Avocado seed starch was extracted to prepare a biodegradable plastic film with enset cellulosic reinforcement and glycerol plasticiser. The characterisations were made in detail. Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimeter (DSC), scanning electron microscopy (SEM), X-ray diffraction (XRD), rheometer, and Rapid Visco Analyser (RVA) techniques were employed to characterise the physicochemical properties of avocado seed starch, enset cellulosic, and the blend. Starch and enset cellulosic extraction yields are 18.3 ± 0.02% and 67.5 ± 0.08%, respectively. As a result, avocado seed starch and enset cellulosic blend can be used as alternative raw materials to develop biodegradable plastics.

1. Introduction

The avocado (Persea americana Mill.) is native to Central America. Mexico is the world’s largest producer of avocado, with a total production of more than three million metric tonnes per year. Avocado is a popular fruit that is enjoyed in many nations. As a result of this, consumers, avocado processing industries, juice houses, and local traders generate a huge amount of avocado waste. Waste disposal is becoming increasingly challenging due to environmental norms and a lack of landfill space. Avocado seed, on the other hand, is starch-rich and can be enriched and have value added. Starch is widely employed in a variety of industrial applications and is frequently derived from various agricultural crops; thereby, conventional starch sources and industrial demand can be destressed.

Natural cellulose fibres are environment-friendly and used largely in textiles and different industrial applications. Also, cellulose fibres have been used for centuries due to their unique properties and renewable nature. Biopolymers-derived packaging materials are abundant, renewable, eco-friendly, sustainable, safe, biocompatible, and biodegradable. Carbohydrate-based functional blends and composite films are developed in combination with fibres or compatible synthetic polymers like polyvinyl alcohol for food packaging and many other medical applications. It is also common to add other additives like plasticisers, cross-linking agents, nanoparticles, and other application-specific functional additives such as antimicrobial agents targeted at eliminating food preservatives. Alternately, natural polymers have the potential to replace man-made polymer fibres due to the high cost and environmental issues associated with the latter. Khazaei et al. [1] have developed an antibacterial film based on polyvinyl alcohol-pinto bean starch composite film impregnated with cinnamon essential oil for food packaging and biomedical applications. Lacerda et al. [2] have reported moist heat treatment of avocado starch above its gelatinisation temperature and followed by its detailed physicochemical, thermal, and structural characterisations. Mirzaei–Mohkam et al. [3] developed vitamin E (-tocopherol) nanocapsules incorporated carboxy-methyl cellulose film with reduced water vapour permeability and high elongation at break properties for packaging and storing food items containing fats or lipids at room temperature. Starch alone is not a plastic, but heat treatments with moisture and in combination with additives disrupt the three-dimensional and granular structure of the starch so that it is modified into a processable plastic. Natural fibres are widely available. They can be easily extracted from plants and are present in the form of leaves, stems, roots, and fruits [4]. In Ethiopia, the Ensete ventricosum plant is abundant and an important food crop. This plant, in brief, is called enset. This plant’s pseudostem (leaf sheaths) is decorticated to produce starchy pulp, which is used as a food product along with its corm. This process generates scrap cellulosic fibre. The extracted residual fibres are used to manufacture sacks, bags, ropes, cordage, mats, sieves, and construction twig rope.

Nowadays, a variety of petroleum-derived polymers are used in the packaging of foods and dry goods [5]. Because of their unique economic values and environmental benefits, polymers and cellulosic fibres derived from plants have recently played an important role as feedstock in a variety of applications. Plant-derived starch can be used to produce a biodegradable plastic film, which can help to mitigate the environmental problems caused by conventional petroleum-based plastics. Starch is becoming popular to produce biodegradable plastics for a variety of applications. Even though bioplastics synthesized from starch are not strong enough and require reinforcing agents such as natural fibres and clays which are yet again natural by-products and low cost. Cellulose is the most abundant natural polymer found in nature, and it is primarily the source of plant cell walls, which provide structural support and rigidity to plants [6]. They could be used as reinforcing elements in biopolymers and hence improve the compound’s mechanical properties. The synergy between starch and cellulose fibre is important for mechanical efficiency in film applications [7].

Biodegradability and environmental safety have become important considerations in the development of new plant-based materials. Rheological and pasting characteristics of this avocado starch blend with enset cellulosic are discussed in this work. Another detailed process and properties report on this starch-cellulosic plastics film are under preparation.

2. Materials and Methods

2.1. Materials

Avocado seed kernels were collected from a local fruit juice shop in Addis Ababa. Enset pseudostem fibre was sourced from Hawassa, Ethiopia. Sodium metabisulfite (Na2S2O5) and sodium hydroxide (> 98%), ethanol (> 98%), potassium iodide (KI), acetic acid (99.8%), and sulfuric acid (> 98% purity) were purchased from a chemical store in Addis Ababa, Ethiopia.

2.1.1. Equipment

The following laboratory equipment/instruments were used; miller, sieve (BS 410, England), centrifuge (FUNKE GERBER 1205 Berlin), muffle furnace (MF 106, Turkey), Rapid Visco analyser (RVA 4500, Perten instrument Ltd., Sweden), modular compact rheometer (Anton Paar MCR 102), X-ray Diffraction (XRD-7000, SHIMADZU Co., Japan), Fourier transform infrared spectroscopy (FTIR) (Nicolet iS50, Thermo Scientific, USA), UV-visible spectrophotometer (JASCO V-770, Japan), scanning electron microscopy (SEM, Stereo scan 250MK3, UK), and differential scanning calorimeter (DSC, SKZ1052, China).

2.2. Starch Extraction

Avocado seed 1 kg was peeled, washed with clean water before being cut into pieces, and pounded using mortar. Then the crushed avocado seeds were subjected to a wet miller and reduced into a moist, fine powder. The wet, milled, fine avocado seed was mixed with 2 litres of distilled water. Then sodium metabisulfite was added at a concentration of 0.03% and the mixture was left for 24 h [8] to avoid enzymatic oxidation, mainly by peroxidases and phenolates, and to prevent the browning reaction.

The starch slurry was filtered using a muslin cloth, and the filtrate was left for an hour to settle the starch. Starch sediment was separated from the slurry and then washed again with distilled water. Starch sediment was centrifuged at 1200 rpm for 10 min in order to discard by scraping off the reddish supernatant layer with a spatula. This procedure was repeated three times with added distilled water. Later, the starch slurry was dried for 24 h in an oven at 50°C [9]. The dried starch was crushed with a mortar and pestle and sieved through an 80 μm sieve to obtain powdered starch. Hereafter, this will be referred to as avocado starch.

2.3. Extraction and Preparation of Cellulose Fibre

Enset fibre cellulosic was extracted following a reported method [10]. Enset fibre was cleaned with detergent to remove gummy/oil substances and other organic impurities. Subsequently, the fibre was soaked in copious distilled water at room temperature for 10 min. The fibre was filtered, rinsed, dried, cut into 2 cm pieces, and collected for further treatments.

2.3.1. Alkaline Pretreatment

Alkaline pretreatment was carried out in a 600 ml beaker with a 1 : 10 (w/v) fibre to 10% NaOH solution ratio in a hot water bath at 70°C for 3 h [11]. This treated fibre was soaked in a 1 : 10 (w/v) fibre to 10% acetic acid solution ratio at 70°C for 10 min to neutralise the alkali. Later, the fibre was filtered and rinsed with hot distilled water many times until a neutral pH was noticed. This alkali-treated fibre was dried and subjected to hydrogen peroxide treatment. Alkali treatment of fibres removes mostly surface pectin, hemicellulose, and extractives and hence enriches cellulosic.

2.3.2. Hydrogen Peroxide Bleaching

Subsequently, bleaching was carried out by treating the fibres with a 10% H2O2 and 10% NaOH solution mixture at a 1 : 10 fibre to solution ratio. The fibre was soaked at room temperature for 30 min, followed by the solution mixture being heated in a water bath at 70°C for 30 min. This treated fibre was copiously washed with hot distilled water, filtered, and dried in an oven at 80°C for 24 h. This bleached fibre was ground into a fine powder. The powder was sieved with an 80 μm size and stored for the blend preparation.

2.4. Avocado Starch Characterisation
2.4.1. Moisture Content Determination

The amount of moisture present in the starch was determined [12]. Avocado starch, 3 g, was weighed into a crucible. The crucible with the sample was dried in an oven at 105°C for 12 h. The sample was then placed in a desiccator to cool before the final weight was measured. The percentage of the moisture content of avocado starch was determined as per the following equation:where MC = moisture content,  = initial weight (g), and  = final weight (g).

2.4.2. pH Determination

The pH of the starch solution was determined following a reported method [13]. 5 g of starch was added to 20 ml of distilled water and mixed carefully for 5 min. The electrode of the pH metre was placed in the starch slurry and the pH of the solution was measured.

2.4.3. Yield of Starch

The yield of extracted starch was mainly dependent on the types of raw materials used, the extracting agent, and the extraction time. The starch yield was determined as the amount of starch isolated from the dried avocado seed kernel, which is measured in percentage as per the following equation [14]:where Y = yield of starch,  = weight of isolated starch (g), and  = weight of seed (g).

2.4.4. Determination of Ash Content

Ash content was determined according to AOAC’s 1984 procedure. Avocado starch, 5 g in a dried crucible, was placed in a furnace, and the temperature was maintained at 540°C for 3 h. Later cooled in a desiccator. The crucible was weighed again to know the weight of the ash after incineration. The ash content was determined as per the following equation:where AC = ash content,  = weight of ash (g), and  = weight of dry starch (g).

2.4.5. Determination of Pasting Properties of Starch

The pasting property of the avocado starch was determined by using a Rapid Visco Analyser (RVA) and following a reported method [15]. 3 g of starch was weighed and added to 22 ml of distilled water. The suspension was then equilibrated at 50°C for 2 min, heated to 90°C at a rate of 10°C/min, and then cooled to 50°C. Pasting temperature (PT), peak viscosity (PV), peak time (pt), trough viscosity (TV), breakdown viscosity (BV), final viscosity (FV), and setback viscosity (SV) were determined from the final pasting curve of the avocado starch.

2.4.6. Determination of Starch Swelling Power and Solubility

The starch swelling power and solubility were evaluated by following a reported method [16]. The starch sample of 2 g was treated in a centrifuge tube with 60 ml of distilled water. The slurry was heated at 70°C in a water bath for 1 h, shaking the slurry every 10 min. After cooling down to room temperature, it was centrifuged at 1200 rpm for 15 min. The supernatant was recovered in a preweighed clean tray, and the swollen starch sediment was weighed separately. The supernatant was evaporated and dried at 100°C for 3 h in an oven until it became a constant weight. Swelling power and solubility were determined using the following equations.where Sp = swelling power, S = solubility,  = weight of the wet sediment (g),  = weight of the dry starch (g), and  = weight of the dry supernatant (g).

2.4.7. Determination of Amylose Content

The amylose content of starch was determined following a reported method [17] and as per equation (6). Starch, 0.1 g, was weighed and mixed with 9 ml of 0.1 N NaOH solution and 1 ml of 95% ethanol. The starch suspension in the solution was continuously stirred and boiled for 10 min. The starch suspension was then transferred to a beaker and diluted with 50 ml of distilled water. A 5 ml aliquot of the starch suspension was added to the volumetric flask. The 5 ml solution was then diluted to 100 ml with 1 ml of 1 N acetic acid and 2 ml of iodine solution and mixed well. The solution was scanned using a UV-Vis spectrophotometer at 620 nm wavelength to determine the absorbance of the starch suspension solution. The amylopectin content in starch was also determined by subtracting the amylose percentage.where Am = amylose content, A = absorbance, Ap = amylopectin content, and 3.06 is the conversion factor.

2.4.8. Dynamic Rheological Properties Analysis

Avocado starch, 6 g, was mixed with 25 ml of distilled water. The suspension sample was heated on a hot plate stirrer at 90°C for 30 min in order to obtain uniformity. The starch paste was cooled to room temperature for 1 h. Rheological parameters such as shear stress, temperature, viscosity, and time are recorded from the viscoelastic profile of the starch paste using the rheometer. The starch paste was gently placed on the Peltier and rotational rod of the rheometer, which is inserted into the tube-like structure in order to prevent evaporation. The sample was heated starting from 20 to 90°C at a rate of 2°C/min.

2.4.9. Blend Film Preparation

The blend film was synthesized by the solution casting method [18]. 10 g starch and cellulosic were measured with a 70 : 30 wt% ratio and glycerol was taken with 0.3 ml/g to the total blend weight. Distilled water was used with a ratio of 1 : 10 (g/ml) of the starch-cellulosic blend to prepare the solution mixture. The solution was heated on the hot plate and stirred continuously at 700 rpm up to the gelatinisation temperature of 85°C. After a 10 min interval, the glycerol was added. The starch, cellulosic, and glycerol mixture was heated and stirred gently for 20 min more. The suspension was cooled down to 50°C before being cast using a Petri dish. The cast solution was dried in an oven at 30–50°C after that the film detached and stored for further characterisation.

2.5. Avocado Starch Characterisation
2.5.1. FTIR Analysis

A pinch of starch powder was gently pressed onto a pellet surface of a diamond crystal using a system press tip flap under a hydraulic press. The FTIR scan was run for a transmittance range between 4000 and 400 cm−1 wave number, and the spectrum was recorded under ambient conditions [19].

2.5.2. DSC Analysis

The starch sample, 5 mg, was mixed with 15 ml of distilled water [20]. The slurry of the starch was stirred well in order to achieve a homogenous solution, and it was prepared for 1 h prior to analysis. The starch sample was encased in a sample pan holder. Then a sealed, empty sample holder was used as a reference. The pan was then heated from 20 to 200°C at a heating rate of 10°C per min under the inert N2 gas atmosphere at a 50 ml per min purge rate. Thermal characteristic parameters such as onset temperature (To), gelatinisation enthalpy (H), conclusion temperature (Tc), and peak temperature (Tp) were determined.

2.5.3. SEM Analysis

The double-sided sticky tape was used to spread the dried avocado starch powder onto a metal stub. The sample was placed onto the stage, and SEM scans were taken at a 5 kV accelerating voltage with magnifications of ×1000 and ×2000.

2.5.4. XRD Analysis

An XRD scan was performed at a scanning speed of 4°/min using 40 kV, Cu-radiation, and 30 mA in the angular (2θ) range 5–80°. A dry powder of starch and cellulosic as well as blend film samples were used.

All numerical sample test results were performed in triplicate, and results were expressed as means with standard deviation.

3. Results and Discussion

3.1. Physicochemical Properties of Avocado Starch
3.1.1. Moisture Content

Moisture content is very important for all types of raw materials and other finished products in order to know the amount of water and other volatile components that will be absorbed. The moisture content of the avocado seed obtained was determined to be 12.7 ± 0.03%, which is slightly higher than the values already reported [21] but in compliance with a range of 10–14% in another reported article [22]. The starch, with a moisture content of 12.7% at room temperature, is not vulnerable to deterioration by bacteria or fungi.

3.1.2. Ash Content

The ash content of the starch was found to be 0.67 ± 0.014%, which is in considerable deviation from a reported value of 0.23% [23] but less than the value reported in another report [24]. This ash content may be due to the small starch being burnt with inorganic impurities [25]. This indicates that any impurities found in the starch are negligible.

3.1.3. pH

The pH is an indispensable parameter that must be measured to ensure the acidity or basicity of substances. Based on the experimental result, the pH of the starch was found to be 5.73 ± 0.09, and this result is in agreement with the findings of other research [26].

3.1.4. Yield

The starch yield is shown in Table 1. The yield, 18.3 ± 0.02%, was found to be slightly lower than the reported yield of 19% [27] but strongly deviated from reported yields of 24.4 to 29.3% by others [28, 29].

3.1.5. Amylose and Amylopectin Content

The amylose and amylopectin content, as well as the granule size, influence starch properties such as water absorption, gelatinisation and pasting, and susceptibility to enzymatic attack. UV spectrophotometry was used to determine the amylose content of avocado starch, and the absorbance of the standard sample was measured at 620 nm. Based on the recorded characteristic absorbance, the amount of amylose content was determined to be 17 ± 0.03%, which is comparable to reported research [30].

The amylopectin content of the starch was obtained to be 83.2 ± 0.11% by subtracting the amylose content from 100%. The variation of amylose and amylopectin contents of avocado starch might be significantly affected by various factors, such as botanical genotypes, soil type, climatic conditions, and harvesting time of cultivation.

3.1.6. Pasting Properties of Avocado Starch and Blend

The pasting profile of avocado starch and the blend are shown in Figure 1. The variation in pasting parameters such as pasting temperature (PT), peak viscosity (PV), peak time (pt), trough viscosity (TV), breakdown viscosity (BV), final viscosity (FV), and setback viscosity (SV) is directly related to the cooking behaviour of starch in the heating and cooling cycle and are shown in Table 2. Final viscosity reveals the tendency of the starch to form a viscous paste after cooling [31]. The viscosity of avocado starch (6165 cP) is significantly higher than the starch-cellulosic blend (2672 cP). The amylose content variation due to blending with cellulosic in the starch leads to a reduction in viscosity. The retrogradation of the starch increases due to the interaction between leached amylose and amylopectin chains. The higher final viscosity of the starch might be due to its higher amylose interactions and water-binding capacity.

3.1.7. Avocado Starch Swelling Power and Solubility

Water absorption capacity (WAC), swelling power (SP), and solubility are correlated directly to increases in temperature. The swelling power of the avocado starch is 7 g of water per g of starch, and this result slightly deviates from a reported value by Chel-Guerrero et al. [30]. The ability of the starch to retain water determines the swelling power via hydrogen bonding. Heating in excess water revealed a disrupted crystalline structure, and this might be the disruption of hydrogen bonds, amylose content, and the amylopectin content of the starch. The solubility of avocado starch was found to be 20.6 ± 0.03%. It is a measure of how much soluble starch leaches out of swollen granules. The higher solubility of the avocado starch may be explained due to structural differences such as chain structure and length distributions compared to maise starch [32].

3.1.8. Rheological Properties of Avocado Starch

Dynamic rheology is the study of the flow and deformation properties of a substance when force is exerted on it. The dynamic rheometer permits the continuous measurement of dynamic moduli such as storage modulus and loss modules during temperature ramp oscillation testing of the starch gelation. Shear stress is a dynamic rheological parameter that measures starch’s ability to flow or resistance to flow as temperature changes. The rheological properties of starch during heating are shown in Figures 2(a) and 2(b). Shear stress rises quickly and then falls abruptly in a short period of time. This might be due to the breakdown of the starch granules, which causes the melting of residual crystallites and increased mobility [33].

As the temperature increases, the gelatinisation of starch increases and, after a while, shows a decrease. The peak shear stress (295 Pa) was obtained at a temperature of 85°C. This might be due to the gelatinisation of the starch, which makes it thicker and more viscous than it was before. After the peak value, the shear stress dropped with an increase in time. while the starch-cellulosic blend at 85°C was found to have 194 Pa, which is lower than that of the starch. This might be due to the weakening of starch particles’ interaction with cellulosic particles. The shear stress of the starch-cellulosic blend decreased almost gradually with increased time. This indicates that more cellulosic impregnate the starch matrix, which reduces its viscosity and also requires less energy to flow. The rheological property of starch is mainly affected by temperature and granular structure, which leads to the leaching out of amylose in the entire process. Leaching of amylose structure from starch granules is a reason behind the shear stress increase before the maximum temperature is reached. High shear stress in avocado starch may also be due to the existence of phosphate groups and the absence of lipids [34].

3.2. Avocado Starch Characterisation
3.2.1. FTIR

The FTIR spectra of samples are shown in Figure 3. The avocado starch and the starch-cellulosic blend exhibited similar patterns in the 4000–400 cm−1 region. The peaks below 800 cm−1 exhibited the skeletal mode vibration of the glucose pyranose ring, whereas peaks in the range of 800 to 1500 cm−1 reflect the characteristic vibration of glucose molecules. Avocado starch and the starch-cellulosic blend exhibited characteristic bands with peaks at 2928.3 cm−1 and 2928.4 cm−1 due to the symmetric stretching of C-H bonds. The band at 1637.3 cm−1 and 1650 cm−1 exhibit the O-H bending vibration of absorbed water molecules, particularly in the amorphous region of starch [35]. Characteristic peak intensities observed in the region of 800–1500 cm−1 at 1344.8, 1329.8, 1147.3, 1150, 1080.6, 985.8, 925.4, and 850.7 cm−1, which all indicate the C-C bond stretching and C-H group bending vibrations.

3.2.2. DSC

DSC analyses determine physicochemical changes in terms of the heat flow to or from a sample under controlled heating conditions. DSC thermograms are shown in Figure 4 for avocado starch and the starch-cellulosic blend. The gelatinisation temperature reveals the heat stability of the crystalline structure, which determines the distribution of the starch granule size range [36]. Thermal parameters of avocado starch are summarised in Table 3. The avocado starch exhibits lower gelatinisation enthalpy (H) compared to that of the starch-cellulosic blend. The gelatinisation enthalpy (H) has been related to the degree of crystallinity. Starch with higher crystallinity had higher hydrogen bonds and lower swelling power [37]. Avocado starch granules exhibit a lower relative crystallinity value, which requires less energy for gelatinisation. The variation in hydrogen bonds in avocado starch among different varieties may be due to differences in the longer-chain amylopectin domain. A similar result is reported by Singh et al. [33]. The second mild endothermic peak of the starch is observed at 179°C, which might correspond to an amylose-lipid complex that has been observed at higher temperatures [38]. It is also expected that the phosphate fraction may destabilise the crystalline structure in the amylopectin portion of the starch granules, which will lower the gelatinisation and melting temperatures of the starch [39].

3.2.3. SEM

SEM morphology of avocado starch is shown in Figure 5. The morphology of the starch, on the other hand, has a smooth surface and granular structure. SEM study revealed that granular morphology was mostly oval and ellipsoid, some elongated, with a very smooth surface and no cracks or surface pores. The nature of granular morphology may be attributed to the biological origin and biochemistry of the amyloplastic physiology of the plant [40].

SEM morphology of the blend film is shown in Figure 6. The reinforced starch-cellulosic biodegradable film has a smooth surface, with cellulosic particles observed on the surface of the film, which is similar to the reported film morphology by Ashori [41]. The incorporation of cellulosic filler in the starch matrix produces a biodegradable film which has a significant impact on the microstructure of the film. This could be because the avocado starch, enset cellulosic, and glycerol were combined to produce the blend film with strong interfacial interactions.

3.2.4. XRD

The XRD spectra features are diffraction peaks, crystal patterns, and relative crystallinity. Diffraction peaks depend on the amylopectin proportion of the starch and the nature of the cellulosic. Figure 7 depicts the XRD spectra of avocado starch, enset cellulosic, and the blend. These starch and enset cellulosic samples exhibited strong diffraction 2θ-peak values of 5.64°, 17.30°, 19.62°, 22.3°, 34.42°, and 15°, 22°, 32°, respectively. Blend film 2θ peaks were recorded as 3.48°, 17°, and 19.7°; which are in agreement with the findings of Kadokawa et al. [42] and Macena et al. [27]. The starch and the cellulosic show similar diffraction patterns with different peaks. Sharp and shallow peaks are characteristics of crystalline and amorphous regions. Starch has three forms of crystalline pattern, whose differences are mainly related to the crystal structure of the amylopectin chain, which has a double helix structure [43]. The relative higher crystallinity of the avocado starch might be due to rich amylopectin chain-based crystallites and the purification steps applied. The reinforced blend film had a strong peak at 3.48° and 19.7°, which could be attributed to the strong interactions between starch and cellulosic via hydrogen bonds [19].

4. Conclusions

Starch was extracted from the avocado seed, and cellulosic was recovered from enset pseudo-stem fibre for biodegradable film applications. The characterisation of the physicochemical, structural, pasting, morphological, thermal, and rheological properties of the starch and the blend were studied. Moisture content, ash content, pH, and yield are reported for the starch. Starch amylose content was recorded at 17%. Pasting property studies reveal that starch’s viscosity is dramatically reduced when blended with cellulosic. Swelling power and solubility were recorded as 7 g water per g of starch and 20.6%, respectively. The dynamic rheology of avocado starch and the starch-cellulosic blend showed peak shear stress values of 295 and 194 Pa, respectively, at 85°C. These results show that gelatinisation of the starch under dynamic conditions is affected and viscosity is reduced by blending with cellulosic. DSC results show the starch exhibited lower gelatinisation onset temperatures compared to the starch-cellulosic blend. A SEM study of avocado starch revealed mostly granular morphology and a smooth surface. Consequently, this avocado starch/cellulosic blend-based biodegradable plastic film can be an alternative for bioplastic applications.

Data Availability

The data used to support the findings of this research can be used to support the findings of the study and are available within the article.

Conflicts of Interest

The authors have declared no conflicts of interest.


The authors acknowledge Addis Ababa Science and Technology University for financial support and Adama Science and Technology University for providing the laboratory and instrumental facilities.