**3. Botanical origin of honey**

honey (originating from *Eucriphya cordifolia*), quillay honey (originating from *Quillaja saponaria*), tevo honey (originating from *Retanilla trinervis*), and others. The botanical origin of honey may be known through a quantitative and qualitative melissopalynological analysis. Honeybees are selective in the use of flower resources. The dominant plant community in Central Chile corresponds to the Matorral, an evergreen sclerophyllous vegetation with quillay and tebo as dominant plant species. The deep south of the country is dominated by temperate forest where ulmo is one of the dominant species. Biodiversity varies along an altitudinal or latitudinal gradient in Chile, so the beekeepers usually maintain their beehives along the native plant communities, so the bee products, as well as their potential biological properties will also be different depending on the botanical and geographical origin. Among the bioactive molecules inherited from a specific floral source, phenolic compounds obtained from honey have been related with the antioxidant and antibacterial properties that they show. Honey as a natural product offers many advantages that classify it as an excellent source of active molecules, which could be used as a treatment of human diseases in the forthcoming years. Among these attractive characteristics of honey, it is important to note that this product has low toxicity and the medicinal properties of honey will help to protect honeybees from disappearance by adding value not only to the significantly important process of pollinating

crops and native plants, but also for the medicinal importance of their products.

**2. Chile's unique geographical features and its endemic flora**

unique characteristics.

64 Honey Analysis

The continental Chilean territory has an area close to 75 million hectares and is situated on the southwest border of South America. It has a length of approximately 4300 km from north to south and the average width is 180 km. Pits, terraces, mountainous regions, and valleys form Chile's diverse geomorphology, which together with the biogeographical isolation of a territory limited by geographical and climatic barriers, has configured a biodiversity characterized by a high level of endemism in ecosystems. Chile's vascular flora contains approximately between 5500 and 6000 species, without including subspecies and varieties. Although the number of species, compared with other South American countries, is not especially high, the most prominent trait of Chilean vascular flora is the presence of close to 50% endemic plant species, which gives the Chilean vascular flora a marked uniqueness [1]. Chilean apicultural production is defined by a high variety of honey types which contain a high percentage of nectar obtained from native plant species. The portion of nectar originating from native plants related to the endemism of Chilean flora result in the production of honey with

The production of native and endemic monofloral honey is segregated into two large geographical areas: the first area corresponds to the central zone of Chile and the second corresponds to a region with a climatic transition from humid Mediterranean (VIII Region) to temperate humid (X Region). The central zone of Chile is of the five regions in the world that has a Mediterranean climate. It is characterized by a high level of endemism and biodiversity. Matorral is the dominant vegetal community in this zone. Characteristic matorral species include *Baccharis concave* (chilca), *Peumus boldus* (boldo), *Lithraea caustica* (litre), Honeybees show great selectivity in the use of the vegetation surrounding their beehives. It has been shown that bees select plants with a high production of nectar, high concentration of sugar and that do not contain toxic compounds like certain alkaloids. Nevertheless, the presence of other secondary metabolites including terpenoids, phenolic acids, and flavonoids confer to honey import medicinal properties [3]. Nectar is an aqueous plant secretion whose content is mainly sugars and amino acids. It is collected by bees, particularly *Apis mellifera* L., and is converted into honey by enzymatic actions and dehydration, producing about 18% water content [4]. Honey is a food that contains about 200 substances and consists mainly of sugars, water, and other substances such as proteins (enzymes), organic acids, vitamins (especially vitamin B6, thiamine, niacin, riboflavin, and pantothenic acid), minerals (including calcium, copper, iron, magnesium, manganese, phosphorus, potassium, sodium, and zinc), pigments, solid particles derived from honey harvesting, a large variety of volatile compounds, and also secondary metabolites characteristic of the origin species like phenolic compounds and terpenes [4, 5].

Melissopalynology is the division of palynology, which studies the botanical and geographical origin of honey by subjecting honey sediment, and therefore pollen grain and the other structures therein, to microscopic analysis (**Figure 1**). Honey pollen profiles indicate floral diversity, forest vegetation, and species composition of plants that honeybees forage. The relative pollen frequency is utilized for tagging purposes and to ensure geographical origin, factors which considerably influence honey's commercial value. Furthermore, relative pollen frequency is also utilized as a traceability tool by food control institutions and to assess correlations with *in situ* climatic parameters such as rainfall and temperature, important external factors influencing pollinators and pollination networks [6, 7]. In Chile, the official policy (NCh2981.Of2005) established by the Standards Division of the National Institute for Standardization [2] indicates that the melissopalynological test must be used to differentiate the botanical origin of honey produced in this country. In agreement with this regulation, honey can be classified according to three types of botanical origins: monofloral, bifloral, or polyfloral. Monofloral or unifloral honeys are those where at least 45% or more pollen grains found in it belong to the same species; bifloral honeys are those where pollens from two species are dominant within the total pollen grains, so that, as a whole, both species cover more than 50% of the total pollen grains, and there is not a difference higher than 5% among them; and finally, polyfloral honeys are those where none of the requirements for monofloral and bifloral honeys are met, that is, those where no species reaches at least 45% of the total pollen grains, nor two of them covers more than 50% of the said total.

**Figure 1.** Plants species and respective pollens (microscope 400x) (a) *Retanilla trinervia* (tevo), (b) *Quillaja saponaria* (quillay), (c) *Escallonia pulverulenta* (corontillo), (d) *Azara petiolaris* (corcolén), (e) *Eucryphia cordifolia* (ulmo), (f) *Weinmannia trichosperma* (tineo), (g) *Gevuina avellana* (avellano chileno), and (h) *Caldcluvia paniculata* (tiaca).

The melissopalynology technique is quite laborious, time-consuming and requires a highskilled and trained technician. Thus, a large number of research groups worldwide have focused their attention and studies on improving the knowledge of honey characterization. The most promising approach appears to be the simultaneous detection of multiple components utilizing spectroscopic methods along with statistical analysis. Chemometrics along with Raman, FTIR, NMR, and NIR spectroscopic methods have been used for defining the floral origin of honey and development of classification models. These procedures promptly provide quantitative information without complex pretreatment of samples and primarily through a single spectroscopic technique [6, 8, 9]. Despite advances in these methods in the last few years, limitations still exist in these studies related to the small number of samples and the validity of the proposed methods are rarely demonstrated [8]. Notwithstanding, the emerging new methods are making way to new frontiers in honey characterization. The most promising strategy appears to be the multidisciplinary one, which focuses on the detection of multiple components assisted by chemometrics. Apicultural industries and small producers will make the most of the advantages of more advanced methods which allow for more scrupulous controls, increasing the quality level and safety of honey and derivatives [9].
