Cryogenic milling-based keratin microparticle production from Anatolian goat fibers and their structural,chemical and thermal properties

Abstract

Fibers procured from Anatolian goat (Capra aegagrus hircus) furs were converted into keratin microparticles by the cryogenic milling method. Single-stage mechanical milling with a 2.5 h optimal grinding time was sufficient enough to generate the desired high-quality microparticles. No chemical solvents were utilized during the production process of keratin microparticles. Structural, chemical and thermal properties of keratin microparticles were assessed using particle size analyzer, stereo microscope, Brunauer–Emmett–Teller™ surface analyzer, field emission scanning electron microscope, X-ray diffractometer, attenuated total reflection Fourier transform infrared spectroscopy, thermogravimetric analyzer and differential scanning calorimetry methods. According to the characterization results, microparticles with amorphous structure in micro size could be produced without any agglomeration during the milling process. Goat farming is usually for dairy products and their fibers have very limited use in the textile industry because of their flat structure. Therefore, the conversion of goat fibers into another product that can be used in other industrial areas is expected to contribute significantly to a sustainable economy.

Keywords

Sustainability Anatolian goat fibers cryogenic grinding keratin microparticles particle characterization

Natural polymer materials are widely preferred due to their biocompatibility, biodegradation, mechanical durability, cellular functionality and low toxicity properties.^1,2 The use of natural polymers in biomedicine and biotechnology for tissue engineering,³ in controlled drug delivery⁴ and medical implant⁵ applications has gained increasing attention.

Nowadays, studies on the use of keratin as a natural protein have gained significant importance. Such studies have been oriented toward developing functional materials and devices with high added values.^6,7 Bird feathers, hairs, hooves, horns, wool and goat fibers can be described as some of the major keratin resources⁸ in nature as biological types of materials.

Goats were among the first livestock to be domesticated and have been contributing to the economic, agricultural and social life of human civilization for more than 10,000 years.⁹ They are also among the rare species that can survive extreme climate conditions with minimal feeding resources. The reproduction methods of goats have been traditionally unchanged for generations, usually substantiated in marginal geographies and harsh environmental conditions. There are 800 million goats in the world and Asian countries harbor nearly 60% of this population. Although the general economics of goat breeding is for meat and milk production, there is a marginal benefit from their fibers.¹⁰ It is possible to use them as thermal insulators in the textile industry due to the physical, thermal stability and mechanical properties of goat fibers.

The Anatolian goat (Capra aegagrus hircus) population in Turkey was over 10 million in 2009, which has increased over the past decade in parallel with the nutritional needs of people. In addition, there has been an increase in the fibrous waste obtained from the hides of these animals.^11,12 Due to their flat structure, it is very difficult to convert these fibers into a value-added product in the textile industry. However, in recent years, there has been a limited number of studies on the conversion of these fibers into value-added keratin resources, which may be useful for many sectors of the economy.^13,14

As a biological-based polymer, goat fiber is comprised of the cuticle, cell membrane complex and cortex. The cortex constitutes 90% of goat fibers by weight. The cortex incorporates keratin with strong sulfate bonds inherent to sulfur-containing amino acids.¹⁵ Keratin comprises well-ordered α-helix and β-sheet structures in combination with some other irregular structures. The α-helix and β-sheet structures of keratin appear in a tightly packed form. Moreover, due to the cysteine content of keratin, a sulfur–sulfur covalent bond is produced between the cysteine molecules.

In addition to intermolecular disulfate linkages between cysteines, intramolecular bonds, such as non-covalent interaction, electrostatic forces, hydrogen bonds and hydrophobic interaction types of non-covalent interactions occur.¹⁶ All of these bonds within the protein induce a strong and stable keratin structure and hence complicate the extraction of keratin by chemical solutions.^17,18

Recent studies on the extraction of keratin particles from Anatolian goat fibers (Capra aegagrus hircus) have been aimed using of (i) chemical solvents, (ii) the combination of chemical solvents and mechanical milling methods and (iii) various mechanical milling methods in a multistage process, mainly.

In one of the studies, keratin extraction was performed by preparing an aqueous solution of cashmere goat fibers and then this keratin was utilized to develop flexible, transparent and comfortable optical devices.¹⁹

In another study based on a chemical method, keratin particles were procured by dissolving goat fibers in an ionic liquid.²⁰ Finally, in a research study based on mechanical grinding methods, keratin powders were produced from cashmere goat fibers by using a wet milling method and a combination of chopping, attritor milling, spray drying and air jet milling methods.^21,22

The methods utilized in the previous studies have two major disadvantages: (i) regarding the use of chemical solvents during the production of keratin particles, there is a strong possibility of some residuals still remaining within the structure of keratin after the washing procedure for the estrangement of chemicals; (ii) increased energy costs and a longer keratin production process due to the use of different mechanical milling methods together. At this point, the pulverization of goat fibers without utilizing any kinds of chemicals and converting the fibers into highly pure particles is very significant for their use in biomaterial applications as a keratin resource.

Due to the elasticity of goat fibers and the complex bond structure of the inherent keratin, the pulverization and conversion of the fibers into a powder form by using a single-type mechanical grinding method is almost impossible. Considering the drawbacks of various conventional approaches in the trituration of goat fibers into keratin particles, here an alternative cryogenic milling method is proposed. Keratin particles were produced by optimizing the process conditions for cryogenic milling of goat fibers. Then the procured keratin particles were characterized by stereo microscope, laser diffraction particle size analyzer, Brunauer–Emmett–Teller™ (BET) surface analyzer, field emission scanning electron microscopy (FESEM), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) analyzer and thermogravimetric analyzer (TGA) measurements.

This study has two critical and innovative aspects: (i) no chemical solvent was used during the course of keratin particle production; (ii) one type of mechanical milling method was sufficient to achieve the required particle form. Furthermore, this is the first study proposing a cryogenic milling method for the trituration of goat fibers into keratin microparticles.

Materials and methods

Specimen preparation

The goat fibers utilized in the experimental studies were procured from a local market in Turkey. The measured dimensions of the goat fibers were, on average, 70.4 µm in diameter and, on average, 300 mm in fiber length.

The fibers were cleaned with a washing solution to remove the oil and dirt residues on the surface before the implementation of the milling process. The washing solution was prepared by mixing 3 g NaOH into 300 mL distilled water. Afterwards, Triton X-100 and 0.45 g citric acid were added to this solution. The bath ratio of the washing solution was prepared with a 50:1 liquor-to-goods ratio. After adding the fibers into the beaker with the washing solution, the fibers were mixed by a magnetic stirrer at 60°C for 1 h. The washed fibers were rinsed and dried at ambient room temperature for 24 h. After the drying process, the fibers were cut manually by SDL Atlas equipment into ∼2–5 mm length dimensions for cryogenic milling. Finally, the fibers were milled with liquid nitrogen to reduce the size of the material down to powder dimensions.

Production and characterization of keratin particles

The preliminary stage of this study consists of the production of keratin particles using the cryogenic milling method. In the second stage of the study, characterization studies were performed to determine the particle size distribution, surface area, morphology, crystallinity, chemical structure and thermal stability of the keratin particles.

Production of keratin particles by cryogenic milling

Chopped goat fiber snippets with a batch size of 4 g were placed into a polycarbonate vial together with a stainless steel magnetic impactor. These wool fibers were milled by placing the vial in liquid nitrogen (–196°C) enclosed by a Spex Certiprep™ Cryogenic Mill 6870-230 (Thermo Fisher Scientific). The goat fibers were pulverized by the cryogenic mill under the prescribed conditions: preliminary freezing in a liquid nitrogen bath for 15 min, milling at a frequency of six cycles per second for 8 min and holding for 8 min between cycles. Total milling was completed in 48 min and this cryogenic milling operation was repeated three times. Samples were kept in the liquid nitrogen bath during the entire milling process. After the completion of the cryogenic milling process, the samples were left for 1 h to warm them to room temperature before opening the lid of the polycarbonate vial. Figure 1 presents a schematic illustration of the cryogenic milling process for obtaining keratin particles from goat fibers.

Particle size distribution

After cryogenic milling of goat particles, the volume weighted particle size distribution of keratin powders was measured by a Malvern™ Mastersizer 3001. Potable water was used as a particle-dispersion medium in this laser diffraction-based particle size analyzer. The average of three measurements taken per each sample is reported here in the coming sections.

Measurement of the BET surface area

BET surface area measurements were performed by a Quantachrome™ Autosorb-1 MP device using the N₂ gas adsorption technique. Some 1 g of goat snippets of 5–10 mm in length were placed in a sample holding glass. Likewise, keratin powder samples were placed in between the sample holding glass, and to prepare for the gas adsorption measurement, both snippet and powder samples were degassed under N₂ at 80°C for 24 h. Each sample was measured three times, and the average BET data are reported here.

Stereo microscope analysis

Three-dimensional depth images of goat fibers and keratin powder particles were captured at low magnification rates using a stereo microscope. The light transmitted to the surface of the samples, reflected back to the objective of the device, forms the basis of surface analysis by microscope. Low magnified images of the fibers and particles were obtained by a high-resolution Zeiss™ Axiocam ERc5 s digital camera combined with a Zeiss™ Discovery.V12 stereo microscope.

Field emission scanning electron microscope analysis

Goat fibers and keratin particle morphologies were observed by an FEI-Quanta™ FEG 250 SEM scanning electron microscope (FESEM). Before capturing SEM images of the samples, double-sided sticky carbon tapes were pasted onto the surface of the aluminum stubs. Goat fibers and keratin particles were placed onto these stubs. Then, the outer surface of the samples was coated with a thin layer of gold before the image capturing procedure by the FESEM measurement device. The chemical compositions of the goat fibers were measured by energy dispersive X-ray spectroscopy (EDS, coupled to FESEM).

X-ray diffraction analysis

The crystal structure of goat fibers and keratin particles was determined by XRD analysis. A Bruker™ AXS Discovery D8 was used to characterize the samples with XRD spectra over a 2θ range of 10–50° and at a scan rate 5°/min.

The crystallinity index (C.I.) of the samples was computed by Segal’s equation²³

C . I . = \frac{I_{9} - I_{14}}{I_{9}} \times 100

(1)

Here, I₉ and I₁₄ are approximately the maximum crystal lattice diffraction at 2θ = 9° and 2θ = 14°, respectively. Generally, high C.I. indicates high crystallinity.

Fourier transform infrared analysis

The surface chemistry of goat fibers and keratin particles were analyzed by FTIR. The FTIR spectrum was recorded on a Thermo Scientific Nicolet IS50 attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) spectrometer adjusted to 2 cm⁻¹ resolution and 400–4000 cm⁻¹ frequency range. The KBr pellets used during the measurements were prepared by mixing KBr with the sample materials in particulate form.

Thermal analysis

The TGA analysis of samples was performed using a TA Instruments SDT Q600 model device to quantify the thermal decomposition properties of the samples. TGA measurements were initiated from 50°C, heated at a rate of 10°C/min and terminated at 600°C under nitrogen atmosphere conditions. Differential scanning calorimetry (DSC) was conducted on a TA/Discovery DSC251 with 5–10 mg of sample in closed aluminum pans at a ramp rate of 10°C per min.

Results and discussion

Stereo microscope images of goat fibers and keratin particles

Figure 2 shows stereo microscope images of goat fibers and keratin particles captured after the cryogenic milling process. Figure 2(a) renders the goat snippet cuts prepared from goat fibers.

Goat snippets possess a straight, unpleated acicular geometry with a black colored appearance. After cryogenic milling, keratin particles were converted into powder form (Figure 2(b)), which then appears brown in color.

Keratin particle distribution and BET surface area

Figure 3 depicts the particle size distribution of keratin particles after the cryogenic milling process. The distribution graphics indicate a monodisperse and highly homogeneous spread of keratin particles. This monomodal distribution is a key evidence to state that there is no tendency for particle agglomeration. Average size of the particles were measured as 4.67 µm. The production of microparticles by the cryogenic milling process was completed in a timeframe of less than 2.5 h, denoting a much more efficient method compared to the conventional alternatives, which usually entail higher energy consumption.²⁴

The lubricants utilized during the trituration of inorganic materials by conventional mechanical milling act as a process control agent for size reduction while preventing the agglomeration of the particles.^25–27 The cryogenic milling-based production method proposed in this study has been sufficient enough to attain microparticles without the need for any lubricant utilization during the process.

This can be elucidated by the effect of the cryogenic milling temperature inducing a reduction in activation energy for elastic fracture and hence the minimization of particle agglomeration.²⁸ Consequently, with short milling periods and without using any lubricant to prevent agglomeration, it has been possible to obtain micro size particles successfully.

The BET surface area of goat snippets and the particles obtained by cryogenic milling were measured as 0.005 and 2.12 m²/g, respectively. After the grinding process, a significantly higher BET surface area was attained.

Morphology of goat fibers and keratin microparticles

The FESEM images and EDS spectrum of the samples are shown in Figure 4. On examining the morphological structure of goat fibers (Figures 4(a) and (b)) at the micro scale, they individually exhibit a random orientation in three dimensions with a flat structure. Besides, the epidermis layer inherent to goat fibers is lucidly visible. There is no impurity on the surface region of the fibers. No deterioration of the cuticle layer has been observed, after chopping the fibers down to 10–15 mm length goat snippets and then subjecting them to the cryogenic milling process.

The elements involved in the goat fibers and their concentration rates were studied by EDS analysis. From the EDS spectrum (Figure 4(c)) the peaks associated with the carbon (C), oxygen (O), nitrogen (N) and sulfur (S) elements inherent to goat fiber can be easily identified.²⁹ The purity of the goat fiber can be justified by the spectrum indicating the sole presence of C, O, N, S elements in the samples investigated.

The morphologic structure of keratin particles produced by cryogenic milling can be seen in Figures 5(a) and (b). After the milling process, the particles have been pulverized into a needle-like and partially arc-shaped geometry. Particle size measurements were conducted by random selection from the FESEM images, indicating an average dimension of 5.65 μm. This result conforms with the range provided by the particle size distribution measurements.

In addition, FESEM images have evinced the fact that the keratin microparticles were crushed into small pieces without any agglomeration. It is clearly visible that the outer cuticle layer of the keratin disappeared, revealing the keratin-containing inner cortex of the samples.

It has been verified by FESEM images that cryogenic milling is a promising keratin microparticle procurement method, with a short period of milling time, no need for chemicals and no tendency toward agglomeration as some prominent advantages.

Crystallinity of goat fibers and keratin microparticles

XRD analysis is the most prominent method to determine the crystal phases in material structures. In this study, the physical change in goat fibers after cryogenic milling and the crystallinity of the samples were assessed by XRD analysis.

Figure 6 describes the XRD patterns for goat fibers and keratin microparticles. Upon investigating the XRD patterns of goat fibers (Figure 6(a)) at 9.8° and 20.6°, 26.9°, 29.7°, 2θ values of half crystal and crystal peaks are evident. The prominent peaks at Bragg angle 2θ = 20.6°, 26.9°, 29.7° and the medium peak at 2θ = 9.8° are associated with the β-sheet and α-helix in the peptide chain structure of the goat fiber.³⁰ The XRD pattern of keratin microparticles indicates the presence of amorphous peaks at 2θ values of 8.2° and 20° (Figure 6(b)). These diffraction peaks are related to the α-helix and β-sheet conformation inherent to the molecular structure of keratin protein.

During the conversion of goat fibers into keratin particles, semi-crystal and complete crystal peaks disappeared, emerging into broader and low-amplitude amorphous peaks. This steady deterioration and conversion into amorphous form is associated with an increase in disoriented areas in the β-sheet configuration.³¹ In a previous study we determined the disappearance of the cuticle layer on the outer surface of the protein fiber inducing the revival of a higher rate of steady crystalline structures in microparticles.³² However, in this study, complete destruction of the outermost layer of the fibers yielded the procurement of keratin microparticles in an amorphous structure.

The C.I.s of the samples were determined by using Equation (1). The ratios for goat fibers and keratin microparticles were measured as C.I. = 42.6% and 30.8%, respectively, indicating a decrease in C.I. for keratin microparticles.

This can be attributed to the destruction of some regions of semi-crystal and crystal regions of keratin particles after cryogenic milling. The proteins containing crystal β-sheet configuration were removed by the cryogenic milling process, producing keratin particles with increased amorphous regions.

Chemical composition of goat fibers and keratin microparticles

The type of bonds in goat fibers and keratin microparticles and the alteration in their bonding mechanisms has been assessed by FTIR analysis. In this study, conformation of the polypeptide chain inherent to the keratin structure and the orientation of the corresponding components were determined from FTIR spectra.

Figure 7 shows the FTIR spectrum for goat fibers and keratin microparticles. By examining FTIR spectra, goat fibers and keratin microparticles exhibit definitely similar vibration behaviors. The characteristic peaks that emerge in the spectrum of goat fibers (Figure 7(a)) are associated with the peptide bonds inherent to the keratin protein structure and such peaks need to exist in a typical production process, which are major indicators of the characteristic bonds of keratin material.

The peak vibrations in peptide bonds are due to the bands that are delineated as Amide A, Amide I, Amide II and Amide III.³³ The medium peak that appeared at 3275.2 cm⁻¹ is related to Amide A titration, associated with both N-H and O-H stretching vibration. The peak observed at 2930.7 cm⁻¹ is related to C-H stretching vibration. An intense absorption peak emerged at 1634.4 cm⁻¹, originating from Amide I, which is associated with C=O stretching vibration, representing the secondary structure of the protein. This peak is essentially an indicator of α-helix conformation in the major chains of the protein within the fiber structure. The strong peak at 1515.5 cm⁻¹ and the medium peaks at 1446.7 and 1393.5 cm⁻¹ are associated with Amide II related N-H bending vibration, respectively. The medium level peak emerging at 1235.1 cm⁻¹ is related to the C-N stretching vibration of Amide III. This stretching vibration originating from Amide III is an indicator that highlights β-sheet conformation in goat fibers.^18,34 The peak at 1079.6 cm⁻¹ is due to the bonding mechanism of cysteine monoxide (-SO-S-).³⁵ The stretching vibration of the disulfide bond (-S-S-) can be ascribed to the existence of a corresponding peak emerging at 513.9 cm⁻¹.³⁶ Examining the spectrum of keratin microparticles (Figure 7(b)), the vibration peaks associated with similar bond structures tend to emerge at similar frequencies, but with some reduction in response amplitudes. The attenuation of peak amplitudes can be attributed to the destruction of the crystal regions of the fiber structure by the cryogenic milling process and, hence, ultimately yielding an increase in amorphous regions.

Using chemical solvents to extract keratin from goat fibers resulted in nascent chemical bond structures.^37,38 However, in our current study, due to the chemical-free particle production process described here, no vibrational peak associated with the bond structure of a new functional group has been observed in the FTIR spectrum of the ultimate keratin material.

Thermal properties of goat fibers and keratin microparticles

The thermal stability, degradation behavior and weight loss ratio of the test samples due to thermal effects has been assessed by TGA measurements. Thermograms of goat fibers and keratin microparticles can be seen in Figure 8.

The degradation of samples characterized by the thermogram measurement is in three stages. The first stage is described by the temperature range that results in a 3–5% weight loss. Here, the initial degradation of goat fibers and keratin microparticles occur in the 25–60°C temperature range. This degradation can be attributed to the detraction of physically adsorbed moisture from the goat fibers after its progress to the surface of the structure.

The maximum amount of degradation occurs in the second stage. Goat fibers and keratin microparticles exhibit the highest weight loss rates in the 283.3–336.6°C and 307–343.7°C temperature ranges, respectively. The second stage of the weight loss is due to denaturation of α-helix and β-sheet proteins.⁴ Finally, the least amount of weight loss for goat fibers and keratin microparticles was observed in the third stage of the thermogram, specifically in the 336.6–650°C and 343.7–650°C temperature ranges, respectively. This third stage of weight loss is due to the termination of oxidative degradation of ash-contained residue formation during the second stage of the degradation.³⁹ Such degradation behavior indicates a marginal increase in the thermal stability of keratin microparticles produced by cryogenic milling. The remaining mass from goat fibers and keratin microparticles after the exposure to 650°C temperatures is 27.6% and 30.2%, respectively.

Phase behavior of goat fibers and keratin microparticles was examined by DSC measurements (Figure 9). Both materials exhibit a low-temperature endothermic peak at 36°C due to evaporation of the adsorbed water by the test samples. In the DSC curves for keratin microparticles, this peak poses a shift to lower temperatures. This indicates a partial alteration in the water sorption capacity of the samples after the cryogenic milling process.

In DSC measurements for goat fibers and keratin microparticles, the exothermic peaks at 206°C and 214°C are related to denaturation and disordering, which emanates after melting of the α-helix protein.⁴⁰ The exothermic peaks that appear in the goat fiber DSC graphic at 307°C temperatures are associated with the melting of sulfur cross-bonds and the β-sheet chain containing microfibrillar matrix structures.³⁶ This peak was not visible in the DSC thermogram of the keratin microparticles. This is due to the transformation of the β-sheet crystal structure into the amorphous form. DSC measurement data comply with the XRD results and the results of the other characterization studies.

Conclusion

Titration peaks associated with the bond structures of goat fibers and keratin microparticles exhibit a constant shape elucidating an intact chemical composition for the protein. However, with the proportional increase in the amorphous region of the resulting microparticles, the intensity of the vibrational peaks of the associated bonds decreased partially. This tendency has also been verified with the result obtained from XRD patterns. In addition, the TGA and DSC results indicate that the keratin microparticles possess better thermal characteristics.

Footnotes

Acknowledgement

The authors would like to thank Nurcan Acar (a BS student in Metallurgical and Materials Engineering, Bursa Technical University) for her drawings of the cryogenic milling schematics.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Merve Küçük

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