Preparation of Sheepskin Unhairing Extracts from Locally Available Plants: Cleaner Leather Processing
Leather is made from animal hides and skins that have passed through several stages of processing, from soaking to finishing. Unhairing is a crucial processing stage in which hair is removed from the animal hide or skin through open up the hair and it facilitates subsequent operations. The conventional sodium sulfide-based unhairing process generates a high volume of effluent, which accounts for 50 to 70% of the total biochemical oxygen demand (BOD) and chemical oxygen demand (COD) load in the tanneries’ effluent. This study aimed to investigate the potential of unhairing agents prepared from locally available plants. The research employed qualitative methods. Plant materials are collected, dried, and ground. In different proportions, unhairing extracts were obtained from Phytolacca dodecandra leaves, Cucurbita foetidissima fruits, and Solanum incanum fruits. In the conventional soaking process, plant extracts were applied in various concentrations to sheepskin. The physical parameters of conventionally processed (control) and experimentally treated leather were examined using FTIR, SEM, tear strength, percentage of elongation, and organoleptic tests. The unhairing solution was prepared from a mixture of 0.5% S. incanum extract, 0.5% P. dodecandra extract, 0.6% C. foetidissima extract, and 260 g/L lime powder lime, and this solution effectively removed the hair from the sheepskin in both hairs saving and hair burning unhairing process. The study revealed that the sheepskin treated with the plant extracts based on an unhairing agent and the conventional unhairing agent showed a comparable tensile strength (42.3 kg/cm and 45.2 kg/cm), tear strength (140.1 kg/cm2 and 143.5 kg/cm2), and percent elongation at break (40.2 and 42.3), respectively, which were above the permissible limit for leather production making. According to the study findings, the plant extracts have a good potential for removing hair from sheepskin, and they are eco-friendly and cost-effective compared to unhairing chemicals such as sodium sulfide.
Tanning is the process of conversion of animal hides and skins into finished leather. In Leather processing, animal skins or hide are passed through different processing stages including the beam house, tanning, post-tanning, and finishing operations. Unhairing is a crucial operation in leather processing that involves removing the hair or wool from the hide or skin. The conventional chemicals being used during this operation are sodium sulfide, sodium hydrosulfide, lime, etc . During the unhairing process, a reductive reaction was carried out. Hydrosulfide (HS−) and hydroxyl (OH−) ions disrupt the disulfide bridge of hair cysteine and convert it to cysteine, allowing for keratin hydrolysis (hair protein) .
The weakening of hair is dependent on the breakdown of the disulfide link of the amino acid cysteine, which is the characteristic of the keratin class of proteins that gives strength to hair and wools. Keratin a fibrous protein typically makes up 90% of the dry weight of hair . The hydrogen atoms supplied by the sharpening agent weaken the cysteine molecular link causing the covalent di-Sulphide bond linkages to rupture and weaken the keratin. To some extent, sharpening also contributes to unhairing, as it tends to break down the hair proteins [4, 5].
The conventional unhairing process in leather processing uses a lot of lime and sodium sulfide, which is hazardous and creates a lot of effluents. Under acidic conditions, sodium sulfide releases large amounts of hydrogen sulfide, which is a common source of fatal incidents . Similarly, the conventional unhairing process emits a significant quantity of pollutants, including biochemical oxygen demand (BOD), chemical oxygen demand (COD), and total solids (TS) .
Previously, several researchers attempted to mitigate the conventional unhairing-related problem by using plant-based and enzyme-assisted unhairing agents, which are environment-friendly options that decrease BOD and COD load in leather processing . However, only a few plant-based and enzyme-assisted unhairing are performed in industrial applications, and currently, there is no commercially available environmentally friendly unhairing agent that can replace sodium sulfide [8, 9]. This research aimed to extract eco-friendly unhairing agents from locally accessible plants in order to minimize tannery effluent and replace conventional unhairing agents.
2. Materials and Methods
The study used both qualitative and quantitative approaches to assess the properties of plants and the tanned leather properties. The research experimental work has been was carried out at the Ethiopian Institute of Textile and Fashion Technology, Bahir Dar University, Ethiopia, and Bahir Dar Tannery, Ethiopia. The study data were analyzed by using tables, pictures, and graphs.
2.1. Plant Collection, Drying, and Grounding
P.dodecandra leaves, S. incanum fruit and C. foetidissima fruit were collected from the regions of Amhara and Oromia states, in Ethiopia. P. dodecandra leaves and S. incanum fruit was cleaned, washed repeatedly with water, and cut into small chips for faster drying. The plant parts were dried for five days at room temperature and oven-dried at 120°C for 2 h. Similarly, C. foetidissima fruit was cleaned, washed repeatedly with water, and cut into small chips for juice preparation. All plants selected for the experimental work were shown in Figure 1.
2.2. Plant Powder and Juice Preparation
P. dodecandra leaves and S. incanum fruit plant parts were individually weighed and ground in a high-speed multifunction grinder with a mesh size of 50–300. The reason behind grounding the plant parts was to maximize the surface area available for extraction, thereby raising the extraction rate. The powdered samples were placed in small plastic bags for further experiment activity. Similarly, C. foetidissima fruit was cut into small pieces and the plant juice was prepared.
2.3. Extracts Preparation
In the study, separate extracts were obtained by mixing 0.5 kg C. foetidissima fruit sap, P. dodecandra leaves powder, and S. incanum fruit powder, and 80 mL of 99.5% methanol were used as a solvent. The plant extracts were prepared from each plant type in triplicate. The mixture was homogenized for 1 h using a magnetic stirrer at room temperature and the mixtures (juices) were separately filtered using a Whatman filter 1 paper, 15 cm disc. Each filtrate portion was then put in a glass beaker with aluminum foil on top and left to stand at room temperature for 48 h. The extracts were further purified using a centrifuge 5810 with 10,000 rpm centrifugation for 10 min at room temperature in order to remove insoluble materials. The filtrate was boiled in a water bath in order to evaporate the methanol and leave behind the extracts which was weighed and the percentage yield was calculated. The percentage of the plant fruit sap was calculated using the following method and their averages were used .
Fruits weight = Wf, Gross juice weight = Wj, Weight of the beaker = Wb, Net juice weight = Wb.
The pH of each extract was measured using a pH meter and their values were recorded. As shown in Figure 2, each extract was then preserved in volumetric flasks in a refrigerator at 4°C, for later experiment usage.
2.4. Yield Percentage of the S. incanum, C. foetidissima, and P. dodecandra Extracts
The amount of extracts from S. incanum fruits, C. foetidissima fruits, and P. dodecandra will depend on the plant age, growing geography, and methods of extraction. The amount of each plant extract was indicated in the following Table 1.
2.4.1. Phytochemical Tests
The plant extract was subjected to preliminary qualitative phytochemical screening for steroids, alkaloids, flavonoids, tannins, saponins, and phenol-based on the following standard methods.
2.4.2. Test for Flavonoids
The alkaline reagent test method was used to detect the presence of flavonoids in the extracts. A small amount of extract was treated with aqueous NaOH and HCl and observed for the formation of the yellow-orange color .
2.4.3. Test for Alkaloids
Mayer's reagent test method was used to process the extracts (1.36 g of mercuric chloride and 5 g of potassium iodide in 100 ml of water). The presence of alkaloids is indicated by the formation of a yellow-colored precipitate .
2.4.4. Test for Phenolic Compound
Ferric chloride test method was used to detect the presence of phenoloic compounds in the extracts. A 3–4 drops of 5% ferric chloride solution were added to the extracts. The presence of phenols is indicated by the formation of a bluish-black color .
2.4.5. Test for Saponins
The Froth test method was used to detect the presence of saponin in the extracts. A 0.5 gm of extracts were mixed with 2 ml of water and shaken for 15 minutes in a graduated cylinder. The presence of saponins is shown by the formation of a 1 cm layer of foam .
2.4.6. Test for Tannins
Ferric chloride test method was used to detect the presence of tannin in the extracts. A 0.5 g of crude of each plant extract was mixed with 10 mL of distilled water and boiled and then filtered. Three drops of 0.10% ferric chloride were added to the filtrate. The formation of brownish, greenish, or blue-black color was an indication of the presence of tannins .
2.4.7. Test for Steroid
Chromatographic test method was used to detect the presence of steroids in the extracts. A 1 ml of the plant extract was added to 1 ml of concentrated tetraoxosulphate (VI) acid (H2SO4). A red coloration confirmed the presence of steroids .
2.5. Expermental Hypothesis: Optimization of Unhairing Agents for Experiments
In the study, unhairing of sheepskin was conducted by the mixture of extracts unhairing solutions prepared from P. dodecandra leaves extract, C. foetidissima fruits extract and S. incanum fruits extract. Initially, the sheepskin was treated with conventional soaking agents (i.e., water and wetting agents) in order to rehydrate the skin, and then the unhairing extracts were applied to the skin. The reaction of a solution occurred at a low temperature. In order to obtain the optimum effective unhairing ratio, three experiments were carried out under different parameters, which are shown in Tables 1–3.
2.6. Preparation of Unhairing Extract Solution
Based on the unhairing recipe indicated in Tables 2–4, a 400 mL separate unhairing solution was prepared by mixing S. incanum extract, P. dodecandra extract, and C. foetidissima extract. After mixing the solution, 260 g/L of lime powder was added and stirred. The biome of the solution was checked. The prepared unhairing solution was kept for 24 h in order to enhance the solution mixture uniformity. The prepared unhairing solution was applied to sheepskin using two different methods: a hair-saving method on the flesh side of the skin and a hair-burning method using a drum.
2.7. Preparation of Soaking Recipe for Unhairing Process through Hair-Saving Method
Purification and defects-free raw sheep skin were used. The sheepskin soaking process was carried out by preparing a soaking recipe for the hair-saving method, as shown in the following Table 5.
2.8. Application of Conventional Unhairing through Painting Method (Control)
Based on the recipe indicated in Table 5, half-side of the sheepskin was soaked in the soaking solution. Then, the skin was weighed and painted with a paste containing 10% lime, 2% sodium sulfide, 15% water, and 0.2% wetting agent. The paint was applied on the flesh side of the skin. Then, the treated sheep skin was covered well with wet gunny cloth and was kept in pile flesh to flesh for 5-6 h. Lastly, unhairing was carried out using mechanical action by using a dull knife.
2.9. Application of Experimental Unhairing through Painting Method (Experiment)
Based on the recipe indicated in Table 6, half-side of the sheepskin was soaked in the soaking solution. Then, the skin was weighed and painted with a solution containing S. incanum, P. dodecandra extract, C. foetidissima extract, and lime powder, which were indicated in Table 1. The paint was applied on the flesh side of the skin and piled for 5-6 h by the flesh to flesh side and grain to the grain side. Lastly, unhairing was carried out using mechanical action by using a dull knife. After the soaking process was completed liming was carried out.
2.10. Preparation of Sheepskin for Unhairing Process through Hair-Burning Method
In the study, purification and defects-free raw sheepskin were used. The soaking process was carried out by preparing a soaking recipe prepared for hair-burning method, as shown in Table 7.
2.11. Application of Conventional Unhairing through Hair-Burning Method (Control)
The hair-burning process was carried out in the drum according to the recipe shown in Table 6. A half-sheep skin was soaked in the drum containing 300% water, 5% lime, and 3% sodium sulfide. Then, the drum was run for 10 min within 2 h and left overnight. By the next day, the skins were unhaired and re-limed for a day with a solution containing 5% lime ad 0.2% caustic soda. When the sheep's skin were properly plumped, they are taken for unhairing and fleshing processes. Drums are used to accelerate the liming process and the rpm of the drum was set to 2-3 in order to avoid too much beating action. Completion of the liming process was decided by the characteristics desired in the final leather as well as the raw materials from which they are produced.
2.12. Application of Experimental Unhairing through Hair-Burning Method (Experimental)
The experimental unhairing (hair burning) was carried out in the drum according to the recipe indicated in Table 8.
3. Results and Discussion
3.1. Phytochemical Analysis of Plants Extracts
Different studies revealed that the presence of bioactive compounds in the plant has a significant antimicrobial activity, which may enhance the quality of the leather treated with the plant extracts. As shown in Table 9, the S. incanum fruits extracts, C. foetidissima fruits extracts, and P. dodecandra leave extracts showed the presence of some bioactive compounds.
3.1.1. Flavonoids Detection
Alkaline reagent test revealed a strong positive (++) presence of flavonoids only in S. incanum extract, as shown in Table 9. The present study investigation was similar to that of Lin et al. and Sbhatu et al [15, 16], who reported the presence of flavonoids in S. incanum.
3.1.2. Saponins Detection
The Froth test approach revealed the existence of strong positive (++) saponins in the extracts of S. incanum, C. foetidissima, and P. dodecandra, which were shown in Table 9. Correspondingly, different studies revealed the presence of saponins in the extracts of S. incanum [17, 18], extracts of C. foetidissima [19, 20], and extracts P. dodecandra [21, 22].
3.1.3. Phenols Detection
As indicated in Table 9, the ferric chloride test revealed the presence of a strong positive (++) phenols in the extracts of S. incanum, C. foetidissima and P. dodecandra. Similary, Prohens et al. and Belayneh et al. [23, 24] reported the presence of phenols in S. incanum, Salehi et al.  reported the presence of phenols in C. foetidissima and Makonnen et al. and Namulindwa et al. [25, 26] reported the presence of phenols in P. dodecandra.
3.1.4. Alkaloids Detection
As shown in Table 9, Mayer’s and Wagner’s tests revealed the presence of strong positive (++) alkaloids in the extracts of S. incanum, C. foetidissima and P. dodecandra. Related findings by Eltayeb et al. and Desta et al. [27, 28] reported the existence of alkaloids in S. incanum, Ferguson  reported the existence of alkaloids in C. foetidissima and Ogutu et al. and Namulindwa et al.[26, 30] reported the existence of alkaloids in P. dodecandra.
3.1.5. Tannin Detection
In ferric chloride test, a strong positive (++) tannins were detected in S. incanum, C. foetidissima and P. dodecandra, which were shown in Table 9. Correspondingly, Sahle, Okbatinsae et al. and Desta et al. [28,31] reported the presence of tannins in S. incanum extracts, Salehi et al. and Pámanes et al. [20, 32] reported the presence of tannins in C. foetidissima and Namulindwa et al.  reported the presence of tannins in P. dodecandra.
The chromatographic test approach detected the presence of strong positive (++) steroids in S. incanum, C. foetidissima, and P. dodecandra, which were indicated in Table 9. Correspondingly, Ferguson  revealed the existence of steroids in S. incanum and C. foetidissima, and Ogutu et al. and Namulindwa et al. [26, 30] revealed the existence of steroids in P. dodecandra.
3.2. Unhairing Effectiveness of the Plants Extracts
The unhairing experiment was carried out with different mixing ratios of S. incanum extract, P. dodecandra extract, C. foetidissima extract, and lime powder under different temperatures, times, and pH values.
Experimental trial 1 was conducted under a constant temperature (25°C), Time (4 h), and lime powder (260 g/L) with a variable ratio of plant extracts. In the current study, the level of unhairing efficiency was judged by subjective by looking at the treated skin carefully. A high level of unhairing indicates that the hair was completely removed from the sheep skin, medium level of unhairing indicates that the hair was removed to some extent whereas low level of unhairing level indicates the hairs were removed in a low extent. As indicated in Table 10, the unhairing mixture ratio of 0.5 S. incanum, 0.5 P. dodecandra, 0.6 C. foetidissima and 260 g/L lime powder resulted in high level of unhairing effectiveness compared to the other experimental trials. As shown in Table 10, when the unhairing extracts mixing proportions were close to each other (0.5, 0.5, 0.6), the effectiveness of unhairing was enhanced.
In the study, Experimental Trial 2 was conducted under a constant temperature (30°C), Time (4 h) and lime powder 260 g/L. As indicated in the above Table 11, the unhairing mixture ratio of 0.5 S. incanum, 0.5 P. dodecandra, 0.6 C. foetidissima, and 260 g/L lime powder resulted in high level of unhairing effectiveness compared to the other experimental trials. Similar to Experimental Trial 1, when the mixing proportion of the extracts was close to each other and the effectiveness of unhairing were enhanced, which were shown in Table 11.
Experimental Trial 3 was conducted under a constant Temperature (35 C), Time (3 h) and lime powder 260 g/L. As indicated in Table 12, the mixture of 0.5 S. incanum, 0.5 P. dodecandra, 0.6 C. foetidissima and 260 g/L lime powder resulted in high level of unhairing compared to the other experimental trials. As indicated in Table 11, when the mixing proportion of the extracts was close to each other and the efficiency of unhairing was enhanced. Based on three Experimental trial results, it is observed that the unhairing mixture ratio of S. incanum, 0.5 P. dodecandra, 0.6 C. foetidissima and 260 g/L lime powder resulted in a high level of unhairing effectiveness. In the three experimental trials, varying the Temperature (25 C, 30 C, and 35 C) and Time (3 h and 4 h) has not affected the unhairing extracts' hair removal effectiveness.
3.3. FTIR Analysis
Fourier transforms infrared (FTIR) spectroscopy is an important technique used for the chemical analysis of biological substances. Based on this, the raw sheepskin, experimentally unhaired sheep skin and conventionally unhaired sheep skin were characterized using in order to determine the sheep skin relative functional group. The raw skin experimentally treated skin and conventionally treated skin FTIR% Transsimitence vs wavelength result images were shown under Figures 3–5.
As shown in Figures 3–5, there is a huge difference between raw skin, treated and conventional treated leather. Similarly, there was a difference between conventional and experimental treated leather that reveal a broad band in the range of 3550–3150 . Raw skin has more single bond, double bond, triple bond, and fingerprints and also has more peaks than experimental treated and conventionally treated leather. The FTIR spectra of untreated raw skin were shown in Figure 3.
The FTIR spectra of Experimentally Treated Leather were shown in Figure 4.
The FTIR spectra of conventionally Treated Leather were shown in Figure 5.
3.4. Comparison of Sheep Skin Treated with Experimental and Conventional Unhairing Agents
The main objective of unhairing were to remove hair from sheepskin. The weakening of the hair depends on the breakdown of the disulfide link of the amino acid and cystine. The experimental and the conventional unhairing process result has been discussed as follows.
3.5. Experimental Unhairing through Hair Saving Method Result Analysis
In the experimental saving (painting) process, the sheepskin was treated with a 400 mL unhairing containing a mixture of extracts of 0.5% S. incanum extract, 0.5% P. dodecandra extract, 0.6% C. foetidissima extract, and 260 g/L lime powder. This unhairing process was resulted in complete removal of hair from sheep skin after exposure of 3-4 h and 25–30°C. The unhairing agent extracted from the plant extracts not only removed the hair but also makes the sheep's skin clean and swell up. The plant-based unhaired sheep skin was to some extent similar to conventionally unhaired sheep skin. Sheepskin before treatment and sheep skin after treatment with unhairing through the hair-saving method was shown in Figure 6.
3.6. Experimental Unhairing through Hair Burning Method Result Analysis
In the hair burning experiment, the sheep skin was treated with a 400 mL solution containing an extract mixture of 0.5% S. incanum extract, 0.5% P. dodecandra extract, 0.6% C. foetidissima extract, and 260 g/L lime powder has resulted in complete removal of hair from sheep skin after exposure of 12 h on a drum, which is indicated in Figure 7.
In the current study, the unhairng solution was prepared from a mixture of 0.5% S. incanum extract, 0.5% S. incanum extract, 0.5% P. dodecandra extract, 0.6% C. foetidissima extract and 260 g/L lime powder have effectively removed the hair from the sheepskin in both Hairs-saving and Hair-burning unhairing process.
According to different studies, P. dodecandra fruits and S. incanum are rich in detergent agents namely saponins, which resulted in a clean leather, reduced smell, and substantially reduced amount of load released on the environment such as sulfide, nitrogen, carbon-oxygen demand.
According to different literetures, the presence of calcium thioglycolate in S. incanum, the presence of zinc in C. foetidissima (pumpkin), and the presence of saponified (antimicrobials) in P. dodecandra leaves have resulted in the breakdown of disulfide bond in keratin and fiber open up. Correspondingly, the presence of lime in the unhairing agent enhanced the hair removal effectiveness from sheep skin. On the other hand, the experimental plant-based unhairing extract has the ability to reduce bacterial attacks and also acts as a preservative in the leather. Therefore, this study clearly indicates that the unhairing solution prepared from the plants has a good potential to replace the conventional sodium sulfide-based unhairing and liming process in the leather industry.
3.7. Physical Test Analysis of Experimentally and Conventionally Treated Sheep Leather
The physical test analysis was carried out after sheepskin was treated with experimental and conventional unhairing recipes/. After the unhairing process, the sheepskins were passed through a similar leather processing stages. The final leathers were tested and their physical test results are presented in Figure 8.
3.8. Tear Strength Determination
Tear strength test methods were intended for the determination of load in kg required to tear the test sample. Tear strength testing was carried out according to ISO 3376 : 2011 and ASTM D2209—00 (2015) test methods. As indicated in Table 6, the sheepskin treated with the experimental extracts and conventional solution showed a comparable tensile strength (42.3 kg/cm) with conventionally unhaired leather (45.2 kg/cm), which was above the permissible limit for leather production making.
3.9. Tensile Strength Determination
Tensile strength test methods were intended for the determination of tensile strength, temporary and permanent elongation at specified load, modulus, and elongation at break of sheep leather. The treated sample pieces were prepared according to ISO 3376 : 2011/ASTM D2209 - 00(2015). As indicated in Figure 6, the study result indicates that the leather treated with plant unhairing extracts has a comparable tensile strength (140.1 kg/cm2 and 143.5 kg/cm2) and percent elongation at break (40.2 and 42.3), which were above the permissible limit for leather production making.
3.10. Grain Crack Strength Load and Grain Crack Strength Distention
Consequently, the study revealed that the sheep skin treated with the experimental unhairing extracts and conventional solution showed a comparable Grain Crack Strength load (23 kg and 23.2 kg) and Grain crack strength distention (11.2 mm and 11.65 mm), which were above the permissible limit for leather production making.
3.11. Organoleptic Property Comparison of Experimentally and Conventionally Treated Leather
Organoleptic property was carried out by nine experienced leather technologists working at an industry level and the average response were taken. As indicated in Figure 9, the organoleptic property of comparison between the conventional and experimental treated leather evaluation highest scores out of 9. The score result strength increases from 1 to 9. In the leather, the technologist response indicated that the conventionally treated leather and experimentally treated leather had the same result on grain pattern, uniformity of color, and fullness, however, they have a difference in feel and general appearance. The experimentally treated leather has shown a better feel and general appearance than the conventionally treated leather.
3.12. Pollution Load Analysis
As indicated in Figure 10, the use of unhairing agents extracted from locally available plants resulted in a reduction in pollution loads such as BOD, COD, TDS, and TSS in comparison with the conventionally treated sample. The conventionally treated sample showed more amounts of pollution loads in the unhairing process. This study revealed that the experiment treated sheep skin resulted in a COD, BOD, TDS, and TSS load of 62.7, 79, 81.8, and 27.2%, respectively. The experimental treated skin also shows complete unhairing along with a 59.3% reduction in the suspended solids level.
Keys: COD: chemical oxygen demand; TDS: total dissolved solid; ppm e parts per million.
3.13. SEM Analysis
To study the effect of the unhairing agents on the structural characteristics of the crust leather, scanning electron microphotographs were used to analyze the effect of experimentally treated and conventionally treated on the crust. The SEM analysis was carried out using FEI-Quanta 200 scanning electron microscope based on the following SEM standards.
In the study, the grain surfaces and the cross-section image of conventionally treated and experimental treated sheep skin crust were analyzed using Scanning Electron Microscope. As shown in Figure 11, it s observed that the experimentally treated crust grain surface appears to be more even, open up, and smoother than the conventionally treated leather. Likewise, the experimentally treated crust pores are free of any hair residues and the fiber of collagen was more open than conventionally treated leather.
Extracted unhairing agents from locally available have a potential of removing hair from sheepskin. The current study result revealed the possibility of eliminating pollution-causing chemicals such as sodium sulfide, lime, and sodium sulfide in the unhairing process. Consequently, the unhairing agent prepared from the plant source not only eliminated sulfide but also enhanced the softness and quality of the final leather. The current study resulted in the treated leather being knit, clean, and reduced the pollution load on the environment. Since lots of plant species are available in the world, the potential of related plant species for an unhairing agent needs to be studied in the future.
The data collected and analyzed during this study are included in the paper and can also be accessed from the authors through a rational request.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
All the authors have contributed to the conceptualization, investigation, data collection, and analysis in the study and have read and approved the final manuscript.
The authors would like to acknowledge the Ethiopian Institute of Textile and Fashion Technology, Bahir Dar University, for the financial and laboratory facilities support in successful completion of the research work. The authors also would like to thank Ethiopian Leather Industry Development Institute for the laboratory facilities support.
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