Medires Publishers - Article

Abstract

Honey quality and authenticity continue to be important factors in honey consumption and marketing. The major concern with honey quality is to keep its natural properties and compositions. This research work is aimed at investigating the botanical origin and physicochemical, antioxidant, rheological, and sensorial quality characteristics of comb, crushed, and processed honey. Accordingly, thirty samples were collected from the Burie district of Ethiopia. Based on Melissopalynology, botanical origin was determined. Physicochemical properties were determined based on the standard methods of the International Honey Commission (IHC) and the Association of Official Analytical Chemists (AOAC). Antioxidant propertiesand diastase activity were determined using UV-visible spectroscopy. All the samples were polyfloral honey. Comb honey was found to have moisture (17.98±0.72), sucrose (2.46±0.77), diastase activity (11.78±1.5 DN), total phenol content (TPC) (120.8±47.4 GAE/100 gm), total flavonoid content (TFC) (53.7±24 CEQ/100g), 2,2-diphenyl-1-picrylhydrazyl (DPPH) inhibition (30.9±14.9%), IC50 (54±5.72 mg/ml), and hydrogen peroxide (H2O2) scavenging (57±16.06%). It was determined that crushed honey contains moisture (19.43±0.40), sucrose (4.21±0.76), diastase activity (9.28±0.25), TPC (84.3±51.86), TFC (29.4±61), DPPH inhibition (33.45±5.07), inhibitory concentration (IC50) (80±6.25), and H2O2 scavenging (52.99±15.9). Analysis of processed honey indicated that moisture (23.76±0.78), sucrose (6.12±0.6), diastase activity (7.76±0.71), TPC (66.7±30.59), TFC (29.47±11.22), DPPH inhibition (33.98±1.84), IC50 (120±8.25), and H2O2 scavenging (56.61±4.86). Correlation analysis indicated that moisture is associated with sucrose content (r=0.88), diastase activity is related to sucrose (r=-0.86), and TPC correlates with H2O2 scavenging activity (r=0.82). Honey samples were found to be a Newtonian fluid. The sensory quality of comb honey is preferable. The result revealed that comb honey adequately satisfies the requirements of national and international standards.

Introduction

Honey production is a very long-standing and deep-rooted household activity in Ethiopia. Ethiopia has a huge potential for beekeeping because it is endowed with diversified honey flora and suitable topography [1]. The apiculture sector has a significant role in generating and diversifying the incomes of smallholder beekeepers [2]. It has been contributing to the national economy through export earnings. Ethiopian honey production is characterized mainly by traditional, backyard, forest beekeeping [3]. In Ethiopia, 6,986,100 bee colonies were found in beehives, of which 95.87% were found in tra- traditional, 2.66% in frame, and 1.47 in transitional beehives [4]. Besides, about 1.4–1.7 million households are engaged in beekeeping, which produces and markets mainly crushed and comb honey. Ethiopian honey varies in color, consistency, and purity depending on geographical location, botanical origin, and harvest season [5]. Ethiopian honey is mainly sold as comb, crushed, and processed. The widespread fraud practices and quality limitations of honey have exerted a negative effect on the use and trust of different types of honey, not only in the study area but also worldwide. According to [6], honey is a natural product having varied physicochemical properties and compositional variations. The geographical locations, the botanical origins, and pre-and post-harvest handlings are the factors of variation. The physicochemical properties of honey can be changed during post-harvest handling, processing, and storage practices. The marketing of comb and crushed honey was also experienced in Africa and some other continents. Evaluating and measuring the botanical origins, physicochemical, antioxidant, rheological, and sensory attributes of comb, crushed, and commercially processed honey for wider communication is fundamentally important to encourage quality product marketing and enhance honey industry development. This will, of course, contribute further to the protection of domestic consumers, promote fair competition among producers, and improve the foreign currency earnings from honey exports. Ripe honey in the beehives is qualified enough for human consumption and can be used for industrial purposes. Honey is one of the most functional foods due to its unique nutritional and medicinal properties, which are attributed to the influence of the different groups of substances it contains [7]. The Codex Alimentarius Commission [8] and other international honey standards stipulate that honey must only comprise honey and that no specific component may be eliminated. Organoleptic characteristics, physicochemical analyses, bio-functional characteristics, and pollen spectrum are now the primary grading criteria for honey [9]. Because of the multiple importance of honey, from food to medicine, it is of great interest to carry out a complete analysis and measure- ment of honey [10]. The composite nature of honey makes it vulnerable to adulteration. It is testing and verification that have been challenging due to the similarity in the composition of adulterants [11]. On the other hand, Bogdanov et al. [12] stated that there was a growing need to assess the quality and authenticity of honey, particularly with regard to quality, geographical and botanical origins. Domestic consumers can learn to trust and feel confident utilizing comb honey rather than processed and smashed honey, claims [13]. As a result, it was explained why there has been an increase in the demand for comb honey. For this reason, beekeepers in and around the study area were experienced in managing the comb honey market supplies. Beyond this, Belay et al. [14], Debela & Belay [15], and other reports were conducted to investigate honey in different parts of Ethiopia. However, no research has been done on the physicochemical, antioxidant, sensory, and rheological properties of comb, crushed, and processed honey from the Burie district. Therefore, the objective of this research is to measure and evaluate the quality merit of comb, crushed, and processed honey using various tools.

Material and methods

Sample collection and preparation

Representative comb honey samples were randomly selected from three honey production potential peasant associations (Shekua, Weynima, and Tiyatiya), and crushed and processed honey samples were also collected from the local market of Burie district, West Gojam, Ethiopia (Figure 1). The geographical coordination of the district is found at X-Y GPS coordinates of 1180000 N, 300000 E. The representative honey samples were collected randomly during the second main honey flow season in the study area (15th of July–20th of August 2021). The honey samples were collected and handled based on the principles used by Belay et al. [14] until laboratory analysis. A total of thirty honey samples were randomly collected, ten from each (combs, crushed, and processed).

Figure S1: HPLC Chromatogram of Sugars in honey (where retention time of fructose = 9.295, glucose = 10.522, sucrose = 13.640, turanose = 14.688, and Maltose 16.290).

Laboratory Analysis

Botanical origin

Botanical origin was determined based on Belay et al. [14], using pollen analysis. Accordingly, 10 g of honey was weighed using a centrifuge tube and dissolved in 20 ml of warm distilled water (20–40°C). The solution was centrifuged at 2060 g (3500 rpm) for 10 minutes, and the supernatant was decanted. Twenty ml of water was added again to completely dissolve the remaining sugar crystals and centrifuged at 2060 g (3500 rpm) for 5 minutes, and the supernatant was removed. The sediment was spread evenly using a sterile micro spatula on the microscope slide, and to dissolve some fat particles from the pollen grain, a drop of glycerin jelly was added and smeared to the cover slip, and the pollen grains were identified using a pollen atlas [3]. The pollen count was done under a microscope (ZEISS, Germany). The frequency of occurrence was determined by counting 500 pollen grains from a single slide. Then, the pollen counts were converted into percentages to calculate the relative dominance.

Physicochemical property Moisture

Moisture was determined with a digital refractometer (Abbe refractometer, Leica Mark II Plus) thermostated at 20°C. The honey sample was homogenized, and the surface of the prism was covered evenly with the sample. After 2 min, the refractive index was read with a refractometer. The refractive index reading was converted to moisture content using the AOAC, 969.38 conversion table [16].

Sugar's profile

Sugars were determined using high-performance liquid chromatography (HPLC-1260 Infinity Series, Agilent Technologies, Germany) equipped with a differential refractive index (DRI) detector AOAC [16]. Glucose, Fructose, Sucrose, maltose, and Turanose sugars were determined using high-performance liquid chromatography (HPLC) 1260 Infinity Series Agilent Technologies at an Ethiopian conformity assessment laboratory. The honey sample (5 g) was dissolved in a 25% methanol in water solution, and the solution was transferred and made into a 100 ml volumetric flask. The solution was filtered through a 0.45 μm syringe and injected into the HPLC. The separation was performed using an NH₂ column (4.6 × 250 mm) (ZORBAX NH₂) with a particle size diameter of 5 μm, and the column was kept at 30°C. The mobile phase composition was 70.

Antioxidant properties

Total phenolic content

The total phenolic content in honey was determined using the Folin-Ciocalteu method in an alkaline environment. The Folin-Ciocalteu method, modified by [20], was used to determine total phenolic content in honey. Gallic acid (0–100 mg/ml) was used for calibration. Total phenolic compound content was expressed as mg of Gallic acid equivalent per kg of honey (mg of GAE/gm). Five gm of honey was dissolved in 50 ml of distilled water, shaken, and kept in a dark place, vortexed, and then filtered using quantitative filter paper 202 moderate 150 mm. One mg of gallic acid with 1 ml of distilled water was dissolved, shaken, and diluted (0–100 μg/mL) with a concentration range of (0, 20, 40, 60, and 100. It was prepared as a standard to establish the calibration curve, and absorbance was measured at 760 nm.

Total flavonoids content

The total flavonoids in honey were determined using a modified photometric method [20]. One gram of quercetin in 1 mL of distilled water was dissolved, shaken, and the stock solution was prepared. To perform different concentrated solutions, a serial dilution of the quercetin standard with a concentration of 0, 20, 40, 60, and 100) was prepared. Absorbance was measured at 510 nm using a UV Visible spectrophotometer (JENWAY 6300). The total flavonoid contents of the honey samples were determined using AlCl₃ according to Wieczorek et al. [21]. Quercetin was used as a standard to establish the calibration curve. A standard curve of quercetin was prepared within a concentration range of 20-100 μg/ml, and the results were expressed as mg of quercetin per kg of honey (mg of QE/kg).

Radical scavenging activity

Radical scavenging activities were determined using Meda et al., [22]. Honey samples were dissolved in distilled water at concentrations of ascorbic acid ranging from 20 to 140 g/ml and were mixed with 4 ml of 0.004% DPPH. A pure L-ascorbic acid standard was used as a reference. The mixtures were shaken vigorously and left for 30 min at room temperature in the dark, and the absorbance of the DPPH was measured at 517 nm against a blank using a UV visible Spectrophotometer (JENWAY 6300). The radical scavenging activities of the DPPH radical, expressed as % inhibition, were calculated from the following equation. Where Abs blank = blank absorbance at 517 nm; Abs sample = sample absorbance at 517 nm.

Hydrogen peroxide (H2O2) scavenging activity

The hydrogen peroxide scavenging activity was assayed according to the method described by [23]. Accordingly, the samples were dissolved in 0.04 M phosphate buffer (pH = 7.4), and 5 mL of the samples were mixed with 0.6 mL of 40 mM H₂O₂ solution (prepared in the same buffer). The absorbance of the mixture and hydrogen peroxide was determined at 230 nm using a UV Spectrophotometer (Biochrom 80-7000-30, Cambridge, England). Ascorbic acid was used as a standard, and the blank was prepared in phosphate buffer without H₂O₂.. The decrease in absorbance value showed the high level of hydrogen peroxide scavenging activity designation. The results were expressed as H₂O₂ SC = micrograms per milliliter. The percentage of scavenged hydrogen peroxide was calculated by using the following equation: Percentage of scavenging H₂O₂: 100 Where Ai = absorbance of control; At = absorbance of test honey

Inhibitory concentration (IC50)

A substance's ability to inhibit a certain biochemical function by 50% is measured by its IC50 (inhibitory concentration), which is calculated using the technique outlined by Al-Farsi et al. [24]. The dose-response curves created by graphing the percentage of inhibition versus concentration were used to get the IC50 value.

Rheological measurement

The rheological properties of comb, crushed, and processed honey samples were measured by the rheometer machine MCR 102 (Anton Paar GmbH, Graz, Austria) with the use of cone-and-plate measuring geometry. The rheometer was adjusted at a temperature range of 20–50°C and with a constant shear rate of 50 s⁻¹. Honey samples were uniformly smeared on the cone and plate of the rheometer. The diameter of the cone is 50 mm at an angle of 10. Measurements were carried out at 20, 30, 40, and 50°C. The effect of temperature on the apparent viscosity of honey was analyzed, and graphic curves were evaluated. After a complete set of readings for each sample, the cone and plate bearing and stirring rod were washed with tap water, rinsed, and allowed to dry before the next sample was applied.

Sensory profile

For sensory analysis, ISO 13299: [25] was used. The test room has been arranged to conform to ISO 8589 (1988) minimum requirements (lighting, temperature, noise, and odors). The assessors were selected based on detection, recognition, and the ability to memorize and communicate sensory impressions. Twenty-three (twenty-two female and eleven male) panelists were selected and trained by the panel leader following international standards [26]. Instructions were also given on how to smell or taste, rinse the mouth between samples, and dissolve the sample in the mouth. The required materials for sensory analysis and evaluation forms were prepared. The trained assessors identified the sensory stimuli on the basis of previously memorized standards (ISO, 8586-1, 1993; ISO, 8586-2). The samples were prepared and given a random three-digit code; about 30 g of the sample from comb, crushed, and processed honey were put into clean, dry, and odor- and flavor-free sampling beakers and served in a single tray for each assessor, Piana et al. [27], established by the International Honey Commission. The samples were tested by 33 assessors and measured using a 1-to-7-line score.

Results and Discussion

Botanical origin

The relative frequency of nectar source plants of honey samples is presented in Table 1. The honey samples collected from the Burie district were found with a variety of nectar distributions. Table 1 shows the comparative pollen distribution of honey samples from Eucalyptus species of comb (31±1.70), crushed (43.7±3.86), and processed (33.5±1.72); as well as Croton macrostachyus of comb (31±3.30), crushed (44.1±3.38), and processed (42±3.83). Trifolium decorum, Plantago lanceolata, Cyperus longusus, Vicia faba, and Coffea arabica were relatively identified in a small proportion (Table 1). The floral origin helps to provide specific information on the color, taste, aroma, chemical compositions, sensorial, and viscosity behavior of honey [12]. Identification and documentation of nectar and pollen source plants are the most limiting factors in the development of the apiculture industry of Ethiopia [28]. Based on the relative frequency of the pollen taxonomy, honey is considered monofloral if the dominance is more than 45% [14-15]. Accordingly, the honey samples collected from Burie were polyfloral. A description of botanical origin is useful to authenticate and register honey for a protected designation of origin (PDO) that helps to promote specific plant-origin honey. At present, consumers and international buyers are demanding branded honey based on botanical origin and geographical indication. To guarantee quality and authenticity, a European Union (EU) council directive [29]. states that the botanical origin of honey shall be declared on the label.

Physicochemical Analysis Moisture

The mean ± SD of moisture content for comb, crushed, and processed honey was 17.98±0.72, 19.43±0.40 and 23.76±0.78 g/100 g, respectively (Table 2). There was a signif- icant difference (p<0>Schefflera abyssinica and 18.90 ± 0.45 g/100gm for polyfloral honey from Ethiopia) [30]. In addition, the findings of this study were in line with the literature values reported by [31]. (18.6–18.8 g/100 g), and [32]. (17.5–23 g/100 g). The significant difference in moisture content between honey samples may occur due to the maturation of honeycombs during harvesting and post-harvest handling practices. The amount of water present in honey determines its stability against fermentation and granulation [29]. Moisture content is related to the process by which the bees synthesize honey, the harvesting and extraction techniques, and economic adulteration, and can vary from season to season [Kumar et al.,[33]. and Belay et al., [31]. The Codex Alimentarius established a limit of 20 g/100 g of honey as per the Codex Alimentarius [8].

Sugar profile

Sugar compositions are important quality aspects for energyvalue, viscosity, hygroscopicity, and granulation of honey. The results of sugars, namely fructose, glucose, sucrose, maltose, and turanose, are presented in (Table 2). The HPLC chromatogram retention times for the sugar profiles of the analyzed honey samples were 9.29 (fructose), 10.22 (glucose), 13.34 (sucrose), 14.68 (turanose), and 16.29 (maltose) minutes. The mean ± SD value of fructose content in the present study was 38.89±0.59, 38.89±0.59,38.29±1.24 and 36.99±0.51 g/100 g for comb, crushed, and processed honey, respectively (Table 2).In the analyzed honey samples, the highest fructose content was identified in comb honey (39.5 g/100 g) and the lowest was reported in processed honey (36.5 g/100 g).The processed honey significantly varied (p<0>et al. [34], (37.70 ± 1.46 g/100g) for Brazilian polyfloralhoney. The mean ± SD value of glucose in the present study is 33.87±1.0, 33.09±1.13 and 33.73±1.22 g/100 g for comb, crushed, and processed honey samples, respectively. There is no significant difference (p > 0.05) between honeysamples in glucose content. The fructose/glucose ratio (F/G) is also one of the honey quality indicators. In this study, the fructose/glucose ratio falls in the range of 1.10 to 1.20 (comb), 1.13 to 1.21 (crushed), and 1.07 to 1.16 (processed) honey samples. The F/G ratios in all the honey samples of this study were greater than 1.0, which indicates slower crystallization. Honey crystallization is slower when the fructose/glucose ratio is more than 1.3 and it is faster when the ratio is below 1.0. The mean ± SD value of the sucrose content of the analyzed honey samples was found to be 2.46 ± 0.77, 4.21 ± 0.76, and 6.12 ± 0.6 g/100 g for comb, crushed, and processed honey samples, respectively (Table 2). The Codex Alimentarius, European Union, and Ethiopian standard allow a maximum sucrose content of 5 g/100 g in honey. The results of this study show that the sucrose content of comb honey was 2.46 ± 0.77 and crushed honey 4.21 ± 0.7 g/100 g and met the requirements of international [8] and national regulatory standards [17]. The sucrose content in processed honey might be affected by the lengthy supply chain between the farm gate and processors. The content of turanose is 0.049±0.014, 0.051±0.025 and 0.068±0.014 g/100 g for comb, crushed, and processed honey samples, respectively. The processed honey was varied (p, 0.05) with a comb and crushed honey. The result of maltose content is 0.15±0.24, 0.14±0.2 and 0.086±0.006 g/100 g of combs and crushed and processed honey samples, respectively. A significant difference was not observed (p > 0.05) between the comb, crushed, and processed honey samples.

Table S1: Botanical origin /pollen analysis of the comb, crushed, and processed honey sample

Diastase activity

The mean ± SD of diastase activities of the comb, crushed, and processed honey samples of the present study were 11.78 ± 1.57, 9.28 ± 0.259, and 7.76 ± 0.71Schade units (DN), respectively (Table 2). A significant difference (p<0.05) was observed among the honey samples. Diastase activity is important to detect and predict honey age and freshness, storage time, and prolonged processing temperature and overheating of honey (Silva et al., [35]. The present diastase result was in agreement with the reports of Debela and Be- lay [15], which were 7. 64 ± 0.84 and 12.5 ± 0.55 Schade for Coffee arabica honey and Vernonia amygdalina honey, respectively. Ash content The mean ± SD of ash content was 0.32±0.08, 0.48±0.06 and 0.55±0.10 g/100g for comb, crushed, and processed honey samples, respectively (Table 2). There was no significant difference (p >0.05) observed in ash content among the honey samples. The ash content in the current study was higher than that reported by Debela and Belay [15], which was 0.28 ± 0.05 g/100g for Coffea arabica and 0.28 ± 0.01 g/100g for Vernonia amygdalina honey. However, the present result was also consistent with Tesfaye et al., [36], who reported a 0.1–1.0% ash content of honey samples. The over- all percentage of ash content was found to be 0.42±0.015 g/100g, which is below the allowable maximum and thus the honey samples conform to the international regulatory stan- dards set for honey quality. The present study result is also consistent with Silva et al.,[35], who reported 0.1–1.0% ash content of Ethiopian honey and is in line with the report of Erturk et al., [37], who reported (0.33 g/100g) for monofloral and (0.42 g/100g) for polyfloral honey. The comb, crushed and processed honeys of this report met the standards set by the Codex Alimentarius [8] and Ethiopian Standard [17], (not more than 0.60 g/100 g) in ash content.

Electric conductivity

The mean ± SD of electrical conductivity for comb, crushed, and processed honey of this finding was 0.70±0.08, 0.8± 0.1 and 0.88±0.04 mS/cm, respectively. The electrical conductivity of honey in this study was within the recommendation of Codex Alimentarius and Ethiopian standards (not more than 0.8 mS/cm) [8-17], Comb, crushed, and processed honey samples were identified as nectarous honey. The electrical conductivity of all the studied honey samples was in line with the report of Gebru et al., [38], (0.25–0.41 mS/cm) and [39] (0.41- 0.72 mS/cm). The measurement of electric conductivity depends on the ash and acid content of honey: the higher their content, the higher the resulting conductivity. The highest electrical conductivity value was observed in crushed and processed honey samples. The observed significant variations in electrical conductivity may be due to the possible variation in the degree of maturity of honey, post-harvest handling practices, botanical origin, and soil type. The current finding is in good agreement with the results of Eyobel et al., [34], who reported 0.55 ± 0.08 mS/ cm. Moreover, the study result is nearly similar to that reported- ed by Belay et al. [31], who reported a mean of 0.70 ± 0.04 mS/cm for honey from the Bale Harenna forest of Ethiopia. Free acidity The mean ± SD level of free acidity in the present study is 36.85±2.04, 41.73±4.22 and 38.08±7.74 meq/kg for comb, crushed, and processed honey samples, respectively (Table 2). The acidity level of the crushed honey sample exceeded the acidity limit set by national standards (40 meq/kg) ES [17]. Such results indicate the presence of unwanted experiences like the age of honey samples and the occurrence of fermentation. However, the overall findings of this study indicate an acceptable range for Codex and EU Directive [8], which is ≤50 meq/kg. Besides, Belay et al. [14], reported that the free acid content of the Harenna forest honeys of Ethiopia ranged from 25.49 to 48.81meq/kg, which is in agreement with the present study range (36.85 to 41.73meq/kg).

Specific rotation

The mean ± SD of the specific rotation of comb,crushed and processed honey samples is -9.81±8.05, 9.08±7.88 and- 8.51±0.81[α] D20), respectively. There was a significant difference (p<0>Coffea arabica and -5.98 ± 0.07 ([α] D20) for Vernonia amygdalina honey. The highest negative value was revealed by comb honey (-9.8), with a mean value of -8.8 ([α] D20). Nectar honey contains a predominance of fructose that results in a negative specific rotation.

Table S2: Physicochemical properties of comb, crushed and processed honey sample Mo= Moisture, Elcd=Electric conductivity, HMF=Hydroxymethylfurfural, Spcr=Specific rotation, Glu= Glucose, Fur= Fructose, Suc =Su- crose, Mal=Maltose, Tur =Turanose, Insolu mat= Insoluble matter, Acid= Acid

Hydroxymethyl furfural

The HMF content of comb, crushed and processed honey was 6.05± 2.3, 8.9±1.8 and 11.8±1.5mg/kg respectively (Ta- ble 2). The Hydroxymethylfurfural (HMF) content of honey is used as an indicator of heating temperature and/or pro- longed storage and it is recognized as an indicator of honey freshness. The findings of this study were under the require- ments of the international Codex [8] and national ES [17], honey quality standards which set maximum of 40 mg/kg. There was a significant difference (p<0.05) in HMF content among the honey samples. The result of the present study of comb, crushed and processed honey was in agreement with Debela H. and Belay A. [15], reported 6.12 ± 2.14 mg/kg for Schefflera abyssinica and 4.37 ± 1.83 mg/kg for polyfloral honey of Ethiopia. Schievano et al., [42] reported an HMF value of 26.2 mg/kg for Coffea arabica honey, which was higher than in the current study.

Water insoluble matter

The mean ± SD of water insoluble matter of the present study for comb, crushed and processed honey samples was 0.13±0.04, 0.16±0.08 and 0.23±0.18 g/kg, respectively. The result of the present study of insoluble matter to combs, crushed and processed honey samples were found to be more than the permitted value by Codex [8], 0.1g/100g. The measurement of insoluble matter is an important means to detect impurities in honey. The mean water insoluble matter content of processed honey samples was significantly higher than that of comb and crushed honey samples. The variation may be due to differences in harvesting practice, processing and storage conditions. The probable water-insoluble solids in honey include wax, pollen, propolis, and other particles of debris.

Antioxidant content

Total phenolic content (TPC)

The mean ± SD of total phenolic content for comb, crushed and processed honey samples was 120.8 ±47.4, 84.3±51.86 and 66.7±30.59 GAE/100g, respectively (Table 2), and a significant difference (p<0>et al., [39], for the phenolic content of monofloral honey (66.45 ± 15.4) and polyfloral honey (59.37±13.3 mg GAE/100g). The phenolic content of the present study was in line with the re- port of Alisi et al. [40] (106 to 130 mg GAE/100g) for Nigerian honey. Total flavonoid content (TFC) The mean ±SD values of flavonoid content are 53.7 ± 24, 29.46 ± 14, and 29.47±11.22 CEQ/100g for comb, crushed, and processed honey samples, respectively. There was a significant difference (p<0.05) among the honey samples. The TFC values of the present study were found to be higher at 2–80 mg CEQ/100g of honey. This is in agreement with the report of Debala H. and Belay A. [15], (2.03±1.49 and 31.07±1.31 CEQ/100g) for Schefflera abyssinica and Vernonia amygdalina honey, respectively. DPPH radical scavenging activity In this study, the DPPH radical scavenging activity of honey was identified for comb 30.9 ± 14.91, crushed 33.45 ± 5.07 and processed honey 33.98 ± 1.84 % (Table 2). Significant differences (p>0.05) were not observed in DPPH radical scavenging activity among the honey samples. The finding of this study is in line with the report of Hailu and Belay [30], who found 44.43 ± 0.97 for Schefflera abyssinica and 37.93 ± 1.14% for polyfloral honey from Ethiopia. Moreover, the result of the present study is nearly similar to the report of Goslinski et al. [41], 40.0 ± 0.3% DPPH scavenging activity value for New Zealand Manuka honey. The unpaired electron of DPPH forms a pair with hydrogen donated by a free radical scavenging antioxidant from honey. Hydrogen peroxide scavenging (H2O2) The hydrogen peroxide scavenging potentials of combs, crushed, and processed honeys were 57 ± 16.06, 52.99 ± 15.9, and 56.61 ± 4.86 % inhibitions, respectively (Table 2). There was a significant difference (p<0.05) between the hon- ey samples. In this study, comb honey samples had higherhydrogen peroxide scavenging activity than crushed and processed honey. The current study result was in agreement with that reported by Hailu and Belay [30], 78.00 ± 4.82 and 67.22 ± 2.93 % for Schefflera abyssinica and polyfloral honey, respectively. The hydrogen peroxide concentration of New Zealand Manuka honey ranged from 16.1 ± 1.2 to 95.8 ± 9.1 Juraj et al. [43], which is in agreement with the current research. Hydrogen peroxide plays a key role in honey's antibacterial activity. The production of H₂O₂ in honey requires glucose oxidase (GOx) that oxidizes glucose and produces hydrogen peroxide [43]. Inhibition concentration The DPPH IC50 value of comb, crushed, and processed honey samples was analyzed with a concentration range of 20–140. The average DPPH IC50 values of comb, crushed, and processed honey in the present study were 54±5.72, 80±6.25and 120±8.25 mg/ml, respectively (Table 2). A lowerIC50 concentration in honey indicates a higher ability to neutralize free radicals [24]. There were significant differenc- es among the honey samples in terms of their scavenging abilities, expressed as IC50 of the DPPH radical-scavenging activities. The increase in concentration shows an increase in the scavenging value of honey samples. The findings of this study on comb, crushed, and processed honey were in agreement with the report of Hailu and Belay [30] 134.60 ± 8.66 for Schefflera abyssinica and 152.84 ± 8.25 mg/ml for polyfloral honey from Ethiopia. Rheological properties In this rheological property study of honey samples, dynamic viscosity with fluidity was measured in a temperature range of 20–50°C with a constant shear rate of 50 s-1. As presented in Table 3, the average viscosity of comb, crushed, and pro- cessed honey samples as a function of temperature is found to be 4125, 2506, and 1543 Pa.s, respectively. The current study's comb, crushed, and processed honey's temperature sensitivity was evaluated by sketching the logarithmic of viscosity (Pa s) versus the reciprocal temperature (1/T) and the results were fitted using the Arrhenius model (Figure 2). The viscosity of all analyzed honey samples decreased with increasing temperature (Figure 2). The model adequatelydescribed the relationship between honey viscosity and temperature. In line with this, an exponential dependence of honey viscosity on temperature was evaluated with the use of regression analyses, and the coefficient of determination for comb, crushed, and processed honey samples was r² = 0.874, r² = 0.853, and r² = 0.877, respectively (Table 3).

Table S3: Antioxidant analysis of comb, crushed, and processed honey. AE = Gallic Acid Equivalent, CEQ = Qurecetin Equivalent SCV=Scavenging value

The temperature dependence of the honey viscosity in the present study agrees with the reports of James et al. [44], for North-Central Nigeria honey, Oroian et al. [45] for Romanian honey; and Belay et al. [46] for Ethiopian monofloral hon- ey. Similar observations to those of the present study were reported by Sopade et al. [47] for Australian honey, Yanni- otis et al., [48], which reported that the temperature effect on viscosity is strong at lower temperatures. The Arrhenius relationship, which states that the higher the temperature, the greater the chemical reaction and molecular mobility of viscous fluids, is what causes the change in viscosity with an increase in temperature [49]. The average viscosity of each honey sample was higher at an initial 20°C and decreased as the temperature increased (Table 3). The viscosity of combs and crushed and processed honeys obtained by fitting of the experimental data as a function of temperature is presented in Table 3). At all operating temperatures (20, 30, 40, 50 o C), the highest viscosity was observed in the comb, and the lowest was in processed honey. Generally, as the temperature increases, the average velocity of the molecules in honey increases, and the amount of contact time with adjacent molecules decreases. Gomez et al. [49] and thus, the average intermolecular forces decrease and the viscosity decreases. The effect of temperature was found to be more pronounced up to 30°C. However, at temperatures above 30°C, the dif- differences in viscosity were very small in most of the analyzed honeys. At higher temperatures, the difference in viscosity among three honey types decreases but still exists even at 40°C, which could be attributed to the natural variations in composition (sugar and water content) (Yanniotis et al. [48]. The analyzed honey samples of the present study exhibited Newtonian behavior (Figure 3), which was described by the linearity of shearing time (1/s) and viscosity (Pa.s).

Figure S2: Calibration graph of Fructose

Figure S3: Calibration graph of Glucose in g/100g Most honeys are Newtonian fluids. Dobre, et al., [50], Oroian et al. [45] characterized it by constant viscosity at a fixed temperature, which can be described by Newton’s law for flow. The finding of the study was in agreement with the report of Vanelle et al. [51] for Brazilian honeys and Mahder and Belay et al. [52]. Sensory test The sensory attributes and descriptors of honey, like color (light brown, dark brown), appearance (color, fermentation, viscosity), flavor (odor, taste), and taste (mouthfeel, body, aftertaste), floral fruits (orange, rose), fresh (eucalyptus, mint), warm/aroma (caramel, smoked), and spoilage (teji) of comb, crushed, and processed honey were presentedin Table 4. The means ± ±SD of comb honey samples for light brown (1.56 ± 0.74), dark brown (5.49 ± 0.76), orange (2.66 ± 1.32), rose (2.81 ± 1.47), mint (2.70 ± 1.16), eucalyptus (4.98 ± 0.89), sweet taste (5.24 ± 1.06), body (5.49 ± 0.78), aftertaste (5.02 ± 0.72), caramel (3.25 ± 1.39), smoked (3.09 ± 1.75), and spoiled teji (1.54 ± 0.70) are in Table 1. The means ± SD of crushed honey samples were found to be light brown (3.29±1.12), dark brown (3.16±1.18), orange (3.01±1.36), rose (3.10±1.38), mint (2.87±1.13), eucalyptus (4.33±0.98), sweet taste (4.58±1.05), body (3.54±1.13), aftertaste (3.42±0.94), caramel (3.15±1.23), smoked (2.92±1.47), and spoiled teji ji (2.43±0.72). The mean ± SD value of processed honey samples was found light brown (5.04±0.76), dark brown (2.55±1.06), orange (3.02±1.33), rose (2.98±1.52), mint (2.45±1.15), eucalyptus (4.01±0.83), sweet taste (4.52±0.89), body (3.36±1.14), after taste (3.27±1.13), caramel (3.68±1.24±1.24), smoked (2.68±1.34), and spoiled teji (3.34±0.94) (Table 4). There was a significant (p<0.05) difference between the comb honey taste (4.05±0.825), odor (4.05±0.759), color (4.30±0.732), and aroma (3.75±0.550) reported by et al. [53].

Table S4: The Viscosity of comb, crushed and processed honey samples at different temperatures (20-50°C)

Association between variables

The physicochemical and antioxidant properties of comb, crushed, and processed honey samples were correlated by Pearson correlation (Table 5). Moisture and sucrose were found to be positively correlated (r = 0.88). This is based on the principle that the moisture and sucrose content of honey are widely related to the level of maturity and the harvesting season. A significant (p<.001) inverse association was observed between diastase activity and sucrose content (r= -0.86). This is based on the prin- ciple of early maturity at harvest and adulteration practice. Correlations were observed between total phenolic content and hydrogen peroxide scavenging activity (r=0.82), total flavonoids and total phenol content (r=0.61). The moisture and sucrose content of comb,crushed, and processed honey from the Burie district correlated significantly (p<0 r=0.88), r=663).>