**3.2. Physical appearance: colour and consistency**

The results obtained for the colour and consistency of honey are illustrated in **Table 2**, and **Figure 3** and **Figure 4**, respectively. The darkest colour was observed for the autumn honey.

**Figure 2.** Spatial maps for (a) population density (adopted with permission from [17]) (b) spring, (c) summer and (d) autumn honey sample collection.


**Table 2.** Mean absorbance values at 560 nm and mean consistency values for honey samples from the three seasons.

**Figure 3.** The colour scale for honey samples from the three honey seasons. SU = summer, SP = spring, AU = autumn.


**Figure 4.** The consistency scale for honey samples from the three honey seasons. SU = summer, SP = spring, AU = autumn.

The autumn honey is characterised by carob and eucalyptus sources. This honey is so distinctive, compared to other honey types (*p* < 0.001), that it is sometimes confused with carob syrup. Spring honey has a more liquid consistency than honey from the other two seasons (*p* < 0.01). As the nectar type determines seasonality, this has no direct impact on the consistency of honey and therefore some other factor might influence this parameter. This can only be determined through the investigation of other physicochemical characteristics.

#### **3.3. Determination of brix and moisture content in honey**

The brix and moisture contents of honey are illustrated in **Table 3**. Although in general there are minimal differences between the brix values for each particular season with year, seasonal statistical analysis reveals a significantly lower brix values for the autumn honey samples as compared to the other two seasons, that is, less than 79.90% (autumn) as opposed to more than 80.33% (spring and summer). The moisture content is practically opposite to the brix value, in which case autumn honey moisture content is significantly higher (*p* < 0.001) than that of the other two seasons. That is, more than 18.52% (autumn) and opposed to less than 18.46% (spring and summer). The main reason for this difference may be due to the abundance of water during the beginning of autumn, which is considered as the rainy season. Although during winter it is likely that 'winter honey' is produced, this is removed before the spring season starts, as this honey is mainly made from syrup. This is mandatory as syrup honey is considered as adulterated honey. The use of syrup during the winter months is only allowed so as to maintain the bee colony alive and healthy, considering that during winter very few plant species flower.


**Table 3.** Mean percentage brix and moisture values for honey samples from the three seasons between 2011 and 2014.

#### **3.4. Determination of pH and free acidity of honey**

**Figure 3.** The colour scale for honey samples from the three honey seasons. SU = summer, SP = spring, AU = autumn.

**Figure 2.** Spatial maps for (a) population density (adopted with permission from [17]) (b) spring, (c) summer and (d)

Colour (560 nm) 1.844 ± 0.242 3.909 ± 0.207\*\*\* 2.143 ± 0.299 Consistency 2.563 ± 0.190 2.227 ± 0.254 1.750 ± 0.083\*\*

**Table 2.** Mean absorbance values at 560 nm and mean consistency values for honey samples from the three seasons.

**Summer Autumn Spring**

**Figure 4.** The consistency scale for honey samples from the three honey seasons. SU = summer, SP = spring, AU =

autumn.

autumn honey sample collection.

\*\*p < 0.01. \*\*\*p < 0.001.

178 Honey Analysis

The acidic nature of honey is important for several reasons. The most important reason is that the low pH inhibits the presence and growth of microorganisms. Other aspects of food technology, permit the honey to be blended with other food products, due to its low pH. The acidic nature also contributed to the flavour of honey particularly in monofloral honeys [18]. **Table 4** shows the mean pH and mean acidity values for honey samples from the three seasons. It was observed that there is slight yearly variation between mean pH values for the separate seasons. However, seasonal statistics reveal significant differences between the three season, the pH being the highest for the autumn honey (pH > 3.95), followed by summer honey (pH < 3.95) and finally spring (pH < 3.84). On the other hand, the total acidity was not statistically different for the three seasons, meaning that the organic


**Table 4.** Mean pH and mean acidity (mM/kg) values for honey samples from the three seasons.

acid content did not seem to differ of these three seasons. pH mirrors the moisture content of the seasonal honeys. This may reflect the mobility of more free protons (H<sup>+</sup> ) with a higher moisture content.

#### **3.5. Determination of electrical conductivity**

The electrical conductivity of honey is measured at 20°C using a 20% solution of honey on dry weight basis. Conductivity is measured in mS/cm or μS/cm, reflecting the presence of ionizable substances, such as minerals [19], typically not exceeding 800 μS/cm. **Table 5** shows the mean conductivity values for honey samples from the three seasons throughout the project period. It was observed that there is slight yearly variation between mean pH values for the separate seasons. However, seasonal variations were significant. Autumn honey has the highest and a significantly different conductivity of all three seasons (ECautumn>963.6 μS/cm compared to the other two seasons (<752.5 and <767.1 μS/cm for summer and spring, respectively). The high salt content for autumn honeys may occur due to the arid summer conditions that result in the salting out of minerals during this period (summer). When precipitation commences in autumn, the high salt content is dissolved leading to a higher uptake in plants, and the accumulation of salt in the nectar. The salt accumulation on autumn plants following a dry period was observed in other studies under local conditions [20].

#### **3.6. The determination of HMF after White**

5-Hydroxymethylfurfural (5-HMF) is an aldehyde, which can be used as an indicator of honey quality deterioration. 5-HMF forms through the Maillard reaction, a complex series of reactions between amino acids and reducing sugars (hexoses). The International Honey Commission [7] recommends three methods for the determination of HMF. The method described by White [8] involves the measurement of UV absorbance of clarified aqueous honey solutions with and without bisulphite. An HPLC method is also described in the IHC harmonized methods [7].

The Codex Alimentarius [21] established that processed or blended honey should not contain HMF levels higher than 80 mg/kg. The European Union [6] adopted the same upper limit for honey coming from Countries or Regions with tropical temperatures. In most cases, an upper limit of 40 mg/kg is applicable in EU member states.

**Table 5** shows the mean HMF values for honey samples from the three seasons throughout the project period. HMF was exceptionally higher in autumn samples as opposed to summer and


**Table 5.** The mean conductivity (μS/cm), mean HMF (mg/kg), mean diastase (Schade units) and mean proline (g/kg) values for honey samples from the three seasons.

spring honeys (*p*< 0.05). It was observed that 2012 honeys from all three seasons exhibited higher HMF content with respect to other years. It was expected that summer honey may contain more HMF. However, with a higher brix level and lower water content, the HMF production is favoured. Honey samples turn darker (browner) in colour due to the accumulation of HMF.

#### **3.7. The determination of diastase activity**

acid content did not seem to differ of these three seasons. pH mirrors the moisture content

**2011 2012 2013 2014**

Spring\*\* 3.77 ± 0.03 32.55 ± 1.39 3.75 ± 0.02 34.12 ± 1.38 3.73 ± 0.08 41.13 ± 5.39 3.84 ± 0.03 33.29 ± 1.16 Summer 3.84 ± 0.03 34.74 ± 1.16 3.87 ± 0.06 45.52 ± 2.82 3.73 ± 0.11 40.04 ± 4.12 3.95 ± 0.02 29.71 ± 1.53 Autumn\*\*\* 4.01 ± 0.04 29.48 ± 2.77 4.04 ± 0.07 43.53 ± 4.73 3.95 ± 0.03 40.85 ± 2.76 3.98 ± 0.09 30.68 ± 3.02

pH Acidity pH Acidity pH Acidity pH Acidity

The electrical conductivity of honey is measured at 20°C using a 20% solution of honey on dry weight basis. Conductivity is measured in mS/cm or μS/cm, reflecting the presence of ionizable substances, such as minerals [19], typically not exceeding 800 μS/cm. **Table 5** shows the mean conductivity values for honey samples from the three seasons throughout the project period. It was observed that there is slight yearly variation between mean pH values for the separate seasons. However, seasonal variations were significant. Autumn honey has the highest and a significantly different conductivity of all three seasons (ECautumn>963.6 μS/cm compared to the other two seasons (<752.5 and <767.1 μS/cm for summer and spring, respectively). The high salt content for autumn honeys may occur due to the arid summer conditions that result in the salting out of minerals during this period (summer). When precipitation commences in autumn, the high salt content is dissolved leading to a higher uptake in plants, and the accumulation of salt in the nectar. The salt accumulation on autumn plants

) with a higher

of the seasonal honeys. This may reflect the mobility of more free protons (H<sup>+</sup>

**Table 4.** Mean pH and mean acidity (mM/kg) values for honey samples from the three seasons.

following a dry period was observed in other studies under local conditions [20].

5-Hydroxymethylfurfural (5-HMF) is an aldehyde, which can be used as an indicator of honey quality deterioration. 5-HMF forms through the Maillard reaction, a complex series of reactions between amino acids and reducing sugars (hexoses). The International Honey Commission [7] recommends three methods for the determination of HMF. The method described by White [8] involves the measurement of UV absorbance of clarified aqueous honey solutions with and without bisulphite. An HPLC method is also described in the IHC harmonized methods [7].

The Codex Alimentarius [21] established that processed or blended honey should not contain HMF levels higher than 80 mg/kg. The European Union [6] adopted the same upper limit for honey coming from Countries or Regions with tropical temperatures. In most cases, an upper

**Table 5** shows the mean HMF values for honey samples from the three seasons throughout the project period. HMF was exceptionally higher in autumn samples as opposed to summer and

moisture content.

\*\*\*p > 0.001 for pH values.

\*\*p < 0.01.

180 Honey Analysis

**3.5. Determination of electrical conductivity**

**3.6. The determination of HMF after White**

limit of 40 mg/kg is applicable in EU member states.

Diastase, also referred to as any α-, β- or γ-amylase, can break down carbohydrates. Hence, diastase is the enzyme that converts the long chain starch to dextrins and sugars. This enzyme is produced by the bees and introduced into honey by the bees themselves. Diastase is used an indication of adulteration as honey that is harvested from hives which are feed sucrose to produce high volumes will have a diastase content which is low.

The α-amylase (alternative names: 1,4-α-D-glucan glucanohydrolase; glycogenase) is a calcium metalloenzyme, completely unable to function in the absence of calcium. As opposed to HMF, diastase activity decreases with time. However, this is another quality parameter where the degradation of honey enzymes indicates a decline in the functionality of the honey as a food supplement and also as a medicine.

**Table 5** shows the mean diastase values for honey samples from the three seasons throughout the project period. Diastase was exceptionally lower in autumn samples (<8.70 Schade units) as opposed to spring honeys (>9.10 Schade units, *p*< 0.01). However, for 2011, the spring diastase level was low compared to the other years. The summer samples showed a varied diastase level, with the lowest values obtained during 2013 (2.98 Schade units) and highest values obtained during 2012 (10.89 Schade units).

Possible heating of honey to skim waxes should be avoided. Unfortunately this is a common local practice amongst beekeepers as the Maltese consumer prefers liquefied honey. It seems that enzymatic activity is more sensitive to heat than HMF and perhaps diastase activity may be considered as a more significant indicator of quality than HMF. However, diastase degradation seems to have less implications on human health than HMF accumulation.
