Antioxidant and antimicrobial properties of Indo-Malayan stingless bee (Heterotrigona itama) honey from different seasons and distribution of flowers

Mahmood, A.L., Lani, M.N., Hassan, Z., Razak, S.B.A. and Ahmad, F.T. Faculty of Fisheries and Food Science, Universiti Malaysia Terengganu, 21030, Kuala Nerus, Terengganu, Malaysia SIG Apis and Meliponine, Universiti Malaysia Terengganu, Terengganu,21030, Kuala Nerus, Terengganu, Malaysia Insititute of Marine Biotechnology, Universiti Malaysia Terengganu, 21030, Kuala Nerus, Terengganu, Malaysia Faculty of Science and Technology, Universiti Sains Islam Malaysia, Bandar Baru Nilai, 71800, Nilai, Negeri Sembilan


Introduction
Throughout history, honey has always been described as a natural product with great importance for human health. It has good culinary qualities and also contains remarkable nutritional and therapeutic properties (Siddiqui et al., 2016;Marfo et al., 2016;Biluca et al., 2020;Keng et al., 2017). The chemical and nutritional properties of stingless bee honey differ significantly from honey produced by Apis mellifera (Duarte et al., 2012;Menezes et al., 2013). Honey produced by stingless bees has a lower viscosity, darker colour and stronger acid taste compared to honey from Apis spp. (Garedew et al., 2003). The composition and properties of honey are strongly affected by the bee species, edaphoclimatic conditions and pollen source (Gheldof and Engeseth, 2002). Ávila et al. (2019) has concluded stingless bee honey can be clustered according to the source of pollen. Hence, designing the source of pollen has become a new method in stingless beekeeping to produce stingless bee honey with different properties. One of the potential pollen sources may come from the stevia plant (Stevia rebaudiana), a non-caloric natural sweetener (Kąkol et al., 2012). Our observations indicated that stingless bees foraged on the pollens of  -Arab et al., 2010;Halim et al., 2019). The effects of pollen from stevia plants on the properties of honey are still unclear and need to be explored. It may be possible that such honey may also contain these compounds which need to be elucidated. A study by Razak et al. (2019) has also found the stevioside in stingless bee honey collected from plant area with stevia.
Seasonal variation has been indicated as one of the major factors that affect the properties of honey. Nascimento and Nascimento (2012) and Oyerinde et al. (2014) showed that the rainy season not only negatively affect honey yields but also may affect the properties of the honey itself. As the Malaysian state of Terengganu is well-known for its tropical monsoon climate, which consistently occurs from November until March (Sang et al., 2016), information on the effect of this climate on the quality of the harvested honey is much needed.
Honey from a stingless bee was reported to have antimicrobial and antioxidant properties (Ahmad et al., 2019;Biluca et al. 2020) and is suggested as an alternative medicine to help in wound healing (Al-Mamary et al., 2002;Mulu et al., 2004). Previous studies have shown that the antibacterial and antifungal properties of stingless bee honey are contributed by the low pH, osmotic pressure (Mandal and Mandal, 2011;Zainol et al., 2013;Garedew et al. 2003;Hasali et al., 2015;Ahmad et al. 2019), total phenolic and flavonoid content present in the honey (Khalil et al., 2011;Iurlina et al., 2019). Moreover, Hasali et al. (2015) stated that the antibacterial and antifungal properties of stingless bee honey have originated from the presence of lactic acid bacteria (LAB) strains.
Furthermore, the pollen source has also been indicated to influence the antioxidant properties of honey (Moniruzzaman et al., 2013). Thus, apart from studying the antioxidant properties of stingless bee honey, the present research is also intended to study the antimicrobial properties of multifloral honey produced by stingless bees that gathered pollen in areas with and without stevia plants.

Stingless bee honey samples
Fresh honey from stingless bee (Heterotrigona itama) with different pollen sources was collected from two different sites (with and without stevia) at Pusat Tunas Stevia (PTS) plant nursery, Besut, Terengganu during the dry (April to July 2017) and rainy (September to November 2017) seasons. Sterile pipettes and glass bottles were used for the honey sampling process. Extracted honey samples were placed in sterile, enclosed sterilized opaque glass containers wrapped with aluminium. Samples were then stored at 4 o C until further analysis.

Dry and rainy season identification
Sample collection of stingless bee honey involved two phases, which were the dry and rainy seasons. Hygrometers were placed at both sampling sites few days before sample collection according to the method of de Figueiredo-Mecca et al. (2013), Nascimento et al. (2012) and Puškadija et al. (2007). Relative humidity and temperature were recorded at 1-hour interval for 3 hrs. The data collected was compared with data from the Terengganu Meteorological Department, Malaysia to determine the season.

Pollen identification
The main objective of pollen identification is to determine the pollen distribution of stingless bee (Heterotrigona itama) honey samples. The multifloral source of stingless bee honey was analysed using the pollen identification method by Marcos et al. (2015) with some modifications. In this method, five stingless bees with pollen on their hind tibia were captured per area for 6 days and then kept in separate glass jars. Pollens were collected from the stingless bees' hind tibia using a needle and then suspended in 1 mL of distilled water. One drop of suspended pollen was been placed onto a glass slide and covered with a coverslip. The pollens were observed under 100× magnification using a light microscope. Photos of the pollens were taken and used for visual identification.

Total phenolic content (TPC)
The total phenol (TPC) content test was determined using a modified version of Folin-Ciocalteu's phenol reagent (Singleton et al., 1999). Samples of 1 gram of stingless bee honey were weighed in test tubes wrapped with aluminium foil and were diluted with 10 mL of distilled water. One millilitre of diluted honey samples was transferred using a micropipette and mixed with 5 mL of 10% (v/v) Folin-Ciocalteu's reagent and incubated for 5 mins at room temperature (25±2°C). After 5 mins, 4 mL of 75% w/v aqueous sodium carbonate solution was added and the mixture was further incubated for 2 hrs. The absorbances of the samples were then measured at 765 nm using a UV-Vis (double beam) spectrophotometer (Shimadzu UV-1700 PharmaSpec, Japan). The TPC of each sample was reported as the mean value of triplicate assays and expressed in mg gallic acid equivalents (GAE)/kg of eISSN: 2550-2166 © 2021 The Authors. Published by Rynnye Lyan Resources FULL PAPER honey sample.

Total carotenoid content
The total carotenoid content of the honey samples was determined as previously reported by Ferreira et al. (2009) using ß-carotene as the standard (Boussaid et al., 2014). The samples were mixed with an acetone-hexane mixture (6:4) and shaken vigorously at room temperature. The mixture was measured at 450 nm using a UV-Vis (single beam) spectrophotometer (Spectroquant Pharo 300, USA).

Free radical scavenging ability
The free radical scavenging activity of the honey samples was determined using the 1,1-diphenyl-2picrylhydrazyl (DPPH) assay (Boussaid et al. 2014). Samples were added with the DPPH and then incubated for 60 min at room temp (25±2 o C). The absorbance of the samples and control were measured at 517 nm with a UV-Vis spectrophotometer (Shimadzu UV-1700 PharmaSpec, Japan). The radical inhibition measurements were expressed as a percentage (%) of DPPH inhibition (Boussaid et al., 2014).

Ferric reducing antioxidant power (FRAP)
The reducing power of the honey samples were determined based on the method described by Benzie and Strain (1996). The FRAP reagent was prepared by mixing 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) solution with 300 mM acetate buffer (pH 3.6) and 20 mM iron (III) chloride hexahydrate. An aliquot of 200 μL of honey solution was mixed with 1.5 mL of FRAP reagent, and incubated at 37°C in a water bath for 4 mins. The absorbance of the mixture was then measured at 593 nm (distilled water as blank) using a UV-Vis spectrophotometer and compared with ferrous sulfate (FeSO 4 ) as the standard.

Antimicrobial activity using well-diffusion method
The effects of different pollen sources and seasons towards the antimicrobial activity of stingless bee honey (Heterotrigona itama) were determined using the welldiffusion method on Mueller Hilton agar against Escherichia coli (ATCC 25922), Listeria monocytogenes (ATCC 13932)., Salmonella enterica serovar Typhimurium (ATCC 13311) and Staphylococcus aureus (ATCC 6538). Suspensions of 24 hrs cultures of food-borne pathogenic bacteria were prepared using saline water and the turbidity was ensured to be equivalent with 0.5 McFarland. Suspensions (100 µL) made were spread onto Mueller Hilton agar plates and then, four wells were made on the agar plates using the back of sterile tips (Ewnetu et al., 2013). Then, 100 µL of each sample was added to the wells. All of the plates were then incubated at 37 o C for 24 hrs in an incubator and the diameter of bacterial growth was inhibition was measured.

Statistical Analysis
All analyses were carried out in triplicate for each sample and the experimental results were expressed in mean value ± standard deviation. Significant analysis of variance (p < 0.05) was performed using the Minitab statistical software (version 14).

Pollen identification from hind tibia
Pollens collected from stingless bee-hind tibia were compared with the reference flowers under 100X magnification using a light microscope (Table 1) for identification. The data obtained from pollen identification is important to determine the differences between the multifloral honey samples. The data obtained clearly showed that the type of pollen collected depends on the species of flowering plants surrounding the nest.

Pollen distribution analysis
The distribution of pollens foraged by stingless bees varies according to the available flower source and preference by the stingless bee itself. This preferential behaviour is normally done by the scouts of stingless bees which will identify high-quality pollen before signalling to the foraging stingless bees to forage for the pollen (Real, 2012). Stingless bees identify the types of flower based on the colour of pollen and also scent (Harder et al., 2004). Previous studies (Steven et al., 2003;Harder et al., 2004) discovered that flower characteristics such as the colour of petals colour, the colour of pollen and the size of petals influence the foraging behaviour of stingless bees. The pollen distribution analysis in the present study showed different types of pollens foraged by stingless bees at nests in both areas (A and B; Figure 1). Only two plant species were identified from the pollen collected from stingless bees in area A. The majority of the pollen was from the flowers of Antigonon leptopus (53%) while the remainder was from S. rebaudiana (47%). A leptopus is a flowering plant that grows with a vine structure. Stingless bees are attracted to A. leptopus which have smaller and more attractive pollen and petal colour compared to the white flowers produced by S. rebaudiana. Meanwhile, there were more (four) plant species identified from the pollen on stingless bees in the area B, which were A. leptopus, Biden pilosa, Caudatus sulphureus and Orthosiphon stamineus. A. leptopus FULL PAPER

Effect of environmental conditions and pollen distribution on antioxidant and antimicrobial properties of stingless bee honey
These analyses were done to study the relationship between environmental conditions and pollen distribution on the antioxidant and antimicrobial properties of stingless bee honey. The environmental conditions of the selected area were determined based on the temperature and relative humidity. The highest recorded relative humidity (%) was at 84.30% during the rainy season while the lowest was at 51.67 % during the dry season ( Figure 2) and the temperature was in the range of 28 -31°C and 21 -22°C for dry and rainy seasons, respectively (Figure 3). The increase in the percentage of relative humidity was because of the rainfall. The relative humidity during the rainy season was within the range as reported by the Malaysian Meteorological Department which ranged from 72% to 90% (Malaysian Meteorological Department, 2009).
From the results obtained, there were significant differences (p < 0.05) in the antioxidant of stingless bee honey obtained during the dry and rainy season from different flower sources (Table 2). The total phenolic content (TPC), DPPH and FRAP scavenging activity (%) in multifloral stingless bee honey from area B were higher compared to the honey collected in area A in both seasons (p < 0.05), where the highest was during the dry season. Compounds that may have contributed towards the antioxidant activity were phenols, catalase, glucose oxidase, phenolic acids, ascorbic acids, flavonoids, carotenoids derivatives, organic acids, Maillard reaction products, and amino acids (Alzahrani et al., 2012). The lower antioxidant activity in honey samples during the rainy season may be due to the negative effect of the season on foraging activity and pollen viability (Puškadija et al., 2007). In rainy seasons, strong winds reaching 10 to 30 knots (Abdullahi et al., 2014) can cause pollens to be blown away while also disturbing the foraging activity of stingless bees (Puškadija et al., 2007). During the rainy seasons, when the food source is insufficient, stingless bees will forage pollen in the range of only 20 meters around the nest (Samsudin, 2016) and some of the foragers will just remain in the hive until the rains stop (Jaapar et al. 2018). During the dry season, a large forager bee can forage up to 2100 meters (Kuhn-Neto et al., 2009). Besides, the presence of water vapour during the rainy season also gives a negative effect on the pollen and the quality of the stingless bee honey. The effects of relative humidity and temperature on the distribution of pollen was clearly explained by Aronne (1999) and subsequently affected the bee foraging activity. Puškadija et al. (2007) observed the most intensive bee activity occurred at a humidity of 40 to 50% while a higher relative humidity (>70%) reduced the bee scavenging activity down to 0%. These suggest that the rainy season reduce the antioxidant properties and the yield of the honey. Observation of stingless bee honey from both seasons also showed differences in the volume of yield and intensity of colour. Honey collected during the dry season was darker and of higher yield compared to those from the rainy season.
Other than the season, the high antioxidant properties of multifloral stingless bee honey samples from area B were clearly due to the higher variety, quantity and quality of flowers surrounding the nest. The plant species identified from the pollen on stingless bees in the area without stevia plants (Figure 1, Area B) may have contributed to the high antioxidant activity in the honey. Previous studies reported that alkaloids, flavonoids, tannins, terpenoids, saponins and phlobatanin in A. leptopus (Govindappa, 2015;Pradhan and Bhatnagar, 2016) are well-known to have potential medicinal and therapeutic properties (Kennedy and   Table 2 also shows a similar trend where multifloral stingless bee honey samples from area B that were collected during the dry season showed the highest total carotenoid content (0.25 µg/mL) compared to other samples. The difference in total carotenoid content in honey is strongly influenced by the flower sources. This can be seen in a study by Boussaid et al. (2014) who showed that honey from citrus flowers having had the highest total carotenoid content (4.72 mg/kg) while the lowest content (1.16 mg/kg) was found in honey from rosemary flower. Meanwhile, a previous study by Jimenez et al. (2016) on Scaptotrigona mexicana honey showed a very low total carotenoid content of 0.56 mg/ kg.
The antimicrobial activities of multifloral stingless bee honey from both areas against four pathogenic bacteria are shown in Table 3 for both seasons. As can be seen, there were significant differences (p < 0.05) in the antimicrobial activities of the honey samples from the dry and rainy seasons. Multifloral stingless bee honey samples from both areas during the dry season showed higher inhibition compared to the rainy season. Positive inhibition results against L. monocytogenes can be seen in the result and again, the larger inhibition was shown by honey from the dry season. Amongst the four pathogenic bacteria, the largest inhibition zone was shown against S. enterica ser. Typhimurium while the lowest was E. coli. The relatively small inhibition zone showed that E. coli was able to withstand the low pH and other non-peroxide antimicrobial properties of the multifloral stingless bee honey. A previous study by Ulusoy et al. (2010) showed a similar inhibitory trend where honey samples bees honey from different flower sources have smaller inhibition zones against E. coli compared to other pathogenic bacteria. However, the present study found that the multifloral stingless bee honey from area B had a larger inhibition zone against E. coli at 11.33 mm compared to honey samples from a previous study by Ulusoy et al. (2010) where the inhibition was only at 6 mm and 8 mm for honey from monofloral (lime flower) and multifloral (rhododendron, chestnut and lime), respectively.
Multifloral stingless bee honey samples from both areas during the dry and rainy seasons were able to inhibit S. aureus higher than those collected during the rainy season. A study by Ulusoy et al. (2010) showed no inhibition of honey from the lime flower, rhododendron and chestnut against S. aureus. This shows that stingless bee honey has a stronger antimicrobial activity compared to honey produced by Apis spp. In another study by Zainol et al. (2013), the inhibition of Gelam and Tualang honey against the S. aureus were 15.52 and 16.34 mm respectively, which were smaller in comparison to the result in the present study (19.33 mm). Table 3 also shows positive inhibition result against L. monocytogenes and again, the larger inhibition was shown by honey from the dry season. Amongst the four pathogenic bacteria, the largest inhibition zone was shown against S. enterica ser. Typhimurium. The large inhibition zone of stingless bee honey against S. enterica ser. Typhimurium may be due to the optimum pH growth of S. enterica ser. Typhimurium being pH 7-7.5 (Podolak et al., 2010) which was not compatible with the much lower pH of the stingless bee honey.
Overall, this study showed a trend where the higher antioxidant values in honey exhibited the larger antimicrobial inhibition zones, and this observation is in agreement with a previous study by Ulusoy et al. (2010). Multifloral stingless bee honey samples from both areas during the rainy season showed a lower inhibition zone  PAPER against all of the tested bacteria compared to honey samples collected during the dry season. As previously discussed, the rainy season reduced the foraging activity of stingless bees and increased environmental humidity which negatively impacted the antimicrobial activity of stingless bee honey. The increase in humidity will also influence the pH of stingless bee honey produced in the nest. Bacteria are sensitive to the hydrogen ion concentration in their environment thus, pH plays an important role in the inhibition of bacteria. Other compounds that contribute towards the antimicrobial activity of stingless bee honey are phytochemical factors such as phenolic compounds, flavonoids, antibacterial peptides, methylglyoxal, methyl syringate, antibiotic-like derivatives and other components present in trace amounts which are classified as non-peroxide antibacterial factors (Mandal and Mandal, 2011). Plants produce a great number of secondary metabolites that have an antimicrobial activity which also contributes to the antimicrobial properties of honey (Cos et al., 2006) and as discussed, all of the identified plants in the study have this property. Although the multifloral honey sample from area A which planted with stevia plants showed lower antioxidant and antimicrobial activities, data from our preliminary study tested on all of the samples (unpublished data) indicated the presence of beneficial sweeteners from stevia plants (stevioside and rebaudioside A) in the honey which could potentially give additional value to the honey. A similar result was also shown by a study on stingless bees honey as reported by Razak et al. (2019).

Conclusion
In conclusion, different flower sources and seasons influenced the antioxidant and antimicrobial properties of multifloral stingless bee honey. The rainy season was proven to reduce antioxidant and antimicrobial properties. Meanwhile, higher flower variety and quantity increased antioxidant and antimicrobial properties. Pollination with the presence of stevia plants may increase other beneficial properties derived from the plant. The differences in antioxidant and antimicrobial activity between the two honey samples strongly showed the potential relationship between the availability of flower sources and its' effect on the antioxidant and antimicrobial activity of the stingless bee honey itself.

Conflict of interest
The authors declare that there are no conflicts of interest.