Honey Volatiles as a Fingerprint for Botanical Origin—A Review on their Occurrence on Monofloral Honeys

Honeys have specific organoleptic characteristics, with nutritional and health benefits, being highly appreciated by consumers, not only in food but also in the pharmaceutical and cosmetic industries. Honey composition varies between regions according to the surrounding flora, enabling its characterization by source or type. Monofloral honeys may reach higher market values than multifloral ones. Honey’s aroma is very specific, resulting from the combination of volatile compounds present in low concentrations. The authentication of honey’s complex matrix, according to its botanical and/or geographical origin, represents a challenge nowadays, due to the different sorts of adulteration that may occur, leading to the search for reliable marker compounds for the different monofloral honeys. The existing information on the volatiles of monofloral honeys is scarce and disperse. In this review, twenty monofloral honeys and honeydews, from acacia, buckwheat, chestnut, clover, cotton, dandelion, eucalyptus, fir tree, heather, lavender, lime tree, orange, pine, rape, raspberry, rhododendron, rosemary, strawberry tree, sunflower and thyme, were selected for volatile comparison purposes. Taking into consideration the country of origin, the technique of isolation and analysis, the five main volatiles from each of the honeys are compared. Whereas some compounds were found in several types of monofloral honey, and thus not considered good volatile markers, some monofloral honeys revealed characteristic volatile compounds independently of their provenance.


Introduction
Honey is defined as "the natural sweet substance produced by Apis mellifera bees from the nectar of plants or from secretions of living parts of plants or excretions of plant-sucking insects on the living parts of plants, which the bees collect, transform by combining with specific substances of their own, deposit, dehydrate, store and leave in honeycombs to ripen and mature", according to the Council Directive 2001/110/EC relating to honey produced and marketed in the European Union (EU) [1]. Both the EU [1] and the Codex Alimentarius Commission [2] set compositional criteria for honey, which basically comprises a concentrated water solution of two main sugars, fructose and glucose, with small amounts of various complex sugars, as well as other constituents such as enzymes, amino acids, organic acids, carotenoids, vitamins, minerals, volatile compounds, pollen and wax [3][4][5][6][7][8][9]. Honey has been reported to also contain a variety of flavonoids and phenolic acids that exhibit a wide range of biological properties and are responsible for its antioxidant and anti-inflammatory properties [10][11][12][13][14].
According to the geographical origin of production, honey can be linked with specific areas within the EU, under the labels of Protected Designation of Origin (PDO) and Protected Geographical Identification (PGI). Honeys stamped with these labels generally present characteristics that are essentially or exclusively related to a specific region or a particular local environment with inherent natural and human factors [22]. At present, Portugal is the country with the highest number of honeys registered in the EU (nine PDO honeys), followed by Spain (five PDO and one PGI honeys) and France (two PDO and three PGI honeys) [22].
Honey from bees which collect most of the nectar from a certain type of flower is called monofloral and possesses distinctive organoleptic characteristics, like highly distinguishing aromas, probably derived from the nectar, indicating the presence of volatile components responsible for their characteristic fragrances, and thus is considered as a premium product [7,[23][24][25]. Contrarily, multifloral honey is obtained when bees collect nectar from different types of flowers. In addition, we may also find honey from sugar exudates, so called honeydew or forest honey, which is normally collected from insect's sweet exudates. However, the most widely available honey in the market is blended honey, consisting of a homogenous mixture of two or more honeys with different characteristics such as geographical origin, botanical source, colour, flavour or density [25,26].
The consumer demand for monofloral honey has increased in recent years, due to its particular flavour and pharmacological properties, increasing its commercial value [11,27]. This fact may induce adulterations with low-cost and nutritionally valuable substances or mislabelling concerning the botanical origin [28]. To overcome this fraud, the scientific community has strengthened the research on the development of reliable methodologies and chemical markers that may contribute for honey discrimination, indicating floral and/or geographic origin [29,30]. These would allow the obtaining of a standard of quality and authenticity for honey, protecting the consumer from fraudulent mislabelling of inferior honey, trying to raise its market value [21,31,32].
The International Honey Commission (IHC) is encouraging the development of harmonized analytical methods of quality certification for different honeys. The assessment of honey'sbotanical origin is of great importance in food analysis, particularly in honey, since authenticity guarantees the quality [33].
In order to evaluate the organoleptic quality and authenticity of a food product, the aroma profile is one of the most typical characteristics; the volatile compounds are the main responsible for this organoleptic feature, contributing to flavour, along with taste and physical factors [25,34]. Honey's aroma is one important factor for its differentiation as a function of botanical origin.
Research on honey volatiles began in the early 1960s and it was found that volatiles could originate from a number of factors, such as: a) nectar or honeydew collected by honeybees and linked to the plant characteristics, b) the transformation of plant compounds by the honeybee or directly generated by honeybee, including during honey processing or storage, or c) from microbial or environmental contamination [25,33,35]. Therefore, the volatile compound profile can potentially be used as a fingerprint for honey authentication, which could enable the identification of a honey's origin [11,21,36,37].
The present review details the key factors that influence honey volatiles, the isolation and analytical procedures, and compares the volatile constituents of specific monofloral honeys from different countries. Aiming at trying to unravel the importance of botanical source, geographical origin and isolation/analysis techniques in monofloral honey volatiles characterization, the selected twenty monofloral honeys, Table 1, were chosen considering a minimum requirement of two different countries of provenance, for comparison purposes on marker volatile compounds.

Factors that Influence Honey Volatiles
Different research works have shown that it is difficult to establish reliable volatile compounds as chemical markers for honeys obtained from several botanical sources [38,39]. This is mainly due to the chemical composition of honey being dependent not only on the botanical source, but also on geographical origin, harvesting season, storage conditions, possible interactions between chemical compounds in the honey that occur naturally and also during thermal processing [30,40]. Furthermore, compounds belonging to different chemical classes are dependent on a honeybee's metabolism but also on technical processing, which includes volatile extraction and analysis [41].
Moreover, different volatile isolation procedures and analysis techniques may lead to different results, obtaining more than one class of marker compounds in different proportions for honeys with the same botanical origin [23,35,42]. The importance of these factors on honey volatiles is discussed in the three following sections.

Botanical Source-Honey's Characteristic Aroma Profiles
The chemical composition of honey is highly dependent on the floral origin of the nectar foraged by bees. However, honey is often marketed as mixed-flower honey with a blend of flavours. In order to determine the legitimacy of the botanical source of honey, analyses of pollen (melissopalynology) and organoleptic or physicochemical properties are traditionally employed [43]. Melissopalynology requires a very experienced analyst, is very time consuming, and depends on the expert's ability and judgment. Honey melissopalynological analysis, based on the identification and quantification of the pollen percentage by microscopic examination, is a very useful method to determine the geographical origin of honeys [44]. However, pollen analysis may not give enough information for several honey types obtained from plant genus with underrepresented or overrepresented pollen, such as Citrus spp., Robinia pseudoacacia, Arbutus unedo or Castanea sativa [45][46][47][48]. Consequently, there is a need for detailed chemical characterization of these honeys, and the identification of volatile compounds can provide an additional tool in its authentication [49][50][51].
Owing to the high number of volatile components in honey, the aroma profile may represent a fingerprint of the product, which could be used to determine its origin [9,52]. Several authors have argued that the careful analysis of honey´s volatile compounds could be a useful tool for characterization of botanical origin [53]. This determination based on the aroma profile is particularly dependable for a flavour-rich product such as honey and has led to the development of techniques for measuring its volatile fraction [52].

Honey Volatiles Not Derived from Bee Processing or from Honey Botanical Source
Several factors may contribute to the presence of volatile compounds in honey, which are not directly related to the bee processing or to the honey botanical source, namely (a) thermal processing, (b) storage conditions (temperature and storage period), and (c) extraction techniques. During thermal processing or prolonged storage, labile compounds may be oxidized or destroyed, and volatile compounds could be produced by Maillard reactions and by Strecker degradation reactions [20,54].
Volatiles extraction and analysis techniques may also contribute to the formation of certain compounds not related with the honey botanical source. Heating during an hydrodistillation, might induce the formation of artefacts, mainly due to the thermal degradation of sugars [55].
Carbohydrates and free amino acids are responsible for the generation of furan and pyran derivatives, mostly hydroxymethylfurfural (HMF), whilst Strecker degradation reactions occurring between amino acids and dicarbonyl compounds can produce aliphatic and aromatic aldehydes [20,30,56]. The use of extraction methods that apply heat during this procedure as in hydrodistillation (HD) or microsimultaneous steam distillation-solvent extraction (MSDE), could also contribute to the formation of these furan and pyran derivatives [55,56].

Isolation and Analysis of Honey Volatiles
The isolation of volatile compounds from a complex matrix like honey is very difficult and so different methods can be used, with different degrees of selectivity and efficacy [35,57]. Humid-heatbased extraction procedures, such as hydrodistillation (HD) or distillation-extraction [58,59] are common techniques for volatile isolation. Other methods, such as dynamic headspace extraction (DHE) and headspace solid-phase microextraction (HS-SPME), perform a partitioning of volatiles between honey and the above vapour phase [35,59]. Ultrasound-assisted extraction (USE) is a methodology also mentioned in the literature, which significantly reduces extraction times and improves it, when compared with traditional methods, due to the mechanical effect of the ultrasound on the extraction solvent, enhancing the penetration of the solvent into the matrix via cavitation [60].
Most studies herewith reviewed used HS-SPME to isolate the volatile fraction, because it has some advantages over other methods, such as its simplicity, being free of organic solvents, allowing for the quantification of a large number of molecules with little or no handling of samples whilst significantly decreasing the extraction time [20,61]. Other studies used different extraction techniques [HD, liquid-liquid extraction (LLE), solid-phase extraction (SPE), USE], that provide complementary information concerning honey volatiles, as they are based on diverse principles [60,[62][63][64].
The techniques most used to identify and quantify isolated honey volatiles compounds, following the extraction procedure, are gas chromatography (GC) and gas chromatography-coupled mass spectrometry (GC-MS). In this review, the data is compared based on the frequency of occurrence of specific compounds for each monofloral honey, since a direct comparison based on the quantification of the identified volatiles was not possible: some studies performed quantification by GC or GC-MS, others reported the data in percentages or in absolute amounts and, in other cases, no quantification was made.

Main Volatile Compounds in Monofloral Honeys from Different Geographical Origins
Aroma compounds are present in honey at very low concentrations and are seen as complex mixtures of volatile components of different functionalities and relatively low molecular weights [57]. Among these are compounds from distinct biosynthetic pathways, such as terpenoids and phenolic derivatives, obtained via mevalonic and shikimic acid pathways, respectively, with several functional groups, such as alcohols, aldehydes, ketones, esters, carboxylic acids, benzene derivatives and nitrogen containing compounds [65]. Currently, more than 600 volatile compounds have been identified in honeys from different botanical origins [66]. In recent years, several works have been published describing the volatile profiles of different monofloral honeys, which highlights the growing interest in this subject [50,[52][53][54]57]. As stated in Section 2.1, the percentage of pollen grains needed to classify a honey as monofloral is variable, ranging from 8% to 20% of Arbutus pollen for strawberry tree honey, >45% of Erica pollen for heather honey and >86% of Castanea pollen for chestnut honey [44]. Given the new challenges in the international market or domestic regulations, which may demand a higher percentage of dominant pollen grains for the characterization of particular monofloral honey types, volatile compound analysis may provide a faster and more accurate tool for botanical and geographical origin identification, by identifying the specific volatile metabolites [67].
In this section, the characteristic aroma profiles of honeys from twenty different botanical sources are reviewed (Table 1). Different isolation procedures and quantification methods were used to describe each honey´s volatiles in the different studies. Therefore, herein the comparison will be performed in terms of the frequency of occurrence of specific compounds in each monofloral honey from different countries of provenance, in order to summarize the most important volatile components that characterize them (Tables 2-21). Therefore, each table corresponds to a monofloral honey or honeydew type and is organised according to the country of origin of the samples as well the botanical source (in some cases discriminating honey from honeydew), number of samples studied, melissopalynological examination, volatile isolation procedure and corresponding analysis. Finally, the five dominant volatile compounds identified for each sample are described (the five main quantitative compounds independently of the measure units used by the authors-percentage, µg/100 g, µg/kg or other units), which do not always correspond to the marker compounds of the analysed honey types. Although in Tables 2-21 only the five dominant compounds are presented for each sample belonging to a specific monofloral honey, other compounds that are not among the dominant ones, but which are common to honeys from different countries, are also mentioned.

Acacia Honey
Honey from acacia, characterized by a sweet, beeswax and sourish flavour [41], produced in thirteen countries from three continents, namely Europe, Africa and Asia, was studied, aiming at the identification of characteristic volatile compounds ( Table 2). The oxygen-containing monoterpene cis-linalool oxide, the alcohol 3-methyl-3-buten-1-ol and the aldehyde heptanal, detected in acacia honey samples from twelve of the thirteen countries [52,[68][69][70][71][72][73], were considered to be marker volatile compounds. cis-Linalool oxide was identified in honeys from Austria, Czech Republic, France, Germany, Italy, Morocco, Poland, Romania, Slovakia and Spain. 3-methyl-3-buten-1-ol was isolated in honeys from Austria, Czech Republic, Romania and Spain. Heptanal was detected in honeys from France, Germany, Hungary, Italy, Poland, Slovakia and Romania. Unlike most of the samples, acacia honey from China [74] did not present any of these three volatile compounds.
Benzaldehyde, furfural, hexanal, octanal, nonanal, decanal were compounds frequently reported in this type of honey. Ethanol was also identified in several honeys, which could indicate a fermentation process [75].

Clover Honey
Trifolium repens (white clover) and Trifolium pratense (red clover) are two species of the same genus; however, different volatiles were identified in honeys obtained from each species as the main botanical source. According to the literature (Table 5), white clover honey is characterized by an abundance in benzene derivatives, namely methoxybenzaldehyde, benzyl alcohol [89], phenylacetaldehyde, benzaldehyde [55,76], methyl benzoate, methyl 2-methoxybenzoate, benzoic acid and 2-hydroxy-3-phenylpropionic acid [90]. Red clover honey presents as its main volatile compounds lilac aldehyde isomers, followed by phenylacetaldehyde and benzaldehyde [91].

Cotton Honey
The literature has reported only four studies concerning cotton honey's volatile compounds, from Greece, Palestine and Spain (Table 6) [87,88,92,93]. This honey has a mild aroma and very sweet taste [93]. Despite the small number of studies, compounds like nonanal, phenylacetaldehyde and phenylethyl alcohol were common to all samples from the three countries.

Heather Honey
Nectar from the genera Erica and Calluna contribute to the production of heather honey [100], its flavour being characterized by sweet and candy-like notes [105]. The identified volatile compounds in this honey comprise several ones also present in other types of monofloral honey, including carboxylic acids (2-methylpropanoic acid, phenylacetic acid, 2-hydroxy-3-phenylpropionic acid, butyric acid phenylacetic acid, decanoic acid) and benzene derivatives (benzaldehyde, benzyl alcohol, benzoic acid, benzeneacetic acid) (Table 10).

Pine Honey and Honeydew
Pine honey, produced in Greece and Turkey, shows no incisive taste or aroma [122]. This type of honey is mainly obtained from honeydew secreted by the scale insect Marchalina hellenica that sucks the sap of pine trees, mainly of Pinus brutia Ten and Pinus halepensis Miller [122,123]. Nonanal, nonanol, decanal and octanal were the volatile compounds most frequently reported by three different studies on the profile of this honey (Table 14) [88,122,124].

Rape Honey
Rape honey is characterized by a sweet, musty and slightly fermented flavour [41]. The chemical composition of this type of honey, produced in nine different countries (Table 15) has been reported. Dimethyl disulfide was the most characteristic constituent detected in rape honeys produced in Denmark, France, Germany and Poland [52,71]. Besides this, there were several other compounds with variable presence depending on the honey origin: dimethyl trisulfide was observed for samples from Austria, Estonia and Germany, phenylacetic acid in samples from Estonia and Germany [41,105,125], both butyrolactone and pantolactone in Poland and Slovakia samples [70,71], benzyl alcohol in samples from Denmark, France, Germany and Poland [52,72], and benzoic acid in Estonia, Lithuania and Poland rape honey samples [55,72,105].

Raspberry Honey
Raspberry honey is not frequently commercially available and thus only two references were reported, concerning volatile compounds of this honey produced in Estonia and Slovakia (Table 16). This honey can be characterized by a large number of green notes and lack of honey notes [105]. According to Špánik et al. [70] 2-ethenylbuten-2-al, 3-methylhexane, 3-methylnonane, 3-pyridinemethanol, β-myrcene, cyclopentanemethanol, norbornane, and undecanal are characteristic of raspberry honey volatiles, although Seisonen et al. [105] didn´t find these compounds. It is clear that there is a need for further studies to ascertain the existence of a common volatile profile for this type of honey.

Rhododendron Honey
Rhododendron honey, characterized by having a woody and floral-fresh fruit aroma, originates from the species and natural hybrids spread in the Alps and Pyrenees, specifically Rhododendron ferrugineum L., Rhododendron hirsutum L. and their hybrid Rhododendron x intermedium [48]. In Turkey, this honey is obtained from the nectar of Rhododendron ponticum growing on the mountains of the eastern Black Sea and is usually known as "mad honey" or "toxic honey", due to the presence of the toxic diterpenoids and grayanotoxins in the leaves, flowers, pollen and nectar of many Rhododendron species. However, these compounds were not detected in this honey type [126].
The description of volatiles in rhododendron honey, produced in five countries (Table 17) [76,77,79,87], did not highlight the presence of marker compounds or even common ones between them.

Strawberry Tree Honey
Strawberry tree honey is produced in the south of Europe and has peculiar organoleptic characteristics, a distinct fragrance and a bitter aftertaste being particularly appreciated, although there are few data concerning its volatile composition [104,127] (Table 19). Table 19. Strawberry tree honey's main volatiles, with reference to the country of origin, number of samples, isolation and analysis procedures and five main volatile components. Unless otherwise specified, the honeybee type was A. mellifera.

CHO
# MPA VIP VA Dominant Volatile Compounds Ref.

Sunflower Honey
Sunflower is mainly cultivated for its oily seeds in several European countries, especially in eastern and southern ones, representing an important source of nectar and pollen to bees, contributing to the production of honey characterized by a floral-fresh fruit (fruity), warm and vegetal aroma [48].

Conclusions
Nowadays, the marketing of monofloral honeys, particularly from a specific geographical region, assumes great importance on the part of the consumers, and the beekeeping sector is aware of this. Guaranteeing authenticity and differentiated quality in monofloral honeys reinforces the usefulness of identifying volatile compounds in order to provide the correct labelling of these honeys.
Although there are already studies on this topic, these are few and disperse, as the information is difficult to gather, as there are several variables to consider related to monofloral honey production such as geography, local flora, soil or climate and corresponding volatile analysis, including compound isolation and analytical procedures.
Aware of this variability, this review attempted to indicate as putative markers the volatile compounds that were most often reported in the several existing studies, from twenty selected monofloral honeys, highlighting the five dominant volatiles identified for each honey sample. However, these main components do not always correspond to markers for the analysed honey types, as other compounds, although present in smaller amounts, may be more often referred in a specific monofloral honey obtained from different countries. For this reason, some of the main volatile compounds could not be used as reliable markers, due to their ubiquity in different monofloral honeys (Figure 1), namely benzaldehyde, furfural, octane, nonane, 2-phenylethanol, nonanal or phenylacetaldehyde.
On the other hand, some specific volatile compounds may be used as markers for particular monofloral honeys, such as cis-linalool oxide, 3-methyl-3-buten-1-ol and heptanal for acacia honey, 3-methylbutanal, 2-methylbutanal and isovaleric acid (3-methylbutyric acid) for buckwheat honey, 2-aminoacetophenone, acetophenone and 1-phenylethanol for chestnut honey, α-isophorone and isophorone derivatives such as 2-hydroxyisophorone for heather honey, lilac aldehyde isomers and methyl anthranilate for orange honey and α-isophorone, β-isophorone and 4-oxoisophorone for strawberry tree honey (Figure 1). However, many more studies are needed to validate the importance of these compounds as volatile markers for the six mentioned monofloral honey types.
Despite the number of studies, the variability in the reported data did not allow for the recognition of marker compounds for some honey and honeydew types, such as those of clover, cotton, fir tree, pine, raspberry, rhododendron or thyme. In contrast to the above, each of the monofloral honeys of dandelion, eucalyptus, lavender, lime tree, rape, rosemary and sunflower showed common compounds. Nevertheless, these compounds In contrast to the above, each of the monofloral honeys of dandelion, eucalyptus, lavender, lime tree, rape, rosemary and sunflower showed common compounds. Nevertheless, these compounds could not be considered reliable markers, since they did not occur in most of the samples of each honey type from different provenances.
Volatile markers for a specific monofloral honey from different regions may be rather different due to the presence of specific compounds in the flora of one country and their absence in another country. For a better understanding of this variability, comparative studies on the volatiles of local natural flora and the corresponding honey are desirable to better understand the relationship between both. Moreover, further research in this area should include both melissopalynological information and physicochemical data to understand to what extent volatile compounds can be used to classify with success the valuable monofloral honeys.