*3.12. Polyphenols Composition*

Polyphenols are powerful antioxidants that can reach more than 0.8% (by weight) in bee products [77]. Phenolic acids and flavonoids were extensively investigated in honey [78] and were used to evaluate its quality. The correlations between antioxidant activity and total concentration of phenols was confirmed for seven types of honey from Italy [79] and four honey types from Romania [37]. In another study on Portuguese honey, it was shown that polyphenols in honey were responsible for its antimicrobial effects [80]. Phenolic compounds can be used to classify honey according to its botanical origin [78]. The composition of honey in polyphenols was found to be mostly dependent on the botanical origin due to the fact that these compounds mostly originate from the nectar collected by bees; the nature and quantity of phenolic compounds can also vary with the season, climatic conditions and processing factors [63,79].

In the studied samples, 12 polyphenols were analyzed, which were mostly found in all samples in different concentrations (Table 4). Gallic acid was found at a high concentration (1.55 mg·100 g<sup>−</sup>1) in mint honey, while the lowest value was identified in thyme honey (0.57 mg·100 g<sup>−</sup>1). The protocatechuic and 4-hydroxybenzoic acids were identified in higher concentrations in mint (2.04 mg protocatechuic acid·100 g<sup>−</sup><sup>1</sup> and 1.20 mg 4-hydroxybenzoic acid·100 g<sup>−</sup>1) and raspberry honey (2.57 mg protocatechuic acid·100 g<sup>−</sup><sup>1</sup> and 2.33 mg 4-hydroxybenzoic acid·100 g<sup>−</sup>1). Compared to other honey types, mint also had a high content of vanillic acid (3.03 mg·100 g<sup>−</sup>1) and chlorogenic acid (1.48 mg·100 g<sup>−</sup>1). These two phenolic acids were also found in sunflower and thyme honey in large quantities. Caffeic acid predominated in polyfloral honey (1.20 mg·100 g<sup>−</sup>1) and did not exceed the level of 0.38 mg·100 g<sup>−</sup><sup>1</sup> in other honey samples. Thyme honey had the highest content of *p*-coumaric acid, while myricetin predominated in rape honey, although it was found in all types of honey. Rosmarinic acid was found only in raspberry honey at a very small concentration of 0.03 mg·100 g<sup>−</sup>1, while kaempferol was determined only in polyfloral honey in a concentration of 0.38 mg·100 g<sup>−</sup>1. Quercetin was quantified only in 3 types of honey: mint, polifloral and sunflower honey and luteolin was not determined in any sample.

Gasic et al. [81] observed that quercetin and eriodictiol can be used for sunflower honey authentication and we observed too that quercetin is presented in the sunflower honey analyzed.

### *3.13. Organic Acids Composition*

Organic acids are found in honey in small quantities (<0.5%), but are important chemical components because of their significant contribution to the stability and preservation of the physicochemical and sensory properties of honey [11].

The total acid content increases due to the fermentation phenomena and aging that may occur during storage [82]. Some authors have suggested that organic acid profiles are useful for identifying the botanical and/or geographical origin of honey [83].

As shown in Table 5, the predominant acid in all the honey samples analyzed was gluconic acid. The maximum gluconic acid content was determined in raspberry honey (4.83 <sup>g</sup>·kg−1) and the lowest value in rape honey (3.59 <sup>g</sup>·kg−1). Brugnerotto et al. [84] also identified gluconic acid as the predominant acid in all the honey samples that they studied. Gluconic acid is predominant in both honeydew and floral honey and its concentration can be influenced by the botanical source and the pollen and nectar of the flowers collected by bees. In our study, the concentration of gluconic acid was not influenced by botanical origin or year of production (*p* > 0.05).

Romanian mint and thyme honey were also high in propionic acid (2.67 <sup>g</sup>·kg−<sup>1</sup> and 2.36 <sup>g</sup>·kg−1). Honey samples had a succinic acid content that ranged from a minimum value of 0.05 <sup>g</sup>·kg−<sup>1</sup> in raspberry honey to a maximum value of 0.13 <sup>g</sup>·kg−<sup>1</sup> in mint honey. In the study conducted by Suarez-Luque et al. [85] on 50 honey samples from Galicia (Santiago de Compostela, Spain), the succinic acid content was much higher. Formic, acetic, lactic and butyric acid were determined in low concentrations in all honey samples. The content of honey samples in both propionic and acetic acids was strongly influenced (*p* < 0.001) by botanical origin.

The quantification of malonic and glycolic acids in floral honey was firstly reported in a study by Brugnerotto et al. [84] who determined concentrations of 82.2–134 mg malonic acid·100 g<sup>−</sup><sup>1</sup> and 27.8–43.7 mg glycolic acid·100 g<sup>−</sup>1. Acetic, lactic, formic, and propionic acids were identified in lower concentrations, while fumaric and tartaric acids were not detected. In their study, citric and malic acid concentrations were of 48.2–506 mg·100 g<sup>−</sup><sup>1</sup> and 19.9–132 mg·100 g<sup>−</sup>1, respectively [84].

Suarez-Luque et al. [85] also observed variations in the composition of organic acids in honey that were attributed to its botanical origin. The concentration of citric, malic, succinic and fumaric acid was high in chestnut honey and low in eucalyptus honey. Polyfloral honey had a high content of maleic acid, while clover honey did not contain malic and succinic acids.

The concentration and content of organic acids, as well as ketones and benzene compounds such as 2-hydroxy-2-propanone, 2-phenylethanol, butanoic acid or benzyl alcohol, which were identified in fresh honey, increase with temperature and storage time [86].

### *3.14. Principal Component Analysis (PCA)*

Principal component analysis (PCA) is a statistical procedure that is used to perform a comparison of the results of analytical methods applied to a group of samples. In this study, PCA was applied to analyze and identify the honey samples that share similar characteristics from a total number of 45 samples of di fferent honey types from various regions in Romania. The first principal component (PC-1) accounted for 82% of the variance, while the second principal component (PC-2) accounted for 9% of the variance; together, the first two principal components accounted for 91% of the initial variability. The separation of the honey samples according to botanical origin is shown in Figure 1. As seen in Figure 1, there are three ellipses which represent the rape, sunflower and thyme honey, which are not overlapped with other honey samples, except the polyfloral honey. Regarding the mint and raspberry honey, it can be observed that the raspberry honey ellipse is placed in the mint honey ellipse so a clear separation cannot be observed in this sample. Polyfloral honey was not perfectly grouped due to the fact that this honey type has a wide variety of pollen grains.

In Figure 1, the honey types are marked as: RA—rape, T—thyme, P—polyfloral, S—sunflower, M—mint, and R—raspberry honey. In Figure 2, the parameters used for the projection are abbreviated as: Pf—Pfund color, pH, Fa—free acidity, EC—electrical conductivity, Mo—moisture, HMF, TPC—total

polyphenols content, TFC—total flavonoids content, DPPH, GA—gallic acid, PA—protocatechuic acid, 4-hA—4-Hydroxybenzoic acid, VA—vanillic acid, CA—chlorogenic acid, CafA—caffeic acid, p-CA—p-coumaric acid, RA—rosmarinic acid, My—miricetin, Qu—quercetin, Lu—luteolin, Ka—kaempferol, F—fructose content, G—glucose, S—sucrose, Tu—turanose, Ma—manose, Tr—trehalose, Me—melesitose, Ra—raffinose, GluA—gluconic acid, ForA—formic acid, AcetA—acetic acid, ProA—propionic acid, LacA—lactic acid, ButA—butyric acid, and SucA—succinic acid.

In Figure 2, the parameters which are in the outer ellipse have a greater contribution to variability than the parameters located in the inner ellipse. The rape honey samples were correlated with L\* values, pH, c\*ab, h\*ab, turanose content, manose content and HMF content. The thyme honey samples were correlated with trehalose content and mint honey with caffeic acid, *p*-coumaric acid, vanillic acid, rosmarinic acid and chlorogenic acid content. Regarding the physicochemical parameters, it seems that the moisture content was in opposition to the rest of the parameters.

There was a clear differentiation between Mo variable, the variable groups Tu, L\*, Ma, HMF, pH and h\*ab (PC-1 direction) and variable groups TPC and SucA (PC-2 direction). Between variable groups from the PC-1 direction (Tu, L\*, Ma, HMF, pH, h\*ab and M), there was no correlation with variables TPC and SucA. Between Tu, L\*, HMF, pH and h\*ab, and variable Mo, there was negative correlation and the highest fraction of explained variance among these variables was 82%. Furthermore, the small distance between Tu, L\*, HMF, Ma and h\*ab showed a strong correlation between variables.



F—fructose, G—glucose.


### *Foods* **2020**, *9*, 306



**Figure 2.** Principal component analysis—loadings: L\*, h\*ab, c\*ab, Pf—Pfund color, pH, Fa—free acidity, EC—electrical conductivity, Mo—moisture, HMF, TPC—total polyphenols content, TFC—total flavonoids content, DPPH, GA—gallic acid, PA—protocatechuic acid, 4-hA—4-Hydroxybenzoic acid, VA—vanillic acid, CA—chlorogenic acid, CafA—caffeic acid, p-CA—p-coumaric acid, RA—rosmarinic acid, My—miricetin, Qu—quercetin, Lu—luteolin, Ka—kaempferol, F—fructose content, G—glucose, S—sucrose, Tu—turanose, Ma—manose, Tr—trehalose, Me—melesitose, Ra—raffinose, GluA—gluconic acid, ForA—formic acid, AcetA—acetic acid, ProA—propionic acid, LacA—lactic acid, ButA—butyric acid, and SucA—succinic acid.
