Introduction

Honey is a natural sweetener with health-promoting properties. Two of the main species  of honeybees include Apis mellifera and Apis dorsata. A. dorsata is larger in size than A.  mellifera, with a wingspan of up to 6 cm and 4.5cm in A. dorsata and A. mellifera, respectively. A. dorsata also has distinctive yellow and black stripes on its abdomen,  while A. mellifera can have various patterns and colours on its abdomen. Honeybees  collect nectar from plants, break down the nectar into simpler sugar using enzymes, and deposit the nectar into honeycomb cells. Honeybees then fan their wings to evaporate water content in nectar and seal the honeycomb cell with wax capping. The produced  honey is packed with antimicrobial capabilities to help relieve sore throats and promote  wound healing. Packed with antioxidants, it fights free radicals and improves general health. Moreover, it encourages healthier eating choices than processed sugar as  processed sugar lacks significant nutritional value unlike honey that contains trace amounts of vitamins, minerals and antioxidant. Additionally, honey’s lower glycemic index  makes it a more effective alternative for those with diabetes or those aiming to regulate  their blood level. Honey also contains flavonoids and phenolic acids, leading it to contain  anti-inflammatory and antioxidant properties which are absent in processed sugar. Overall, honey's nutritional, therapeutic, and ecological benefits confirm its significance  on a global scale.

Bioactive properties of honey are greatly affected depending on factors such as the  honey's floral source, place of origin, processing techniques, and storage conditions  (Anjum et al., 2019). Its antioxidant activity and other health-promoting qualities are  mostly dependent on the presence of bioactive components like flavonoids and phenolic compounds, which are greatly influenced by the floral source (Khalil et al., 2019).  Furthermore, due to the differences in climate, soil, and vegetation, the geographic area  where honey is produced has a significant impact on determining its composition and  bioactivity (Anjum et al., 2019). Thus, knowing these elements are crucial to preserve and  optimize honey's bioactive potential for a range of uses. As the maturity of honey plays a  vital role in determining its antioxidant property, both mature (capped) and immature  (uncapped) honey produced by Apis dorsata were analyzed for their antioxidant  capability through 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. Additionally, due to the  increasing market needs to obtain processed sugar substitute with beneficial properties,  attempts to convert honey into a powder form was conducted in this experiment and its antioxidant capability was further analyzed.

Materials

• Apis dorsata and Apis mellifera honey were purchased from a local bee farm. Mature (capped) honey and immature (uncapped) honey were harvested from the beehive in 1.5 to 2 months and 3 weeks, respectively.

• 2,2-diphenyl-1-picrylhydrazyl (DPPH) was purchased from PT Smart Lab Indonesia.  Methanol and ascorbic acid were purchased from Merck. 

• Food-grade arabic gum was purchased from a local market.

Creating Honey Powder using Arabic Gum

Honey powder was produced with the help of arabic gum. Firstly, 6.4 grams of arabic  gum was dissolved in 10 mL distilled water and mixed thoroughly. Arabic gum solution was then added to 10 grams of honey and further mixed. This gave a total of 60% honey  concentration in the concoction with 40% of arabic gum concentration. Arabic gum-honey  solution was then poured into petri dishes and frozen overnight before further lyophilized  for 1-2 days.

Antioxidant Activity through 2,2dipheyl-1-picrylhydrazyl (DPPH) method

The antioxidant properties of honey samples were determined by the 2,2-diphenyl-1 picrylhydrazyl (DPPH) assay as described by Isla et al. (2011) with modifications. DPPH  stock solution of 0.2 mM was prepared in 70% methanol solution. All honey samples  were prepared at varying concentrations, ranging from 100-40,000 mg/L, and were  individually added with 10 mL DPPH stock solution to obtain a final DPPH concentration  of 0.04 mM. The absorbance was then measured at 517 nm after 30 minutes of  incubation in a dark room at 25°C. Ascorbic acid was used as positive control. Honey's  capability to scavenge DPPH free radicals was measured as follows:

Radical Scavenging Activity (%) = (Acontrol - Asample) / Acontrol x 100%.

where:

• Acontol : the absorbance of control

• Asample : the absorbance of sample

Result and Analysis

Result can be seen on the image section.

Analysis:

Two mature honeys produced by Apis mellifera and Apis dorsata displayed differing  antioxidant capability with A. mellifera honey displaying better IC50 value at 4,630 mg/L  than A. dorsata honey at double the IC50 value at 8,755 mg/L, suggesting twice as  weak the antioxidant capability than A. mellifera honey. The significantly different IC50  values may have been attributed by the different bee involved during the honey  production. Nevertheless, both mature honey successfully displayed high IC50 values.  Upon comparing between mature and immature A. dorsata honey, uncapped (immature)  A. dorsata honey displayed the weakest IC50 value when compared with the previously  analyzed honey at 58,312 mg/L, 6.6 times weaker from its capped (mature) form. This  result then suggests the importance of honey maturity and ripening prior to harvesting  process to further improve the quality of the harvested honey. Flavonoids and phenolic  acids are two examples of the chemicals that are changed into antioxidants by the  honey's enzymes during the ripening process of honey (Nayaka, 2020.). Since the  enzymatic process has not yet taken place due to the shorter period of honey storage,  immature honey lacks these antioxidant components, hence the weak IC50 value.  

Looking further into the lyophilized form of both A. mellifera and A. dorsata honey, both  honey successfully retained their antioxidant capabilities as they displayed IC50 values  of 6,322 mg/L and 16,068 mg/L, respectively. These antioxidant capabilities are not  directly linear with the amount of honey being diluted in the presence of arabic gum as  A. mellifera honey powder displayed stronger antioxidant capability than the theoretical  antioxidant activity of A. mellifera honey powder in the presence of arabic gum.  Contrastingly, this was not the case with A. dorsata honey powder as it showed slightly  weaker antioxidant capability than the theoretical antioxidant activity of A. dorsata  powder in the presence of arabic gum. The stronger antioxidant activity in A. mellifera  honey powder was mainly due to the antioxidant power of arabic gum as its  encapsulating agent. The antioxidant capacity of arabic gum is due to its higher content of flavonoids (Noorsabrina et al. 2021). Moreover, the antioxidant properties of arabic  gum may be attributed to its hydroxyl group, polypeptide, and its highly branched  structure which may improve the stability and antioxidant properties of honey (Kong et  al. 2014). On the other hand, the weaker antioxidant capability in A. dorsata honey  powder may have been due to the greater amount of moisture in its native honey than  A. mellifera honey. Should both honey showed linearity with the native honey,  lyophilized A. mellifera honey would have shown a total of 7717 mg/L, while lyophilized  A. dorsata would have shown 14,592 mg/L.

Conclusion and Future Work

• Mature honey produced by Apis mellifera showed almost twice as high the  antioxidant capability of mature honey produced by Apis dorsata.  

• Uncapped (immature) Apis dorsata honey displayed a very weak IC50 value of  58,312 mg/L, suggesting a 6.6 times weaker antioxidant capability than its capped  (mature) form.

• Lyophilization process of both mature honey by Apis mellifera and Apis dorsata  successfully produced honey powder with retained antioxidant capabilities.  

It is then imminent that factors such as the type of honeybee involved and the harvesting  period of honey played a significant role in determining the final bioactive property of  honey such as its antioxidant capability. Furthermore, this experiment suggests the  possibility of turning a thick, dense honey to a easy-to-carry honey powder with the aid  of an encapsulating agent such as arabic gum. As a future outlook of this study,  attempts to understand and replicate the fabrication process of bioactive properties in  honey should be performed. Additionally, other bioactive properties of both honey and  its powder form such as anti-inflammatory capability should be analyzed.