Alkaline-Enzymatic Hydrolysis of Wool Waste for Different Applications

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1 Alkaline-Enzymatic Hydrolysis of Wool Waste for Different Applications MARIANA DANIELA BERECHET 1, MIHAELA DOINA NICULESCU 1 *, CARMEN GAIDAU 1 *, MADALINA IGNAT 1, DORU GABRIEL EPURE 2 1 National Research and Development Institute for Textile and Leather-Leather and Footwear Research Institute, 93 Ion Minulescu Str., , Bucharest, Romania 2 ProbstdorferSaatzucht Romania SRL, Bucharest, Romania Wool waste represents a valuable and renewable material with low level of valorization and high potential to be integrated in bioeconomy. The extraction of keratin from wool by-products generated by sheep breeders and furskin industry represents a valuable approach for reducing the environmental pollution with organic and heavy biodegradable waste and a possibility to use a renewable product in agriculture or different industries. Keratin hydrolysates were obtained by alkaline and alkaline-enzymatic hydrolysis with extraction yields of %. The obtained keratin hydrolysates were characterized by physical-chemical analysis (dry substance, nitrogen content, ph, ash etc), FT-IR spectra, Dynamic Light Scattering (DLS), electrophoresis (SDS-PAGE) and surface tension (VCA Optima XE). Alkaline and alkaline-enzymatic hydrolyses of wool waste showed the possibility to obtain different keratin polypeptides with suitable properties for application in leather industry or in agriculture. Keywords: wool waste, keratin hydrolysate, alkaline and enzymatic hydrolysis, recovery and reuse The purpose of these experiments is to obtain keratin hydrolysates from by-products of the leather industry. The recovering of these natural resources leads to the reduction of the amount of stored waste and prevents environmental pollution. Keratin hydrolysates can be used to develop new biomaterials, with multiple applications, as well as for environmentally-friendly leather and fur treatments in order to obtain specific functionalities. This research consists in transforming low economic value (sheep wool remnants) into a product, with potential for exploitation in various fields of activity: leather processing, cosmetics, agriculture. Wool is a source of organic nitrogen, as a macronutrient, and a source of sulfur, as a mesonutrient, both essential for the growth of crop plants. In accordance with European legislation, waste from the leather industry must be subject to reuse, recycling, energy recovery and valued through chemical and biochemical degradation for recovery of useful organic compounds [1,2]. The study of bio-materials follows the trajectory for discovering new materials in order to provide principles and mechanisms harmonized with micro and macro multifunctional design requests [3-10]. Many biological materials are composites based on biopolymers and minerals. These materials with remarkable properties and functionalities are mainly made of constituents with low hardness. However, wood, spider web, bone and deer horn have a higher resistance as an order of magnitude than their mineral constituents [11,12]. The specific properties exhibited are usually due to a complex hierarchical structure that incorporates biopolymers and minerals [13]. Keratin is the basic polymer of hair, wool, claws, hooves, horns and feathers that gives them structure, mechanical behavior, and specific physical-chemical properties [13, 14]. This biopolymer is used in the composition of ones products in cosmetic industry [15,16], in medical field as new biocompatible materials for tissue engineering [17], injury and trauma care [18] or in surgical interventions [19]. Products derived from chemically modified keratin may also be used for adsorbents in water pollution control [20-22]. * mihaelaniculescu59@yahoo.com, carmen_gaidau@hotmail.com, Enzymatic processing of keratin leads to development of materials for various applications, such as the improvement of wool fabric properties [23] or for gentle dyeing of hair and wool [24-27]. Keratinized materials are rich in cysteine, which differentiates them from other biopolymers, being usually durable, rigid and non-reactive with the natural environment [3]. In some applications, keratin is used as gels, films or similar products. The aim of this paper is to obtain keratin hydrolysates from wool waste through alkaline or alkaline-enzymatic hydrolysis with various characteristics, suitable for reintegration in the bioeconomic circuit. The originality of hydrolysis processes in discontinuous or continuous operation ensures versatile properties of keratin hydrolysates and their applicability in the leather industry or in agriculture. Experimental part Materials Wool waste was collected from sheep breeders who do not fully recover the produced wool resource. Materials used for wool degreasing were: ammonia solution 25% p.a, anhydrous sodium carbonate p.a (Chimreactiv SRL) and ethoxylated alkyl non-ionic detergent (Borron SE). Chemical hydrolysis of wool was performed using hydrated calcium oxide p.a (SC Cristal R Chim SRL), sodium hydroxide p.a. and potassium hydroxide p.a (Lachner). Alcalase 2.4L (Novozymes) was used for enzymatic hydrolysis. These is a protease extract from Bacillus licheniformis culture and it has 2.4 U/g activity under optimal hydrolysis conditions (ph , temperature of 60 C). Obtaining keratin hydrolysates Raw wool was degreased in 35 0 C water, at a 1:2000 (weight/volume) float ratio, by stirring in automated drum (FAVE), with 1g/L ammonia solution 25% p.a, 1g/L anhydrous sodium carbonate p.a and 1g/L Borron SE, for 12 h. The degreased wool was rinsed to neutral ph, dried and ground. The extraction of keratin from sheep wool REV.CHIM.(Bucharest) 69 No

2 aimed to highlight the influence of the chemical hydrolysis procedure (chemical and chemical-enzymatic) and the type of process ( discontinuous or continuous as presented in figs. 1 and 2) on characteristics of keratin hydrolysate. The hydrolysis was carried out at 80 0 C, in aqueous medium with various alkali (10% CaO, 5% NaOH or 5% KOH), for 12 h with stirring and then statically for remaining 8 hours. Identification of optimal hydrolysis yields was the research aim. The keratin hydrolysate was filtered under vacuum, with 388 grain filter paper. The following products were obtained: KHA1 (hydrolysate with CaO), KHA2 (hydrolysate with NaOH) and KHA3 (hydrolysate with KOH). Hydrolysis yields were calculated according to the formula: where: N wool represents the nitrogen determined by washed raw wool analysis N keratin represents the nitrogen determined by keratin hydrolysate analysis The alkaline keratin hydrolysates (KHA1 and KHA2) were subjected to enzymatic hydrolysis at ph=8.5, 60 o C, with 1% Alcalase 2.4L, under stirring, for 4 hours. The aim was to obtain lower molecular polypeptides, more easily available to agricultural vegetables metabolism. The resulting products were named KHAE1 and KHAE2. The continuous process of chemical-enzymatic hydrolysis was carried out without separation of the keratin hydrolysate obtained by chemical hydrolysis with calcium oxide, by adjusting the ph value followed by the enzymatic hydrolysis. The resulting product was named KHAE3. Figure 1 and figure 2 present schemes of principal operations (SOP) used in this study. Characterization of keratin hydrolysates Physical and chemical analyses of raw, washed wool and keratin hydrolysates included analysis referring to: moisture (SR EN ISO 4684:2006), fatty substances (SR EN ISO 4048:2002), ash (SR EN ISO 4047:2002), water soluble Fig. 1. SOP of discontinuous alkaline-enzymatic wool hydrolysis Fig. 2. SOP of continuous alkaline-enzymatic wool hydrolysis substances (ICPI Method), ph status (STAS 8619/3:1990), total nitrogen respectively protein (SR ISO 5397:1996), amine nitrogen (ICPI Method), residual calcium oxide (ICPI Method). The average molecular weight of keratin hydrolysates was determined by the Sorensen method [28], SDS-PAGE gel-electrophoresis analysis was conducted according to the method described by Laemlli [29] with a vertical electrophoresis unit (VWR) and compared to a marker that enables highlighting of proteins with molecular weights between Da range. The structural modifications of the keratin, molecule hydrolysated through various chemical-enzymatic processes were highlighted by FTIR-ATR spectroscopy (4200 JASCO) on keratin films. Other characteristics of the obtained keratin polydispersions were considered referring to their potential applications in agriculture. It is the case of size of keratin polydispersities, determined by dynamic light scattering measurements with Nanosizer Nano ZS (Malvern). The surface tension of polydispersions was also measured (VCA Optima XE). Preliminary investigations on the use of keratin hydrolysates as auxiliaries for retanning and fatliquoring leather were conducted at laboratory scale on neutralized bovine hides according to the technology outlined schematically below (table 1). The materials used are technical, specific to the processing of natural leather, supplied by chemical producers (Elton Corporation SA). The main analyses to identify the effects of keratin hydrolysates on leather properties were: softness (ST300 Softness Tester, SR EN 17235:2002), wet and dry friction resistance of dyes (SR EN ISO 11640:2013) and water droplet resistance (SR ISO 15700:2012). Results and discussions The analysis of raw and washed wool revealed (table 2) that the degreasing process was efficient, the fatty substances were removed up to 93.6% and the watersoluble materials up to 65.2%. Chemical sheep wool hydrolysis processes (table 2) resulted in yields of 16.2% (KOH), 36.2% (CaO) and 43.5% (NaOH). The chemical hydrolysis variants with CaO and NaOH were selected for the discontinuous enzymatic hydrolysis step and the CaO variant for the continuous chemical-enzymatic hydrolysis REV.CHIM.(Bucharest) 69 No

3 Table 1 FRAMEWORK TECHNOLOGY FOR PROCESSING BOVINE LEATHER WITH KERATIN HYDROLYSATE (KHA2) Table 2 PHYSICO-CHEMICAL CHARACTERIZATION OF RAW WOOL, WASHED WOOL, KERATIN HYDROLYSATES OBTAINED BY ALKALINE HYDROLYSIS AND YIELDS OF KERATIN CHEMICAL HYDROLYSIS process. The selection was based on analysis of hydrolysis Alkaline-enzymatic hydrolysis (table 3) in discontinuous yields and cost of CaO variant. The amount of organic process enabled refinement and a more advanced cleavage nitrogen contained in hydrolysates ranged between 11.57% of keratin because the lowest molecular weights were and 14.97%, which recommends them for use as organic obtained (4650 Da). The similar process in continuous flow, fertilizer. Hydrolysis with CaO led to the most advanced led to polypeptides with higher molecular weights, of cleavage of the keratin molecule resulting in Da. The keratin hydrolysate obtained by using NaOH and polydispersions with an average molecular weight of the Alcalase 2.4L enzyme showed a molecular weight of Da, while hydrolysis with NaOH resulted in higher Da. The values of the mean molecular weights molecular weights, of Da. REV.CHIM.(Bucharest) 69 No

Table 3 PHYSICO-CHEMICAL CHARACTERIZATION OF KERATIN HYDROLYSATES OBTAINED BY ALKALINE-ENZYMATIC HYDROLYSIS IN DISCONTINUOUS (KHAE1, KHAE2) AND CONTINUOUS PROCESS (KHAE3) determined by the Sorensen

Here the band 1 corresponds to keratin hydrolysate obtained through alkaline enzymatic hydrolysis with CaO in discontinuous process (KHAE1) showing the presence of low average molecular weight

4 Table 3 PHYSICO-CHEMICAL CHARACTERIZATION OF KERATIN HYDROLYSATES OBTAINED BY ALKALINE-ENZYMATIC HYDROLYSIS IN DISCONTINUOUS (KHAE1, KHAE2) AND CONTINUOUS PROCESS (KHAE3) determined by the Sorensen method, were confirmed by the SDS-PAGE electrophoresis and these are shown in figure 1. Here the band 1 corresponds to keratin hydrolysate obtained through alkaline enzymatic hydrolysis with CaO in discontinuous process (KHAE1) showing the presence of low average molecular weight proteins in the Da range. The band 3 corresponds to keratin hydrolysate (KHAE3) extracted with calcium oxide by continuous-flow alkaline-enzymatic hydrolysis, which is more intense in the Da range. Band 2 corresponding to keratin hydrolysate extracted with sodium hydroxide, by continuous-flow alkaline-enzymatic hydrolysis (KHAE2) reveals proteins with average molecular weights especially in the Da range. These results make that it possible to envisage applications in obtaining foliar fertilizers containing amino acids and peptides with sulfur and organic nitrogen (low molecular weight hydrolysates). The FT-IR/ATR spectra (fig. 4 and table 4) show slightly deviated bands in the keratin hydrolysates obtained by alkaline-enzymatic hydrolysis in the amide I specific range ( cm -1 ), cm -1 for KHAE1 and cm -1 for KHAE2. The second band at cm -1 is for KHA1 and KHA2, obtained by alkaline hydrolysis. All analysed samples reveal the absence of any signal Fig. 3. SDS-PAGE electrophoresis for 1-KHAE1, 2-KHAE2 and 3-KHAE3 Fig. 4. FTIR/ATR spectra for keratin hydrolysates obtained by alkaline hydrolysis (KHA1, KHA2) and alkaline-enzymatic hydrolysis (KHAE1, KHAE2) in discontinuous flow Table 4 ATTRIBUTION OF MAIN CHARACTERISTIC FREQUENCIES RECORDED BY FTIR/ATR ANALYSIS REV.CHIM.(Bucharest) 69 No

5 corresponding to the vibrations of amide II, namely in-plane N-H bending vibrations and C-N stretching vibrations, but wagging vibrations were recorded out of the plane of CH 2 groups, γ rch2, specific to amino acids, in the case of keratin hydrolysates from hydrolysis using calcium oxide as alkaline agent, KHA1 and KHAE1. The bands in the cm -1 range attributed ν C-S stretching vibrations are specific to sulfur compounds [30, 31] and as expected, were identified in all four hydrolysates. The structural analysis of keratin hydrolysates highlight the fact that chemical hydrolysis of wool lead to its destructuring, particularly in the presence of enzymes; relevant in this regard is hydrolysate KHAE1, that stands out through the appearance of spectral bands at cm -1 and cm -1, in areas specific to amino acids in correlation with the low molecular weight (table 3). The measurements on average particle size of keratin polydispersions, obtained by hydrolysis with CaO, NaOH and by discontinuous-flow enzymatic hydrolysis (table 5), reveal a different composition depending on the alkaline agent of hydrolysis. While in the case of hydrolysis with CaO the particles are very large (2566 nm), enzymatic hydrolysis reduces at 302 nm with a reduction in polydispersity (from 1 to 0.835). In case of hydrolysis with NaOH, the average particle size is smaller (590 nm) and increases by enzymatic hydrolysis (746 nm) with a decrease in the polydispersity value, from to The number of particle populations is also different for the two types of hydrolysis, CaO hydrolysis generates a single particle population (fig. 5a) and 4 particle populations after enzymatic hydrolysis (fig. 5b). In case of hydrolysis with NaOH, 3 particle populations (6a) and a lower number of populations after enzymatic hydrolysis (6b) were identified. Particle size investigations in protein polydispersions are original [32] and highlight the diversity of hydrolysate Table 5 AVERAGE SIZES AND POLYDISPERSITIES FOR KERATIN HYDROLYSATES: KHA1, KHAE1, KHA2 AND KHAE2 properties. This study shows the variation of keratin polydispersions depending on the type of hydrolysis agent. Surface tension analysis of keratin hydrolysates obtained through various chemical-enzymatic processes allows the estimation of the display properties on the plants surface in the case of potential foliar applications. In this regard, the results of analyses, presented in table 6, indicate low surface tensions, knowing that protein hydrolysates show numerous free hydrophilic groups with tenside properties [33]. Low surface tension values indicate potential Table 6 SURFACE TENSION DETERMINED FOR ALKALINE KERATIN HYDROLYSATES Table 7 THE INFLUENCE OF KERATIN HYDROLYSATE ON LEATHER DYEING RESISTANCE applications as dispersants and surfactants in various industries and in agriculture technologies. Experiments on the use of keratin hydrolysate as a retanning agent for bovine leather, intended for footwear, were based on literature investigations on the ability of collagen or keratin hydrolysates [34] to provide fullness, finesse and firmness to leather uppers, intensity to dyeing. This is a procedure for introduction of additional carboxyl and amine groups in bovine leather, capable of fixing dyes and additional fatliquoring agents. The results of analyses showed an increase in leather softness (SR EN 17235:2002) treated with 27% keratin hydrolysate compared to untreated leather, opening the prospect for further experiments in this direction. The influence of Fig. 5. Distribution of particle population size in keratin hydrolysates KHA1 (a), KHAE1 (b) a b Fig. 6. Distribution of particle population size in keratin hydrolysates KHA2 (a), KHAE2 (b) a REV.CHIM.(Bucharest) 69 No b

6 keratin hydrolysate on leather dyeing resistance is slightly improved, as table 7 shows. Research is ongoing, given the high interest in replacing chemicals from oil sources with renewable materials. The paper presents original results on the wide range of keratin polydispersions that can be obtained by alkaline or alkaline-enzymatic hydrolysis of wool waste. Conclusions Recovery of keratin from sheep wool waste is a major objective in the context of the sustainable development strategy for addressing biotechnologies as an alternative to the use of synthetic chemicals from oil resources. The paper presents possibilities for recovery of keratin from sheep wool by means of alkaline hydrolysis processes with yields, ranging between 16.4 and 43.5% and chemicalenzymatic hydrolysis processes in continuous or discontinuous flow. The results of analyses highlight the possibility of obtaining keratin hydrolysate polydispersions with different molecular weight and particle size properties by approaching chemical hydrolysis with various alkaline agents or by refining hydrolysis through enzymatic processes which opens the perspective of wide-range applications in various industries and in agriculture. Keratin hydrolysates obtained by alkaline hydrolysis processes show low surface tensions ( mj/m 2 ) which confirms the specific surfactant properties of protein hydrolysates, with the prospect of multiple applications. Electrophoresis (SDS/PAGE) and FTIR/ATR assays confirm the wide range of polypeptides with various molecular weights (4650 Da Da) and amino acid content (chemical-enzymatic hydrolysis with calcium oxide). The high molecular weight keratin hydrolysates (35800 Da) suggest the possibility of using them as retanning agents to improve the softness and dyeing resistance of bovine leather. Acknowledgements: These works were supported by grants of Romanian Ministry and the Romanian National Authority for Scientific Research and Innovation, CNCS/CCCDI-UEFISCDI, project number PN-III-P2-2.1-PTE within PNCDI III, contract 55PTE and of Romanian Ministry of Research and Innovation under Nucleus contract no. 26 N/ , project PN References 1. ZAHARIA, M., MAFTEI, D., DUMITRAS-HUTANU, C. A., PUI, A., BLAGOBO ZOMI,C., PINTILIE, O., GRADINARU, R., Rev. Chim. (Bucharest), 64, no. 4, 2013, p PANTAZI, M., STEFAN, D. S., CONSTANTINESCU, R., ANGHEL, R., MEGHEA, A.,VASILESCU, A. M., Rev. Chim. (Bucharest), 65, no. 2, 2014, p SARIKAYA, M., Microsc Res Tech. 27, no. 5, 1994, p SRINIVASAN, A.V., HARITOS, G.K.,HEDBERG, F.L, Appl. Mech. Rev. 44, no. 11, 1991, p MAYER, G., Exp Mech. 42, no. 4, 2002, p BAER, E., HILTNER, A., MORGAN, R.J., Phys Today, 45, no. 10, 1992, p MEYERS, M.A., CHEN, P.Y., LIN, A.Y.M., SEKI, Y., Prog. 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STUDY ON OBTAINING KERATIN EXTRACTS FROM LEATHER INDUSTRY BY-PRODUCTS

ICAMS 2016 6 th International Conference on Advanced Materials and Systems STUDY ON OBTAINING KERATIN EXTRACTS FROM LEATHER INDUSTRY BY-PRODUCTS MIHAELA-DOINA NICULESCU, MARIANA DANIELA BERECHET, CARMEN