## 4 Discussion
### Identification of Main Fluorophores in Clover, Acacia, Marruca, and Lavender Honey  
Among these botanic origins, only acacia [25; 35; 36; 39] and clover [40] honey samples were investigated by means of fluorescence spectroscopy in previous works. In our study, the emission/excitation spectral profile of acacia honey (AC1) is rather similar to those obtained for three marruca honey samples (MA1, MA2 and MA3) produced in the south of Tuscany. As reported in Figure 10a,b, the emission spectra obtained by exciting the samples at \(\lambda_{\rm ex}\) = 280 nm, present a typical maximum at 345 nm, with a spectral profile very similar to that of tryptophan, thus suggesting the presence of a large fluorescence contribution from proteins, enzymes, and free amino acids (mainly tryptophan residues). A second less intense emission band centered around 440-450 nm, which can be ascribed mostly to ferulic acid or to chlorogenic acid, has a very similar fluorescence profile (see Figures 6b and 7a). A very good reproduction of these spectral profiles is obtained by including smaller contributions of 4-hydroxybenzoic, p-coumaric, and vanillic acids, which were found in these samples in smaller amounts by means of chromatographic methods [17]. Although ellagic acid was found only in acacia honey [17], it seems that its fluorescence contribution (see the typical asymmetric emission band centered at 420 nm, in Figure 5a) is not evident in our case. At larger excitation wavelengths, both acacia and marruca honey samples present emission profiles similar to other samples, such as clover and lavender honeys, with a typical band centered at 420-435 nm (\(\lambda_{\rm ex}\) = 340 nm) and 475-490 nm (\(\lambda_{\rm ex}\) = 420 nm). In previous FFF studies, the emission bands of acacia honey were explained with the presence of flavonoids [35] or Maillard reaction products [36]; however, our investigations [47] indicate that these compounds, with the exception of quercetin, have very low fluorescence emission. As shown for other honey samples, it is likely that vitamins may significantly affect fluorescence intensity of honey at \(\lambda_{\rm ex}>\) 330 nm. The case of clover honey, which was studied by fluorescence spectroscopy only in a previous paper dealing with honey from New Zealand [26], is rather significant. The emission spectra recorded by exciting at \(\lambda_{\rm ex}=280\) nm in our samples from Tuscany (CL1, CL2, and CL3) present different profiles with a common spectral feature: an emission band centered at 335-340 nm with a shoulder (more or less pronounced depending on the sample) around 320 nm (Figure 10c,d). This peak can be ascribed to 4-hydroxybenzoic acid (see Figure 6a), which was found in large amount in these samples [17, 47]. Interestingly, the differences among the emission/excitation properties of these three clover honey samples can be explained by the different concentrations of caffe, vanillic, ferulic, syringe, and p-coumaric acids, in agreement with the different phenolic profiles of the same clover honey samples determined by chromatographic techniques [17, 47]. The good reproduction of the emission spectra of clover honey samples by using the sole phenolic acids' spectral contributions is shown in Figure 10c,d. On the other hand, the presence of large amounts of phenolic acids is also confirmed by the very intense band (**I**) in the synchronous spectrum (see Figure 3). To our knowledge, lavender honey was never investigated by fluorescence spectroscopy before. In Figure 11, the emission spectra of lavender honey from Tuscany (L1) obtained by exciting at \(\lambda_{\rm ex}=280\) nm (a) and at \(\lambda_{\rm ex}=340\) nm (b) are reported. The emission profile of lavender honey at \(\lambda_{\rm ex}=280\) nm (Figure 11a) is very similar to that of vanillic acid. A good reproduction of this spectrum can be indeed reached by using a combination of the emission spectra of vanillic and syringe acids, with the addition of smaller contributions from caffe, p-coumaric, and 4-hydroxybenzoic acids.