.78||22.1||52.3||40.3|
|Esters||1.4 ± 0.96||0.2||3.5||0.2 ± 0.29||0||0.7||0.3 ± 0.27||0||0.8||0|
|Alcohols||9.7 ± 6.17||3.3||27.8||9.2 ± 3.3||4.9||12.7||12.8 ± 10.40||3.0||40.2||2.8|
|Acids||6.8 ± 3.31||0.4||15.4||9.7 ± 5.23||3.0||15.1||10.2 ± 5.28||5.6||27.0||10.9|
|Oxides||5.2 ± 2.76||2.5||12.6||6.3 ± 2.45||3.0||9.3||5.5 ± 2.64||3.3||14.3||0|
a Order of elution is given on apolar coloumn (Rtx-1). b Retention indice on the Rtx-1 apolar column. c Group number was given in “Chemical variability of Corsican “spring” honeys”. d Means ± SD, Min. and Max. values expressed as percentages. e Total peak area was expressed in arbitrary units.
3.4. Botanical Origin and Volatile Composition of Corsican “Spring” Honeys
The 17 samples of “clementine” honeys (group I: 1–17) could be distinguished from other “spring” honeys (group II) by the presence of three lilac aldehydes (C25, C27 and C28) and two p-menth-1-en-9-al isomers (C31 and C32) (Table 3, Table S2-supplementary materials). These honey samples were dominated by phenolic compounds (12.6%–59.3%), followed by furan compounds (12.2%–38.6%) and linear compounds (11.3%–36.6%). The main components were phenylacetaldehyde C14 (0.8%–39.1%), methyl-benzene C2 (1.5%–15.6%), (2R,2′S,5′S)-lilac aldehyde C27 (4.9%–16.5%), (2S,2′S,5′S)-lilac aldehyde C25 (1.3%–8.9%), benzaldehyde C9 (2.4%–17.9%) and (2R,2′R,5′S)-lilac aldehyde C28 (2.2%–8.1%). Low amounts of methyl anthranilate C38 (0.2%–3.5%) were found in the volatile fraction of the “clementine” honeys analyzed. This component is a known chemical marker of Citrus (species not specified) unifloral honey . Additionally, various linalool derivatives, such as linalool oxides, lilac aldehydes and/or p-menth-1-en-9-al isomers, have also been reported as characteristic compounds of citrus unifloral honeys from Spain and Greece [15,16,30,31,32,33]. These compounds were also identified in the volatile components of Corsican “clementine” honeys. Conversely, some other linalool derivatives, such as lilac alcohol isomers (previously reported in the Spanish and Greek citrus honeys), were not detected in our honey samples. Alissandrakis et al.  showed that methyl anthranilate and lilac aldehydes could be found in honeys of mixed botanical origin with the presence of citrus nectar. These volatile compounds were also detected in the honey samples 2–4 in which were found the RFmax of Trifolium sp. T9 and Echium sp. T13 taxa. Chemical investigation showed that these honey samples displayed the similar volatile composition of “clementine” honey. As these two taxa could provide great quantity of nectar and pollen , it appeared that they played only a polleniferous role in these honey samples.
The volatile composition of C. sinensis × reticulata flowers has not been reported previously. The HS-SPME fraction of clementine flowers is characterized by 29 compounds, which accounted for 75.5%–87.0% of the volatile composition (Table 4). Linalool (9.6%–22.6%), sabinene (13.4%–19.6%), dihydrolinalool (8.5%–14.8%) and myrcene (5.6%–6.5%) were identified as the main compounds. Linalool and dihydrolinalool were also found in low concentrations in the volatile fraction from “spring clementine” honey samples. Methyl anthranilate was detected in the volatile fraction of Corsican clementine flowers (0.1%–0.3%) and corresponding honeys.
The decrease in linalool amount and the occurrence of other linalool derivates (hotrienol, linalool oxides, lilac aldehyde isomers and p-menth-1-en-9-al isomers) in honey samples could be explained by the enzymatic degradation of linalool by some pathways : (1) linalool can be transformed to 8-hydroxylinalool isomers by enzymatic hydroxylation at the C8 position, and then hotrienol; (2) 8-hydroxylinalool can be transformed to lilac aldehyde via (E)-8-oxolinalool and lilac alcohols, or p-menth-1-en-9-al via 8-hydroxygeraniol and (3) linalool can also be transformed via 6,7-hydroxylinalool into furanoid linalool oxide isomers under acidic conditions or by heating. These results were in accordance with those previously reported on the volatile fraction of citrus flowers and corresponding honeys . It demonstrated that the flowers from Citrus species (orange, tangerine and sour orange) had high amounts of linalool (51.6%–80.6%) and that the honeys consisted of more than 80% of linalool derivatives (lilac aldehydes and lilac alcohols).
|Table 4. Chemical composition of volatile fraction of clementine and asphodel flowers.|
|Components a||RI(Lit) b||RI c||Clementine Flower d||Asphodel Flower e||Identification g|
|Mean ± SD f||Min.||Max.||Mean ± SD f||Min.||Max.|
|3-Furaldehyde||799||800||-||-||-||1.0 ± 0.87||0.5||2.7||RI, MS|
|Furfural||831||836||-||-||-||3.5 ± 1.26||1.7||5.2||RI, MS|
|2-Furanmethanol||839||842||-||-||-||2.1 ± 1.48||0.8||4.7||RI, MS, Ref|
|Heptanal||882||876||-||-||-||5.4 ± 2.46||3.1||9.5||RI, MS|
|α-Thujene||924||922||1.2 ± 0.21||1.0||1.4||-||-||-||RI, MS|
|α-Pinene||932||931||3.6 ± 2.46||2.0||6.4||-||-||-||RI, MS|
|Benzaldehyde||929||933||-||-||-||2.7 ± 0.78||1.4||3.7||RI, MS|
|Tetrahydro-citronellene||937||935||6.8 ± 4.90||3.3||12.4||-||-||-||RI, MS, Ref|
|β-Citronellene||943||940||2.2 ± 0.15||2.0||2.3||-||-||-||RI, MS|
|Octen-3-ol||962||955||-||-||-||0.2 ± 0.05||0.1||0.2||RI, MS|
|Furfuryl acetate||964||959||-||-||-||0.7 ± 0.31||0.5||1.3||RI, MS, Ref|
|Sabinene||973||958||16.8 ± 3.14||13.4||19.6||-||-||-||RI, MS|
|2-Pentylfuran||973||966||-||-||-||0.8 ± 1.00||0.2||2.8||RI, MS|
|β-Pinene||978||972||1.5 ± 1.36||0.4||3.0||-||-||-||RI, MS|
|Myrcene||987||979||6.1 ± 0.45||5.6||6.5||-||-||-||RI, MS|
|Octanal||981||980||-||-||-||7.0 ± 3.12||3.5||12.6||RI, MS|
|(Z)-3-Hexenyl acetate||989||984||-||-||-||21.6 ± 14.27||5.2||41.8||RI, MS|
|(E)-3-Hexenyl acetate||1002||994||-||-||-||0.8 ± 0.54||0.1||1.5||RI, MS|
|α-Phellandrene||1002||995||1.5 ± 0.23||1.4||1.8||0.3 ± 0.12||0.1||0.4||RI, MS|
|α-Terpinene||1013||1008||0.6 ± 0.44||0.3||1.1||-||-||-||RI, MS|
|Phenylacetaldehyde||1012||1009||-||-||-||0.9 ± 0.67||0.2||2.1||RI, MS|
|p-Cymene||1015||1011||0.6 ± 0.10||0.5||0.7||-||-||-||RI, MS|
|p-Menth-1-ene||1017||1018||0.5 ± 0.15||0.4||0.7||-||-||-||RI, MS|
|Limonene||1025||1020||1.5 ± 0.70||0.8||2.2||-||-||-||RI, MS|
|(Z)-β-Ocimene||1029||1024||0.1 ± 0.06||0.1||0.2||-||-||-||RI, MS|
|(E)-2-Octenal||1034||1034||-||-||-||0.4 ± 0.29||0.1||0.8||RI, MS|
|(E)-β-Ocimene||1041||1036||2.6 ± 2.21||0.9||5.1||-||-||-||RI, MS|
|γ-Terpinene||1051||1047||1.1 ± 0.51||0.5||1.5||-||-||-||RI, MS|
|trans-Sabinene hydrate||1053||1050||1.0 ± 0.36||0.7||1.4||-||-||-||RI, MS|
|1-Octanol||1063||1057||-||-||-||6.0 ± 1.93||2.8||8.8||RI, MS|
|Terpinolene||1082||1078||0.1 ± 0.06||0.1||0.2||-||-||-||RI, MS|
|Nonanal||1076||1081||-||-||-||25.8 ± 10.1||16.5||38.2||RI, MS|
|Linalool||1086||1086||17.8 ± 7.14||9.6||22.6||1.7 ± 0.21||1.5||1.8||RI, MS|
|Tetrahydrolinalool||1099||1095||4.1 ± 3.07||0.7||6.7||-||-||-||RI, MS, Ref|
|Dihydrolinalool||1118||1114||10.8 ± 3.50||8.5||14.8||-||-||-||RI, MS, Ref|
|(E)-2-Nonen-1-ol||1149||1153||-||-||-||2.2 ± 1.65||0.6||4.6||RI, MS|
|1-Phenylethyl acetate||1166||1163||-||-||-||0.1 ± 0.05||0.1||0.2||RI, MS|
|Terpinen-4-ol||1164||1164||0.3 ± 0.20||0.1||0.5||-||-||-||RI, MS|
|Decanal||1180||1182||-||-||-||1.6 ± 0.74||0.7||2.5||RI, MS|
|Undecanal||1285||1285||-||-||-||1.0 ± 1.03||0.2||2.8||RI, MS|
|Methyl anthranilate||1308||1302||0.2 ± 0.10||0.1||0.3||-||-||-||RI, MS|
|(E,E)-α-Farnesene||1498||1492||0.1 ± 0.00||0.1||0.1||-||-||-||RI, MS|
|Heptadecane||1700||1698||0.2 ± 0.10||0.1||0.3||-||-||-||RI, MS|
|Total identification (%)||81.2 ± 5.75||75.5||87.0||85.9 ± 2.66||82.4||90.1|
|Hydrocarbons||48.0 ± 8.16||39.7||56.0||-||-||-|
|Oxygenated compounds||33.1 ± 11.88||19.5||41.3||85.9 ± 2.66||82.4||90.1|
|Phenolic compounds||0.2 ± 0.1||0.1||0.3||3.7 ± 0.99||1.8||4.5|
|Furan compounds||-||-||-||8.2 ± 4.42||4.3||16.7|
|Linear compounds||0.2 ± 0.1||0.1||0.3||73.9 ± 5.41||65.0||79.3|
|Terpenic compounds||80.8 ± 5.75||75.1||86.6||0.8 ± 0.85||0.1||1.9|
|Aldehydes||-||-||-||43.8 ± 13.47||31.2||62.8|
|Esters||0.2 ± 0.1||0.1||0.3||23.2 ± 14.58||5.8||43.1|
|Alcohols||32.9 ± 11.8||19.4||41.1||11 ± 4.13||4.3||17|
a Order of elution is given on apolar coloumn (Rtx-1). b Retention indice of literature on the apolar column reported from references [27,28]. c Retention indice on the Rtx-1 apolar column. d Six clementine flower specimens were collected from Corsica oriental plain. e Six Asphodele flower specimens were collected from six localities of Corsica. f Means ± SD, Min. and Max. values expressed as percentages; tr trace (< 0.05%), g RI, Retention indice; MS, mass spectra in electronic impact mode. Ref., compounds identified from commercial data libraries: Konig et al.  (Samples 8, 34 and 35) and NIST  (Samples 3 and 11).
The 23 “not-clementine” honey samples (group II) were dominated by phenolic compounds (23.2%–60.4%) followed by linear compounds (11.3%–53.4%). The main compounds were phenylacetaldehyde C14 (3.7%–36.2%), benzaldehyde C9 (2.5%–18.4%) and methyl-benzene C2 (1.5%–17.3%). Furanic compounds (average: 7.5%) were less abundant than in “clementine” honeys (average: 26.2%), and acid components (average: 10.3%) were more abundant than in the “clementine” honeys (average: 6.8%). To our knowledge, only one previous report focused on the volatile fraction of asphodel unifloral honeys from Sardinia . Methyl syringate was detected in asphodel nectar in high concentrations and was therefore considered a marker of asphodel honeys . A low content of this component (C42: 0.1%–4.1%) was reported in the volatile fraction of “spring” honey samples (18–21, 24–30, 32, 33 and 36–41). Additionally, the amount of methyl syringate was unrelated to the presence of Asphodelus pollen in the pollen spectrum. This result could be explained by the extreme “under-represented” type of Asphodelus pollen in Corsican “spring” honeys and/or by other nectar contributions in these honeys. The sample 18 exhibited the association of Citrus sp. and A. sinensis; it was grouped with the “not-clementine” honey. In this sample, the citrus nectar contribution was less important than in “clementine” honeys in accordance with the lower concentrations of lilac aldehyde isomers.
To our knowledge, the volatile composition of A. ramosus subsp. ramosus flowers is reported here for the first time (Table 4). The HS-SPME volatile fraction of asphodel flowers was dominated by oxygenated compounds, especially linear compounds. Nonanal (16.5%–38.2%), (Z)-3-hexenyl acetate(5.2%–41.8%), octanal (3.5%–12.6%), 1-octanol (5.7%–8.8%) and heptanal (3.1%–9.5%) were identified as major compounds. The two main components of the honey volatile fraction (phenylacetaldehyde and benzaldehyde) were detected in low concentrations in the flowers. Moreover, methyl syringate (a marker of asphodel honey) was not detected in the flowers analyzed. This result showed that a direct relationship between the volatile fractions of asphodel flowers and the corresponding “spring” honeys could not be established using HS-SPME analysis.
Finally, the characteristic compounds of the volatile fraction of Corsican “chestnut grove” (acetophenone and 2-aminoacetophenone)  and “Erica arborea spring maquis” (p-anisaldehyde and 4-propylanisol) honeys  were found in low concentrations or not detected in the “spring” honeys studied.
3.5. Correlation of Melissopalynological and Chemical Data
To identify relationships between the melissopalynological analysis and volatile composition data of honey samples, CCA was applied on the matrix linked the relative amounts of the 17 volatile compounds (previously used in section “Chemical variability of Corsican “spring” honeys”) and the relative frequency (explanatory variables) of eight nectariferous taxa (T7–T9, T11, T13, T14, T18 and T22).
The correlations between the volatile composition and melissopalynolgical data were show in Figure 2. The first CCA axis was negatively related Trifolium sp. T9, Prunus form T11, Echium sp. T13 and Citrus sp. T18 to methyl-benzene C2, 2,2,4,6,6-pentametylheptane C12, three lilac aldehydes (C25, C27 and C28), two p-menth-1-en-9-al isomers (C31 and C32), methyl antranilate C38 and cis-p-mentha-1(7),8-dien-1-hydroperoxide C39. The second axis negatively related Trifolium sp. T9 and Asphodelus T22 to methyl-benzene C2, 3-furaldehyde C5, Benzaldehyde C9, 2,2,4,6,6-pentametylheptane C12, phenlyacetaldehyde C14 and methyl syringate C42.
The sample distribution showed the occurrence of two main groups, group I (17 samples: 1–17) and group II (24 samples: 18–41), which correspond to the groups defined in “Determination of geographical and botanical origins of Corsican “spring” honeys” Group I was characterized not only by the significant presence of lilac aldehyde isomers (C25, C27 and C28), p-menth-1-en-9-al isomers (C31 and C32) and methyl anthranilate C38, but also by the high abundance of taxa: Citrus sp. T18, Echium sp. T13 and Prunus form T11 (group I: 6.3%, 9.5% and 3.8% versus group II: 0.1%, 4.9% and 0.8%, respectively). According to the literature [15,16,30,31,32,33], all these compounds had been considered as characteristic components of citrus honey. From these results, it appeared that the other nectariferous taxa Echium sp. and Prunus form displayed a polleniferous role in these honey samples.
Group II included 24 samples that had great diversity. According to the sample distribution, we could distinguish 20 honey samples (18–34, 37, 40 and 41), which had higher values of phenylacetaldehyde C14 and methyl syringate C38 (21.4% and 0.7%, respectively). These honeys were also characterized by numerous herbaceous taxa with potential for nectar contribution, such as Lotus sp. T7, Salix sp. T8, Apiaceae T14 and Asphodelus T22 (3.5%, 4.4%, 3.2% and 0.6% versus I: 2.0%, 4.6%, 0.4% and 0.03%, respectively). As previously reported in literature data , the nectar contribution of Asphodelus T22 in these honey samples was characterized by the presence of methyl syringate C38. In the same way, phenylacetaldehyde C14 was reported as main volatile compound of Salix honeys  and Asphodelus honey . For the other nectariferous species Lotus sp. T7 and Apiaceae T14, no chemical markers of nectar contribution was reported in previous studies.
Variables: taxa number corresponding to those of Table 2 and Table 3; volatile components number corresponding to those of Table 3. In the CCA plot, location of each sample indicated its compositional similarity to each other; volatile components locations indicated the similarity of their distribution to each other; length of taxa indicated the importance to the ordination, and the direction of taxa vector indicated its correlation with each axes. The perpendiculars drawn from volatile components to taxa give approximate ranking of volatile components response to the taxa variables.
Corsican “spring” honeys can be classified into two categories according to melissopalynological analysis: (1) honeys characterized by the association of cultivated plants, especially C. sinensis × reticulata with other Citrus species, A. sinensis and other fruit trees; (2) honeys without cultivated taxa, but with herbaceous species (A. ramosus subsp. ramosus, Trifolium sp., Echium sp., Apiaceae, Brassicaceae, Lotus sp., etc.), low shrub species (Rubus sp. and Lavandula stoechas) and some polleniferous taxa with precocious flowering (P. lentiscus and Phillyrea sp.).
Analysis of the volatile fraction of “spring” honeys also demonstrated the existence of two main groups in this range. The volatile fractions were often characterized by high amounts of phenylacetaldehyde, benzaldehyde and methyl-benzene. However, the chemical composition of “clementine” honeys was dominated by three lilac aldehyde isomers that were absent in the “not-clementine” honeys. The statistical analysis showed clearly that the “clementine” honeys were characterized by high volatile content (total peak area), methyl anthranilate, lilac aldehydes, p-menth-1-en-9-al isomers and some cultivated taxa, while the “not-clementine” honeys were characterized by phenylacetaldehyde, methyl syringate and complex taxa associations. The richness of linalool derivatives in the volatile fraction of clementine flowers suggested biochemical transformation occurring during honeybee activity or honey conservation in the hive.
Finally, it appeared that melissopalynological analysis was necessary for the certification of geographical origin and was useful for the determination of botanical origin. Moreover, analysis of the volatile composition could be used to specify the characteristics of volatile compounds in relation to the predominance and/or complexity of botanical origins of the product, especially when nectariferous species have an “under-represented” pollen type in the pollen spectrum, such as Citrus sp. or Asphodelus sp.
The authors are indebted to the Délégation Régionale à la Recherche et à la Technologie de Corse (DRRT), the Collectivité Territoriale de Corse (CTC) and European Community for partial financial support.
Conflicts of Interest
The authors declare no conflict of interest.
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