**2.1. Moisture**

Water content is one of honey's most important characteristics as it influences its viscosity, specific gravity, maturity, crystallization, flavor, preservation, shelf life and palatability [11– 13]. It depends on several factors such as bee species, floral source, honey harvesting time, the degree of maturity achieved in the hive (complete dehydration) and climatic factors [14].

Moisture is analyzed to determine the safety of the product, giving a quality criterion that determines the ability of the honey to remain stable and free of fermentation. A high moisture content can lead to crystallization of the product and promote the development of osmophilic microorganisms responsible for fermentation, negatively affecting its sensory characteristics and nutritional properties and reducing the shelf life of the product [15].

*Method*: humidity is determined according to the method described by the Atago Co [16], using the refractometric method. The method is based on the relationship between the speed of light in a vacuum and a substance through which an incidental beam of light is passed, through a honey solution [8, 16]. This device is adapted from the Abbe refractometer and features a scale, which expresses the value in brix, from which the humidity value is calculated. Three drops of honey are placed in the refractor device and, after adjusting the angle limit, the reading of the refractive index is taken directly from the scale [8]. The refractive measurement provides the dry matter content in all cases where there are pure sugar solutions. When the sugar solution is mixed with other substances, such as honey, the value found is usually very close to the total for the dry matter [17]. Therefore, to obtain the moisture from the honey, the value of the refractive index is checked with a correlation table showing the relationship between the refractive index and the moisture of the honey (**Table 1**). **Table 2** presents the result of an equation developed by Wedmore from the data of Chataway [15].

The refractive index of liquids is also temperature dependent. Generally, refractometers are regulated at 20°C [17]. If the temperature of honey is exactly 20°C, the refractive index obtained directly from **Table 1** can be applied. However, for measurement at different temperatures, the refractive index should be increased or decreased by a value of 0.00023 for each degree Celsius above or below 20°C, depending on the sample temperature. In the case of refractive index values not included in **Table 2**, the desired value can be calculated using Eq. (1).

$$\mathbf{y} = \mathbf{61} \mathbf{4} \times \mathbf{60} \mathbf{-400} \times \mathbf{x},\tag{1}$$

where y = moisture, x = refractive index.


**Table 2.** Determination of honey moisture from the refractive index.

#### **2.2. pH**

The pH determined refers to the hydrogen ions present in a solution of honey and can influence the formation of other components such as the production of hydroxymethylfurfural— HMF [19].While pH analysis is useful as an auxiliary variable to estimate the quality of the product and as a parameter for evaluating total acidity, it is not directly related to free acidity due to the actions of the buffer acids and minerals present in honey [20].

The pH of honey ranges between 3.5 and 5.5 depending on its botanical source, the pH of nectar, soil or plant association, and the concentration of different acids and minerals such as calcium, sodium, potassium and other ash constituents [2, 15]. Altered values may indicate fermentation or adulteration [12, 21]. Mandibular substances added to the nectar may also change the pH of honey, a process that begins with the transport of nectar to the hive in the honey vesicle [22].

*Method*: pH is determined according to the method described by De Moraes and Teixeira [23]. Weigh 10 g of honey in a 100 mL beaker using an analytical balance and homogenize the sample in 75 mL of distilled water. Thereafter, using a pH meter calibrated with appropriate buffers (pH solution of 7.0 and 4.0 pH solution) for each honey sample, a direct reading is taken from the device.
