**3.8. The determination of proline**

Honey is very low in protein. As a matter of fact it contains less than 1%. The protein portion is mainly made up of several amino acids. A chemical marker that represents proteins, i.e. amino acids, is proline. Proline is not an amino acid as there is no free amine group in its structure. Although proline content is not considered as one of the main indicators of honey quality, legal issues can be resolved by taking into consideration this parameter. A honey that contains less than 180 mg of proline per kilo of honey is an altered honey [7].

**Table 5** shows that all mean proline values for honey from the three seasons (>240 mg/kg) were well above the 180 mg of proline per kilo of honey standard, except for the summer 2013 honeys (160 mg/kg). The years 2012 and 2014, showed a very high content of proline throughout the three seasons. Compared to the other two seasons, autumn 2012 and 2014 showed exceptionally high proline content. Combining all the 4 years for the three seasons, the highest proline content was observed for spring. However, the difference in proline contents was not significant with the other two seasons.

#### **3.9. Determination of sugar content**

Honey is made up of a matrix of sugars. Although the brix content provides a good indication of the content of sugars, the individual sugars are not identified by this method. Honey sugars are formed by the action of several honey bee enzymes on the floral nectar. The result is a complex mixture composed of 70% of monosaccharides and 10–15% disaccharides. Honey is also used as a sweetener in hundreds of products manufactured [22]. The oligosaccharide content of honey contributes to its prebiotic properties, promoting the growth of Bifidobacteria and Lactobacilli [23]. Many scientists attempted to characterize the sugars in many honey types [24–26]. Different techniques such as HPLC [27, 28] or GC-MS [29] were used. These methods have been standardized by the "International Honey Commission" [7]. The HPAEC-PAD (high-performance anion-exchange chromatography with pulsed amperometric detection) is one of the most used techniques for the analysis oligosaccharides [30].

In some studies [31, 32], the disaccharide content was used to characterize the type and origin of the honey. Moreover, maltose, turanose and nigerose were useful for differentiating Brazilian honey in several other geographic areas [33]. The sugar profiles are also used to


<sup>\*\*\*</sup>p < 0.001.

**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

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 degra-

Honey is very low in protein. As a matter of fact it contains less than 1%. The protein portion is mainly made up of several amino acids. A chemical marker that represents proteins, i.e. amino acids, is proline. Proline is not an amino acid as there is no free amine group in its structure. Although proline content is not considered as one of the main indicators of honey quality, legal issues can be resolved by taking into consideration this parameter. A honey that

**Table 5** shows that all mean proline values for honey from the three seasons (>240 mg/kg) were well above the 180 mg of proline per kilo of honey standard, except for the summer 2013 honeys (160 mg/kg). The years 2012 and 2014, showed a very high content of proline throughout the three seasons. Compared to the other two seasons, autumn 2012 and 2014 showed exceptionally high proline content. Combining all the 4 years for the three seasons, the highest proline content was observed for spring. However, the difference in proline contents was not

Honey is made up of a matrix of sugars. Although the brix content provides a good indication of the content of sugars, the individual sugars are not identified by this method. Honey sugars are formed by the action of several honey bee enzymes on the floral nectar. The result is a complex mixture composed of 70% of monosaccharides and 10–15% disaccharides. Honey is also used as a sweetener in hundreds of products manufactured [22]. The oligosaccharide content of honey contributes to its prebiotic properties, promoting the growth of Bifidobacteria and Lactobacilli [23]. Many scientists attempted to characterize the sugars in many honey types [24–26]. Different techniques such as HPLC [27, 28] or GC-MS [29] were used. These methods have been standardized by the "International Honey Commission" [7]. The HPAEC-PAD (high-performance anion-exchange chromatography with pulsed amperometric detection) is

In some studies [31, 32], the disaccharide content was used to characterize the type and origin of the honey. Moreover, maltose, turanose and nigerose were useful for differentiating Brazilian honey in several other geographic areas [33]. The sugar profiles are also used to

dation seems to have less implications on human health than HMF accumulation.

contains less than 180 mg of proline per kilo of honey is an altered honey [7].

one of the most used techniques for the analysis oligosaccharides [30].

values obtained during 2012 (10.89 Schade units).

**3.8. The determination of proline**

182 Honey Analysis

significant with the other two seasons.

**3.9. Determination of sugar content**

**Table 6.** Mean percentage sugar values for honey samples from the three seasons throughout the project period.

differentiate honeydew honey from flowers. Indeed, the honeydew presents lower concentrations of glucose and fructose and higher oligosaccharides including melezitose or erlose [34]. It is worth noting that the concentrations of fructose and glucose are used to classify floral honey [7]. Two disaccharides of importance are sucrose and maltose. According to Council Directive [6], the content should not exceed 5% of the total sugars.

**Table 6** provides the mean percentage sugar values for honey samples from the three seasons throughout the 4-year period. **Figure 5** shows a typical chromatogram for the Maltese honey samples. The most abundant sugar is fructose followed by glucose. The disaccharides, sucrose and maltose, are found in lower concentrations. Maltose is more consistent than sucrose. Melezitose is only present in autumn honey. The content of glucose and fructose for the three seasons, as shown in **Table 6**, is well above the content stated in Ref. [5], which is a minimum of 60 g/100 g. For the three honey types the amount exceeded 84 g/100 g.

Fructose is significantly high in autumn honey (48.64%) and least in spring honey (44.07%). Glucose is relatively similar in all seasonal honey types ranging from 39.89% for summer honey to 41.90% for autumn honey. Sucrose varies significantly between the three seasons; in spring honey being the highest (11.69%) and in autumn honey being the lowest (5.41%). This goes in accordance with the brix results, which partially concluded the possible feeding of a sucrose syrup to bees during winter, when flora is scarce, and the possible incorporation of this 'syrup honey' within the spring honey. Therefore, beekeepers should be advised that the honey produced from the artificial syrup should be discarded prior to the commencement of honey production in spring. Melezitose (**Table 6**) was only present in autumn honey. However, not all autumn honey samples contained this saccharide. This sugar is typical of honeydew honey. Honeydew is a sugar-rich sticky liquid,

**Figure 5.** A typical chromatogram for honey samples.

produced mainly by aphids and some scale insects as they feed on plant sap. According to Mifsud et al. [35], aphids are present on carob and citrus during autumn, particularly *Aphis* (Aphis) *gossypii* on carob, *Aphis* (Aphis) *craccivora* and *Toxopteraaurantii* on citrus. This may explain the presence of honeydew honey within the floral honey during autumn. Honeydew honey may be produced during summer and autumn [36]. However, due to limitations in tree numbers, this cannot be produced on a large scale in Malta. Therefore, a possible indicator of pure autumn honey may be melezitose, although this needs to be further investigated.

#### **3.10. Polyphenolic content**

The mean total polyphenolic content in mg TAE/100 g honey for spring (56.943 ± 7.027) was significantly lower (*p*< 0.05) than for the other two seasons (AU: 79.692 ± 8.000 and SU: 69.598 ± 3.208 mg TAE/100 g honey) (**Table 7**). It was observed that the darker the honey colour, the higher was the total polyphenolic content. This was the case with autumn honey samples [12].
