A green approach for modification and functionalization of wool fabric using bio- and nano-technologies

Native keratinaceous materials, chicken feathers and sheep wool, were prepared using the method of Friedrich et al. Native chicken feathers and sheep wool had been cut into small pieces, rinsed thoroughly with water, and defatted in chloroform–methanol (1:1 v/v) for four days. Once a day, the solvent was changed. Then, these materials were thoroughly washed, dried for 3 d at 50 °C and ground. Before being used as a source for powdered keratin, the powder was sifted throughout a 0.5 mm screen. Preparation of soluble keratin had been achieved by heating feathers in dimethyl sulphoxide (DMSO) at a temperature of about 100 ~ C for 2 h (Badrulzaman et al. 2021). In this manner, a 1.2 percent keratin protein solution in DMSO was ready. Acetone was used to precipitate the protein from the solution. For 1 volume of protein solution, 2 volumes of acetone were used. Phosphate buffer (0.1 M, pH 8) was used to re-suspend the keratin protein precipitate.

Homogenized soft corals, and sea sediments were subjected to serial dilution technique (10–1 to 10-6dilutions). Sterilized agar plates (15 cm) each having 50 ml of solidified isolation medium (containing g/l: yeast extract 5, glucose 10, peptone 5, KH₂PO₄ 5, MgSO₄. 7, H₂O 1, Na₂CO₃ 10 and agar 20). Na₂CO₃ was sterilized separately and added to the medium just before solidification giving final pH of 9.0 were prepared. Actinobacterial strains were isolated by inoculating the prepared agar plates with 200 µl from each dilution. The appeared actinobacterial colonies were picked and maintained on starch-nitrate slants after incubation at 30 °C for 7–14 days and then kept at 4 °C till the next use.

For the 11 actinomycetes, primary screening experiments were conducted using the agar plates technique. The isolates’ protease activity was assessed in this stage through their inoculation onto agar plates with the following ingredients (g/l): yeast extract 4, skim milk 10, starch 10, peptone 2 and agar 15 adjusted at pH 10 (Demir et al. 2015). Overflowing the Coomassie brilliant blue (CBB) R-250 solution, which was being destained with glacial acetic acid (10%) and methanol (40%), revealed activity as a clear zone all around the colony (Anbu et al. 2008). Good potential protease producers were chosen based on the clear zone diameter to colony diameter ratio.

Actinobacterial isolates C4, S33 and S11 (high protease/keratinase producers) had been identified using 16S rDNA sequence analysis, which includes the following steps: extraction of genomic DNA, polymerase chain reaction (PCR) and 16S genes sequencing for amplification of genomic DNA. For genomic DNA extraction from 18 h old actinobacterial culture, DNA purification kit from GeneJET Genomic (Thermo Fisher Scientific, USA) was used. Biometra Thermal Cycler (Labrepco, Germany)was used for the amplification of 16S rDNA using two 16S rDNA universal primers 27F (AGAGTTTGA TCM TGG CTC Ag) and 1492R (TAC GGY TAC CTT GTTACG ACT T). PCR products were purified using a Montage PCR Clean-up kit (Millipore). The Big Dye terminator cycle sequencing kit (Applied Biosystems, USA) was used to complete the sequencing. The automated DNA sequencing system (Applied BioSystems, model 3730XL, USA) was used to resolve the sequencing products. The NCBI website’s basic local alignment search tool (BLAST) was used to deposit the nucleotide sequence (1420 bp) into Genbank and compare to nucleotide sequence database. Alignment of 16S rDNA sequences was performed and phylogenetic trees were constructed. The taxonomic positions of strains based on 16S rRNA gene sequences of C4, S33 and S11 isolates are observed in the phylogenetic tree. This was determined using a workflow that began with a multiple sequence alignment using maximum parsimony (MP) trees and MUSCLE. The alignment with RAxML and TNT yielded maximum likelihood (ML).

For ML, rapid bootstrapping was used in connection with the auto-MRE bootstrapping characteristic, followed by a search for the best tree;1400 bootstrap replicates were used for MP, along with tree-bisection-and-reconnection branch swapping. To verify the sequences for compositional bias, the X2 test, as applied in PAUP, was used (Abdel-Aziz et al. 2019).

Alkaline protease was produced by cultivating the selected strains (with a CFU value of about 2.1 × 10⁸ cells/ml) on a production medium (g/l, w/v): 10 glucose, 5 casein, 5 yeast extract, 2 KH₂PO₄, 2 K₂HPO₄ and 1 MgSO₄.7H₂O (Sedrah et al. 2021). The medium pH was adapted to 9.0–9.5 using Na₂CO₃. The culture was grown for 5 and 7 days in an incubating shaker incubator (150 rpm) at 30 °C after inoculation with 10% of cells/ml. At the end of the fermentation period, the produced culture was centrifuged at 8,000 rpm and 4 °C for 20 min. The collected supernatant was used as the crude enzyme.

In addition, using the chicken feathers as the sole carbon and nitrogen source, chosen strains were tested for feather degradation capacity. These strains were grown in mineral salt medium (50 ml/ 250 ml Erlenmeyer flask) supplemented with 1% chicken feather as a keratin source. The mineral salt medium composed of (g/l, w/v): 1.5 K₂HPO₄, 0.05 MgSO₄.7H₂O, 0.025 CaCl₂, 0.015 FeSO₄.7H₂O, 0.005 ZnSO₄.7H₂O, pH 7.5 (Anbu et al. 2008). Fermentation broth was centrifuged at 5000 rpm for 3 min to remove the produced biomass and leftover solid substrate. Extracellular keratinase and protease activities were assayed in the cell-free fermentation broth.

Following Peng et al. (Peng et al. 2019) with a minor modification, 0.1 ml of culture supernatant was mixed with 0.4 ml of 1% (w/v) keratin (or other previously mentioned keratinaceous materials) in 50 mM Glycine/NaOH buffer (pH 10), and it was incubated at 40 °C for 1 h. The reaction was halted by the addition of 0.5 ml trichloroacetic acid (TCA, 10%). After 30 min at room temperature, the non-digested proteins (precipitated) were separated by centrifugation for 20 min at 10,000 × g. For Folin–Ciocalteu method, 200 μl Folin–Ciocalteu reagent and 1 ml Na₂CO₃ (4%) was added to 200 μl supernatant and incubated at 20 °C for 1 h. Its absorbance was measured at 660 nm. TCA solution was added before the enzyme reaction as a control. Under specific conditions, one unit of keratinase activity was expressed as the enzyme proportion requisite to release 1 g of tyrosine/min. Keratinase activity is the average of at least two determinations where the difference in values is less than 5%. Using a Shimadzu spectrophotometer (UV-1601) the amount was calculated from standard tyrosine solution with a concentration range of 50–500 µgm/ml.

The alkaline protease activity was determined in the cell-free supernatant using the previously described keratinase activity assay method, but with 1% (w/v) casein as a substrate in Glycine–NaOH buffer (50 mM; pH 10). Under standard conditions, one unit of protease activity was expressed as the enzyme proportion requisite to liberate 1 μg of tyrosine/min.

Except when differently specified, a sample containing 1% wool powder, chicken feather or keratin in 50 mM Gly/NaOH buffer (pH 10.0) was mixed with an appropriate dilution of enzyme. After 24 h at 40 °C, the reaction was terminated with the addition of 4% TCA. The hydrolysis yield was monitored as tyrosine by the Folin-Ciocalteu reagent. To test the hydrolysis in the presence of the reducing agent, the substrates were suspended in 50 mM Gly/NaOH buffer (pH 10.0) containing 0, 0.5 or 1% Na₂SO₃. The hydrolysis was done at 40 °C and the hydrolysis yield was evaluated by monitoring the increase in absorbance at 280 nm at a scheduled time (3, 6, 24, 48 and 72 h).

Initially, a sample of a grey wool fabric was washed (W) using Na₂CO₃: NaHCO₃ (0.1 M: 0.1 M) buffers (pH 9), along with nonionic wetting agent (2 g /l), in aqueous solution with liquor to material (20: 1) at (50 °C), for (30 min). The treatment bath was carried out in IR–machine for dyeing followed by rinsing in tap water followed by distilled water.

Grey fabric samples were washed and H₂O₂ -bleached in one step (W/B) using H₂O₂ (4% owf), along with nonionic wetting agent (2 g/L), at pH (9), and material to liquor ratio (1/20), for one hour at 50 °C, using IR-dyeing machine, followed by neutralization and washing for three times using distilled water. After washing, the treated fabric samples were oven dried to a constant weight.

Portions of grey (G), washed (W), and washed-bleached (W/B) wool samples were bio-treated with the produced alkaline keratinolytic protease enzymes using (5 ml/g fabric) enzyme dose, (2 g/l) sodium silicate, (2 g/l) nonionic wetting agent, and L/R (1/20), at pH (8) and temperature (50 °C) for time (60 min) in IR-dyeing machine. The enzyme-treated fabric samples were neutralized, using acetic acid to terminate the activity of the used enzyme, followed by thoroughly washing and drying.

Portions of enzymatically treated wool fabric samples were post finishing using ZnONPs (5% owf), ZrO₂NPs (5% owf), ascorbic acid (10% owf), or vanillin (10% owf) at 60 °C for 30 min in a sonicator bath with 55 kHz frequency, followed by padding twice to 80% wet pick-up and microwave fixation at 400 watts for 6 min.

Cotton and cotton/polyester (50/50) were used in this study. Specimens of the used fabric were stained with blood and egg stains. The samples were washed with alkaline keratinolytic protease enzyme S11-E (1 g/l) in presence of a nonionic wetting agent (1 g/l). The washing process was carried out in an ultrasonic bath using 47 kHz at 50 °C for 30 min. After washing the washed specimens were thoroughly rinsed with cold running water to remove the removed stains and used chemicals.

Applications of locally produced alkaline keratinolytic proteases, under appropriate conditions in surface modification of wool substrates followed by subsequent functional finishing using eco-friendly active ingredients namely ZnONPs, ZrO₂NPs, ascorbic acid and vanillin individually to impart highly demanded antibacterial, and UV-shielding functionalities to the biotreated substrates have been investigated.

The data in Table 3 revealed that pre-washing and pre-washing/H₂O₂ bleaching of grey wool samples result in increment in weight loss, decrement in nitrogen content, a slight improvement in antibacterial activity against the tested pathogenic E. coli bacterium along with a remarkable decrease in UPF values. The variation in the tested properties reflects the differences between the tested pretreatment steps in the extent of hydrophobic contaminants removal, e.g. fatty acids, grease, wax, natural colorants, etc., along with attacked, ruptured and removal of wool scales and the amino acids in the keratins due to oxidative effect of H₂O₂, which in turn resulted in increased weight loss and decreased nitrogen content of pretreated substrates (Ibrahim et al. 2012; Madhu and Chakraborty 2017; Radhakrishnan 2014; Wang et al. 2012).

It is also observed that addition of H₂O₂-bleaching agent to the prewashing formulation improves both hydrophilicity and wettability along with partial oxidation of natural colorants, i.e. improved whiteness, which in turn resulted in minimizing the UV-protection functionality, expressed in UPF value (Shen 2019).

On the other side, the positive role of H₂O₂ species like per hydroxyl species in attacking the keratins-amino acids and rupturing of the disulfide linkages of cysteine and hence increases the number of available active sites, e.g. ‒COOH, ‒NH₂, ‒SH, etc. consequently results in inhibiting the growth of pathogenic bacterium via altering the microbial cell membrane and thus damage the cell (Ibrahim et al. 2019).

Also, the data represented in Table 3 clearly show that post-enzymatic treatment of the pre-washed fabric samples using the produced proteolytic enzymes C4-E and S11-E results in a reasonable loss in weight, a slight decrease in nitrogen content, an increase in antibacterial activity and a significant reduction in UV-shielding functionality of bio-treated fabric samples. The degree to which the tested physicochemical and functional properties decrease or increase is governed by kind of substrate, G, W, or W/B and type of enzyme, i.e. its source, enzymatic activity and stability, active sites, bio-catalytic efficiency, molecular size and hence the extent of diffusion and penetration within the wool structure (Madhu and Chakraborty 2017).

As far as change in WL (%), N (%), BR (%) and UPF values of bio-treated substrates vary according to enzyme type. Table 4 data show that the decrease in weight, nitrogen content and UV-shielding ability follows the decreasing order: S11-E > C4-E, keeping other parameters constant. The experimental results demonstrate the variation in surface modification and functionalization of bio-treated substrates, the extent of degradation of cuticle scales and removal of hydrophobic matters, subsequent improvement in hydrophilicity and wettability, along with the creation of more accessible cationic active sites like amine terminal groups as a direct consequence of the enzymatic attack (Ibrahim et al. 2012).

The enhancement in antibacterial functionality with the combination of pre-and bio-treatments reflects the positive role of enzymatic post-treatment on enhancing the cationic sites on/within the wool structure thereby enabling the electrostatic interaction with the negatively charged bacterial cell membrane and consequently leading to leakage on intercellular components of the bacterial cell (Sheikh and Bramhecha 2018). Also, partial immobilization of the used proteolytic enzyme onto/within the bio-treated wool structure via adsorption, entrapment and/or chemical bonding makes the pathogenic bacterial protein more susceptible to subsequent attack, and hence the imparted antibacterial functionality is improved (Shen 2019).

Figure 6 depicts the EDX spectra and SEM images of untreated (a, b), prewashed then bio-treated using S11-E enzyme (c&d), and prewashed/H₂O₂ bleached then bio-treated (e, f) selected wool fabric samples. As seen incorporation of H₂O₂ bleaching along with post-enzymatic treatment with S11-E enzyme are accompanied by a noticeable modification and improvement of the surface of wool scales as a direct consequence of removal and/or modification of these scales via an oxidative treatment using H₂O₂ followed by proteolysis (e, f). The extent of descaling and surface modification follows the decreasing order: W/B → enzymatic treatment (Fig. 6e) > W → enzymatic treatment (Fig. 6c) >  > untreated (Fig. 6a) (Ibrahim et al. 2012; Jajpura 2018).

On the other hand, EDX elemental analysis (Fig. 6b,d,e) demonstrates the decrease in the S- element of the treated substrates as a direct consequence of pre‒H₂O₂ bleaching and subsequent enzymatic treatment which in turn resulted in the rupture of some cysteine disulfide bonds (Wang et al. 2012). The extent of decrease in S-content follows the above-mentioned order.

As shown in Fig. 7a post-treatment of bio-treated wool fabric samples with ZnONPs or ZrO₂NPs leads to a reasonable increase in the antibacterial functionality against the tested E. coli bacterium, which is one of the most common pathogenic negative bacteria, regardless of the used alkaline keratinolytic protease enzyme and type of nanometal oxide (NMO). The type of alkaline keratinolytic protease determines the extent of antibacterial functionality enhancement that follows the reducing order: S11-E > C4-E, which reflects their differences in the extent of modification of wool structure, generating of accessible active sites e.g. ‒NH₂, ‒COOH, ‒SH etc. and consequently uptake, fixation and immobilization of NMO onto/ within the wool structure. Figure 7a also demonstrates that the imparted antibacterial activity to the wool substrate follows the descending order: ZnONPs > ZrO₂NPs >  > None, keeping other parameters constant.

The imparted antibacterial functionality could be discussed in terms of the photocatalytic activity of loaded NMO under UV/Vis irradiation and subsequent generation of highly reactive species, e.g. H₂O₂, ^·OH, ^·O₂^‾, single oxygen (El-Nahhal et al. 2020; Ibrahim 2015; Ibrahim et al. 2018a, 2018b) as follow: which have the ability to inhibit the normal metabolism of the pathogenic bacteria, and oxidize the organic components in the bacterial cell thereby contributing to the imparted antibacterial efficacy. Additionally cell wall damage and change in the cell wall permeability due to the abrasion of the loaded NMO particles along with releasing of metal cations leading to the death of pathogenic E.coli bacterium (El-Nahhal et al. 2020; Prasad et al. 2016).

It is quite clear from Fig. 7b that loading of ZnONPs or ZrO₂NPs onto the bio-treated wool fabric samples results in a significant increase in UV-protection functionality, demonstrated as UPF value, irrespective of the used alkaline keratinolytic protease and follows the decreasing order: ZnONPs > ZrO₂NPs >  > None. On the other hand, bio-treated fabric samples using S11-E, in the absence and presence of NMO particles, demonstrate better UPF in comparison with C4-E bio-treated ones, which could be discussed in terms of the degree of the nano-finished materials distribution onto/within the nano-finished fabric structure. The substantial improvement in the UV-shielding capacity of the bio-treated then nano-finished fabric samples, (UPF > 50⁺, excellent), reflects the capability of the loaded ZnONPs and ZrO₂NPs in shielding and blocking the harmful UV radiation most probably as a result of their greater surface area and intense absorption in the UV-range (Harifi and Montazer 2017; Ibrahim et al. 2018d).

On the other hand, Fig. 7a and b demonstrate that even after 15 consecutive washing cycles, the imparted functional properties remain > 90% and > 50⁺ for antibacterial and UV-protection functional respectively, which reflects the high extent of fixation and immobilization of the used ZnO and ZrO₂ NPs onto/within the bio-treated then nano-finished wool fabric samples.

Once again, the so obtained results ultimately indicate that enzymatic treatment of wool fabric with the prepared enzymes results in surface modification, degradation of cuticle scales, removal of hydrophobic contaminants, enhancing fabric hydrophilicity and wettability along with creation of more accessible active sites, e.g. –NH₂, –COOH, –NH, thereby improving the extent of loading/fixation and immobilization of ZnONPs and ZrO₂NPs onto/within the wool structure and hence upgrading its antibacterial and anti-UV functionalities compared with the untreated ones.

Figure 8 demonstrates the surface morphology and EDX spectra of selected wool fabric samples bio-treated and then nano-finished with ZnONPs and ZrO₂NPs respectively, as shown in Fig. 8a, b, c and d). SEM images Fig. 8a and c confirmed the deposition of ZnONPs and ZrO₂NPs onto the surface of nano-finished wool fabric samples. Additionally, EDX spectra of nano-finished bio-treated wool fabric samples Fig. 8b and e showed peaks of Zn and Zr-elements in their pattern confirming the immobilization and loading of both ZnONPs and ZrO₂NPs onto the bio-treated then the nano-finished wool fabric samples in comparison with the biotreated ones Fig. 6e and f.

Changes in the antibacterial properties along with UV-shielding capacity of bio-treated wool fabric samples as a function of the type of environmentally sound active ingredient, the data in Fig. 9a and b demonstrate that individual incorporation of ascorbic acid and vanillin in the finishing bath as active phenolic compounds leads to a substantial enhancement in both antibacterial activity against E. coli (pathogenic Gram-negative bacteria), and UV-protection functionality. The extent of improvement in the aforementioned functionality demonstrates the following order: S11-E > C4-E and ii) vanillin > ascorbic acid >  > None, keeping other parameters constant.

The imparted antibacterial activity to the bio-treated fabric samples loaded with ascorbic acid and vanillin most probably is attributed to the negative impacts of the loaded phenolic bioactive compounds on DNA and disrupting effect on the bacterial cell which in turn adversely affected its growth and survival (Gegel et al. 2018; Hong 2014, 2015; Ibrahim et al. 2019, 2018c; Kumaraswamy et al. 2019).

On the other hand, the inclusion of any antibacterial ingredients, ascorbic acid and vanillin phenolic compounds, in the post-functional finishing of bio-treated wool samples is accompanied by a remarkable enhancement in their UV-protection capacity, expressed as UPF value, reflecting the positive role of bonded phenolic compounds in blocking the fabric porosity, in shielding and/or absorbing the extra UV-harmful radiation (Hong 2015; Ibrahim et al. 2019; Kim 2015). Additionally, loading of vanillin fragrant onto/within the bio-treated wool structure adds the fragrance functional property to the finished substrate.

Moreover, Fig. 9a and b further demonstrate that, all post-finished fabric samples, after 15 wash cycles, exhibited a small reduction in both BR (< 15%) and UPF (< 10%), irrespective of the loaded phenolic compound, due to the release of unfixed, physically bonded and/ or entrapped active ingredients by increasing the wash cycles. The degree of fixation and immobilization of the nominated phenolic compounds is governed by chemical structure, molecular size, functional groups as well as a mode of interaction and chemical fixation onto/within the bio-treated wool accessible active sites as follows:

which in turn enhances the degree of fixation and immobilization of the used active ingredients, i.e. high durability to wash.