Wet Blue Enzymatic Treatment and Its Effect on Leather Properties and Post-Tanning Processes

Due to their variety, specific activity, and mild reaction conditions, enzymes have a wide application in beam house processes such as soaking, dehairing, bating, and de-greasing. Recently, due to improvements in biotechnology, re-bating after chroming has received increased attention. The aim of this work was to investigate the application of enzyme preparation in the re-bating process and its effect on the semifinished and finished product, as well as its influence on post-tanning operations. The enzymatic treatment of chromed semifinished leather (wet blue) led to a higher shrinkage temperature (1–6 °C), greater water vapour absorption (0.3–5.5%), better chromium compounds exhaustion during re-chroming (4–21%), and better dye penetration. Moreover, collagen was affected during the enzymatic process; the results showed a greater concentration influence in the operation compared to the process time. On the other hand, no effect on the physical and mechanical properties and fat-liquoring process was observed. Overall, these results indicate that some properties and processes are improved; however, before use for re-bating, every enzyme should be carefully investigated.


Introduction
The leather industry employs waste from the food industry to produce diverse products, such as garments, footwear, upholstery, etc. However, these operations use a significant amount of chemicals and water, producing huge amounts of solid and liquid waste and leading to a negative effect on the environment [1]. To decrease environmental impact, new technologies are being developed; these can be classified into two main groups: (1) Wastewater treatment and cleaner solid waste processing; (2) Cleaner technologies that reduce the pollution load or do not use hazardous chemicals in the processing of leather [2].
Currently, studies are investigating enzyme employment in leather processing, not only to reduce the impact on the environment, but also to improve the properties of the leather. Enzymes find wide applications in beamhouse processes (soaking, dehairing, bating, de-greasing) because of their variety, specific activity, and mild reaction conditions [3].
Despite the improvements in biotechnology, enzymes are mainly used in the bating of hide/skin after unhairing and the opening of derma; for this step, the use of ferments is crucial. During bating, non-collagenous substances such as albumin, elastin, globulin, and proteo-glycan are removed. This step is necessary to create a soft finished product, increase the area, and prepare leather for tanning [4][5][6]. Furthermore, non-collagenous proteins remaining in hide can cause a lack of flexibility and firmness in dried leather [4]. For interfibrillar substances, the removal of alkaline or neutral proteases is carried out; nevertheless, new studies show the possibility of acid protease application in bating [7][8][9][10]. Currently, proteases used in bating are derived from non-hazardous microorganisms.

Analysis Methods
The caseinolytic activity of EP was determined using the Anson method [19]. Sodium caseinate was used as a substrate. For the determination of collagenolytic activity, the modified method of Xian et al. [20] was used. Soaked leather fibres from lyophilized hairless hide were prepared and chosen as a substrate. An amount of 300 ± 1 mg of fibre was accurately weighed in a 20 mL test tube, followed by adding 9 mL of B-R buffer, (5 pH) and stirred in a shaker for 10 min at 40 • C with 200 r/min. Then, 1 mL of enzyme solution (diluted to a certain concentration with the same B-R buffer) was added and stirred for another 30 min at 40 • C with 200 rev/min, precisely. Finally, the reaction liquor was filtered with a qualitative filter paper, and the reaction solution was hydrolysed using 6 N of HCl at 120 • C for 10-12 h. The concentration of hydroxyproline in the digested liquor was tested according to the photo-colorimetric method [21].
The amount of collagen proteins removed was estimated from the amount of hydroxyproline in the pickling solution using a photo-colorimetric method [21]. Samples of the wet blue bating solution after the process were hydrolysed using 6 N of HCl at 120 • C for 10-12 h. A formation of coloured soluble product was based on a reaction of hydroxyproline with p-dimethylaminobenzaldehyde. The absorption was measured with a spectrophotometer GENESYS-8 (Spectronic Instruments, Cheshire, UK) at a 558 nm wavelength.
The shrinkage temperature after the re-bating and post-tanning processes was determined as described in the literature using special equipment and replacing the distilled water with glycerol [22]. An image of the equipment and working principle is presented in Figure S1.
Chromium compound exhaustion was estimated by determining the concentration of chromium in the initial re-chroming solution, and in a mixture of the used re-chroming solution and washing (after re-chroming) solution. The concentration of chromium in solution was determined according to the method described in the literature [22]. The method prescribes oxidation of the chromium presented in the solution into a hexavalent state using hydrogen peroxide, and an analysis of the solution by iodometric titration.
For IR spectroscopy, a Perkin-Elmer FTIR Spectrum GX (Waltham, MA, USA) spectrometer with a horizontal attenuated total reflectance accessory was used. The wavelength interval was 4000-650 cm −1 ; the resolution was 4 cm −1 , and the scan number was 10 times. Before the IR-spectroscopy analysis, the leather samples were dehydrated with acetone [23].
The exhaustion of the dye was determined by the colorimetric method by measuring the absorbance of the dye solution. The dye absorbance was measured using GENESYS-8 (Spectronic Instruments, Cheshire, UK) at a 495 nm wavelength. Distilled water was used as a solvent. Dye consumptions were calculated using the calibration curve.
The penetration of dye through the hide was evaluated using a special optical microscope with scale (magnification 15 times) MPB-2 (Izyum Instrument Making Plant, Izyum, Ukraine).
The strength properties, water vapour absorption, amount of chrome compounds in the leather, the matter soluble in dichloromethane, and the volatile matter were determined according to the standards [24][25][26][27]. Before the mechanical tests, samples of wet blue were dehydrated (fixed) with acetone [23] and dried in fume board at ambient temperature (23 ± 2 • C) for 24 h. The finished leather samples were placed on a table and dried in a free state for 48 h at a temperature of 23 ± 2 • C. After drying, all the test pieces were stored at a minimum of 24 h prior to testing in the standard climatic conditions at a temperature of 23 ± 2 • C and a relative humidity of 50% ± 5%.

Statistical Analysis
All data were expressed as the average value of measurements performed in triplicate. One sample was used for one measurement. Standard deviations did not exceed 5% for the values obtained.

Results and Discussion
The conventional bating process uses alkaline enzymes, which are most active at higher pH values. However, due to the relatively low pH value of wet blue, for re-bating, it is important to use enzymes that have proteolytic activity in acidic medium. For this, caseinolytic and collagenolytic activity were assessed for the four EPs that were used in the following bating process ( Table 2). The results show different proteolytic activities depending on the substrate used. All the EPs were more active on casein, whereas their activity on collagen was relatively low. This is not necessarily a negative finding, collagenolytic activity that is too high can cause damage to the leather structure. NovoBate and Zime SB showed the greatest results with both substrates, although the differences between both EPs were significant; Zime SB activity was more than four times lower on casein and almost eight times lower on collagen substrate in contrast to NovoBate.
NovoBate WB had the highest proteolytic activity on both substrates compared to other EPs; its caseinolytic activity was more than 166 times higher compared to the activity of Oropon WB, and the collagenolytic activity was 39 times higher compared to the lowest activity of Oropon DVP. As mentioned before, the lowest activity on casein was obtained using Oropon WB; despite this, the activity on the collagen substrate was closer to the Zime SB value. The lowest collagenolytic activity was shown using Oropon DVP EP.
After the proteolytic activity was evaluated, the EPs were used for the WB treatment. Sixteen re-bating variants were tested for chromed leather bating (Table 3).  It is important to determine the effect on collagen, as greater collagen hydrolysis can have an impact on the physical and mechanical properties of leather [28]. Accordingly, after re-bating, the removed collagen amount and shrinkage temperature were assessed ( Table 3). The results show a different effect on collagen depending on the EP that was used in the process. EP Zime SB and NovoBate WB had a greater effect on collagen in wet blue. Increasing these EP concentrations led to a higher amount of removed collagen in the re-bating solution; using 5% EP, the removed collagen amount was more than three times higher compared to using 1%. Process times had less influence than concentration. However, the results indicate that Oropon DVP and Oropon WB had a lower impact on collagen in wet blue; the amount removed was very similar, despite the process time or concentration.
Despite the EP effect on collagen, the shrinkage temperature increased after treatment using Zime SB. The results obtained in Table 3 can be explained in that during re-bating, collagen and other substances in wet blue were affected; they were washed out during this process. Enzymatic bating led to better leather fibre opening. Due to this, the gaps between the fibrils narrowed and the shrinkage temperature increased. After re-bating, all samples had a higher shrinkage temperature than the control sample. An increase in shrinkage temperature was also observed in wet blue after treatment with enzymes used in the work of G.C. Jayakumar et al. [17].
After evaluating the amount of removed collagen and the shrinkage temperature, the re-bating process was repeated, and physical properties such as water vapour absorption, tensile strength, and relative elongation at the strain of 10 N/mm 2 and at the break were determined. As the EP concentration had a greater influence on the effect on collagen than the process time, the physical properties were assessed for samples that were re-bated for 1 h with EP concentrations of 1 and 5% ( Table 4).
The results in Table 4 indicate no apparent dependency on physical properties, such as tensile strength, relative elongation at break, and at the strain of 10 N/mm 2 . However, after enzymatic re-bating, the water vapour absorption increased; this can be explained by fibre opening and the removal of non-collagenous substances during wet blue treatment. Enzymatic re-bating results in an increased area; after wet blue bating, more gaseous water can bind to leather [3]. To evaluate how deeply the enzymatic treatment affected WB, an FTIR analysis was performed. The IR spectra after the EP process with all eight variants and control were recorded and analysed (Figure 1). In the control sample, a broad peak was observed at around 3600-3000 cm −1 , which is usually associated with adsorbed water (O-H stretching vibrations), as well as hydrogen bonds in amino acids (N-H stretching) [29][30][31]. After enzymatic treatment in the 3600-3000 cm −1 range, a higher number of low-intensity peaks were observed; this could be explained by the EP effect on intermolecular water and on hydrogen bonds in amino acids in protein. Furthermore, in all experimental samples, new peaks appeared at around 2989-2981 cm −1 and 1078-1055, which can be assigned to -CH 2 /NH 2 vibrations and C-O/C-O-C stretching, respectively. New peaks indicate an EP effect on WB and the formation of new bonds. Typically, 1100-1000 absorbance bands are assigned to carbohydrate moieties in leather [32,33]; however, carbohydrates did not form during the enzymatic treatment. Instead, EP may have affected the leather by hydrolysing the bonds between collagen and other substances. Nevertheless, the FTIR spectra showed that, regarding the formation of new bonds, the absorbance bands for amide (I), amide (II), and amide (III) remained the same.
After re-bating, other post-tanning operations must be performed to achieve the desired properties of the finished product. In this study, first, re-chroming with all 16 variants was performed, and wet blue properties such as the shrinkage temperature and chromium oxide content in leather were determined. Furthermore, the exhaustion of chromium compounds was estimated (Table 5). Today, chromium is the most used chemical in the tanning process; over 90% of leather goods are produced through chroming. Nevertheless, only 55-70% of the chromium salt is fixed in the leather; the remaining salt is fixed to the effluent [34]. The results in Table 5 reveal the influence of enzymatic bating in re-chroming. Enzymatic bating led to higher chromium exhaustion; using 5% Zime SB consumption of chromium compounds can reach up to 79-80%. The study by Zhang et al., showed a similar effect after a pickling-bating process. Using enzymes for pickling-bating, a higher uptake of chrome and post-tanning chemicals was reached. Fibre relaxation enables other tanning and re-tanning substances to better penetrate the hide [15]. This effect was obtained not only after bating, but also after re-bating [17].
The shrinkage temperature after re-chroming became similar to that of the control samples; the differences that were obtained after re-bating disappeared. This can be explained by the fact that although the consumption of chromium increased, crosslink formation with collagen was similar to that of the control; the remaining chrome oxide may bond to collagen without formatting crosslinks. Only when using Oropon DVP at 1% for 3.5 h was the shrinkage temperature lower than the other variants, even though Cr 2 O 3 exhaustion was greater than 72%.
were observed; this could be explained by the EP effect on intermolecular water and on hydrogen bonds in amino acids in protein. Furthermore, in all experimental samples, new peaks appeared at around 2989-2981 cm −1 and 1078-1055, which can be assigned to -CH2/NH2 vibrations and C-O/C-O-C stretching, respectively. New peaks indicate an EP effect on WB and the formation of new bonds. Typically, 1100-1000 absorbance bands are assigned to carbohydrate moieties in leather [32,33]; however, carbohydrates did not form during the enzymatic treatment. Instead, EP may have affected the leather by hydrolysing the bonds between collagen and other substances. Nevertheless, the FTIR spectra showed that, regarding the formation of new bonds, the absorbance bands for amide (I), amide (II), and amide (III) remained the same. Figure 1. FTIR spectra of untreated wet blue (1) and after enzymatic treatment of wet blue with Zime SB 1% (2); Zime SB 5% (3); NovoBate WB 1% (4); NovoBate WB 5% (5); Oropon DVP 1% (6); Oropon DVP 5% (7); Oropon WB 1% (8); Oropon WB 5% (9).
After re-bating, other post-tanning operations must be performed to achieve the desired properties of the finished product. In this study, first, re-chroming with all 16 vari- Figure 1. FTIR spectra of untreated wet blue (1) and after enzymatic treatment of wet blue with Zime SB 1% (2); Zime SB 5% (3); NovoBate WB 1% (4); NovoBate WB 5% (5); Oropon DVP 1% (6); Oropon DVP 5% (7); Oropon WB 1% (8); Oropon WB 5% (9). To influence the determination of the post-tanning processes, six different enzymatic treatments were chosen and performed (Table 6). These series were chosen because the results of the amount of collagen removed, the shrinkage temperature, and the exhaustion of Cr 2 O 3 showed a greater concentration influence on re-bating compared to the processing time. Moreover, NovoBate WB was excluded due to too high of an effect on collagen. Fibre opening during re-bating process should loosen the fibers and due to this improve tanning agents, dye and fat-liquors diffusion into the skin [6]. Few studies have been performed with the aim of evaluating dyeing or fat-liquoring efficiency after using EP in re-bating. Song et al. [35] applied different types of proteases in crust leather dying; study demonstrated better dye absorption in treated leather. Other studies [36,37] with protease in leather dyeing showed high dye exhaustion and better distribution of colour in the finished product.
However, during this study a greater amount of fat-liquoring substances were not obtained in the leather or the exhaustion of dyes during the post-tanning processes ( Table 6). The highest dye consumption was achieved with the control sample; despite this, all values are similar. The results indicate that re-bating did not have an influence on dyeing consumption. Fat-liquoring process was analogous to dyeing; in finished product the amount of matter soluble in dichloromethane were similar between experimental samples. No apparent dependence on post-tanning processes was observed despite different effects on removed collagen amount, shrinkage temperature, and chromium exhaustion. Despite similar dye exhaustion, different depth of dye penetration was observed using different EP and its concentrations (Figure 2).
In control sample the total penetration of dyes was lowest. Furthermore, a greater diffusion of dyes was achieved on the flesh side, due to well-known structural differences between the grain and the flesh side; flesh side has more open fiber structure that helps dye penetrate better [38]. A tendency to better diffusion through the flesh side was also observed with Oropon WB, different EP concentrations leaded to a similar effect. Nevertheless, diffusion of dyes on the flesh side with Oropon WB increases significantly compared to control samples. Using 1% Oropon WB achieved 35.4% of dye penetration through flesh and with 5-37%. This indicates that although dye exhaustion was similar, the penetration of substances in the leather was deeper.
Treated with Zime SB and Oropon DVP leather showed different dye penetration tendencies. Using these EP led to better dye diffusion on grain side; wet blue affected by rebating behaved differently during dyeing than control sample. Using higher concentration of Zime SB led to similar dye penetration in both layers; total diffusion in leather was similar to the control.
values are similar. The results indicate that re-bating did not have an influence on dyeing consumption. Fat-liquoring process was analogous to dyeing; in finished product the amount of matter soluble in dichloromethane were similar between experimental samples. No apparent dependence on post-tanning processes was observed despite different effects on removed collagen amount, shrinkage temperature, and chromium exhaustion. Despite similar dye exhaustion, different depth of dye penetration was observed using different EP and its concentrations (Figure 2). Physical and mechanical properties after post-tanning were also determined ( Table 7). The control sample grain layer broke with less strain applied compared to other samples; only variant treated with a higher concentration of Oropon DVP grain layer broke at lower value. Furthermore, relative elongation of control sample was also the highest. This can be explained that during re-chroming more chromium oxide bonded to experimental samples, however, bonding might appeared more on the surface and because of that the grain layer is stronger than the strength of the samples. Other mechanical properties did not have apparent dependency based on performed process. Lastly, FTIR analysis was performed after post-tanning. After enzymatic treatment spectra differences between control sample and experimental were observed; however, after post-tanning processes FTIR spectra are almost identical (Figure 3). This means that differences appeared after re-bating are eliminated during later operations.  (1) and after enzymatic treatment of WB with Zime SB 1% (2); Zime SB 5% (3); Oropon DVP 1% (4); Oropon DVP 5% (5); Oropon WB 1% (6); Oropon WB 5% (7).

Conclusions
The enzymatic WB treatment led to an additional fibre opening; the process changed the WB properties as well as influenced later processes. During the study, a greater influence of EP concentration was observed compared to the process time. Depending on the enzymes used, the amount of removed collagen varied. Moreover, after the process, all samples showed greater thermostability and higher water vapour absorption compared to the control. Furthermore, the effect of the EP caused changes in the supermolecular structure, which was reflected in the FTIR spectra.
After the enzymatic treatment, WB bonded more chromium in the re-chroming process and deepened the penetration of dyes; using EP Zime SB 1% and Oropon DVP, the dyes penetrated better through the grain layer. On the other hand, there were no significant improvements in the mechanical properties, dye exhaustion, and fat-liquoring process.
Overall, these results indicate that some properties and operations are improved, while other are similar to conventional processing. Nevertheless, every enzyme application in re-bating should be carefully investigated in order to achieve the best process conditions and desired product properties.

Conclusions
The enzymatic WB treatment led to an additional fibre opening; the process changed the WB properties as well as influenced later processes. During the study, a greater influence of EP concentration was observed compared to the process time. Depending on the enzymes used, the amount of removed collagen varied. Moreover, after the process, all samples showed greater thermostability and higher water vapour absorption compared to the control. Furthermore, the effect of the EP caused changes in the supermolecular structure, which was reflected in the FTIR spectra.
After the enzymatic treatment, WB bonded more chromium in the re-chroming process and deepened the penetration of dyes; using EP Zime SB 1% and Oropon DVP, the dyes penetrated better through the grain layer. On the other hand, there were no significant improvements in the mechanical properties, dye exhaustion, and fat-liquoring process.
Overall, these results indicate that some properties and operations are improved, while other are similar to conventional processing. Nevertheless, every enzyme application in re-bating should be carefully investigated in order to achieve the best process conditions and desired product properties.

Conflicts of Interest:
The authors declare no conflict of interest.