The study areas

Dandi and Meta Robi are districts located in the West Shewa Zone of the Oromia Regional State in Ethiopia. These two districts were purposefully selected from the West Shewa Zone due to their high potential for stingless bee honey production and ease of access during data collection. According to the 2021/22 Annual Reports from the Agricultural Offices, Dandi district is located at 8°54’0.11"N latitude and 38°25’0.35"E longitude, and experiences minimum and maximum temperatures ranging from 9.3°C to 23.8°C, with annual rainfall between 750–1,300 mm. Meta Robi district is located at 9°19’0.6"N latitude and 38°09’0.6"E longitude and experiences minimum and maximum temperatures ranging from 20°C to 30°C, with annual rainfall between 750–1,300 mm). The locations were purposively chosen for the study representing the high potential areas of stingless bee, Meliponula beccarii, and with diverse floral composition.

Sample acquisitions

Careful investigation and excavations of 24 wild stingless bee nests (Meliponula beccarii) (8 from Dandi district and 16 from the Meta Robi district) were conducted. From each nest, fresh honey samples ranging from 0.25 to 0.5 liters were collected using disposable 50 ml syringes. In addition, three honey samples were purchased from the market to be used for comparison, making the total honey samples 27. The collected honey samples were then stored in separate, airtight, and labeled jars (Fig 1) and properly refrigerated at 4°C until the dates of the analysis.

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Fig 1. Excavated feral stingless bee nest.

https://doi.org/10.1371/journal.pone.0311725.g001

Methodologies and protocols of the honey analysis

The analysis of the collected honey samples was done at Ethiopian Conformity Assessment Enterprise (ECAE) [19, 20] in Addis Ababa in accordance of the codex Alimentarius commission of the joint FAO/WHO food standards program [21]. The analyzed parameters included sugar content, moisture content, free acidity, pH, electrical conductivity, ash, and HMF each with three replications. Determination of sugars was performed with high-performance liquid chromatography (HPLC) equipped with a differential refractive index (DRI) detector [22] with the separation using NH₂ column (4.6 x 250mm, ZORBAX NH₂) and a particle size diameter of 5μm with the column was kept at 30°C throughout the analysis. The mobile phase composition was 70% acetonitrile in water. The injection volumes of the samples were10μl, with a flow rate of 1.3ml/min. Retention times were identified using standards of peak. The honey samples were also spiked with standards in order to verify the identity of the chromatographic peaks. Triplicate injections were performed and for the peak quantification average peak areas were used. Monosaccharaides (glucose, fructose) and disaccharides (sucrose, maltose, turanose) were used as standards to determine the sugar content of the honey samples. A 5g of honey sample was dissolved with 25% methanol in water solution. The solution was transferred to a 100ml volumetric flask. Solution was filtered through 0.45μm syringe and injected to HPLC. A standard curve was used to determine sugar content (fructose, glucose, sucrose, turanose and maltose) and the values were calculated as gram/100gram of honey sample.

A digital refractometer (Abbe refractometer, Leica Mark II Plus) thermo stated at 20 °C with the refractive index and regularly calibrated with distilled water was used [23] to determine the moisture content.

The honey samples were homogenized and the surface of the prism was covered evenly with the sample. After 2 minutes, the refractive index was read with refractometer. Each honey samples were measured triplicate and the average value was taken [23]. Free acidity and pH measurement according to [23]. A pH meter was used to measure the pH of a 10% (w/v) solution of honey prepared in deionized water. A 10g honey sample was dissolved in 75 ml of carbon dioxide-free water. It was stirred with the magnetic stirrer, then pH electrode immersed in the solution and the volume of titrate was recorded when titrated with 0.1M NaOH to pH 8.30. The reading was recorded and the free acidity was calculated using the following formula.

Electrical conductivity was measured using a conductivity meter (HANNA, HI 2550, EC/TDS/NaCl meter). 20% (w/v) honey solution was suspended in deionized water. Potassium Chloride solution (0.1M) was used for the cell constant. The electrical conductance of this solution was read in mS after the temperature has been equilibrated to 20°C (International Honey Commission, 2009). 20g anhydrous honey was dissolved in distilled water and quantitatively transferred to a 100ml volumetric flask. The electrical conductivity of the honey solution was calculated using the following formula:

Where:

SH = electrical conductivity of the honey solution in mS.cm^-1

K = cell constant in cm^-1

G = conductance in mS

For temperatures above 20°C: subtract 3.2% of the value per °C

For temperatures below 20°C: add 3.2% of the value per °C

Ash content was determined according to the methods of International Honey Commission (2009). Accordingly, 5 g of honey was placed in combustion pots, which required preheating to darkness. Then, the samples incinerated at high temperature (550°C) in a burning muffle (THERMO CONCEPT, KLS 45/13, Germany) for 1 h. After cooling at room temperature, the obtained ash weighed and the proportion of ash (Ac) in g/100g honey was calculated using the following formula: -

Where:

m₃ = weight of honey

m₁ = weight of dish + ash,

m₂ = weight of dish

Hydroxymethylfurfural was identified as Standard solution: 5-(hydroxymethyl-) furan-2-carbaldehyde (HMF) 1, 2, 5, 7 and 10 mg /L aqueous solution was prepared. The absorbance A of the prepared standard solution was determined using an UV spectrophotometer (CECIL, CE 7500, 7000 series) at 285 nm in 1 cm quartz cells with water in the blank cell. The concentration of the standard solutions was calculated from the literature values for molar absorptivity, ε = 16830 or absorptivity.

Where: A is the absorbance of the standard solution

HMF content was determined with HMIHC (International Honey Commission, 2009) with some modifications. A high-performance liquid chromatography was used with an absorbance scale 285nm using DAD detector. 10g of prepared honey sample was dissolved with 50ml deionized water. The solution was filtered through a 0.45μm syringe filter and read on a HPLC (Agilent, 1260infinity) using a C18 column (3.0 x 250mm) (ZORBAX SB-C18) kept at 30°C throughout the analysis with a particle size diameter of 5μm. The mobile phase composition was 90% deionized water (1% acetic acid) with methanol. The injection volumes of the samples were 10μl, with a flow rate of 0.7ml/min (International Honey Commission, 2009). The HMF content of the samples was calculated by comparing the corresponding peak areas of the samples and those of the standard solutions taking into account the dilution. The collected data were systematically organized, coded, tabulated, and analyzed using the Statistical Package for Social Science (SPSS) software version 20. One-way ANOVA was employed to compute the variations between the means of each variable in every studied parameter and 0.05% tests were considered significance.

Free acidity

In this study, the overall mean free acidity values of the stingless bee honey samples were 306.64 ± 87.95 meq/kg, ranging from 200–424.74 meq/kg (mean 338.79 ± 72.46) for the Dandi district, and 188.25–456.30 meq/kg (mean 274.49 ± 93.10) for the Meta Robi district. The mean free acid value for the purchased honey sample was 348.5 ± 12.0 meq/kg, and there was no significant difference (P>0.05) between the freshly collected honey samples from the two districts and the purchased sample (Table 1).

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Table 1. Mean physicochemical properties of stingless bee honey samples (N = 27).

https://doi.org/10.1371/journal.pone.0311725.t001

The mean free acidity values in this study were well above the acceptable limit of international standards for Apis mellifera honey (<50 meq/kg) [21], further confirming the acidic nature of the stingless bee honey [6]. Reports from Ethiopia indicate mean free acidity levels of 92.39 ± 4.45 meq/kg [18] and 7.3 ± 0.36 meq/kg [3] for stingless bee honey samples from various parts of the country, both of which are significantly lower than our findings. Our value is also notably higher than the free acidity of stingless bee honey from Southeast Asia (40 to 60 meq/kg) and South America (50 to 70 meq/kg). The elevated free acidity of *Meliponula beccarii* honey in our samples is likely attributed to a combination of diverse floral sources with high organic acid content [24], specific environmental conditions that promote fermentation [25], and unique soil characteristics influencing nectar composition [26], enzymatic activity and low pH content [27].

Honey free acidity is an important quality parameter, as it indicates the presence of organic acids, which contribute to the flavor and aroma of honey [11]. Acidity values can also be useful for discriminating honeys of different floral origins, and honey adulterated with sugar syrup typically has very low free acidity (<1) [28]. The acidity levels in stingless bee honeys can vary significantly based on factors like bee species, geographical origin, and other variables. The free acidity values were reported 26.5 to 66.4 meq/kg for Melipona scutellaris honey (Souza et al., 2006), 29.7 ± 4.4 meq/kg for Plebeia saiqui honey, and 42.7 ± 4.2 meq/kg for Tetragonisca angustula honey (Vit et al., 1998), and 22.4 to 34.6 mEq/kg for Melipona beecheii honey (Ruiz-Argueso and Rodriguez-Navarro, 1973). The Meliponula beccarii honey studied appears to have unusually high free acidity and low pH compared to stingless bee honey from Brazil (Melipona scutellaris), Venezuela (Plebeia saiqui, Tetragonisca angustula), and Mexico (Melipona beecheii), which typically range from 22.4 to 66.4 meq/kg. This suggests the high free acidity may be due to increased fermentation, potentially stemming from factors like differences in bee species and geographical origin [29], improper handling, or high moisture content [30], which requires further investigation. The higher free acidity in honey is directly linked to its enhanced medicinal potential, and honey with higher free acidity (lower pH) exhibits stronger antimicrobial activity against pathogens [31, 32], greater antioxidant capacity [7, 10], improved anti-inflammatory and wound healing properties [33], and effective antibiofilm activity that can disrupt and prevent microbial biofilms [34, 35], collectively highlighting the direct relationship between the high free acidity in honey and its potential for medicinal and nutritional valueof honey.

Moisture content

Honey moisture content is an important quality parameter due to its impact on fermentation. The overall moisture content of the honey samples was reported to be between 23.45% and 36.75%.(mean 28.05±3.52%). The recorded moisture contents ranged from 25–36.75% (mean 29.34±4.02%) for the Dandi district and 23.45–30.80% (mean 26.77±2.49%) for the Meta Robi district. The purchased honey samples had a moisture content of 34.20–36.90% (mean 35.40±0.79%), and there was no statistical difference (P > 0.05) between the honey samples from the two districts (Table 1). The moisture content of freshly collected honey was lower than the in marketed honey and this lower moisture content helps prevent fermentation, indicating better quality preservation of the freshly collected honey sample [25].

This finding is consistent with previous studies [3] reported a moisture content of 25.1–35.0% (mean 29.6±1.4%) for honey samples of the same stingless bee species collected from four districts of Ethiopia [36] also reported similar results. Studies have shown that stingless bee honey from Southeast Asia (Malaysia) typically has a moisture content ranging from 30% to 35% [26] and in contrast, honey from South America has been observed to have slightly lower moisture content, between 28% and 30% and Brazilian stingless bee honey exhibited moisture levels around 28% [24].

Variations in moisture content among honey samples can be affected by factors such as adulteration, humidity, floral origin, soil type, collection period, and processing aspects [12, 37–39]. The moisture contents noted in this study are lower than what was reported for stingless bee honeys from Mexico, Guatemala, and Venezuela (31.4% to 38.74%) [13], but higher than the 13.86±1.06% reported for Melipona species in Nigeria [40].

The standard moisture content limit for Apis mellifera honey is 20% or less, as per the Ethiopian [26] and European [23] standards.

The pH value

The mean pH values ranged from 3.0–3.42 (mean 3.24±0.13) for the Dandi district and 3.0–3.60 (mean 3.35±0.18) for the Meta Robi district. The purchased honey samples had a pH range of 3.27–3.35 (mean 3.31±0.023). The pH parameter showed no statistically significant differences (p>0.05) among all the sampled honeys (Table 1).

All honey samples showed low pH values, which is characteristic of stingless bee honeys [5, 6]. This finding is consistent with previous studies [3] reported an overall pH range of 3.4–3.9 (mean 3.7±0.15) for stingless bee honey from four districts in Ethiopia [40] reported pH values of 3.75 and 4.21 for stingless bee honey (Melipona species) in Nigeria, and [41] presented similar findings for the genus Melipona in Brazil [24]. The pH of honey from Southeast Asia typically varies from 3.5 to 4.5, as reported by [26] and honey from South America exhibits a pH range of 3.0 to 4.0 according to findings by [24], indicating the Meliponula beccarii honey from Ethiopia has a pH, which falls within the lower spectrum of both Southeast Asian and South American honeys. Lower pH may enhance the honey’s preservation and antibacterial properties, contributing to its quality [25].

The standard pH value for Apis mellifera honey is 3.42–6.10 [42, 43]. The low pH value of Melipona honey has great significance for its extraction and storage, as it inhibits the occurrence and development of microorganisms [44, 45] and reduces some infectious diseases [46, 47].

Ash contents

In this study, the ash contents ranged from 0.40–1.0 (mean 0.65 ± 0.25g) for the Dandi district, 0.36-1g (mean 0.63±0.24g) for the Meta Robi district, and 0.54–0.83g (mean 0.64± 0.09g) for the purchased honey samples. There was no significant difference (P < 0.05) in the ash content among all the samples (Table 1).

The mineral composition of honey could be influenced by the geographical and botanical origin of the flora, the type and activity of the bees, the extraction technique, and the storage conditions [48]. The results of this study are consistent with the findings of [3], who reported ash contents ranging from 0.21 to 0.57% for stingless bee honey. Although the overall average ash content of stingless bee honey is similar to the purchased honey from the market (0.5 ± 0.00%), the results of the present study suggest that honey produced from the stingless bee (M. beccarii) in Ethiopia is richer in mineral content.

This is significant, as the mineral content in honey can have important implications for its for various aspects; used as a marker to identify the geographical origin of the honey, allow for honey traceability and authentication [49]; and indicates the predominant floral sources that the bees have visited, as different plant species accumulate varying levels of minerals in their nectar [50]; used to assess the quality and authenticity of honey, as adulteration or improper processing can alter the natural mineral profile [51]; and it is relevant to the honey’s nutritional value, as it provides information about the bioavailability of essential minerals, such as potassium, calcium, and magnesium [52]. It is also established that the ash content of stingless bee honey varies by region, with Southeast Asia ranging from 0.1% to 0.3% [26] and South America between 0.2% and 0.5% [24]. The lower ash eontents for Meliponula beccarii honey from Ethiopia suggests that Ethiopian honey may have a reduced mineral profile, likely due to the specific flora and soil characteristics in the region and such differences highlight the impact of local biodiversity on honey composition and its potential nutritional value [24–26].

Sugar contents

The mean sugar concentrations reported were glucose (9.39 ± 4.26 g/100g), sucrose (0.24 ± 0.01 g/100g), maltose (10.81 ± 4.95 g/100g), fructose (16.57 ± 2.55 g/100g), and turanose (0.20 ± 0.00 g/100g). Interestingly, the only significant difference (p<0.05) between the two districts was observed for the fructose concentration. Comparing these results to a previous study, the sugar concentrations recorded were generally lower are fructose (36.48 ± 0.54 g/100g), glucose (27.67 ± 0.43 g/100g), and sucrose (1.24 ± 0.18 g/100g) in stingless bee honey samples [36]. The Melipona honeys, typically contain small quantities of maltose [53]. The sugar composition reveals that Southeast Asian honey has fructose levels ranging from 35% to 45% and glucose levels from 25% to 35%. South American honey also shows high fructose levels of 40% to 50%, which aligns closely with the Southeast Asian findings. The balance of these sugars influences the taste, sweetness, and overall quality of the honey [24, 25].

Previous studies has reported maltose levels in stingless bee honey generally range from lower values, typically around 1% to 5% [24, 25]. However, the high maltose content in our stingless bee honey may be due to several factors like specific floral sources can significantly influence sugar composition, with certain plants containing higher maltose concentrations [24], fermentation processes within the nest may also increase maltose levels if honey is stored for extended periods [25], the foraging behavior of stingless bees that could lead to the preferential collection of nectar from maltose-rich plants and environmental factors, such as soil composition and climate, that may further impact floral diversity and nectar quality, resulting in elevated maltose levels [54], processing and storage methods might alter sugar profiles, with prolonged storage or heat affecting composition [26], variability among stingless bee species [25].

These differences in sugar composition could be attributed to various factors, such as the geographical and botanical origins of the honey, the species of stingless bees involved, and the extraction and processing methods employed [29, 49, 51, 52, 55].

Hydroxymethylfurfural (HMF)

In this study, 0.06 and 4.2 values were recorded as minimum and maximum with an overall mean of 0.89±1.14 mg/kg (Table 1). The highest HMF value was recorded in honey samples collected from the Dandi district (4.20 mg/kg), and the difference in HMF value recorded in honey samples collected from both districts is not significant (p>0.05). This finding disagrees with [3] that stated a high overall HMF range (11.2–22.4mg/kg) and the value reported by [36] (6.58±0.36mg/kg) for pooled honey samples from 14 districts of Oromia, Ethiopia. [21] permits a maximum of 40 mg/kg for the HMF standard. The low HMF content including the purchased honey sample in this study indicates the freshness and good quality of the honey [56–58]. The Hydroxymethylfurfural (HMF) is used as an indicator of poor quality due to adulteration or poor handling like inappropriate storage and the lower the HMF, the freshness and good quality of the honey [9]. HMF levels in Southeast Asian honey are reported at 5 to 10 mg/kg, while in South America, they can range from 10 to 20 mg/kg, indicating Ethiopian honey has an HMF content within the acceptable limits for freshness [25, 54].

Electrical conductivity

The overall mean calculated value of electrical conductivity of the 24 honey samples of stingless bees from the two study districts was 1.31±0.44 mS/cm, ranging from 0.72–1.99. The electrical conductivity values ranged from 0.72–1.99 mS/cm (mean 1.38±0.48) for the Dandi district samples, 0.75–1.85mS/cm (mean 1.24±0.40) for the Meta Robi district and 0.14–1.50mS/cm (mean 1.79±1.34) for purchased honey (Table 1). The electrical conductivity difference of the two-district sampled honey is not significant (P>0.05), suggesting similarities of flora compositions between the districts. The electrical conductivity in the current study is in line with [3, 36] on stingless bee honey in Ethiopia. The conductivity values of the Melipona honeys vary from 0.32–0.44mS/cm [59]. The electrical conductivity values of the explored honey samples were within the range of allowable value of international standard value (i.e, greater than 0.8 mS/cm) [21]. The significance differences in electric conductivity of honey samples between the two locations suggest its usefulness for differentiating honey of different botanical origins and purity [28].

The unique findings from the data are that the stingless bee (Meliponula beccarii) honey from the Dandi and Meta Robi districts of Ethiopia exhibits high acidity, low pH, low HMF, and a distinctive sugar profile, differentiating it from other places studied stingless bee honey [3] indicating potential medicinal properties.