REV.CHIM.(Bucharest)♦ 69♦ No. 7 ♦ 2018 http://www.revistadechimie.ro 1649

Alkaline-Enzymatic Hydrolysis of Wool Waste for Different Applications

MARIANA DANIELA BERECHET1, MIHAELA DOINA NICULESCU1*, CARMEN GAIDAU1*, MADALINA IGNAT1,DORU GABRIEL EPURE2

1National Research and Development Institute for Textile and Leather-Leather and Footwear Research Institute, 93 Ion MinulescuStr., 031215, Bucharest, Romania2ProbstdorferSaatzucht Romania SRL, Bucharest, Romania

Wool waste represents a valuable and renewable material with low level of valorization and high potentialto be integrated in bioeconomy. The extraction of keratin from wool by-products generated by sheep breedersand furskin industry represents a valuable approach for reducing the environmental pollution with organicand heavy biodegradable waste and a possibility to use a renewable product in agriculture or differentindustries. Keratin hydrolysates were obtained by alkaline and alkaline-enzymatic hydrolysis with extractionyields of 16.4-43.5%. 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 woolwaste showed the possibility to obtain different keratin polypeptides with suitable properties for applicationin 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 keratinhydrolysates from by-products of the leather industry. Therecovering of these natural resources leads to the reductionof the amount of stored waste and prevents environmentalpollution. Keratin hydrolysates can be used to develop newbiomaterials, with multiple applications, as well as forenvironmentally-friendly leather and fur treatments in orderto obtain specific functionalities.

This research consists in transforming low economicvalue (sheep wool remnants) into a product, with potentialfor exploitation in various fields of activity: leatherprocessing, cosmetics, agriculture. Wool is a source oforganic nitrogen, as a macronutrient, and a source of sulfur,as a mesonutrient, both essential for the growth of cropplants.

In accordance with European legislation, waste fromthe leather industry must be subject to reuse, recycling,energy recovery and valued through chemical andbiochemical degradation for recovery of useful organiccompounds [1,2].

The study of bio-materials follows the trajectory fordiscovering new materials in order to provide principlesand mechanisms harmonized with micro and macromultifunctional design requests [3-10]. Many biologicalmaterials are composites based on biopolymers andminerals. These materials with remarkable properties andfunctionalities are mainly made of constituents with lowhardness. However, wood, spider web, bone and deer hornhave a higher resistance as an order of magnitude thantheir mineral constituents [11,12]. The specific propertiesexhibited are usually due to a complex hierarchicalstructure that incorporates biopolymers and minerals [13].

Keratin is the basic polymer of hair, wool, claws, hooves,horns and feathers that gives them structure, mechanicalbehavior, and specific physical-chemical properties [13,14]. This biopolymer is used in the composition of onesproducts in cosmetic industry [15,16], in medical field asnew biocompatible materials for tissue engineering [17],injury and trauma care [18] or in surgical interventions[19]. Products derived from chemically modified keratinmay also be used for adsorbents in water pollution control[20-22].* email: mihaelaniculescu59@yahoo.com, carmen_gaidau@hotmail.com,

Enzymatic processing of keratin leads to developmentof materials for various applications, such as theimprovement of wool fabric properties [23] or for gentledyeing of hair and wool [24-27].

Keratinized materials are rich in cysteine, whichdifferentiates them from other biopolymers, being usuallydurable, rigid and non-reactive with the naturalenvironment [3]. In some applications, keratin is used asgels, films or similar products.

The aim of this paper is to obtain keratin hydrolysatesfrom wool waste through alkaline or alkaline-enzymatichydrolysis with various characteristics, suitable forreintegration in the bioeconomic circuit. The originality ofhydrolysis processes in discontinuous or continuousoperation ensures versatile properties of keratinhydrolysates and their applicability in the leather industryor in agriculture.

Experimental partMaterials

Wool waste was collected from sheep breeders whodo not fully recover the produced wool resource. Materialsused 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 hydratedcalcium oxide p.a (SC Cristal R Chim SRL), sodiumhydroxide p.a. and potassium hydroxide p.a (Lachner).Alcalase 2.4L (Novozymes) was used for enzymatichydrolysis. These is a protease extract from Bacilluslicheniformis culture and it has 2.4 U/g activity underoptimal hydrolysis conditions (pH 6.5-8.5, temperature of60°C).

Obtaining keratin hydrolysatesRaw wool was degreased in 350C water, at a 1:2000

(weight/volume) float ratio, by stirring in automated drum(FAVE), with 1g/L ammonia solution 25% p.a, 1g/Lanhydrous sodium carbonate p.a and 1g/L Borron SE, for12 h. The degreased wool was rinsed to neutral pH, driedand ground. The extraction of keratin from sheep wool

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aimed to highlight the influence of the chemical hydrolysisprocedure (chemical and chemical-enzymatic) and thetype of process ( discontinuous or continuous as presentedin figs. 1 and 2) on characteristics of keratin hydrolysate.The hydrolysis was carried out at 800C, in aqueous mediumwith various alkali (10% CaO, 5% NaOH or 5% KOH), for 12h with stirring and then statically for remaining 8 hours.Identification of optimal hydrolysis yields was the researchaim. The keratin hydrolysate was filtered under vacuum,with 388 grain filter paper. The following products wereobtained: KHA1 (hydrolysate with CaO), KHA2 (hydrolysatewith NaOH) and KHA3 (hydrolysate with KOH). Hydrolysisyields were calculated according to the formula:

where:Nwool represents the nitrogen determined by washed raw

wool analysisNkeratin represents the nitrogen determined by keratin

hydrolysate analysis

The alkaline keratin hydrolysates (KHA1 and KHA2)were subjected to enzymatic hydrolysis at pH=8.5, 60oC,with 1% Alcalase 2.4L, under stirring, for 4 hours. The aimwas to obtain lower molecular polypeptides, more easilyavailable to agricultural vegetables metabolism. Theresulting products were named KHAE1 and KHAE2. Thecontinuous process of chemical-enzymatic hydrolysis wascarried out without separation of the keratin hydrolysateobtained by chemical hydrolysis with calcium oxide, byadjusting the pH value followed by the enzymatichydrolysis. The resulting product was named KHAE3. Figure1 and figure 2 present schemes of principal operations(SOP) used in this study.

Characterization of keratin hydrolysatesPhysical and chemical analyses of raw, washed wool

and keratin hydrolysates included analysis referring to:moisture (SR EN ISO 4684:2006), fatty substances (SR ENISO 4048:2002), ash (SR EN ISO 4047:2002), water soluble

substances (ICPI Method), pH status (STAS 8619/3:1990),total nitrogen respectively protein (SR ISO 5397:1996),amine nitrogen (ICPI Method), residual calcium oxide (ICPIMethod). The average molecular weight of keratinhydrolysates was determined by the Sorensen method [28],SDS-PAGE gel-electrophoresis analysis was conductedaccording to the method described by Laemlli [29] with avertical electrophoresis unit (VWR) and compared to amarker that enables highlighting of proteins with molecularweights between 3-198000 Da range. The structuralmodifications of the keratin, molecule hydrolysatedthrough various chemical-enzymatic processes werehighlighted by FTIR-ATR spectroscopy (4200 JASCO) onkeratin films.

Other characteristics of the obtained keratinpolydispersions were considered referring to their potentialapplications in agriculture. It is the case of size of keratinpolydispersities, determined by dynamic light scatteringmeasurements with Nanosizer Nano ZS (Malvern). Thesurface tension of polydispersions was also measured(VCA Optima XE).

Preliminary investigations on the use of keratinhydrolysates as auxiliaries for retanning and fatliquoringleather were conducted at laboratory scale on neutralizedbovine hides according to the technology outlinedschematically below (table 1). The materials used aretechnical, specific to the processing of natural leather,supplied by chemical producers (Elton Corporation SA). Themain analyses to identify the effects of keratin hydrolysateson leather properties were: softness (ST300 SoftnessTester, SR EN 17235:2002), wet and dry friction resistanceof dyes (SR EN ISO 11640:2013) and water dropletresistance (SR ISO 15700:2012).

Results and discussionsThe analysis of raw and washed wool revealed (table 2)

that the degreasing process was efficient, the fattysubstances were removed up to 93.6% and the water-soluble materials up to 65.2%. Chemical sheep woolhydrolysis processes (table 2) resulted in yields of 16.2%(KOH), 36.2% (CaO) and 43.5% (NaOH). The chemicalhydrolysis variants with CaO and NaOH were selected forthe discontinuous enzymatic hydrolysis step and the CaOvariant for the continuous chemical-enzymatic hydrolysisFig. 1. SOP of discontinuous alkaline-enzymatic wool hydrolysis

Fig. 2. SOP of continuous alkaline-enzymatic wool hydrolysis

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process. The selection was based on analysis of hydrolysisyields and cost of CaO variant. The amount of organicnitrogen contained in hydrolysates ranged between 11.57%and 14.97%, which recommends them for use as organicfertilizer. Hydrolysis with CaO led to the most advancedcleavage of the keratin molecule resulting inpolydispersions with an average molecular weight of 13000Da, while hydrolysis with NaOH resulted in highermolecular weights, of 35800 Da.

Alkaline-enzymatic hydrolysis (table 3) in discontinuousprocess enabled refinement and a more advanced cleavageof keratin because the lowest molecular weights wereobtained (4650 Da). The similar process in continuous flow,led to polypeptides with higher molecular weights, of 19200Da. The keratin hydrolysate obtained by using NaOH andthe Alcalase 2.4L enzyme showed a molecular weight of13800 Da. The values of the mean molecular weights

Table 1FRAMEWORK

TECHNOLOGY FORPROCESSING BOVINE

LEATHER WITH KERATINHYDROLYSATE (KHA2)

Table 2PHYSICO-CHEMICALCHARACTERIZATION

OF RAW WOOL,WASHED WOOL,

KERATINHYDROLYSATESOBTAINED BY

ALKALINEHYDROLYSIS AND

YIELDS OF KERATINCHEMICAL

HYDROLYSIS

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determined by the Sorensen method, were confirmed bythe SDS-PAGE electrophoresis and these are shown infigure 1. Here the band 1 corresponds to keratin hydrolysateobtained through alkaline enzymatic hydrolysis with CaOin discontinuous process (KHAE1) showing the presenceof low average molecular weight proteins in the 3000-6000Da range. The band 3 corresponds to keratin hydrolysate(KHAE3) extracted with calcium oxide by continuous-flowalkaline-enzymatic hydrolysis, which is more intense inthe 3000-19000 Da range. Band 2 corresponding to keratinhydrolysate extracted with sodium hydroxide, bycontinuous-flow alkaline-enzymatic hydrolysis (KHAE2)

Table 3 PHYSICO-CHEMICAL CHARACTERIZATION OF

KERATIN HYDROLYSATES OBTAINED BYALKALINE-ENZYMATIC HYDROLYSIS INDISCONTINUOUS (KHAE1, KHAE2) AND

CONTINUOUS PROCESS (KHAE3)

Fig. 3. SDS-PAGE electrophoresis for1-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

reveals proteins with average molecular weights especiallyin the 3000-14000 Da range.

These results make that it possible to envisageapplications in obtaining foliar fertilizers containing aminoacids and peptides with sulfur and organic nitrogen (lowmolecular weight hydrolysates).

The FT-IR/ATR spectra (fig. 4 and table 4) show slightlydeviated bands in the keratin hydrolysates obtained byalkaline-enzymatic hydrolysis in the amide I specific range(1700-1600 cm-1), 1637.27 cm-1 for KHAE1 and 1648.84cm-1 for KHAE2. The second band at 1644.02 cm-1 is forKHA1 and KHA2, obtained by alkaline hydrolysis. Allanalysed samples reveal the absence of any signal

Table 4ATTRIBUTION OF MAIN

CHARACTERISTIC FREQUENCIESRECORDED BY FTIR/ATR

ANALYSIS

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corresponding to the vibrations of amide II, namely in-planeN-H bending vibrations and C-N stretching vibrations, butwagging vibrations were recorded out of the plane of CH2groups, γrCH2, specific to amino acids, in the case of keratinhydrolysates from hydrolysis using calcium oxide asalkaline agent, KHA1 and KHAE1.

The bands in the 600-700 cm-1 range attributed νC-Sstretching vibrations are specific to sulfur compounds [30,31] and as expected, were identified in all fourhydrolysates.

The structural analysis of keratin hydrolysates highlightthe fact that chemical hydrolysis of wool lead to itsdestructuring, particularly in the presence of enzymes;relevant in this regard is hydrolysate KHAE1, that standsout through the appearance of spectral bands at 1407.78cm-1 and 1165.48 cm-1, in areas specific to amino acids incorrelation with the low molecular weight (table 3).

The measurements on average particle size of keratinpolydispersions, obtained by hydrolysis with CaO, NaOHand by discontinuous-flow enzymatic hydrolysis (table 5),reveal a different composition depending on the alkalineagent of hydrolysis. While in the case of hydrolysis withCaO the particles are very large (2566 nm), enzymatichydrolysis reduces at 302 nm with a reduction inpolydispersity (from 1 to 0.835). In case of hydrolysis withNaOH, the average particle size is smaller (590 nm) andincreases by enzymatic hydrolysis (746 nm) with adecrease in the polydispersity value, from 0.804 to 0.699.The number of particle populations is also different for thetwo types of hydrolysis, CaO hydrolysis generates a singleparticle population (fig. 5a) and 4 particle populations afterenzymatic hydrolysis (fig. 5b). In case of hydrolysis withNaOH, 3 particle populations (6a) and a lower number ofpopulations after enzymatic hydrolysis (6b) were identified.Particle size investigations in protein polydispersions areoriginal [32] and highlight the diversity of hydrolysate

Table 5 AVERAGE SIZES AND POLYDISPERSITIES FOR KERATIN

HYDROLYSATES: KHA1, KHAE1, KHA2 AND KHAE2

a b

a b

Fig. 5. Distribution of particlepopulation size in keratin

hydrolysatesKHA1 (a), KHAE1 (b)

Fig. 6. Distribution ofparticle population size in

keratin hydrolysatesKHA2 (a), KHAE2 (b)

properties. This study shows the variation of keratinpolydispersions depending on the type of hydrolysis agent.

Surface tension analysis of keratin hydrolysates obtainedthrough various chemical-enzymatic processes allows theestimation of the display properties on the plants surfacein the case of potential foliar applications. In this regard,the results of analyses, presented in table 6, indicate lowsurface tensions, knowing that protein hydrolysates shownumerous free hydrophilic groups with tenside properties[33]. Low surface tension values indicate potential

Table 7THE INFLUENCE OF KERATIN HYDROLYSATE ON LEATHER DYEING

RESISTANCE

Table 6SURFACE TENSION DETERMINED FOR ALKALINE KERATIN

HYDROLYSATES

applications as dispersants and surfactants in variousindustries and in agriculture technologies.

Experiments on the use of keratin hydrolysate as aretanning agent for bovine leather, intended for footwear,were based on literature investigations on the ability ofcollagen or keratin hydrolysates [34] to provide fullness,finesse and firmness to leather uppers, intensity to dyeing.This is a procedure for introduction of additional carboxyland amine groups in bovine leather, capable of fixing dyesand additional fatliquoring agents. The results of analysesshowed an increase in leather softness (SR EN17235:2002) treated with 27% keratin hydrolysatecompared to untreated leather, opening the prospect forfurther experiments in this direction. The influence of

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keratin hydrolysate on leather dyeing resistance is slightlyimproved, as table 7 shows. Research is ongoing, giventhe high interest in replacing chemicals from oil sourceswith renewable materials.

The paper presents original results on the wide range ofkeratin polydispersions that can be obtained by alkaline oralkaline-enzymatic hydrolysis of wool waste.

ConclusionsRecovery of keratin from sheep wool waste is a major

objective in the context of the sustainable developmentstrategy for addressing biotechnologies as an alternativeto the use of synthetic chemicals from oil resources. Thepaper presents possibilities for recovery of keratin fromsheep wool by means of alkaline hydrolysis processes withyields, ranging between 16.4 and 43.5% and chemical-enzymatic hydrolysis processes in continuous ordiscontinuous flow. The results of analyses highlight thepossibility of obtaining keratin hydrolysate polydispersionswith different molecular weight and particle size propertiesby approaching chemical hydrolysis with various alkalineagents or by refining hydrolysis through enzymaticprocesses which opens the perspective of wide-rangeapplications in various industries and in agriculture. Keratinhydrolysates obtained by alkaline hydrolysis processesshow low surface tensions (40.77-46.49 mJ/m2) whichconfirms the specific surfactant properties of proteinhydrolysates, with the prospect of multiple applications.Electrophoresis (SDS/PAGE) and FTIR/ATR assays confirmthe wide range of polypeptides with various molecularweights (4650 Da - 19000 Da) and amino acid content(chemical-enzymatic hydrolysis with calcium oxide). Thehigh molecular weight keratin hydrolysates (35800 Da)suggest the possibility of using them as retanning agentsto improve the softness and dyeing resistance of bovineleather.

Acknowledgements: These works were supported by grants ofRomanian Ministry and the Romanian National Authority for ScientificResearch and Innovation, CNCS/CCCDI-UEFISCDI, project numberPN-III-P2-2.1-PTE-2016-0214 within PNCDI III, contract 55PTE and ofRomanian Ministry of Research and Innovation under Nucleus contractno. 26 N/14.03.2016, project PN 16 34 01 11.

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Manuscript received: 12.09.2017