From leather wastes to leather: enhancement of low quality leather using collagen recovered from leather tanned wastes

Leather industry produces huge amounts of solid wastes. In the last decade, several methods for the recovery and valorization of these wastes were developed, mainly focused on the extraction of collagen using chemical methods. The extracted collagen, due to its poor quality, is mostly used in agriculture as a nitrogen source ingredient of fertilizers. This study aims to apply collagen, extracted from leather tanned solid wastes using a recently reported new process based on enzymatic hydrolysis, as filling agent for low quality leather. Thanks to the enzymatic hydrolysis, collagen can be extracted without affecting its integrity and, therefore, its quality. In order to use the extracted collagen as filler for low quality leather, an enzymatic mediated cross-linking reaction between collagen and casein was developed. The enzymatic cross-linking reaction was added as an additional phase of the re-tanning process or as a replacement of one of the re-tanning steps. To evaluate the filling effect, thickness of the treated leather was measured and infrared and microscopy analyses were performed, comparing the new methods to the traditional standard one. The mechanical properties of the filled leather were tested and the sensorial features, such as fullness and touch feelings, were estimated through a panel test. Results suggest the high potential of extracted collagen to be employed back in leather processing both as additive and as substitutive filler. Aims and goals of the research study: comparison between re-designed (green workflow) and traditional re-tanning process (grey workflow). Aims and goals of the research study: comparison between re-designed (green workflow) and traditional re-tanning process (grey workflow).


Introduction
Leather making process is long and complex: animal hides and skins derived from meat industry are undergone to a series of operations to be converted in leather (Maina et al. 2019).Leather processing is commonly divided in three main phases: pre-tanning, tanning and post-tanning.The pre-tanning process comprises activities to clean and store hides; the tanning process converts hides in leather, by modifying the collagen present in the skin fibers and the post-tanning steps are responsible of the final aspect and of the esthetic and mechanical properties of leather (Thanikaivelan et al. 2005;Muralidharan et al. 2022).
Among post-tanning procedures, the re-tanning process is crucial not only to achieve uniform leather products, but also to confer new properties to leather, thus increasing its quality (Yorganicioglu et al. 2020): frequently, the addition of fillers is necessary to minimize the imperfections present on hides, such as veiny and loose areas (Taylor et al. 2009;Li et al. 2019).Filling phase is, therefore, the introduction of substances able to penetrate leather and fill the voids of the fibers (Taylor et al. 2006;Liu et al. 2020).The formulation of the filling agents determines the final properties of the treated leather (Danylkovych and Korotych 2019): the ideal filler has to be highly compatible with leather, well soluble in water, capable to penetrate into leather matrix and to bond itself to collagen, and able to homogeneously fill the loosen parts (Yorgancioglu et al. 2020a).
To achieve these desired characteristics, several inorganic or organic chemicals are generally used simultaneously; among the organic materials, four classes of compounds are the most used in re-tanning process: (i) vegetal tannins, (ii) syntans, (iii) resins and (iv) polymers (Jankauskaite et al. 2012).
In the last decade, the high production costs and the low degree of penetration of vegetal tannins in leather (Mokrousova 2010;Jankauskaite et al. 2012), as well as the potential release of polluting and harmful substances (such as freeformaldehyde) due to the use of synthetic fillers (Yorgancioglu et al. 2020b), have promoted the development and the application of alternative eco-friendly and non-toxic compounds, such as proteins and oligopeptides.
Among proteins, collagen resulted to be a very interesting alternative filler (Chen et al. 2001;Rigueto et al. 2020) thanks to its structural function and its compatibility with leather (Sun et al. 2022).In particular, by its cross-linking combined with other molecules, collagen attains new appealing properties, such as thermal stability, elasticity and plasticity (Zehra et al. 2019;Skopinska-Wisniewska et al. 2021).In particular, collagen-casein cross-linking is the key for collagen application in leather processing, being casein able to make collagen more resistant to the thermal stress caused by the re-tanning process (Wu et al. 2017).
Potential collagen sources are numerous, from mammals to marine organisms (Silvipriya et al. 2015), including tannery wastes: leather solid wastes are, indeed, composed by raw hides and by semi-processed skins, where collagen is the main protein component (Kite and Thomson 2006;Yorganicioglu et al. 2020).
Aiming to a cleaner and circular production flow, applying extracted collagen from leather solid wastes as filler into leather processing has been more than a challenge.Employing back collagen in leather manufacturing has also the advantage of recovering a waste without the need of further purification steps, steps which are necessary for the reuse of collagen in other fields, such as cosmetic and medical applications (Sionkowska et al. 2017).
Moreover, leather solid wastes represent a critical issue for the environmental sustainability of the leather processing industry: tanned shavings result to be high polluting and potentially toxic due to the presence of tanning agents that, bonding to collagen fibers in the skin, make these wastes hard degradable.
Nowadays, several methods, based on chemical extraction for collagen recovery, were developed (Xiaoyan et al. 2014;Ding et al. 2015), but the obtained collagen is completely deconstructed and hydrolyzed in small polypeptides (Nogueira et al. 2011), making it hard to be applied in the filling process.
In our previous work, collagen was extracted from leather tanned wastes by enzymatic hydrolysis, thus allowing to recover high quality structured collagen, and providing a protocol for its cross-linking with casein by enzymatic catalysis.Using of enzymes allow to fine tuning the hydrolysis and cross-linking processes, making extracted collagens suitable for application in leather manufacturing.
In the present study, collagen, extracted from different kind of tanned shavings, is integrated in leather manufacturing as filler for low quality leather, exploiting its ability to cross-link with casein through the mediation of a microbial transglutaminase.The transglutaminase catalyzes an acyl transfer reaction between glutamine and lysine residues, allowing the formation of intra-and inter-molecular cross-links not only between the introduced collagen and casein, but also with the collagen fibers present in leather matrix (Wu et al. 2017(Wu et al. , 2018(Wu et al. , 2019;;Cheng et al. 2019).The re-tanning process was redesigned to apply collagen as filler additive or substitute.

Materials
Vegetable tanned bovine shavings, organic tanned bovine shavings and mineral tanned bovine shavings were collected from the Italian Leather Research Institute (form Veneto district).
Ovine leather and re-tanning reagents were furnished by DMD Solofra Spa-Tanning Company.
Reagents, casein and trypsin were purchased from Sigma-Aldrich (Saint Louis, Missouri, USA).Microbial transglutaminase Activa WM was furnished by Ajinomoto (Hamburg, Germany).

Extraction of collagen
Collagen was extract from vegetable tanned bovine shavings, organic tanned bovine shavings and mineral tanned bovine shavings through an enzymatic treatment as reported in our previous work (Gargano et al. 2023a): shavings were incubated with 0.1% w/v of NaOH at RT for 4 h, then 0.2% w/v of trypsin was added and incubated at 50 °C for 2 h.

Application of collagen as filler
Collagen with molecular weights in the range of 25-150 kDa (5% w/w for each gram of treated leather, i.e., 50 mg of collagen for 1 g of leather), casein (0.5% w/w for each gram of treated leather, i.e., 5 mg of casein for 1 g of leather) and transglutaminase (50 U/g of collagen) were applied as additive or as substitutive filler as reported in Fig. 1 during different steps of re-tanning process.

Scanning electron microscopy
Test samples and control were cut in small pieces.A sputter coater (Dynavac) was used for coating a layer of gold on the surface of all samples that were fixed on aluminum stub.
A Zeiss EVO MA10 Scanning Electron Microscope equipped with INCA X-act ENERGY-250XT detector was used for the analyses (magnification 10x-200000x, resolving power 2 µm).

Stereomicroscopy
Test samples and control were set in distilled water overnight and then freezed to cut thin sections by a micrometer.Each section was analyzed by Wild Heerbrugg Stereoscope equipped with OPTIKA Room B3 Digital Camera.

Filling efficiency
Leather thickness was measured before and after re-tanning treatment by a Thickness Tester IG/MS, according to the standard EN ISO 2589:2016.Filling efficiency was calculated using the following formula (Chen et al. 2001)   where T a is the thickness after the treatment and T b is the thickness before the treatment.

ATR-IR (Attenuated total reflection-Fourier transform infrared)
ATR-IR (Attenuated Total Reflection-InfraRed) spectral measurements were performed by Spectrum One ATR-IR Spectrometer on test samples and control.Samples were pressed on the ZnSe crystal and spectra were recorded with a resolution of 4 cm −1 at the range from 600 cm −1 to 4000 cm −1 .

Application of extracted collagen and casein as filling agents
In our previous study (Gargano et al. 2021), the enzymatic cross-linking reaction between collagen and casein was optimized ex situ, in terms of time of reaction, enzymatic units used and collagen-casein ratio.To apply the extracted collagen and casein as filling agents, the enzymatic cross-linking was carried out in situ in the conditions optimized for the ex situ reaction.
The re-tanning process was re-designed to spread extracted collagen and casein as fillers additive or substitute on low quality leather (characterized by the presence of holes, wrinkles and inhomogeneity of the surface) through the mediation of transglutaminase (Fig. 1).The re-tanning process was performed on ovine neutralized wet-blue leather and did not included the dying step.
The thickness of all samples was measured before and after the treatments and the related filling efficiencies were calculated (Table 1): thickness increases after the treatments in all samples except Test D, as a consequence of the filling agents penetration in the leather matrix.
In particular, Tests A, B and E have a comparable filling efficiency, doubled respect to the control, showing that not only the addition of collagen improves the filling, but also that the second step of re-tanning process can be effectively replaced by exogenous collagen, contrary to the fatliquoring step (Test D).Moreover, when collagen is added at the end of the re-tanning process (Test C), a higher leather thickness increase is observed, tripled compared to the control, probably due to the presence of tannins that enhances the cross-linking with collagen (Chen et al. 2001).

Microscopy analysis
To visualize the effects of re-tanning processes on leather at different scales, sections and surfaces of tests and control samples were analyzed through a stereomicroscope and by a scanning electron microscope (Fig. 2).
The microscopic characteristics of samples, summarized in Table 2, suggest that the fatliquoring step is needed as the first step of the re-tanning process both to obtain a homogeneous matrix and to allow the filling agents to act (Tests B, C, E and control), resulting Test A and Test D non-homogeneously filled.Moreover, although the filling efficiency of Test B and Test E are comparable, samples appear very different: the addition of collagen before the synthetic tannins and resins (Test B) improves the filling efficiency and therefore the quality of the leather surface, but it has not significant effect on the matrix, since the section results tight, similarly to the control.On the other hand, using of collagen at the end of the re-tanning process, both as additive (Test C) and substitutive filler (Test E), results in well-filled and homogeneous leather with open pores and uniform and smooth surface.
Test C and Test E, therefore, were selected as the best conditions for the application of collagen as additive or substitute filler during the re-tanning process, respectively.

Cross-linking distribution
ATR-IR analyses were performed to evaluate the homogeneity of the filling: the spectra of different areas of Test  The control sample has a lower cross-linking degree with the highest variability (0.78 ± 0.10) respect to the tests (0.89 ± 0.02 of Test C, 0.98 ± 0.01 of Test E), which indicates a weaker and inhomogeneous fixing of the filling agents in leather matrix, in fact when collagen is introduced in the re-tanning process, especially in the Test E that used the extracted collagen as substitute filler, the cross-linking degrees increased and conversely the standard deviations were reduced up to ten times, thus indicating the homogeneous distribution of the collagen in the leather matrix (Online Resource 2: cross-linking degrees of Test C, Test E and control sample).

Properties of filled leather
To evaluate the effect of collagen on the filled leather, mechanical properties (which describe the behavior of leather under the application of a load Garcia et al. 2009;Albu et al. 2014), and organoleptic properties, such as silkiness, softness, smoothness, and any other pleasant touch feelings, were analyzed.The mechanical properties were measured through physical analyses: as shown in Table 3, Test C and Test E exhibited better tensile and tear strength compared to control, moreover the elongation at the break also increased.Therefore, the employment of collagen directly enhances the physical properties of leather.
The organoleptic properties, whereas, were evaluated by a panel test: 30 estimators assigned, in a blind test, five possible degrees of sensorial experience in terms of the pleasure at the touch and the fullness.From these parameters, Touch Pleasure Index and Fullness Index were calculated as the sum of the obtained values provided by the possible maximum value (Jean Serge et al. 2019), (Online Resource 3: Touch Pleasure Index and Fullness Index, formula and degrees).
From blind test it follows that control sample and Test C exhibit comparable feel properties, while Test E results 30% more full and 20% more pleasant at the touch than control sample, probably due to the better penetration and compatibility of collagen to leather (Fig. 3).
Finally, in order to study a correlation between sensorial and technical parameters, physical properties, cross-linking degree and organoleptic properties were reported in a Kiviat-like diagram, where the values obtained by mechanical tests were normalized by the maximum possible value for each parameter (Florio et al. 2015).Figure 4 shows that higher cross-linking degrees correspond to higher tensile strength and better sensorial properties, while the elongation and the tear strength resulted not well correlated with the other parameters.17.0 ± 0.9 22.4 ± 0.8 23.7 ± 0.5 Longitudinal tensile strength (N/ mm 2 ) 19.0 ± 0.5 20.9 ± 0.9 26.9 ± 0.

Costs/benefits analysis
According to its thickness, microscopic characteristics and physical and organoleptic properties, experts from local tanneries have currently evaluated the market value of ovine leather as (i) 15.00 €/m 2 before the re-tanning (leather is characterized by veiny and loosen areas); (ii) 25.00 €/ m 2 after the traditional treatment of re-tanning (control), (leather is non-uniformly filled); and (iii) 35.00 €/m 2 after the innovative collagen-based treatments (Test C and E), (leather is well-filled and uniform).
On the basis of this assessment, a costs/benefits analysis was evaluated for the leather product derived by our treatments (Online Resource 4: costs/benefits analysis).The collagen-based procedure of Test C increase of 27% the process gain, even if the total costs of process result 35% higher than the ones related to the traditional re-tanning (Fig. 5).
Regarding Test E, its processing rises the process gain up to 35%, despite being 25% more expensive than traditional retanning, rising.As a fact, as mentioned above, the filling step is crucial for the final esthetic aspect of leather that have to provide uniform and well-filled leather after the re-tanning process, increasing the usable and, then, marketable area of each piece, thus rising the market value of each piece.
Considering the economic feasibility, the good properties that it confers to leather and, especially, the replacement of synthetic tannins and resins with a bio-based reactant, the re-tanning process of Test E acquires a double value: there is not only an economic advantage, but also an environmental one.

Conclusions
In this study, extracted collagen from leather tanned solid wastes and casein were cross-linked in situ by a microbial transglutaminase during different steps of re-tanning process to fill the veiny areas of leather.Results show that extracted collagen can be applied as additive, as well as substitute filler: leather treated with collagen shows not only comparable mechanical properties but also better organoleptic properties than leather treated with synthetic fillers.
Using of an enzymatic cross-linker avoids the use of synthetic resins or fixatives, making leather processing more environmental sustainable and allows a more homogeneous distribution of the filling agent in the leather matrix.
The employment of collagen derived from leather solid wastes not only assures a very high compatibility between filling agent and leather matrix, but also adds value to leather scraps: the protein components of these wastes can be in this way applied to produce high quality leather, promoting cleaner production methods and circular production flow.

Fig. 1
Fig. 1 Redesigned re-tanning protocols for the application of extracted collagen as additive (Test A, B and C) or substitute filler (Test D and E)

C
, Test E and control sample were collected and compared (Online Resource 1: ATR-IR spectra of Test C, Test E and control sample); cross-linking degrees were then measured as the ratio between the amide I and amide A signals (A I / A A )(Danylkovych et al. 2016).The amide A is characteristic of -NH 2 group, while amide I corresponds to -NH group; the increase of amide I signal and the decrease of amide A signal indicate the conversion of -NH 2 in -NH groups, i.e., the formation of new covalent bonds, thus the ratio of these signals is directly correlated to the cross-linking degree, as reported byAlbu et al. and Garcia et al. (Sommer et al.  2021).

Fig. 2
Fig. 2 Microscopy analyses of A) Test A, B) Test B, C) Test C, D) Test D, E) Test E and F) control sample

Fig. 3 Fig
Fig. 3 Organoleptic properties of control and test samples Mechanical properties of test samples and control were evaluated as follow: (i) shrinkage temperature according to UNI EN ISO 3380:2015; (ii) tensile strength and percentage of elongation according to UNI EN ISO 3376:2016; (iii) tear strength according to UNI EN ISO 3377-2:2016; and (iv) distention and strength of surface according to UNI EN ISO 3379:2015 (ball burst method).

Table 1
Thickness of leather samples and filling efficiency

Table 2
Microscopic characteristics of sections and surfaces of filled leather

Table 3
Mechanical properties of control and test samples