Optimizing the Extraction of Keratin from Cattle Hoof Using Central Composite Design

Cattle hoofs are abundantly available by-product sources of organic material from the slaughterhouse. It can be successfully converted into keratin protein. Keratin extracted using alkali hydrolysis has a better conservancy of keratin structure. The purpose of this study is optimizing the extraction of keratin from cuttle hoof. Designing of the experiment, analysis of the results, and optimization of the process parameters have been conducted by central composite design (CCD). Three factors (temperature (A), time (B), and concentration of NaOH (C)) each at five levels have been used to extract the keratin protein from cattle hoof and two response variables (dissolution % and purity %) have been considered in the study. The results obtained demonstrate that extraction temperature, alkali concentration, and time showed a significant effect on the purity and dissolution of keratin. The regression model shows that all factors have positive and significant relation with dissolution percentage whereas only temperature and concentration of NaOH have significant and negative relation with purity percentage. It is observed that the Biuret and Fourier-transform infrared spectroscopy (FTIR) tests showed better preservation of protein structure in extractive keratin. The FTIR spectra, indicates that amide I and amide II occur at a wave length of 1633 and 1542cm − 1 for raw cattle hoof and at wave length of 1650 and 1542 cm − 1 for keratin protein, respectively. Thus, it can be concluded that the cattle hoof by-product could offer an alternative keratin source. Finally, the extraction process had an optimum value of 0.5M NaOH, 60 minutes reaction time, and 55 ° C temperature with 85% dissolution and 89.6% purity.


Introduction
Activities in slaughterhouses generate a high level of organic waste, such as cattle hoof, horn, hide, and bone that directly or indirectly a ects the health of residents living in the vicinity. Moreover, hooves are a hard keratin source that has very slow decomposition and pollutes the environment for a long period by releasing foul smells. In fact, the presence of a strong disulphide covalent bond in keratin molecule of the keratinous material makes it to be resistant to a common enzyme and chemical attack (long-time degradation) and becomes insoluble in water, weak acid, alkali, and most organic solvent [1,2].
In valorisation of those keratinous wastes by keratin polymer extraction, some of common ways that has been used include, chemicals (reduction and oxidation) [3][4][5][6] enzyme and microbial [7,8], and microwave methods [4,9]. New green solvent called ionic liquid is also another good means of a human hair [10], wool, camel hair, and chicken feather [11] keratin dissolution, while it has low extraction rate [12]. e solubilization is by reducing the stability of keratin by straightening out of disulphide bonds and hydrogen bonds to access agent to dissolve the protein into solution [13]. ere has been growing interest in the use of naturally derived keratin polymer for a multitude of purposes, which is applicable in food, cosmetics, medical, composite, textile, and agriculture industries [6,[14][15][16].
e chemical composition of a cattle hoof shows that it contains largely α-helical conformation with a mixture of β-sheet [5], around 90% crude protein [17], therefore, it is valuable to extract keratin from hoof for biomedical, cosmetics, and health care applications [18]. About 1.5 billion of cattle are slaughtered every year from meat and from each cattle four pieces of hooves are obtained [19] As a result of this, extraction of keratin from cattle hooves has sustainable supply of raw material. Some research demonstrates that cattle hoof yields the highest keratin protein than wool, feather, and hair keratin [16,20]. Potential keratin extraction from animal hooves tissues was introduced in 1860 [21,22]. Up to date, keratin from cattle hoofs is extracted using a reducing agent [16,20,23] and enzymatic method [4]. Hence, alkaline hydrolysis is a good alternative for the extraction of keratin because of its effectiveness and simplicity with a lower loss of amino acids [8]. During the extraction process, sodium hydroxide (NaOH) is used as the main solubilizing agent and urea as denaturing agent [22].
In the alkali hydrolysis process, many factors could affect the extraction process and properties of keratin extractive, however, in this article extraction time, temperature, and concentration of alkali were taken as important factors affecting the extraction process of keratin from biomass [24,25]. Researchers indicate that having heating aided hydrolysis provides a better yield while, higher heating temperature leads to the destruction of amino acids [2]. To the best of our knowledge, this extraction method has not been used for the digestion of cattle hoof and even the effects of extraction variables on the keratin yield and purity of the keratin have not been well studied. us, the current work aims to extract keratin from cattle hoof using sodium hydroxide solvent and urea through a thermo-chemical process, and then optimize the process to get an experimental condition with optimum yield/dissolution and purity of keratin.

Materials.
Cattle hoofs were collected from a slaughterhouse in the Bahir Dar city, Ethiopia. Laboratory grade sodium hydroxide 99.8% (NaOH), hydrochloric acid 36%·w/ w (HCl), and Urea 99.0% (NH 2 CONH 2 ) were obtained from the Ethiopian Institute of Textile and Fashion Technology, chemical.

Proximate Analysis of Cattle
Hoof. After collection, the hoof was washed with distilled water to remove impurities and dried at 100°C for 5 hours in an oven before further process. As shown in Figure 1, the dried cattle hoof was crushed to 10-20 mm diameter by a hammer crusher and pulverized into 1 mm mesh size and below as indexed by [26][27][28]. e pulverized hoof was then collected in zipper plastic bags and stored at the standard atmospheric condition for further proximate analysis.

Determination of Fat Content.
A minimum standard weight of two grams of Pulverized cattle [26] was used. It is then further soaked with diethyl ether and allowed to stand for 2 hours without stirring for sedimentation [16,27,29] and dried at 50°C for 24 h. e fat content of the raw hoof is determined by the following equation:

Determination of Moisture Content.
A hot air oven was used to determine the moisture content of the samples. Two grams of sample were processed according to ASTM D1576-90 standard, and then average moisture content was determined by the following equation: Where, W1 is the weight (g) of the sample before drying and W2 is the weight (g) of the sample after drying.

Determination of Ash Content.
Ash is the inorganic residue obtained after the combustion of hoof biomass. e ash content of the raw hoof was calculated in relation to the dry weight of the original sample after 4 hr ignition of the sample at 575°C using [30]. e ash content of the raw hoof was determined by the following equation: Ash content(%) � Weight of ash * 100 Weight of sample . (3)

Determination of Protein
Content. e protein content was determined by the Kjeldahl method according to [30] using the official method 979.09. is method has been used in nitrogen determination, and then multiplied by a conversion factor as determined by the following equation: Protien content(%) � Nitrogen content in sample * 6.25.

Alkali Extraction of Keratin.
e defatted hoof keratin was extracted and solubilized using 0.3-1 M of NaOH and 0.64 g of urea. During the extraction of keratin, where the agents solubilize the keratin, alkali media was importantly used [31]. Once the keratin was solubilized, turbid in a solution then filtered using a polyester cloth [28]. e solution from the filtrate was precipitated at acidic media (pH 2 Advances in Materials Science and Engineering 4) using isoelectric precipitation of the protein of 1 N HCl [32,33], and then freeze-dried to obtain recovered keratin powder [34]. To find out the dissolution percentage of the extraction process, the weight of undissolved residue has been identified, once it is oven-dried until constant weight has been obtained [35]. While the purity of the keratin powder was identified using the Kjeldahl method [30]. e actual Design factors levels using CCD are illustrated in Table 1.

Characterization of Extracted Keratin
(1) Biuret Test. One molarity copper sulphate solution and sodium hydroxide solution were prepared separately. e solution collected was mixed with sodium hydroxide solution by 1 : 1 ratio. ree drops of copper sulphate solution were added to the mixture solution. e color changes in the solution were observed and recorded [27].
(2) FTIR Spectroscopy. e functional groups present in extracted keratin powder could be recorded using FT-IR (JASCO FT/IR 6600). About 2 mg of the sample would be taken and prepared using KBr, which is characterized under the spectra range 4000-400 cm −1 in a resolution of 4 cm −1 , 400 scans. Infrared radiation has been made to pass through the sample and the resulting spectrum was representing the molecular transmission, creating a molecular fingerprint of a sample.

Analysis and Optimization.
Central composite design (CCD) has been used to design the experiment, analyze the results of the experiment and optimization of the process. Central composite design is an experimental design, useful in response surface methodology. In this design, the center points are augmented with a group of axial points called star points. With this design, quickly first-order and secondorder terms can be estimated. Five levels have been taken for all three factors (low, center point, high, +α, and −α). For each experiment, the average of five replicate have been taken. e optimization of operational factors such as temperature (°C), time (min), and concentration of NaOH (M) is necessary in order to decrease the number of experimental tests and determine the optimum conditions for input factors. In this regard, the response surface methodology (RSM) coupled with central composite design (CCD) (CCD-RSM design) is an efficient tool that has been used to model the correlation between output response and input of the machining process [19].

Proximate Analysis
e pulverized raw hoof had an average of fat, protein, ash, and moisture content as shown in Table 2.
Based on the result about 1.5% crude fat content was determined by soaking the milled hoof sample with diethyl ether. e crude fat content of the raw sample was 1.47%, which is in agreement with Falaye and Sule, (2020). e average value of ash was 6.55%, which is higher than the report made for cattle hoof meal [17]. Higher ash content is usually an implication of higher impurity or inorganic residue. About 75.0534% protein content was achieved through Kjeldahl analysis, which is relatively lower than the report made by [17]. e average moisture content of the hoof is 5.96%. Hence, it was a bit lower value than the report by [3] and a bit proximate to the report made by [36]. e capability of cattle hoof to absorb moisture from the atmosphere has a vital inference for the storage, transport, processing, and durability of materials, which cause damage due to microorganism contamination [36]. As the moisture content in the raw hoof does not exceed 10.5%, it is a good implication of a material stored for a long time with a minimum microbial growth [28].

Alkali Extraction.
Hoof keratin was extracted and solubilized using different concentrations of caustic soda at different extraction times and temperatures. e solubilized keratin was precipitated and freeze-dried as shown in Figures 2(a) and 2(b), respectively. Freshly solubilized keratins are prone to precipitation because of their hydrophobicity and disulphide bridges tendency to reassemble from the newly resulted sulfhydryl groups. As a consequence, a complex mixture of supramolecular aggregates results, which tend to associate and separate as flocks [37].
Hoof alkali hydrolysis experiment performed on various process parameters has a variability of output (Table 3)    dissolution are mainly a function of NaOH concentration and temperature [32].
In the meantime, the lowest (69.1%) and higher (95.25%) purity percentage of the extractives was found at the extractive condition of 120 min and 65°C, 1.24 M and 60 min, and 55°C and 0.3 M, respectively, from the experimental trials as shown in Table 3. Even if, the experiment results yield higher at higher temperature and concentration it is not true for a purity, because of the keratin polymer structure is disturbed at these experimental conditions. e experimental trial also shows that having a minimum extraction temperature of 48.18°C is not guaranty for higher purity, in which it lowers the conversion of cattle hoof in to keratin polymer. e purity of the extractives was highly affected by the concentration and temperature of the hydrolysis process. is is in fact due to the environment of the process affecting the degradation of biopolymer [11]. As the temperature and NaOH concentration increase vigorously there is a loss of keratin structure and even all protein being in the amino acid level.

Analysis of the Significance of Factors on Dissolution
Percentage. Based on the ANOVA in Table 4, the model generated for hoof keratin extraction was significant (P-value is less than 0.05).
is is also true for a model term of   temperature, time, concentration, a combination of temperature and concentration of NaOH, temprature 2 , and concentration 2 , but not for a combined effect of time with temperature and concentration and time 2 . Lack of fit F-value (0.79) is not significant and its insignificance was importantly been good.
As indicated by Table 5, the predicted R square (0.9328) and adjusted R square (0.9724) for dissolution percentage of keratin were in agreement with each other (their difference is less than 0.2). e quality of the fit model for the developed correlations was stated by R squared (0.9862). It implies that    Advances in Materials Science and Engineering experimental variables considered, can attribute about 98.62% to the extraction process. is shows that the regression model equations successfully captured the correlation between process parameters to the dissolution of product, in which all the points are close to the line of perfect fit as shown in Figure 3(a). e lower coefficient of variation (CV � 1.03%) indicates that the results of the fitted model are reliable. e value of the adjusted coefficient (0.9724) is also very high, supporting the significance of the model. As the fitted model provides a good approximation to the experimental condition, the model was employed to find the values of the process variables for optimum dissolution percentage of hoof keratin.
Model (5) shows the empirical relation between dissolution percentage of hoof keratin with factors considered in the extraction process (A = Temperature, B = time, and C = concentration of NaOH, and AC = interaction of temperature concentration of NaOH). As equation (5) clearly shows, dissolution percentage increases as all the factors increase and it increases as the interaction effect AC, and the quadratic terms, A 2 , an d C 2 decrease.
ere is a positive relation between dissolution percentage of keratin and temperature, time, and concentration of NaOH, where as it has negative relation with interaction (temperature and concentration of NaOH), quadratic effect of temperature and concentration of NaOH.
Dissolution � −28.9345 + 2.19639A + 0.0671607B Table 4 the model is significant for analysis of keratin purity percentage, as model F-value is 58.46. All the factors, the interaction of the factors, and quadratic terms are significant (P < 0.05) except time at a 95% significance level. As the model is expected to fit, the nonsignificance of lack of fit F-value was importantly good as shown in Table 4.

Analysis of the Significance of Factors on Purity Percentage. As shown in
As indicated by Table 5 the predicted R square (0.8955) and adjusted R square (0.9664) for purity percentage of keratin agreed with each other as their difference is less than 0.2. From R squared (0.9832) value, the contribution of experimental variables for the extraction process is about 98.32%, in which all the points are close to the line of a perfect fit. So, the developed model equation successfully captures the correlation between process parameters and the purity of the product. e relation between experimental and predicted values was good as presented in Figure 3(b). As the coefficient of variation (1.21%) is lower, it is possible to confirm the reliability of the fitted model. A higher value of the adjusted coefficient (0.9664), supports the significance of the model. As the fitted model provides a good approximation to the experimental condition, the model was employed to find the values of the process variables for optimum purity percentage of keratin protein.
Model (6) shows the empirical relation between the purity percentage of hoof keratin with factors considered in the extraction process (A, B, and C). As clearly indicated in the equation, all the factors and C 2 have a negative relation with the response (purity) and all interaction effects, and quadratic effects such as A 2 , an d B 2 have a positive relationship with the response.  e effect of temperature on the dissolution percentage of hoof keratin was presented in Figure 4(a), with a temperature range from 55 up to 75°C. As shown in Figure 4(a), the dissolution possesses a constructive relationship with that of temperature, and as the temperature increased the dissolution percentage was increased, which is in agreement with [38]. As mentioned in Figure 4(b) purity of keratin extractives under those temperature ranges was plotted. As reaction temperature is increased, the purity of the extractives was reduced.
In fact, heating aided hydrolysis provides a better dissolution while, higher heating temperature leads to the destruction of amino acids [2]. Also, as the temperature increase from 55 to 75°C, the reaction process can get much energy to accelerate the reduction of the chemical and physical state of the hoof. While lowering the extraction temperature causes a lower reduction of the disulphide bond, not the peptide bond. Figure 5(a) presents the effect of varying the extraction time from 60-180 min on the dissolution percentage of hoof keratin. As illustrated in Figure 5(a), increasing the extraction time increases the dissolution percentage. Generally speaking, increasing extraction time shows an improvement in dissolution percentage. e purity of the extractive keratin is reduced as extraction time increased from 60 min to 80 min while the effect is not substantial as shown in Figure 5(b). At lower extraction time the purity was comparatively higher than at the larger time of extraction. Long-time extraction causes the loss of keratin structure (amino acid) and reduces purity.
At lower concentrations, the reaction process had lower keratin dissolution and vise versal (Figure 6(a)).
is is because at lower concentrations there is a high solid to liquid ratio and aggregation of the hoof material, which reduces the dissolution [38]. ere is a direct relationship between the concentration of alkali with the extent of keratin solubility [10,39]. e purity of the extractive reduced significantly as the concentration of NaOH changes from 0.3-1 M as shown in Figure 6(b). Higher dissolution at higher NaOH concentration may cause some extra dissolved material from hoof possessed in a liquid state. In another way, the increasing concentration also causes degradation of the keratin protein, which affects the purity undesirably. 6 Advances in Materials Science and Engineering

Response Surface Analysis.
e three-dimensional response surface plot as a function of the interaction of two factors on dissolution and purity percentage is indicated in Figures 7 and 8, respectively. e interaction effect of the factors as shown in Figures 7(a)-7(c), affect the dissolution positively, as both of those factors were increased the dissolution percentage increased.
is is because, a fast extraction process and solubility were achieved at higher temperatures, concentrations, and time [38]. e purity of the polymer decreased as temperature and time increased, even if the decrement in purity by time factor is insignificant as shown in Figures 4(b) and 5(b). While the interaction effect of each factor is different from the separate effect of factors as can be seen in Figure 8(a). Figures 4(b)  and 6(b) show that, the reaction temperatures and     concentration harm keratin purity. While the interaction effect of those factors shows the probability of getting little improvement in purity (Figure 8(b)).

Numerical
Optimizations. e optimum condition for the extraction of keratin protein from cattle hoof was found to be 0.5 M of NaOH concentration, time of 60 minutes and 55°C temperature with optimum desirability of 85% for dissolution and 89.586% for purity.

Biuret Test.
e presence of protein in keratin extractive solution was confirmed by the biuret test [27]. After the reagent was added, the color of the extractive solution is changed to purple. is is true only if peptide bonds are present in the solution [40]. e higher the peptide bond in the solution, the higher intensity of purple color possessed.
As shown in Figure 9, a darker purple color was offered in sample "a" using 0.65 M, 65°C, and 120 min of the extraction process. While sample "b" and sample "c" are extracted at a minimum concentration (0.061373 M) and maximum concentration (1 M), respectively, shift from purple to blue-purple and pink-purple color, respectively. erefore, having a higher or lower value of NaOH concentration greatly affects the experimental process or the amount of protein being extracted in the solution. is analysis has also been in agreement with ANOVA analysis of factors on purity and dissolution percent.

Fourier Transform Infrared (FTIR).
Numerous characteristic bands have been possessed by keratin, such as amide A (N-H stretching), amide I, amide II, and amide III [41]. Important information about protein conformation and change in protein backbone structure is given by Amide I-III bands. Amide I is mainly originating from the stretching vibration of C�O at the characteristic band from 1700 to 1600 cm −1 [1]. It is widely related to the analysis of the secondary structure of a protein [42].
While amide II is observed at the characteristic band from 1560 to 1500 cm −1 , which is mainly from N-H bending and C-N stretching vibration. e presence of both α-helix and β-sheets structure in hoof keratin resembles bird and reptile structural conformation [43]. Amide III is also    Advances in Materials Science and Engineering present around 1276 and the presence of this band is due to the presence of beta structure [44]. e FTIR spectra shown in Figure 10, indicate amide I and amide II at 1633 and 1542 cm −1 for raw cattle hoof and 1650 and 1542 cm −1 for keratin protein, respectively. e characteristic band of both α-helix and β-sheets are observed in the amide I as well as in amide II. An α-helix conformation in the amide I and amide II are from 1657-1650 cm −1 and 1550-1540 cm −1 , respectively [28]. While the β-sheets structure of keratin was observed from 1635-1615 cm −1 and 1535-1520 cm −1 in the amide I and amide II region. In raw cattle hoof, α-helix and β-sheets conformation been appeared at 1658, 1650, 1633, and 1623 cm −1 in the amide I region and 1545 and 1524 cm −1 in the amide II region as shown in Figure 11. While for keratin powder, the expanded region shows a broader band for α-helix conformation at 1650 and 1532 cm −1 in the amide I and amide II region, respectively. As shown in Figure 10 presence of amide III around 1276 cm −1 confirms the presence of beta configuration in keratin.
Despite amide I, amide II, and amide III, N-H stretching vibration of the peptide bond (-CO-NH-) can be observed at a wavelength range of 3310-3270 cm −1 [20,41]. In the present scenario, N-H stretching occurs at 3291 cm −1 for both raw hoof and powdered keratin ( Figure 11). is shows the nondenaturing of the folded keratin structure at the time of the experimental process.  e amount of moisture in keratin powder has been measured in terms of moisture content. About 11.31% moisture content was achieved from the analysis. e value indicates that keratin material has better resistance to deterioration because of moisture. Researchers indicate that if the moisture content in the keratin material does not exceed 14% [28], it has better resistance to the moisture effect.
As discussed in the proximate analysis of the raw hoof, the moisture content in the raw hoof was about 5.96%. Larger moisture content was recorded for regenerated keratin than the raw hoof sample. is is because of the lower surface area of the keratin, which allows the hydrophilic site of keratin to react with water. Having a lower degree of crystallization in keratin than the raw material permits the formation of easier water bound to the hydrophilic group of keratins [45].

Conclusion
is study realizes that keratin can be extracted from slaughtered cattle hoofs using a solution of sodium hydroxide and urea.   (i) Around 75% protein content of the raw hoof has been a good indication of the material to be a good keratinous source. (ii) Concentration of NaOH and temperature have the most significant effect on the quality and quantity of keratin. (iii) e optimum condition for the extraction of keratin protein from cattle hoof was found to be 0.5 M NaOH and 60 minutes reaction time at 55°C temperature. (iv) e FTIR spectra, indicates that amide I and amide II occur at a wave length of 1633 and 1542 cm −1 for raw cattle hoof and at wave length of 1650 and 1542 cm −1 for keratin protein, respectively. (v) In conclusion, by employing optimum thermochemical conditions, the protein structure/quality can be maintained during extraction. (vi) Further study will entail increasing the possibility of keratin extraction from cattle hoof by using other extraction methods such as reducing, steam explosion, and microwave methods.

Data Availability
All the data is included in the manuscript, however, any additional data can be provided upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.