Italian Honeydew Honey Characterization by 1H NMR Spectroscopy

Abstract

Honeydew honey represents a bee-derived product with different organoleptic characteristics and distinct properties with respect to floral honey. The market interest in honeydew honey has been growing in recent years due to its higher bioactive characteristics with respect to floral honey. The need for a deeper chemical characterization aimed to evaluate a possible botanical differentiation attracted the use of different analytical approaches. The present work aims to distinguish the botanical honeydew origin by using Nuclear Magnetic Resonance (NMR) spectroscopy and a multivariate approach. Two different data pretreatments have been considered to obtain the best sample discrimination. The saccharide content significantly affects the differentiation of the botanical variety consisting of fir, oak, citrus fruits, eucalyptus, and forest mainly by using a classification approach taking advantage of the Orthogonal Signal Correction filters. Notwithstanding the botanical diversity of the honeydew honey (HDH) samples, fir honeydew (F-HDH), oak honeydew (O-HDH), and eucalyptus honeydew (E-HDH) resulted always well discriminated among all the botanical varieties investigated, while citrus fruits honeydew (CF-HD) and forest honeydew (FO-HDH) did not. In particular, F-HDH resulted characterized by sucrose, erlose, maltose, maltotriose, maltotetraose, and melezitose, E-HDH resulted enriched in α, β-glucose and β-fructose in furanosidic form, and O-HDH enriched in β-fructose in furanosidic form, isomaltose.

Keywords: NMR spectroscopy; botanical origin; honeydew honey; multivariate statistical analysis.

Figure 1

Selected ¹H NMR spectral (a) aliphatic, (b) anomeric, and (c) aromatic regions of the HDH samples. Acronyms stand for: Ace, acetate; Ace-ace, acetoacetate; Ala, alanine; Asp, aspartate derivative; Eth, ethanol; For, formate; βFru_F, β-fructose in furanosidic form; βFru_P, β-fructose in pyranosidic form; Fum, fumarate; αGal, α-galactose; βGal, β-galactose; αGlu, α-glucose; βGlu, β-glucose; αGlu_F, α-glucofuranose; His, histidine; HMF, 5-(hydroxymethyl)furfural; Iso, isomaltose; 2Prop, 2-propanol; Kyn, kynurenic acid; Lac, lactate; Leu, leucrose; Mal, malate; Mel, melezitose; Phe, phenylalanine; 2Phpr, 2-phenylpropanol; Pro, proline; Que, quercitol; Qui, quinic acid; Raf, raffinose; Shi, shikimate; Suc, succinate; Thr, threalose; Tri, Trigonelline; Tyr, tyrosine; U, unknown; Uri, uridine.

Figure 1

Selected ¹H NMR spectral (a) aliphatic, (b) anomeric, and (c) aromatic regions of the HDH samples. Acronyms stand for: Ace, acetate; Ace-ace, acetoacetate; Ala, alanine; Asp, aspartate derivative; Eth, ethanol; For, formate; βFru_F, β-fructose in furanosidic form; βFru_P, β-fructose in pyranosidic form; Fum, fumarate; αGal, α-galactose; βGal, β-galactose; αGlu, α-glucose; βGlu, β-glucose; αGlu_F, α-glucofuranose; His, histidine; HMF, 5-(hydroxymethyl)furfural; Iso, isomaltose; 2Prop, 2-propanol; Kyn, kynurenic acid; Lac, lactate; Leu, leucrose; Mal, malate; Mel, melezitose; Phe, phenylalanine; 2Phpr, 2-phenylpropanol; Pro, proline; Que, quercitol; Qui, quinic acid; Raf, raffinose; Shi, shikimate; Suc, succinate; Thr, threalose; Tri, Trigonelline; Tyr, tyrosine; U, unknown; Uri, uridine.

Figure 2

Anomeric spectral regions for ¹H-¹³C HSQC spectra of HDH samples. In particular, red and blue arrows indicate melezitose and raffinose anomeric signals, respectively.

Figure 3

Score plot and the first component of loading plot (anomeric region) of two classes OSC PLS-DA models are represented on the left and right side, respectively, as following: (a) Fir and Forest honeydew (3LCs, LC1 = 28.7%, LC2 = 16.9%, R²X = 68%, R²Y = 96.4%, Q² = 87.2%); (b) Forest and Oak honeydew (3LCs, LC1 = 33.9%, LC2 = 13%, R²X = 63.1%, R²Y = 97.4%, Q² = 91.7%); (c) Eucalyptus and Forest honeydew (2LCs, LC1 = 33.9%, LC2 = 21.2%, R²X = 55%, R²Y = 98.8%, Q² = 98%); (d) Citrus fruit and Oak honeydew (3LCs, LC1 = 34.1%, LC2 = 16.7%, R²X = 64.9%, R²Y = 97.5%, Q² = 80.3%); (e) Eucalyptus and Oak honeydew (2LCs, LC1 = 34.8%, LC2 = 10.1%, R²X = 44.9%, R²Y = 95.6%, Q² = 83.4%); (f) Fir and Oak honeydew (2LCs, LC1 = 48.4%, LC2 = 17.9%, R²X = 66.4%, R²Y = 95.9%, Q² = 88.1%); (g) Citrus fruit and Eucalyptus honeydew (3LCs, LC1 = 44.4%, LC2 = 31.6%, R²X = 84.9%, R²Y = 99.7%, Q² = 98.8%); (h) Citrus fruit and Fir honeydew (2LCs, LC1 = 38%, LC2 = 22%, R²X = 60%, R²Y = 98.1%, Q² = 88.5%); (i) Eucalyptus and Fir honeydew (2LCs, LC1 = 64.1%, LC2 = 18%, R²X = 82.2%, R²Y = 99.9%, Q² = 99.6%). Yellow dots, red triangles, purple pentagons, green diamonds, and light blues boxes stand for Forest, Fir, Oak, Eucalyptus, and Citrus fruit honeydew, respectively.

Figure 3

Score plot and the first component of loading plot (anomeric region) of two classes OSC PLS-DA models are represented on the left and right side, respectively, as following: (a) Fir and Forest honeydew (3LCs, LC1 = 28.7%, LC2 = 16.9%, R²X = 68%, R²Y = 96.4%, Q² = 87.2%); (b) Forest and Oak honeydew (3LCs, LC1 = 33.9%, LC2 = 13%, R²X = 63.1%, R²Y = 97.4%, Q² = 91.7%); (c) Eucalyptus and Forest honeydew (2LCs, LC1 = 33.9%, LC2 = 21.2%, R²X = 55%, R²Y = 98.8%, Q² = 98%); (d) Citrus fruit and Oak honeydew (3LCs, LC1 = 34.1%, LC2 = 16.7%, R²X = 64.9%, R²Y = 97.5%, Q² = 80.3%); (e) Eucalyptus and Oak honeydew (2LCs, LC1 = 34.8%, LC2 = 10.1%, R²X = 44.9%, R²Y = 95.6%, Q² = 83.4%); (f) Fir and Oak honeydew (2LCs, LC1 = 48.4%, LC2 = 17.9%, R²X = 66.4%, R²Y = 95.9%, Q² = 88.1%); (g) Citrus fruit and Eucalyptus honeydew (3LCs, LC1 = 44.4%, LC2 = 31.6%, R²X = 84.9%, R²Y = 99.7%, Q² = 98.8%); (h) Citrus fruit and Fir honeydew (2LCs, LC1 = 38%, LC2 = 22%, R²X = 60%, R²Y = 98.1%, Q² = 88.5%); (i) Eucalyptus and Fir honeydew (2LCs, LC1 = 64.1%, LC2 = 18%, R²X = 82.2%, R²Y = 99.9%, Q² = 99.6%). Yellow dots, red triangles, purple pentagons, green diamonds, and light blues boxes stand for Forest, Fir, Oak, Eucalyptus, and Citrus fruit honeydew, respectively.