**5. Physicochemical analysis of honey: fundamentals and objectives of the analysis and results for some Brazilian samples**

The honey chemical composition is intrinsically related to factors as bee species, geographical origin, flora, climate conditions, seasons, processing, manipulation, and storage conditions [4]. Brazil that presents a large biodiversity is able to offer several different types of honey, as shown in **Figure 7**, bees visiting "pau‐Brasil" flowers and "cipó‐uva" honey in the comb. As mentioned previously, in general, honey consists of approximately 200 substances including sugars, amino acids, proteins, organic acids, flavonoids, phenolic acids, volatile compounds, vitamins, minerals, pigments, wax, enzymes, pollen grains, and other phytochemicals [4, 29].

**Figure 7.** (A) Honeybees collecting nectar from "Pau‐Brasil" (*Caesalpinia echinata*) flowers, showing the high biodiversity and infinite possibilities of floral honeys. (B) "Cipó‐uva" (*Serjania* spp.) honey in honeycomb. Photography (A) was taken and gently donated by Mr. Antônio Carlos Meda, and photograph (B) was taken and gently donated by Lucas Eduardo Meda, both from Apis Flora Indl. Coml. Ltda, Ribeirão Preto, São Paulo, Brazil.

Quality control analyses are extremely important in the evaluation of origin, quality, adulteration, storage conditions, and contamination of honey. The physicochemical properties of a honey sample may provide important information about its biological and geographic origin [30]. Honey adulteration, mainly due to the addition of sugar derived from sugar cane, corn, and beet or even by providing sugar as a food source for bees, occurs due to its limited availability and high cost [31]. Suitable storage conditions are essential to ensure honey quality, as its chemical composition may change due to the thermal process, oxidation, and fermentation reactions [4]. Nowadays, the increasing use of pesticides in agriculture makes contamination of honey by its residues a public health issue [32].

Taking it into consideration, analytical methods are essential to provide reliable results. In the literature, there are several methodologies employed in honey quality control analyses, which are complementary for honey samples appreciation. In 1990, the International Honey Commission (IHC) was created with the goal to generate a new world honey standard. All employed honey analyses methods were then collaboratively tested and published as "Harmonised Methods of the European Honey Commission" [33]. Based on this fact, the EU Honey Directive and Codex Alimentarius Standards were revised for honey analyses. Since then, IHC continuously aims to improve and develop new analytical methods for honey analysis.

## **5.1. Color**

**5. Physicochemical analysis of honey: fundamentals and objectives** 

The honey chemical composition is intrinsically related to factors as bee species, geographical origin, flora, climate conditions, seasons, processing, manipulation, and storage conditions [4]. Brazil that presents a large biodiversity is able to offer several different types of honey, as shown in **Figure 7**, bees visiting "pau‐Brasil" flowers and "cipó‐uva" honey in the comb. As mentioned previously, in general, honey consists of approximately 200 substances including sugars, amino acids, proteins, organic acids, flavonoids, phenolic acids, volatile compounds, vitamins, minerals, pigments, wax, enzymes, pollen grains, and other phytochemicals [4, 29].

**Figure 7.** (A) Honeybees collecting nectar from "Pau‐Brasil" (*Caesalpinia echinata*) flowers, showing the high biodiversity and infinite possibilities of floral honeys. (B) "Cipó‐uva" (*Serjania* spp.) honey in honeycomb. Photography (A) was taken and gently donated by Mr. Antônio Carlos Meda, and photograph (B) was taken and gently donated by Lucas Eduardo

Meda, both from Apis Flora Indl. Coml. Ltda, Ribeirão Preto, São Paulo, Brazil.

**of the analysis and results for some Brazilian samples**

150 Honey Analysis

The color of honey is an important quality parameter for commercialization as it is its first attractive attribute [4]. The color is directly related to its chemical composition, ash content, temperature of the hive, and it changes during storage time [34]. The main compounds related to the color of honey are phenolic compounds, pollen and mineral contents, which may vary widely according to its botanical and geographical origin [30]. During storage, the color of honey may change due to the fermentation process such as caramelization and Maillard reactions or due to the thermal process, which may change its chemical composition and consequently its color [35] or according the package used. To determine the color of honey, a photometer with direct readout in mm Pfund may be used. The Pfund scale compares an analytical standard scale of reference on the graduation of glycerin in order to provide repeatable and accurate results [30]. According to the Codex Alimentarius Committee on Sugars [8], color of honey may vary from nearly colorless to dark brown.

Regarding Brazilian honey color analysis, Sodré et al. [36] studying 36 honey samples from north coast of Bahia found predominance of the light amber color (75%) followed by amber color (16.6%) and in minor proportion, dark amber, extra light amber and extra white colors (with 2.8% each one). Moreti et al. [37] analyzed 52 samples of honey from several counties of Ceará state and found colors as water white (26.92%), white and extra white (17.31% each one), light amber (15.38%), extra light amber (11.54%), amber (9.61%), and dark amber (1.92%). **Figure 8** presents the different colors observed in only five samples studied here showing how different honey could be from Brazil especially because of the large biodiversity and extension of the country.

**Figure 8.** Color and botanical source according beekeepers about the samples used in this work, respectively, from left to right: Polifloral honey (Apiary Joel Souza, Altinópolis/São Paulo—batch 019400916) and orange (*Citrus sinensis*) honey (Apiary Hugo Charnet, Galvão Peixoto/São Paulo—batch 019300815), coffee (*Coffea Arabica*) honey (Apiary Roberto Quintino, Minas Gerais) samples gently donated by Apis Flora Indl. Coml. Ltda, Ribeirão Preto/São Paulo, Brazil. The second orange (*Citrus sinensis*) honey sample (Baldoni, batch 1607) followed by "Cipó‐uva" (*Serjania* spp.) honey (Baldoni, batch 1484) were produced in Baldoni, Campinas/SP, Brazil and were acquired in Santa Terezinha Empório, Ribeirão Preto/São Paulo.

#### **5.2. Moisture**

The water content in honey samples varies according to botanical origin, climate conditions, processing techniques, and storage conditions [4]. Moisture influences honey's properties such as viscosity, crystallization, solubilization, color, and flavor [38]. The moisture may increase during processing and storage time and should be evaluated since its increase makes honey more susceptible to the fermentation process [39]. Determination of moisture in honey samples can be performed employing a refractometric method, which is based on the increases of refractive index related to solid content, and so it is possible to determine indirectly moisture of honey. According to the Codex Alimentarius Committee on Sugar [8], the moisture content in honey should not exceed 20% [8].

Several authors described the moisture content found in Brazilian honey samples. Périco et al. [40] analyzed 30 samples from Toledo, Paraná and found values ranging from 8.7 ± 0.3 to 17.6 ± 6.8/100 g. In the Rio Grande do Norte, Soares et al. [41] analyzed 24 samples from 12 commercial points of Apodi, RN, and found higher values of moisture, ranging from 16.5 to 21.5/100 g. In turn, Paulino et al. [42] found similar values of moisture (15.2–20.33/100 g) when analyzed 13 samples from various cities of Ceará state. Some examples of moisture and parameters described are shown in **Tables 2** and **3**.


**Table 2.** Presentation of results obtained with different geographic and floral honey found in Brazil.

**5.2. Moisture**

152 Honey Analysis

The water content in honey samples varies according to botanical origin, climate conditions, processing techniques, and storage conditions [4]. Moisture influences honey's properties such as viscosity, crystallization, solubilization, color, and flavor [38]. The moisture may increase during processing and storage time and should be evaluated since its increase makes honey more susceptible to the fermentation process [39]. Determination of moisture in honey samples can be performed employing a refractometric method, which is based on the increases of refractive index related to solid content, and so it is possible to determine indirectly moisture of honey. According to the Codex Alimentarius Committee on Sugar [8], the

**Figure 8.** Color and botanical source according beekeepers about the samples used in this work, respectively, from left to right: Polifloral honey (Apiary Joel Souza, Altinópolis/São Paulo—batch 019400916) and orange (*Citrus sinensis*) honey (Apiary Hugo Charnet, Galvão Peixoto/São Paulo—batch 019300815), coffee (*Coffea Arabica*) honey (Apiary Roberto Quintino, Minas Gerais) samples gently donated by Apis Flora Indl. Coml. Ltda, Ribeirão Preto/São Paulo, Brazil. The second orange (*Citrus sinensis*) honey sample (Baldoni, batch 1607) followed by "Cipó‐uva" (*Serjania* spp.) honey (Baldoni, batch 1484) were produced in Baldoni, Campinas/SP, Brazil and were acquired in Santa Terezinha Empório, Ribeirão Preto/São Paulo.

Several authors described the moisture content found in Brazilian honey samples. Périco et al. [40] analyzed 30 samples from Toledo, Paraná and found values ranging from 8.7 ± 0.3 to 17.6 ± 6.8/100 g. In the Rio Grande do Norte, Soares et al. [41] analyzed 24 samples from 12 commercial points of Apodi, RN, and found higher values of moisture, ranging from 16.5 to 21.5/100 g. In turn, Paulino et al. [42] found similar values of moisture (15.2–20.33/100 g) when analyzed 13 samples from various cities of Ceará state. Some examples of moisture and

moisture content in honey should not exceed 20% [8].

parameters described are shown in **Tables 2** and **3**.



\*\*HMF was determined using spectrophotometry UV methodology.

**Table 3.** Physical‐chemical analysis of different floral sources of Brazilian honeys (*n* = 3).

#### **5.3. Ash content and electrical conductivity**

Ash content and electrical conductivity are parameters mainly used to measure mineral content, which may be an indicative of environment pollution, the geographic and botanical origin of the honey [4, 39]. Mineral content is also associated with sensorial properties as color and flavor, which are important for honey commercialization [38]. Ash content provides important information about the quality of honey, as floral honey has lower ash content than honeydew honey [30]. Determination of ash content is performed by a gravimetric method [43]. The Codex Alimentarius Committee on Sugars [8] does not recommend a specific value for ash content. Electrical conductivity is related to the presence of ions, organic acids, and proteins in honey [4]. The determination of this parameter is based on the measure of the electrical resistance, which is reciprocal of the electrical conductivity [43]. According to the Codex Alimentarius Committee on Sugars [8], it is recommended a maximum value of 800 mS/cm for the electrical conductivity of honey samples.

Paulino et al. [42] found ash content in Brazilian honey ranging from 0 to 1.34%. According to Brazilian legislation, the ash content in blossom honey should be at maximum 0.6%, and at maximum 1.2% for honeydew honey [7]. Rodrigues‐Evangelista et al. [44] found values from 0.17 to 0.20% of ash when analyzed honey samples from Paraíba state. Sodré et al. [36], in turn, found an average of 0.3 ± 0.10% of ash content in honey from Bahia state. In another study, the same group found values ranging from 0.01 to 0.41% of the total ash.

Bendini and Souza [45] analyzed 24 samples of blossom honey derived from cashew flowers from Ceará state and found electrical conductivity values from 179 a 198 μS/cm with an average of 187 ± 4.8 μS/cm. When 13 honey samples from Ceará state were analyzed by Paulino et al. [43], values ranging from 120 to 750 μS/cm were found. Sodré et al. [36] found an average of 780.7 ± 302.70 in 36 samples of bee honey from Bahia state and when honey samples from Ceará were analyzed by the same group, values between 192.00 and 798.67 μS/cm<sup>1</sup> were found [46].

#### **5.4. pH and free acidity**

The presence of organic acids in honey is responsible for its natural acid pH value. Determination of pH in honey samples is important to confirm its authenticity, as an addition of sugar in honey significantly increases pH values [47]. Free acidity is characterized by the presence of organic acids in equilibrium with their respective lactones, esters, and inorganic ions [29, 48]. It is a parameter used to evaluate honey deterioration, as fermentation of sugar into organic acids increases its value [30]. The determination of free acidity in honey is performed by a potentiometric titration method and the results are expressed in milliequivalents of acid per kg of honey [43]. The Codex Alimentarius Committee on Sugars [8] recommends a maximum value of 50 mEq/kg for free acidity in honey.

When 30 samples of honey from Paraná state were analyzed, Périco et al. [40] found pH values ranging from 3.53 to 4.60. Soares et al. [41] determined the acidity value in 24 bee honey samples from Apodi, Rio Grande do Norte and found results ranging from 26.73 to 126.77 mEq/kg. In turn, Sodré et al. [36] determined the pH and acidity value in 20 bee honey samples from Ceará state and their average were 3.77 ± 0.25 and 29.10 ± 7.04 mEq/kg, respectively. Marchini et al. [49] also analyzed the same parameters in 205 honey samples from different localities from São Paulo state and found pH values of 2.90–5.10 to eucalyptus honey, 2.30–5.00 to wild honey, 2.70–4.60 to orange honey, and acidity values of 12.5–55 mEq/kg of eucalyptus honey, 14–75.5 mEq/kg to wild honey, and 14–57 to orange honey.

## **5.5. Sugars**

**5.3. Ash content and electrical conductivity**

\*\*HMF was determined using spectrophotometry UV methodology.

**Table 3.** Physical‐chemical analysis of different floral sources of Brazilian honeys (*n* = 3).

\*Gently donated by Apis Flora Company and #

**Honey Physicochemical Parameters**

154 Honey Analysis

(%w/w)

Insoluble material

Reducing sugars (%w/w)

Apparent sucrose (%w/w)

for the electrical conductivity of honey samples.

**5.4. pH and free acidity**

Ash content and electrical conductivity are parameters mainly used to measure mineral content, which may be an indicative of environment pollution, the geographic and botanical origin of the honey [4, 39]. Mineral content is also associated with sensorial properties as color and flavor, which are important for honey commercialization [38]. Ash content provides important information about the quality of honey, as floral honey has lower ash content than honeydew honey [30]. Determination of ash content is performed by a gravimetric method [43]. The Codex Alimentarius Committee on Sugars [8] does not recommend a specific value for ash content. Electrical conductivity is related to the presence of ions, organic acids, and proteins in honey [4]. The determination of this parameter is based on the measure of the electrical resistance, which is reciprocal of the electrical conductivity [43]. According to the Codex Alimentarius Committee on Sugars [8], it is recommended a maximum value of 800 mS/cm

Baldoni Company.

**Polifloral\* Orange\* Coffee\* Orange# "Cipó‐uva"#**

0.05 ± 0.02 0.00 ± 0.00 0.02 ± 0.01 0.00 ± 0.00 0.01 ± 0.01

71.1 ± 0.60 70.2 ± 0.60 69.1 ± 0.60 70.9 ± 0.70 77.0 ± 1.80

3.47 ± 0.43 2.80 ± 0.02 2.76 ± 0.02 3.48 ± 0.03 4.12 ± 0.37

Paulino et al. [42] found ash content in Brazilian honey ranging from 0 to 1.34%. According to Brazilian legislation, the ash content in blossom honey should be at maximum 0.6%, and at maximum 1.2% for honeydew honey [7]. Rodrigues‐Evangelista et al. [44] found values from 0.17 to 0.20% of ash when analyzed honey samples from Paraíba state. Sodré et al. [36], in turn, found an average of 0.3 ± 0.10% of ash content in honey from Bahia state. In another

Bendini and Souza [45] analyzed 24 samples of blossom honey derived from cashew flowers from Ceará state and found electrical conductivity values from 179 a 198 μS/cm with an average of 187 ± 4.8 μS/cm. When 13 honey samples from Ceará state were analyzed by Paulino et al. [43], values ranging from 120 to 750 μS/cm were found. Sodré et al. [36] found an average of 780.7 ± 302.70 in 36 samples of bee honey from Bahia state and when honey samples from Ceará

The presence of organic acids in honey is responsible for its natural acid pH value. Determination of pH in honey samples is important to confirm its authenticity, as an addition

were found [46].

study, the same group found values ranging from 0.01 to 0.41% of the total ash.

were analyzed by the same group, values between 192.00 and 798.67 μS/cm<sup>1</sup>

Sugars are intrinsically related to the flowers used by bees to produce honey, climate, and geographical conditions. Monosaccharides are the most common sugar in honey and fructose (38.5%) and glucose (31.0%) are the major sugars in honey [47]. The ratio of fructose and glucose in honey samples are used to evaluate the degree of crystallization of the honey sample [50]. Determinations of reducing sugars and apparent sucrose are based on a titrimetric method employing Fehling's reagent. The method is a titration of a Fehling's solution at boiling point by reducing sugars in honey using as indicator methylene blue [43]. Determination of the ratio of fructose and glucose may be performed by quantification of sugars in honey samples by GC methodology employing a sugar derivatization process or by HPLC methodology employing a refractive index detector or a pulsed amperometric detection [43, 51, 52]. The Codex Alimentarius on Sugars [8] stipulates that the minimum content of reducing sugars in floral honey is 60 /100 g.

In 2003, Sodré et al. [36] found an average of 69.20 ± 1.82% of reducing sugars and 2.40 ± 1.42% of apparent sucrose. The same group analyzed in 2006, 20 samples from different regions of Ceará state and found 78.84 ± 2.71% of reducing sugars and 2.71 ± 2.40% of apparent sucrose. Soares et al. [41] found a reducing sugar content of 62.89–86.93% and apparent sucrose from 1.13 to 10.12% in 24 samples of 12 providers from Apodi, the Rio Grande do Norte.

#### **5.6. 5‐HMF**

Sugars present in honey may alter during storage time due to nonenzymatic reactions such as Maillard reaction, caramelization, and sugar degradation [47]. The compound 5‐hydroxymethylfurfural (5‐HMF) is a decomposition product of monosaccharides present in honey. Factors such as temperature, heating, floral origin, pH, and storage conditions may significantly influence in 5‐HMF content [53]. Therefore, 5‐HMF content is a parameter used to determine the freshness of honey, as it is absent in fresh honey and its concentration increases during storage time [30]. Furthermore, high 5‐HMF content may indicate adulteration of honey by the addition of invert syrup [47]. Determination of 5‐HMF content may be performed employing a spectrophotometric method [43], or a chromatographic method by HPLC using calibration curves of 5‐HMF analytical standards to quantify this compound in honey [43, 53]. The Codex Alimentarius Committee on Sugars [8] stipulates 5‐HMF content at the maximum value of 40.00 mg/kg and, if honey is from a tropical region, accepts a maximum value of 80.00 mg/kg.

The HMF values found in Brazilian honey are higher than those found in nontropical countries, as Paulino et al. [42] that encountered 7.00–355.50 mg/kg in 13 samples from several cities of Ceará. Périco et al. [40] also found high HMF values (31.28 ± 0.2 to 581.4 ± 4.2 mg/kg) when analyzed 30 samples from Toledo, Paraná, and Soares et al. [41] found values ranging from 70.62 to 150.27 mg/kg. When honey from two distinct regions (São João do Cariri and Areia, both in Paraíba state) were analyzed by Rodrigues‐Evangelista et al. [44], the HMF content was between 20.70 and 23.90 mg/kg.

#### **5.7. Diastase**

Diastases are enzymes present in honey, which are sensitive to heat and consequently, may be used to evaluate honey overheating [47]. Therefore, the measure of diastase activity is an indicative of honey's freshness and is useful to detect improper storage conditions [30]. Diastase activity may be also an indicative of honeybees fed artificially with glucose, as a diastase enzyme deficiency is observed in this case [54]. The determination of diastase activity is based on a spectrophotometric kinetic method, which measures the activity of diastasis enzymes present in honey, in order to monitor adulteration by the addition of sugar and evaluate storage time and conditions [4, 43]. For that, under specific conditions, the activity of diastase enzymes of honey is measured in a standard solution of starch. The Gothe unit is used to express diastase activity and is defined as the amount of enzyme which will convert 0.01 g of starch in 1 hour at 40°C [43]. The Codex Alimentarius Committee on Sugars [8] stipulates a minimum value of 8.00 Gothe; however, a minimum value of 3.00 Gothe is accepted for honey with low diastase activity if the 5‐HMF content is lower than 15 mg/kg.

The diastase activity was determined in 20 samples of honey from Ceará state by Sodré et al. [36] and found an average of 34.11 ± 8.41 (in Gothe scale). Sodré et al. [36] analyzed 36 honey samples from Bahia and found the value between 5.30 and 43.39. Marchini et al. [49] analyzed 205 honey samples from different localities of São Paulo state and found values ranging from 1.10 to 38.50, with an average of 8.14 for orange honey, 15.77 for eucalyptus honey, and 17.32 for wild honey. For different floral sources, the authors found values ranging from 7.80 to 19.00.

In complement to pollen microscopical analysis, physical‐chemical results for these Brazilian honey samples were conducted and which is presented below, where it is possible to demonstrate the identity and quality of some floral sources of Brazilian samples studied here.
