Multidisciplinary analysis of Italian Alpine wildflower honey reveals criticalities, diversity and value

From the phytosociological relevés performed in each sampling area it is evident that hives were positioned in grasslands rich in Alpine herbaceous species (Table S1). In fact, among the 169 identified species, 85% were herbaceous species common in meadows (of Arrhenatherion elatioris and Triseto flavescentis-Polygonion bistortae phytosociological alliance) and acidophilus pastures (Siversio-Nardetum). 15% of the species were trees and shrubs (not abundant in the floristic relevés of the apiary areas considered), including some of beekeeping interest such as: Rhododendron ferrugineum, Castanea sativa and Rubus idaeus. From the MDS biplot (Fig. 2) elevation is the main ecological variable that differentiates sampling areas. In particular, the relevés of stations B and F are characterized by a floristic composition which is different from the areas at higher elevation (characterized by a higher presence of microthermal alpine species). This is due to the separation between the sub-montane belt and the high mountain belt vegetation on the 1.300 m a.s.l. line in the study area²⁵.

Although the beehives were positioned in mountain grasslands, melissopalynological analysis presented a different picture. The pollen of numerous species detected through the floristic relevés were found in the honey samples via melissopalynological analysis, although the latter did not totally overlap with the floristic characterization of the area, in particular from a “quantitative” point of view. In fact, the floristic relevés showed a relative richness of herbaceous species (Table S1) peculiar of mountain grasslands that would seem promising for the production of wildflower honeys. Conversely, in the melissopalynological analysis the species considered interesting but not predominant in the botanical description were relevant (Fig. 3 and Table S2).

The premises to produce wildflower honey is that the botanical species contributing must be different and sometimes very numerous, without any of them assuming a dominant character. However, this was not fully evident in our research: although it was possible to identify more than seventy species through melissopalynological analysis and even more through the floristic characterization of the areas, most of them were defined as minor or sporadic pollen (Table S2). Even though apiaries were in mountain grasslands, the most relevant role was played by some woody species/shrubs: Rubus (presumably Rubus idaeous L., identified in the floristic relevés) and rhododendron (Rhododendron ferrugineum L.) for the mountain/subalpine belt and Castanea and Ericaceae (heather) in the submountain belt. Following the rules to define ‘‘unifloral honey’’, three of the wildflower honeys could be defined unifloral or bifloral:

• Rhododendron unifloral: honey A (Rhododendron 47.18%), and honey C (Rhododendron 62.93%);

• Raspberry unifloral: honey B (Rubus 67.12%)

• Raspberry and Rhododendron bifloral: honey D (Rhododendron 34.27% and Rubus 34.74%) as well as honey E (Rubus 44.25%, Rhododendron 34.14%).

Honey F, due to the contribution of pollen from Tilia genus (that was detected only in this sample as an important sporadic pollen, 3.5%) Castanea (96.4% in honey F, but it should be noted that chestnut pollen is an overrepresented pollen) and in the second count Ericaceae (32.45%, that was considered a secondary pollen together with Rubus, with a percentage of 38.59% in honey F) differed from the other honeys (Fig. 3).

Rubus pollen was anyway present in good amounts in all the samples considered, and was a dominant pollen in honey B, a secondary pollen in honeys C, D, E and F and a minor pollen in honey A. Sorbus and Tilia pollens were detected only in honey F, while no rhododendron was detected in honey F. Honey D was characterized by a percentage higher than the “rare pollen” category of some important alpine essences, such as Liliaceae, Centaurea, Campanulaceae, Anthyllis f., Polygonum bistorta, Lotus alpinus and Potentilla/fragaria (Table S2).

Although wildflower honeys are intrinsically characterized by a high variability compared with unifloral honey, this shows the importance of the formal characterization of honey to obtain a product which satisfies consumer expectations, and it was demonstrated that the botanical origin of honey cannot be based on the claims of local beekeepers by considering the predominant flowers surrounding the hive.

Although honeybees are considered supergeneralists in their foraging choices, there are certain key species or plant groups that are particularly important in honeybee foraging², and many were identified in the botanical characterization of the area, including Rubus idaeus L., Calluna vulgaris L., rhododendron and some present in the broad-leaved woods mentioned such as chestnut (Castanea sativa Mill.) or plants of Tilia genus. In the research work by Hawkins et al.², Rubus fruticosus L. was among the frequently found species and tree pollen belonging to Castanea sativa L. as well as, for example, species of Malus, Salix and Quercus spp, was frequently seen. These kinds of preferences could relate to the ease of availability and abundance of the plant, the quality and abundance of the nectar and pollen and/or specific nutrients or trace elements provided by these species or neurological aspects (as will be discussed further). As referred by beekeepers, over the last decades the production of mountain wildflower honey, that often does not meet the characteristics expected and presents flavours that are reminiscent of other kinds of honey such as rhododendron or linden or chestnut, is becoming more and more critical and this was absolutely confirmed by this study.

This could be linked to the fragmentation of an important habitat of the Alps—mountain grasslands (meaning pastures and meadows) for anthropic and climatic reasons^8,9. Honeybees from the same colony forage across areas spanning up to several hundred square kilometres, and at linear distances as far as 9 km from the hive⁴¹. Onlooker bees are those in charge of finding nectar sources and of giving instructions to the employed bees, the other foraging bees, that communicate the necessity to look for new resources of food to the onlookers through continuous dance communication⁴². Among the onlookers, there is a difference between the bees that scout for different nectar sources or recruit to well known floral resources⁴³ and there is an optimal ratio of scouts to recruits, for the most effective collective foraging⁴¹. However, this balance may change based on the structure of the landscape in which the bees forage for food^44,45,46. Theoretical models^47,48 and empirical tests⁴⁹ suggest that when resources are concentrated into a small number of highly rewarding patches, colonies perform best with few scouts and many recruits, while when resource patches are small, evenly distributed, and easy to locate, successful colonies invest more in scouting than in recruitment. This is strictly linked to climate and social changes in the mountains: mountain grasslands are no longer evenly distributed and easily localizable, as they are scattered among expanding areas of shrublands and forests⁹ and, for the above-mentioned reasons, it is more efficient for the colony to invest in more recruiters than scouters, as recruiters will identify a small number of highly rewarding patches, such as raspberry or rhododendron shrublands or linden and chestnut woods, that are highly rewarding and very different in quality.

This overlaps with individual and collective honeybee behaviour driven by proximate physiological mechanisms that involve the tryptophan metabolism via kynurenine pathway that is one of main neuroprotective mechanisms. In this research, many of the differences/similarities among the samples might be attributed to metabolic alterations within this pathway, represented by relative amounts of kynurenic acid. However, different quinoline structures have also been identified (Fig. 4). Neurotransmitters play a central role in several of the biological processes that honeybees require to perform activities such as foraging behaviour⁵⁰. A considerable amount of literature highlights the involvement of the neuroprotective kynurenine pathway (KP) final product kynurenic acid (KinA) in the regulation of the stress-related hormone dopamine in the honeybee as well as in other animal species^51,52. The major known source of dietary KynA are pollen and nectar produced by sweet chestnuts^53,54 and it has been verified that this compound is found in high concentrations in chestnut flowers⁵⁵. This is coherent with the results of this study: chestnut pollen was found in honey F, produced in the lower station where chestnuts also appear in the floristic relevés, and KynA was found to be a dominant compound in honey F. Interestingly, chestnut pollen was found as sporadic pollen in all the other samples, even those produced in the highest apiary stations (Table S2).

Further, KinA may possess positive properties in a number of pathologies of the gastrointestinal tract, especially colitis, colon obstruction or ulceration^56,57. It has been proposed that KinA may also possess antioxidative properties^56,57,58,59. This was confirmed by this study, since the wildflower honey with a high component of chestnut pollen was the one with the highest antioxidant properties at the FRSA test (66.61 ± 4.77%), even if lower than manuka honey (84.21 ± 1.04%), a dark honey that is a well-known nutraceutical product and has recently attracted attention for its biological properties, especially for its antioxidant and anti-microbial capacities⁶⁰. Honey A showed the lowest power (22.40 ± 0.28%) while the other honeys ranked around 40% (Fig. 5). Interestingly, metabolomic analysis revealed the presence of 3-hydroxyquinaldic acid (Fig. 4), which is a kynurenic acid isomer and, although its function has not been elucidated in detail, a few literature data indicate its role as a precursor of naturally occurring peptide antibiotics from the quinomycin family⁶¹.

In order to evaluate the ability of honey to induce wound closure, a scratch wound assay was performed (Fig. 6)⁶². Scratch assay creates a gap in confluent keratinocyte monolayer to mimic a wound. It has already been demonstrated that honeys are able to induce wound closure⁶³ to different extents depending on honey origins and properties.

The six honeys were compared to manuka honey⁶⁰. Results suggested that honeys A-E induce a significant effect on wound closure, allowing a positive action on the wound edge closure after 24 h of exposure. The results were comparable to that obtained with manuka at the same concentration. Only sample F did not induce a relevant effect if compared to control condition (i.e. complete medium).

The efficacy of natural honey in wound care has been attributed to its anti-inflammatory activity, for which specific flavonoids and polyphenols, as for example kempherol⁶⁴, are considered to be partly responsible. Honey C was the richest in terms of some specific flavonoids, among which tricin, luteolin, pectolinarigenin, naringenin and kempherol (Figure S1), together with a specific glycoside (dihydroxyfenchone 6-O-d-glucoside). Majtan et al.⁶⁵ discovered two other flavonoids in aqueous extract of honey, one of which, kempherol, suppresses the activity of TNF-α-induced Multiple Medical Problems (MMP)-9 expression in HaCaT and according to Budovsky et al. in⁶⁶ in terms of plant phytochemicals, the activity of alkaloids, flavonoids, terpenes, and glycosides in promoting wound healing, has been better researched than other bioactive plant compounds. It is known that the chemical composition of honey predominantly depends on its botanical source⁶⁷. Among all substances in honey, the ones that are the most depending on floral sources are phenolic compounds, that are also important for their antioxidant activity⁶⁸ while processing, handling, and storage have a lesser impact on the phenolic profile and composition of honey. Phenolic acids and flavonoids can be then considered important markers for the identification of botanical origin for different honey^69,70.

Heatmaps, PCAs and pathway molecular networks, very different but complementary visualization techniques, confirmed and complemented one another providing an additional perspective to recognize the results from honey phenolic compounds analysis (the total ion current chromatograms obtained for each honey samples are showed in Figure S3). The heatmap and PCA (Fig. 7) were performed to compare the overall distribution and variation within and between the honey samples. The PCA and hierarchical cluster analysis indicated that the different floral origins caused significant metabolic changes in the honey samples. A clear separation was observed between sample F (chestnut honey) and other samples. Also, C (prevalently rhododendron) and E (rubus and rhododendron) exhibited specific profiles regarding flavonoid composition. On the other hand, samples B and D, despite the different rhododendron/rubus ratio, exhibited a substantial similarity in their phenolic compound profiles.

The most important compounds found are presented in Table S4 and the possible metabolic transformation is presented in Figure S2. The flavonoid fraction was accentuated in C and it corresponds to the heatmap results while phenolic acid and hydroxy-fatty acids were predominant in F. For each type of honey, the most important candidates for differentiation are presented in Figure S1.

Looking at the volatile profiles, analysing the six samples, 255 compounds were detected (Table S3), belonging to the following groups: alcohols, aldehydes, anidrides, aromatics, carboxylic acid, esters, eters, furanoids, hydrocarbons, ketones, nitrogenates, sulphurated, terpenes and other (Table 2). Data analysis showed that honeys A, B, C, and E were very similar while D and F were richer in volatile compounds and presented a high dissimilarity from the other samples. Honeys D and F were the most interesting in terms of volatile fingerprints, showing the highest quantity in terpenes and terpenoids (both around 45 ppb compared to the other honeys, ranging from 13 to 15 ppb). Honeys D and F also had the highest amount of carboxylic acids (Table 2). The single compounds were then analysed to identify the compounds significantly different in each sample (Fig. 8).

Carboxylic acids are responsible of different scents, depending on carbon chain length. Short chain acids such as acetic acid have for example spicy flavours, while butanoic acid and hexanoic acids determine a rancid aroma¹⁸. Honey F was the one containing the highest quantity of acetic acid (81.89 ± 4.069 ppb) and the one with a consistent presence of chestnut pollen. Honey F was described having the peculiarly spiced flavour of chestnut together with the vegetable/fruity flavour of wildflower and the bitterness of linden tree and chestnut. Honey F was also the richest in nitrogenated compounds (Fig. 8) such as 2-Methylfuran, detected only in honey F. Hydrate furanoids such as 2-Methyltetrahydro-3-furanone have already been found in chestnut honeys of different countries⁷¹. Shikimate pathway derivatives such as 2,4-Di-tert-butylphenol, 3-Methylacetophenone and 3,4-Dimethylacetophenone, and some specific monoterpenes such as z-Rose oxide, α-terpinene and cymene, in higher quantities in honey F, were previously associated to linden honey or in general to the genus Tilia⁷¹, that includes different species generally called linden trees for the European species. These species contribute to the production of linden tree honey, which has a taste which is sweet, bitter, medicinal, floral, woody and hay-like⁷². Honey F is the one produced at the lower station, where the linden and chestnut botanical belt starts, and is more definable organoleptically as a mix of Tilia and Castanea honey than a mountain wildflower honey. Honey F is the only one in which Tilia pollen was detected. Tilia was not detected by the floristic relevé but is present in the production area of honey F; this could mean that bees are willing to travel far to find this essence. 1-Phenylethanol, 2-Phenylethanol, 2-aminoacetophenone and acetophenone content distinguished honey F with a high content of chestnut pollen coherently with a previous report⁷³. Considered as a product related to the shikimic acid pathway as well⁷⁴, acetophenone is formed during phenylpropane metabolism by enzymatic reactions from hydroxy-substituted aromatic acids⁷⁵. According to⁷³, 1-Phenylethanol can be associated to a floral odour and may contribute to the characteristic floral aroma of chestnut honey in synergy with other compounds, such as phenylacetaldehyde or 2-phenylethanol. 2-aminoacetophenone is probably not issued by the shikimate pathway but might be the result of tryptophane degradation⁷⁶. In this study tryptophane was not detected by GC–MS but by HPLC Orbitrap. 2-aminoacetophenone is a strong scent⁷³ that can also contribute to the flavour of chestnut honey. Moreover, honey F had the highest concentrations of products produced by the non-enzymatic browning of sugars such as 2-acetylfuran, 3-Methylfuran, 2-Ethylfuran, menthofuran, 4,7-Dimethyl-benzofuran and 2,3-Dihydro-benzofuran. Only 4,5-Dimethyl-2-formylfuran was highest in honey E. These results fit in with the large amounts of furan and methylfuran previously measured in⁷⁷ with a headspace dynamic method in the same origin. These furan derivatives cannot be considered specific floral markers because they are associated to specific thermal treatment and storage conditions⁷⁷ but they can be useful to authenticate honey of chestnut origin that is characterized by a bitter, sweet, burnt caramel and woody flavour⁷⁸. Considering instead benzene derivatives, benzoic acid is considered a marker of nectar from the genera Erica and Calluna and was found predominantly in honey F (besides honey D, 10 ppb, against less than 1 ppb in the other honeys). Honey F, as well as being chestnut pollen prevalent, it is also the only honey with a dominant presence of Ericaceae pollen. Nectar from the genera Erica and Calluna contribute to the production of heather honey⁷⁹, its flavour being characterized by sweet and candy-like notes⁸⁰. Benzaldehyde was also found in a quantity higher than in the other honeys in honey F (29.16 ± 6.36 ppb) and has been previously described as a chestnut honey marker⁷¹, while in honeys B, D and E it was around 10 ppb and in the remaining less than 2 ppb.

Different compounds that were responsible for the dissimilar profile in honey D were a category that are considered both “non-specific” (deriving from the degradation of carotenoid precursors producing different C9- norisoprenoids) and markers of some specific essences such as Ericaeae (heather honey) and were coherently found in higher quantities also in honey F (Fig. 8). For example, significantly higher quantities of α-Isophorone and 4-Oxoisophorone were found only in honeys D and F, while in the other samples they were present only in traces. Other non-specific compounds found in higher quantities in honeys D and F were numerous carboxylic acids (Hexa-2,4-Dienoic acid isomer, Hexanoic acid, Butanoic acid and Acetic acid) and the terpenes Linalool, trans-Linalool oxide and cis-Linalool oxide, Lilac Aldheide and its isomers. These kinds of compound are among the most common in honey, and many VOCs considered nonspecific such as linalool and linalool oxide have been seen to be involved in communication between flowers of fruit crops and their pollinators⁸¹. Moreover, honeys D and F were the ones containing higher quantities of Hotrienol, a regular monoterpene derived from geranyl pyrophosphate (GPP), among the most common honey terpenes. β-Damascenone, defined as a Rhododendron honey and Ericacee in general honey marker^71,75,82 was found in a higher quantity in honey D compared to the other honeys (2.49 ppb while in the other honeys it was always lower than 1 ppb) (Fig. 8).

A significant amount of nicotinaldehyde was found in honeys B (6.65 ppb), D (2.6 ppb), E (7.88) and F (3.01 ppb), while this compound was not detected in honeys A (almost pure rhododendron) and C where it was found in lower quantities (0.16 ppb) (Table S3). This is remarkably coherent with the melissopalynological analysis where Rubus pollen was found mainly in honeys B (67.12%), D (34.74%), E (44.25%) and F (38.59%), while it was found in far lower amounts in honeys A (11.83%) and C (17.24%). The high correlation between nicotinaldehyde content and percentage of Rubus pollen (r = 0.853, p < 0.05) qualifies this molecule as a promising marker for Rubus pollen occurrence. A lower content in nicotinaldehyde (0.16 ppb) was found in sample C that was characterized by 17.24% of Rubus pollen, whereas it was not detected in sample A that was characterized by 11% of Rubus pollen. Therefore, cut-off values for nicotinaldehyde as a Rubus pollen marker can preliminarily be set at 17%. Raspberry honey is quite rare and only few references exist concerning volatile compounds of this honey produced in Estonia and Slovakia⁷¹, although without mention of the compound nicotinaldehyde. However, nicotinaldehyde was reported in the chemical composition of volatiles released by the flowers and fruits of the red raspberry (Rubus idaeus) by⁸³. Nicotinaldehydes could be involved in the synthesis of quinolones, the class of naturally occurring compounds that was abundantly present in samples according to our HPLC Orbitrap analysis. It is clear that there is a need for further studies to ascertain the existence of a common volatile profile for this type of honey⁷¹. Quinoline alkaloids, sought by bees in both chestnut and raspberry, could therefore have an important role in insect-plant communication. It is important to consider the volatile composition of honey as these substances are the main factors responsible for aroma which, together with other factors such as taste and physical factors, contribute to flavour¹⁷. Nevertheless, the volatile fingerprint of honey as a recognition method for floral origin is a debated topic because it is rather difficult to find reliable chemical markers for the discrimination of honey collected from different floral sources. Therefore, scientific publications may report different floral markers for honey of the same floral origin.

The organoleptic properties of honey (flavour, colour, aroma, and texture) are important factors in consumer’s choice of together with the new concept of functional food. Some kinds of honey can have higher market values because of peculiar sensorial properties or for the content in compounds with antioxidant and antimicrobial activity^67,84,85. Therefore, the characterisation of different honeys could increase their commercial value. Mountain wildflower honeys, for example, can be considered particularly valuable, as they are the expression of meadows and pastures biodiversity. In fact, they are included in the presidia to protect following the rules of the Slow Food Foundation for Biodiversity¹⁰. When transhumance was widespread and regular in the Alps, bees (wild and domesticated) also benefited: the pastures were cared for and cleaned to the benefit not only of the animals, but also of the vegetation, and then the bees¹⁰.

As exposed, the chemical composition and the consequent properties of honey depend largely on its floral source which is very dependent on the geographical origin of honey¹⁴. As well, potential contamination sources can characterise different honey production areas⁸⁶, also in the case of organic honey⁸⁷. The production of honey in uncontaminated areas as mountain areas, can be a way of promoting this product so difficult to obtain, together with the promotion of bioactive properties of a sustainable product from marginal territories.

Nevertheless, the honey of the study area, although produced in a limited territory, produced very different honeys according to the different apiary stations and, most importantly, the honeys were not totally definable as wildflower. This highlights the importance of formal analysis (at least melissopalynological analysis if phytochemical is not possible) to characterize honey, that could often reveal food products even more interesting than expected. A better definition of honeys, in addition, could give important cues for beekeeping management, as there are certain key species or plant groups that are particularly important² and a valuable area of further research is to discover why these particular species are important and how landscapes affect bee foraging in different environments. An understanding of the reasons why honeybees target certain plants could help to provide guidance on what constitutes a balanced honeybee diet². The characterization of honeys, furthermore, could lead to the discovery of important compounds both for bees, in particular considering bee behaviour and bee-plant communication, and for human nutrition.

Source: Ecology - nature.com