Effects of Various Adulterants Materials on Properties of Beeswax

Deressa Kebebe; Meseret Gemeda; Teferi Damto; Gemechis Lagasse

doi:doi:10.11648/j.ab.20251302.15

Research Article |

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Effects of Various Adulterants Materials on Properties of Beeswax

Deressa Kebebe^*

, Meseret Gemeda

, Teferi Damto

, Gemechis Lagasse

Published in Advances in Biochemistry (Volume 13, Issue 2)

Received: 19 February 2025 Accepted: 22 April 2025 Published: 30 June 2025

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Abstract

Beeswax is one of the best valued bee products and its demand is high these times in the world market. Adulteration of beeswax with inexpensive materials has become a challenge for its quality and marketing. The objective of this study was to characterize the physicochemical properties of adulterated beeswax and then identify better methods to detect adulterated beeswax. In this paper, the properties of beeswax-adulterant mixtures, such as beeswax with candle, beeswax with animal tallow, beeswax with soap, beeswax with maize flour, beeswax with lime stone, beeswax with salt, and beeswax mixed with powder of “kocho” in different ratios were prepared based on previous survey study results and quality parameters were analyzed using the official methods. The results indicated that the density of beeswax adulterated with different adulterants such as soap, animal tallow, limestone, “kocho”, salt, candle and maize flour for different proportions were significantly different at p<0.05. Parameters such as acid value of animal tallow, saponification point, ratio number and ester value of maize flour, ratio number, acid value and saponification point of “kocho”, melting and saponification point of candle, ash content of lime stone, melting point, ash value and saponification point for salt, and ash content and ratio number for soap were found to be better methods to identify beeswax adulterated with the respective adulterants mentioned above. It is important to characterize beeswax quality parameters using more sensitive analytical instruments. Pure beeswax is important for bee’s colony health and to attract good market.

Published in	Advances in Biochemistry (Volume 13, Issue 2)
DOI	10.11648/j.ab.20251302.15
Page(s)	51-60
Creative Commons	This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited.
Copyright	Copyright © The Author(s), 2025. Published by Science Publishing Group

Keywords

Adulterants, Adulterated Beeswax, Demand, Kocho, Pure Beeswax

1. Introduction

A natural wax that worker bees secrete from glands under their abdomen is called beeswax. Beeswax is secreted by the honey bee workers in the form of wax scales and it is further being chewed by their mandibles which results in the transformation of texturally anisotropic scale wax into isotropic comb wax

[1]

. The term beeswax is often limited to wax produced by honeybees (Apis species) and many would specify Apis mellifera L. as a source

[2]

. It is one of the most valuable bee products and it is also one of the oldest items used by mankind

[3]

. Beeswax, with its unique characteristics, is now being used in the development of new products in various fields such as cosmetics, foods, pharmaceuticals, engineering and industry

[4]

. Specifically, most of the wax produced nowadays are used in the manufacture of cosmetics, such as hand and face creams, lipsticks and depilatory wax and many other uses. Moreover, the pharmaceutical industry uses the wax in various ointments, for coating pills and suppositories and other miscellaneous industrial products

[5]

. Bee wax is widely used in many branches of the national economy

[6, 7]

, as well as in metallurgical, electrical, chemical, pharmaceutical

[8-10]

food industry, in particular in the production of cheese and sweets, as well as the coverage of fruits and vegetables in order to increase their shelf life

[11, 12]

Some physico-chemical parameters used to analyze oils and fats and described in the official methods of analysis from many pharmacopoeias and countries are commonly applied to evaluate the beeswax quality and detect possible adulterations

[13]

. Melting point, acid, saponification, ester and ratio number values are the most commonly determined parameters, whereas the peroxide value and iodine absorption number have also been considered in a few works

[14, 15]

. The proposed value ranges for the parameters in pure beeswax differ from one country to another

[16, 17]

. These differences could be related to the point of origin of the beeswax because the environmental and geographical factors play a significant role in the bee adaptation and, as a result, in beeswax composition

[14, 15]

The relatively high cost of beeswax in comparison with other industrial waxes and lower-price products, such as microcrystalline wax or paraffin, tallow, stearine and stearic acid, promotes its adulteration with these mentioned fatty products, which is also favored by a lack of regulations

[2]

. Adulteration of beeswax with these cheaper materials has become a problem for beeswax quality and its marketing, especially adulteration of beeswax with paraffin

[2, 18]

. In Ethiopia, beeswax adulteration has been reported repeatedly and is becoming a critical problem

[19, 20]

. These investigations indicated that materials used to adulterate beeswax include animal tallow/fat, candle/paraffin, soap, maize flour and lime stone

[20]

The presence of adulterants in beeswax affects the values of some physicochemical parameters of the wax

[2, 14, 15, 21]

. Thus, several authors have discriminated the adulterated beeswaxes according to the anomalous values of some analytical parameters, for instance the melting point, the saponification value or the iodine value

[15]

. Physicochemical measurements like density, melting and saponification values, ester value, ratio number and iodine value have helped to detect adulteration of beeswax with paraffin, stearic acid and animal tallow

[22]

. Similarly, a melting point and saponification cloud point could equally serve in the detection of as little as 2% animal tallow used in beeswax adulteration

[19]

. However, as reported by

[20]

, more adulterant materials have been utilized by the actors and the techniques of adulteration are also ever changing. Therefore, physical and chemical characterization of the adulterated beeswax using different adulterants at different proportions is important to identify which measurements are more appropriate to be employed for a given beeswax adulterant material and at what proportion level it should be applied. In this study, adulterant materials identified in earlier survey works were used to adulterate beeswax deliberately in different proportions of mixing and beeswax physical and chemical parameters were quantified to see how the adulterants were affect their measurements. The objective of this study was to characterize the physicochemical properties of pure and adulterated beeswax, then identify better methods to detect adulterated beeswax.

2. Materials and Methods

2.1. Beeswax Sample Collection and Purification

Crude beeswax were collected directly from beekeepers found in West, Southwest and Central zones of Oromia region in Ethiopia in two years, from 2022 to 2023 based on the time of availability. Pure and natural beeswax samples were obtained by extracting the crude beeswax using submerged beeswax extracting method by purifying by boiling in water and filtering using cheese cloth, method of beeswax processing.

2.2. Adulterant Material Collections

The adulterant materials were those reported by

[20]

. These include animal tallow, candle, soap, maize flour and lime stone will be acquired from the local market and other available sources.

2.3. Preparation of the Adulterant-beeswax Mixtures

The mixtures of pure beeswax and adulterants were prepared in proportions identified by survey work earlier. The adulterants were candle, maize flour, animal tallow, limestone, soap, salt, and “kocho”. For each adulterant material, five levels of mixtures, 1:1, 2:1, 3:1 and 4:1, including pure beeswax were prepared in the respective orders of pure beeswax for the adulterant and analyzed for each parameter indicated. To achieve a good mixture and homogeneity in the sampling, the beeswax-adulterant mixtures were heated in an aluminum vessel up to the total melting of the mixture, which required a temperature of about 70 ^°C and a time close to 5 min. After that, the mixtures were allowed to cool at room temperature for 24 hours and kept at room temperature and in darkness until their analysis.

2.4. Determining the Physicochemical Parameters of Pure Beeswax, and Beeswax Adulterated with Different Adulterants

2.4.1. Density

A calibrated pycnometer was weighed empty first and then full of water. Both determinations were made at 25°C and in triplicate to calculate the mass mean values. Then, an amount of 10.0 + 0.1 mg of beeswax was placed into a beaker containing water. As beeswax is less dense than water, the first one floats on the surface of the liquid. Next, small volume of methanol was added from a burette until beeswax was suspended in the liquid. At that moment, the density of the liquid matched up with that of the beeswax sample, so the beeswax density (in g/mL) at a temperature of 20°C could be determined with the pycnometer by means of the expression:

Density=0.

9982 \frac{[(m 1 - m 2) + A]}{m 3 - m 2 + A}

Where, A = 0.0012 (m1-m2), with m1 being the mass, in g, of the pycnometer containing the hydroalcoholic mixture; m2 the mass, in g, of the empty and completely dry pycnometer; and m3 the mass, in g, of the pycnometer containing ethanol. Three different portions of the beeswax sample was weighed to calculate the corresponding density values and an average for each sample.

2.4.2. Saponification Value

A beeswax amount of 0.300 + 0.001g was weighed and placed into a 10-mL glass vial. Then, 4 mL of a 4 M NaOH aqueous solution was added to the vial. The vial will be closed and put into an oven to carry out the saponification at 100°C for 1hour. The solution, still hot, was titrated with a 0.5 M HCl solution, using phenolphthalein as an indicator and constant manual shaking. A blank assay was also conducted. The determinations were made in triplicate. The saponification value, expressed as mg KOH/g, was calculated as follows:

Saponificationvalue=

56.1 \frac{M (V - V^{'})}{W}

where V is the volume, in mL, of HCl solution required by the blank;

V^{'}

is the volume, in mL, of HCl solution required for the sample; M is the molarity of the HCl solution; and w is the mass, in g, of the beeswax sample.

2.4.3. Acid Value

A beeswax amount of 1.00 + 0.01g was dissolved in 50mL of chloroform with the help of an ultrasonic bath. Then, two drops of phenolphthalein were added and the solution was titrated with 0.05 M NaOH in methanol with continuous manual shaking until the indicator turns. A blank assay was also titrated to correct solvent acidity. The acid value (in mg NaOH/g) was calculated by the formula:

Acidvalue=

56.1 \frac{M (V - V^{'})}{W}

where V is the volume, in mL, of NaOH solution in methanol required by the sample;

V^{'}

is the volume, in mL, of NaOH solution required for the blank; M is the molarity of the NaOH solution; and w is the mass, in g, of the beeswax sample. The NaOH solution was previously normalized against an aqueous solution of 0.05 M potassium acid phthalate with phenolphthalein as an indicator. The analyses were also done in triplicate.

2.4.4. Ester Value and Ratio Number

The ester value was determined by subtracting the acid value from the saponification value, and the ratio number was calculated as the ester value divided by the acid value.

2.4.5. Melting Point

This index was determined following the “capillary tube method.” Melted beeswax was introduced in a 10 cm long x 2 mm internal diameter thin-wall hollow capillary tube, until reaching a height of about 1 cm. Once the beeswax solidified, the tube was kept at room temperature for about 24 h. After that, the capillary tube containing the beeswax was introduced into a water bath that was slowly warmed at 1-2°C/min; the temperature was checked with a thermometer, whose bulb had to be as close as possible to the beeswax column introduced in the capillary tube. The melting temperature was that at which the beeswax was completely molten; the beeswax liquid is entirely transparent without turbidity.

2.4.6. Ash Content

To tare the porcelain capsules, they were kept in the muffle at 600°C for 30 minutes then cooled within a desiccator until they reached a constant weight. An amount of 2.000+ 0.001g was placed in the capsules, heated prudently until inflammation point in the muffle port and finally heated at 600°C in the muffle for 1.5 h. After that, the capsules with the ashes were cooled in a desiccator until they reached a constant weight. The ash percentage will be determined as follows:

Ashcontent=

100 \frac{(w 1 - w 2)}{W}

Where W1 is the mass, in g, of the porcelain capsule with the ash content; W2 is the mass, in g, of the empty capsule; and w is the mass, in g, of the beeswax sample. Sample analyses will be done in triplicate.

2.5. Data Management and Analysis

Data was entered into Microsoft excel sheet and transported to analysis software. Multiple variance analysis (MANOVA) was run with SPSS software. Each type of beeswax-adulterant mixture was considered an independent variable.

3. Results and Discussion

In this paper physicochemical properties of the beeswax-adulterant mixtures (beeswax with soap, beeswax with maize flour, beeswax with lime stone, beeswax with salt, beeswax with “kocho”, beeswax with animal tallow and beeswax with candle in different ratios were prepared and analyzed for density, ash content, melting point, ester value and ratio number, acid value and saponification value. The results are shown in Tables 1, 2, 3, 4, 5, 6 and 7 for beeswax-candle, beeswax-animal tallow, beeswax-soap, beeswax-lime stone and beeswax-“kocho” adulterated material laboratory analyis.

Table 1. Results of physicochemical properties of beeswax-candle mixtures (Mean±SD).

Beeswax-candle ratio	ρ (g/cm³)	AC (%)	MPt (°C)	EV	RN	AV	SV (°C)
1:1	0.85±0.003^c	0.148±0.04^abcde	62.00±0.00^cbd	87.33±28.50^ab	2.58±1.39^c	15.43±0.84^bcd	102.85±28.05^ab
2:1	0.322±0.001^e	0.367±0.14^abc	59.75±0.25^e	99.48±9.35^a	5.80±2.44^a	13.37±0.00^e	28.05±9.35^d
3:1	1.135±0.004^a	0.49±0.28^a	63.00±0.00^bc	57.33±18.50^d	1.41±0.45^e	16.27±1.40^b	74.80±18.70^abc
4:1	0.44±0.003^d	0.44±0.02^ab	63.00±0.00^b	27.00±9.00^e	5.08±1.19^ab	18.51±0.56^a	46.75±9.35^de
Natural beeswax	0.96±0.001^b	0.33±0.13^abcd	64.75±0.25^a	33.00±4.04^abc	2.28±1.61^cd	15.29±0.98^bc	125.62±29.04^a
ESQA	-	-	61-66	70-80	-	17.0-24.0	-

Different letters down the column show significant difference (p =0.05). SD=standard deviation, ρ = density, AC= Ash content, Mpt = melting point, EV=Ester value, RN=ratio number, AV=acid value, SV= Saponification value. ESQA =Ethiopian Standard and quality Authority.

Table 2. Results of physicochemical properties of beeswax- animal tallow mixtures (Mean±SD).

Beeswax- tallow ratio	ρ (g/cm³)	AC (%)	MPt (°C)	EV	RN	AV	SV (°C)
1:1	0.473±0.003^e	0.34±0.003^b	59.50±0.50^e	98.00±9.00^ab	4.4925±3.13^ab	47.70±2.80^a	102.85±9.35 ^abcd
2:1	0.699±0.005^d	0.22±0.04^bcde	62.00±0.00^bc	99.33±9.50^a	2.7210±2.11^abc	23.42±0.14^b	102.85±9.35^abcd
3:1	0.662±0.004^c	0.24±0.08^bcd	62.75±0.75^b	67.67±29.50^abcde	4.6246±0.36^a	14.59±0.56^e	105.75±19.20^abc
4:1	0.756±0.001^b	0.76±0.73^a	61.75±0.25^bcd	93.67±19.50^abc	1.0544±0.39^abcde	15.43±0.28^c	112.20±18.70^ab
Natural beeswax	0.959±0.001^a	0.33±0.13^bc	64.75±0.25^a	86.33±4.04^abcd	2.28±1.61^abcd	15.29±0.98^d	125.62±29.04^a
ESQA	-	-	61-66	70-80	-	17.0-24.0	-