Evaluation of physicochemical and antioxidant properties of two stingless bee honeys: a comparison with Apis mellifera honey from Nsukka, Nigeria

Several physical, biochemical and antioxidant properties of two Nigerian stingless bee honey varieties (Melipona sp. and Hypotrigona sp.) were compared with Apis mellifera honey using standard analytical procedures. The mean pH of Apis mellifera, Hypotrigona sp. and Melipona sp. honeys were 4.24 ± 0.28, 3.75 ± 0.11 and 4.21 ± 0.37 respectively. The mean moisture contents of the honeys were 11.74 ± 0.47, 17.50 ± 0.80, and 13.86 ± 1.06%. Honey samples from Hypotrigona sp. when compared with other honey samples had the highest mean total dissolved solids (370.01 ± 22.51 ppm), hydroxymethylfurfural (16.58 ± 0.37 mg/kg), total acidity (35.57 ± 0.42 meq/kg), protein content (16.58 ± 0.37 g/kg), phenol content (527.41 ± 3.60 mg/kg), and ascorbic acid (161.69 ± 6.70 mg/kg), antioxidant equivalent—ascorbic acid assay value (342.33 ± 0.78 mg/kg) as well as ferric reducing power (666.88 ± 1.73 μM Fe(II)/100 g) (p < 0.05). Several strong correlations were observed among some of the parameters of the honeys. This is the first study to compare the properties of Nigerian honey bees. Our results suggested that these honeys (specifically Hypotrigona sp. honey) is a good source of antioxidants comparable to A. mellifera honey.


Introduction
Honey is a natural sweet sticky and viscous solution produced by honey insects from the nectar of flowers or from living parts of plants. The nectar is transformed into honey by honey bees and stored in the pot-like comb or waxy honeycomb inside the hive to ripen and mature for further use. Honey is a mixture of substances composed of mainly sugars and water, as well other compounds (less than 1%) such as proteins, minerals, enzymes, vitamins, 5-hydroxymethylfurfural (HMF), volatile compounds, flavonoids, and phenolic acids [1].
Even though honey is produced worldwide, its composition and antimicrobial activity can be variable, and are dependent primarily on their botanical origin, geographical and entomological source [2]. Other certain external factors, such as harvesting season, environmental factors, processing and storage condition, also play important roles [3]. Honey contains significant antioxidant compounds including ascorbic acid, flavonoids, glucose oxidase, amino acids, proteins phenolic acids, organic acids, carotenoid derivatives, and maillard reaction products [1,3]. The biological properties that make it ideal as a medicine are: antibacterial, bacteriostatic, anti-inflammatory, wound and sunburn healing effects, antioxidant activity, radical scavenging activity and antimicrobial activity [3,4].
Previous studies have reported some of the physicochemical properties of Nigerian honey from Apis mellifera [5][6][7][8][9] and stingless bee [10] but to our knowledge there is scanty or no report on antioxidant properties of honeys from stingless bees usually found in Nsukka, Nigeria. Therefore, the aim of this study was to evaluate and compare the physico-chemical and antioxidant properties of three varieties of honeys from A. mellifera, Hypotrigona sp. and Melipona sp.

Collection of honey samples
Nine multi-floral honey samples, two each from Apis mellifera, Hypotrigona sp. (locally called Okotobo in Igbo, Nsukka, Nigeria) and Melipona sp. (Ifufu or Ihuhu) were collected from different bee-keepers at Igbo Eze North Local Government Area (Enugu Ezike), in Enugu State (Nigeria) between June and July, 2015. The honey samples were harvested and kept at 5 °C.

Physicochemical analyses
The pH of a 10% (w/v) solution was measured according to Association of Official Analytical Chemists methods [11]. The moisture content was determined based on the refractometric method of International Honey Commission [2]. A 20% (w/v) solution of honey was prepared and using conductivity meter, electrical conductivity (EC) and total dissolved solids (TDS) of each sample were measured [12]. The colours of the honey samples were determined by spectrophotometric method according to the method of White [13] and the colour intensities (ABS 450 ) were determined using the method of Beretta et al. [14].

Biochemical analyses
The refractometric technique was used to determine the total sugar contents (TSC) of a 25% (w/v) solution of honey [15] and using glucose standard solutions (100-600 μg/mL), the reducing sugar content was measured according to Saxena et al. [16] and the amount of sucrose content (%) was measured [15]. A technique based on the method described by Winkler was used to determine the HMF content of the samples [17] and it was calculated (mg/kg) according to Auerbach and Borries [18]. Using Lowry's method [19], the protein content of honey was determined while the free acidities and lactones contents were determined by equivalence point titration according to IHC [2].

Analysis of antioxidant potentials
By using spectrophotometric Folin-Ciocalteu method [20], total phenolic content was determined based on gallic acid standard solutions (20-100 μg/mL; y = 0.0017x − 0.0104). The colorimetric assay used by Zhishen et al. was used to measure the flavonoid content [21] using a standard solution of catechin (20-100 μg/mL; y = 0.0136x − 0.0092). A method established by the IHC was used to estimate proline content [2] and the ascorbic acid content was determined based on the method described by Ferreira et al. [22], using a standard solution of pure l-ascorbic acid (50-400 µg/mL; y = 3.2460x − 0.0614). The antioxidant content was determined by measuring antioxidant equivalent ascorbic acid content (AEAC) values using the method described by Meda et al. [23] and a standard solution of pure l-ascorbic acid (1-8 µg/mL; y = 0.2746x − 0.0943). The ferric reducing/antioxidant power (FRAP) assay (μMFe(II)) was determined using the procedure described by Bertoncelj et al. [24].

Statistical analyses
Results were reported as the mean ± standard deviation of duplicate experiments. Using SPSS statistical package (version 23), ANOVA and post hoc multi-comparison test were used for comparison of means (p < 0.05). Several parameters were correlated using Pearson's correlation coefficient (r) (p < 0.01).

Results
All the honey samples tested were acidic and the pH values of the honey samples varied (Table 1). There were significant differences between the mean values of pH (F (2,24) Table 1). A. mellifera and the other two honey varieties were amber and light amber in colour (Pfund scale table), respectively.

Correlation among some physicochemical and antioxidant parameters
The correlation matrixes showed significant correlations between some of the physicochemical and antioxidant  parameters. In A. mellifera honey samples, a strong correlation was found between colour intensity and some parameters (Additional file 1: Table S1). Also, correlation matrixes of Hypotrigona sp. honey samples showed strong correlations between ABS 450 and phenolics flavonoid, protein, ascorbic acid and AEAC. The proline content had positive correlations with total phenol content, AEAC, FRAP and flavonoid content (Additional file 1: Table S2). Several strong positive correlations were established between ABS 450 of Melipona sp. and protein and AEAC. The flavonoid contents had correlation with proline, AAC, AEAC and FRAP (Additional file 1: Table S3).

Discussion
All honey varieties analysed were acidic. The observed pH values in this study are comparatively similar to previous reports for Nigerian stinging bee honey (3.32 and 4.90) [6,9] and higher than stingless bee honeys (1.40-3.97) [10]. None of the investigated samples exceeded the allowed limit of international standards (pH of 3.42-6.10) [25]. The moisture content of the honey samples are within the recommended international standard limit (not more than 20%) [25,26]. The moisture content of the tested honeys may be related to the harvest season and the level of maturity in the hive [11]. While similar findings have been reported by Omoya et al. [7] and Eleazu et al. [9], and a higher moisture content values (25.43-26.51%) has been reported for Nigeria stingless bee honey [10]. Hypotrigona sp. honey showed the highest mean EC value and the highest mean content of TDS, which indicates that it is rich in both organic and inorganic substances [12]. The EC values of the investigated honey samples are within the allowed limit of international standards (not more than 0.8 mSs/cm) [25]. This is the first report on ABS 450 of Nigeria honeys and similarly, the ABS 450 values for Slovenian, Algerian and Indian honeys were reported to be 70-495 mAU [14], 724-1188 mAU [27], and 524-1678 mAU [16]), respectively. Comparatively, a smaller strong correlation was found between the colour intensity of Malaysian [28] and Algerian [27] honey samples and the other antioxidant parameters, like flavonoid. In this study, Hypotrigona sp. and Melipona sp. honeys had the highest and lowest mean total sugar content value, respectively. The sugars contents values are within the limits (Reducing sugar-not less than 60% (g/100 g) and sucrose-not more than 5% [g/100 g)] set by the Codex Alimentarius [25] and European Commission [26]. Hypotrigona sp. had the highest mean HMF value and our results were within the recommended standard set by Codex Alimentarius [25] and EU [26] (maximum of 40 mg/kg). These findings are similar to other published levels for HMF on Nigerian honeys (5.0-17.22 mg/ kg) [8], Algerian honeys (15.23-24.21 mg/kg) [27] and Pakistan honeys (27.69-36.08 mg/kg) [29]. The low HMF concentrations confirmed that these samples are of good quality. The free acidity values of the analysed honey samples are within the international standard (not more than 50 meq/kg) [25].