Physicochemical properties of Ethiopian Beeswax, the case of South Wollo zone, Amhara Region

The study was conducted in three purposively selected districts of South Wollo Zone, Tehulederie, Kalu and Dessie Zuria, intended to analyze the physicochemical properties of beeswax produced in 2015/6. Twenty-six beeswax samples were collected being sourced from honey extract, ‘Tej sefef’, old combs and purchased beeswax blocks and analyzed at Sekota Dry land Agricultural Research Center laboratory according to the standard protocols of Ethiopian Beeswax specifi cation ET-1203-2005 developed by Quality Standard Authority of Ethiopia in 2005. The laboratory result showed that the compositional content of beeswax collected from the study areas falls within the range of good quality parameters set for national and world standards. Except for ash content, there is no signifi cant compositional content variation (P>0.05) between the sampled districts. However, there is signifi cant variation (P<0.05) between beeswax samples based on the source from which they were obtained. The mean values are specifi c gravity (0.9552±0.0034), melting point (61.5628±1.50080C), refractive index (1.4439±0.0004), ash content (0.0345±0.0429%), total volatile matter (0.5491±0.2488%), acid value (18.9155±2.7735), saponifi cation value (91.1901±22.3015), ester value (72.0619±20.2859), and ester to acid ratio (3.7211±0.8569). However, melting point, saponifi cation value, ester value and ester to acid ratio values of purchased beeswax samples and beeswax samples from old combs of absconded colonies showed lower result than the national and international limit. In general, this study identifi ed physicochemical properties of the beeswax in the study areas and contributed to the information on beeswax quality of Ethiopia suggesting legal intervention on controlling purchasing of beeswax in bulk. Research Article


Introduction
Ethiopia is a home of diversifi ed fauna and fl ora, which favor the existence of a number of bee colonies and bee subspecies. In Ethiopia, about 1.9 million farm households are involved in beekeeping and there are about 10 million colonies out of which about 5.92 million are hived [1], and it is estimated that the country has the potential to produce 500,000 tons of honey and 50,000 tons of beeswax per annum.
However, currently the country produces 50,790.58 tons of honey in 2015/6 [1], and about 5,344 tons of beeswax in 2013 [2]. This shows that the country is still producing only about 10% of its potential. Beeswax is one of the most valuable and oldest bee products primarily used to construct foundations in beekeeping [3,4]. Besides, humankind is still using it in various fi elds such as cosmetics, foods, pharmaceuticals, engineering and industry [5]. The quality of beeswax is one of the main concerns of apiarists and a determinant factor in the beekeeping development. Generally, beeswax product quality has always been low, leading to high domestic utilization and low export earnings. Besides, due to its high demand in the global market, adulteration of beeswax with cheaper materials and lack of traceability turn out to be common challenges in beeswax quality [6]. On the other hand, due to prolonged over heating throughout rendering, there has been report of quality deterioration and compositional alteration of natural beeswax [7]. Hence, the beekeepers in particular and the country, in general, were not benefi ting from the sector. For that reason, to take the advantage of opportunities from beeswax, interventions to verify the quality of beeswax produced are very crucial. Beeswax is a natural product, no additives are permitted, and it needs no longer heating or higher temperatures that lead to greater degradation and loss of esters. Beeswax is an extremely complex material containing over 300 different substances [8]. It consists mainly of esters of higher fatty acids and alcohols and small quantities of hydrocarbons, acids and other substances. In addition, approx. 50 aroma components have been identifi ed. Currently, adulteration and contamination are main quality issues [9].
Quality control of beeswax is important to determine its suitability for processing and to meet the market demand.
Examination of the sensory characteristics (e.g. odor and color) of beeswax allows a simple and quick quality check but this does not guarantee that the beeswax has not been adulterated. Thus, determination of physicochemical characteristics at laboratory is signifi cant in this regard. Therefore, with this rationale, this study was designed to analyze the physicochemical properties of beeswax produced in the study area with the purpose of confi rmation against physicochemical indicators of national and international standards.

Description of the study area
South Wollo zone is one of the 11 zones of Amhara region, having an area of 17,067.45km 2 [10], located 10.20 0 -11.71 0 N and 38.41 0 -40.02 0 E North of Ethiopia [11], whose main capital is Dessie town. The zone consists of 20 districts from which the three districts namely, Tehulederie (midland), Kalu (lowland) and Dessie Zuria (highland) purposively selected for this study based on the beekeeping potential, accessibility and their proximity to honey and beeswax marketing and processing routs. The zone has a long-term mean (1162mm) rainfall per annum. The monthly minimum and maximum temperature is 12.6 0 C and 26.4 0 C respectively.

Beeswax Sample Collection and Preparation
Twenty-six beeswax samples were collected from three agro-ecologically different districts (Tehulederie, Kalu and Dessie zuria) from December 2015 to March 2016. The collected beeswax samples were extracted from honey purchased from beekeepers and local market, old combs collected from absconded colonies, 'Tej sefef' purchased from local honey mead houses and beeswax blocks purchased from verandah. Crude beeswax samples of 1kg were made from each source. Before physical and chemical analysis, all beeswax samples were rendered, refi ned and purifi ed. Physical and chemical analysis was done at Sekota Dry land Agricultural Research Center laboratory following the protocols of the Ethiopian Beeswax Specifi cation ET-1203-2005 [12]. All chemicals and reagents used were analytical grade.

Specifi c gravity at 200C
Approximately 2g of the beeswax sample melted in a porcelain crucible at a temperature of about 100 0 C and allowed to cool to room temperature. The sample weighed suspended with a tarred thread after it was stored for 2 hours at a temperature of 20 ±1 0 C. The mass of the sample was determined, fi rst in air and then in rectifi ed spirit maintained at 20±1 0 C. The specifi c gravity at 20 0 C/20 0 C of the rectifi ed spirit was measured by means of the specifi c gravity bottle to determine the specifi c gravity of the beeswax sample.

Melting point, 0C
The melting point is an important physical property of beeswax used to identify as an indication of its purity.
The melting point of solid is defi ned as the temperature at which the solid exists in equilibrium with its liquid under an external pressure of one atmosphere [9]. The beeswax sample was melted and a capillary tube was dipped into melted beeswax and let stay for 24 hours. The capillary tube inserted into melting point apparatus (with digital thermometer indicator, model LMP-11). After the sample has attained the melting temperature, the melting point read and recorded. Each beeswax sample was analyzed in triplicate.

Refractive index at 750C
The refractive index determination is a method to measure the ratio of the velocity of light in air to that in the sample. Bench top digital ATAGO ® Abbe refractometer was used to measure refractive index. The sample melted and fi ltered through fast fi lter paper to remove any impurities and last traces of moisture. The temperature of the refractometer was adjusted at 75±1 0 C by circulating water from the water bath. Few drops of the sample were placed on the lower prism and the prism closed tightened fi rmly allowed to stand for one to two minutes. After the sample has attained the test temperature, the reading of refractive index of the sample recorded.

Ash content, % by mass, max
The platinum dish was heated to redness, cooled to room temperature in a desiccator and weighed. About 50g of the material was taken in a watch-glass and weighed accurately. About three-quarter of this quantity was transferred to the platinum dish and heated on a hot plate so that the material burns gently at the surface. When about half of the material is burnt away, heating stopped, cooled and the remainder of the material was added. The dish heated again as before, until the material completely charred. After that, the material was incinerated in a muffl e furnace at 550°C to 650°C for 1 hour, cooled to room temperature in desiccators and weighed. Incineration, cooling and weighing were repeated until the difference between two successive weighing was less than one milligram. The ash content of the sample calculated with the following formula.

Ash, percent by mass
M2= mass in g of the ash, and Ml = mass in g of the material taken for the test.

Total volatile matter, % by mass, max
About 10g of the material was weighed accurately in a suitable dish, previously dried and weighed, and placed in an oven maintained at 105 2 0 C for 6 hours. After 6 hours, the dish was cooled in a desiccator and weighed. The dish heated again in the oven for 30 minutes. The process repeated until the loss in mass between two successive weighing was less than one

Acid value, max
The material was mixed to make entirely liquid and accurately about 5 g of the material was weighed in a 250-m1 conical fl ask. 75 ml of a mixture of two parts of benzene and one part of rectifi ed spirit was added. The sample was heated under refl ux until it dissolved, allowed to cool to room temperature and titrated with standard potassium hydroxide solution using phenolphthalein as indicator until pink color is observed. The acid value (in mg KOH/g) was calculated by the following formula.

Saponifi cation cloud value, min
The saponifi cation value is the number of milligrams of potassium hydroxide required to hydrolyze 1g of sample beeswax. Determining the saponifi cation cloud point is an easy, sensitive and best method for determining adulteration of beeswax. However, the method is limited to detecting quantities greater than 1% of high melting point (80-85 °C) paraffi n waxes, or more than 4-5 % of low melting (50-55°C) paraffi n [9].
Accurately about 2g of beeswax was weighed in a tarred conical fl ask, 25 ml of methyl ethyl ketone added, followed by 25 ml of alcoholic potassium hydroxide solution. Few pieces of pumice stone were added and the refl ux condenser was connected to the fl ask. The fl ask heated on a water-bath or electric hot plate for about 2 hours to boil steadily but gently. The inside of the condenser washed down with about 10 ml of rectifi ed spirit after the fl ask and condenser have cooled. 1 ml of phenolphthalein was added and the residual potassium hydroxide was titrated with 0.5 M standard hydrochloric acid. A blank assay or titration was also performed with 25 ml of 0.5 M alcoholic potassium hydroxide. The following formula was used to determine the Saponifi cation Value according to

Ester value
The ester value calculated as the acid value determined subtracted from the saponifi cation value.

Ester value = Saponifi cation value -Acid value
Ester to acid ratio The ester to acid ratio calculated by dividing the ester value to the acid value.

Data management and Statistical Analysis
The obtained data were analyzed using SAS software

Specifi c gravity at 200C
The specifi c gravity of sample collected from the study areas ranges from 0.9485 to 0.9624 with a mean value of 0.9552 (

Melting point, 0C
The average melting point of each beeswax sample from the study area recorded as 61.56 0 C ranging from 58.3 to 68.5 0 C (

Refractive index, at 750C
The mean refractive index of beeswax samples collected from the study areas was 1.4439 at 75 0 C ( Table 1) and this agree with fi nding of [13], that reported similar fi gure of refractive  Table 2). Generally, the current study indicated that the result was within the limits of Ethiopian and International quality standards, ( Table 2) showing it was free of contamination.

Ash content, % by mass, max
Ash content determination of beeswax is important because it represents its mineral content [14]. The mean value for ash content of current study result (0.0345%) is lower than the maximum limit set by the Ethiopian standard (0.2% by mass) fulfi lling the national requirement [12]. This implies that, on the other hand, there was a signifi cant difference (P<0.05) in ash content between beeswax from Tehulederie (0.5794) and Dessie Zuria (0.0173) ( and honey extract (0.0213) ( Table 2). The higher ash content in old combs might be due to the higher accumulation of minerals because of repeated brood rearing (aging) and there is a fi nding that shows mineral element traces in honey and wax were signifi cantly correlated with comb age [16]. Similarly, [14] reported ash content of 0.0367 and 0.0267 for honey extracted beeswax and 'Tej sefef' respectively.

Total volatile matter, % by mass, max
In beeswax, volatile matters are those substances, other than moisture, that is given off as gas and vapor during combustion in the dry oven out of air contact. The total volatile matter of the collected beeswax samples ranged from 0.2331 to 1.2450% with the mean value of 0.5491% ( showed lower value as compared to the required national and international limit, indicating that beeswax from these sources contains less amount of saponifi able matter. Generally, the overall mean ester value result of the current study meets the quality standard limits of national and international (Table 2).

Ester to acid ratio
The ratio of ester values to acids, a parameter determined in the pharmacopoeia gives information whether pure natural beeswax is changed signifi cantly by prolonged or excessive heating leading to greater degradation and loss of esters [7].
According to this study, the mean value of ester to acid was 3.7211 ranging from 2.1328 -4.9360 (Table 2) and this result agree with [4,12] that reported ester to acid ratio of 3.64 and 3.38 for Holeta and Bale natural forest respectively. The eater to acid ratio of beeswax samples collected from honey extract (4.0615) was signifi cantly (P<0.05) higher than that of purchased beeswax block samples (2.7035) ( Table 2) suggesting beeswax is changed signifi cantly by prolonged or excessive heating. This is because longer heating or higher temperatures lead to greater degradation and loss of esters (Bogdanov, 2016

Conclusions and Recommendations
Based on this study fi ndings, the overall mean values of the compositional content of beeswax in the study areas falls in the range of good quality compared to national and world standards set for beeswax quality determination. Furthermore, the study identifi ed beeswax samples which were obtained through purchase as blocks from city markets as having lower melting point, saponifi cation, ester and ester to acid ratio values suggesting lower quality due to existence of foreign materials. This study identifi ed the physicochemical properties of beeswax in South Wollo zone of Amhara region and contributed to increased knowledge keeps all the stakeholders vigilant on its development, quality and market.