Extraction of native collagen from limed bovine split wastes through improved pretreatment methods

Journal of Chemical Technology and Biotechnology J Chem Technol Biotechnol 83:1041–1048 (2008)

Extraction of native collagen from limedbovine split wastes through improvedpretreatment methodsDong Li, Wei Yang and Guo-ying Li∗The Key Laboratory of Leather Chemistry and Engineering of Ministry of Education, Sichuan University, Chengdu 610065, PR China

Abstract

BACKGROUND: The large amount of limed bovine split wastes discharged by the leather industry has raisedconcerns regarding their environmental effect. The objective of this work was to perform pilot plant trials toextract high-value native collagen from these wastes through improved pretreatment methods.

RESULTS: EDTA- and HCl-pretreatment gave similar removal percentages of inorganic substances. Owing tothe open structure of fibers, the collagen yield of HCl-pretreated splits (HPS) (41.31%) was higher than that ofEDTA-pretreated splits (EPS) (10.42%). Furthermore, HCl-pretreated split collagen (HPC) had a more acidicisoelectric point, lower content of primary amino groups, larger Z-average particle size and higher relativeviscosity than EDTA-pretreated split collagen (EPC). Electrophoretic analysis and circular dichroism spectrarevealed the maintenance of polypeptide and triple helix conformation, respectively. In addition, the transitiontemperatures of EPC (34.7 ◦C) and HPC (34.6 ◦C) detected by differential scanning calorimetry (DSC) were closeto that of commercial collagen from calfskin (CCC) (35.7 ◦C).

CONCLUSION: A process of native collagen extraction from limed bovine split wastes was proposed. While bothEPC and HPC represented similar physicochemical properties to those of CCC, the collagen yield of HPS wasmuch higher than that of EPS. 2008 Society of Chemical Industry

Keywords: native collagen; pretreatment; limed bovine split wastes; physicochemical properties; collagen yield

NOMENCLATUREEPS, EDTA-pretreated splits; HPS, HCl-pretreatedsplits; EPC, EDTA-pretreated split collagen; HPC,HCl-pretreated split collagen; CCC, commercialcollagen from calfskin

INTRODUCTIONHides come to the tanners as a by-product of themeat industry, while the leather industry, in turn,generates considerable amounts of waste, causingconcerns regarding its environmental effect.1 Thewastes discharged during the entire leather-makingprocess are categorized into chromium-containingsolid waste, chromium-free solid waste, and otherscomprising dischargeable fat, soluble protein and solidsuspended pollutants. One metric ton of wet saltedhides/skins yield 200 kg of end product, along withabout 250 kg of chromium-containing solid waste,about 350 kg of chromium-free waste and about 100 kglost in wastewater.2

Chromium-containing solid wastes have applica-tions in a wide range of products, including feedadditives,3 fertilizers4 and chemicals.5 In contrast,reports concerning the utilization of chromium-freesolid waste are rare with the exception of gelatin andcollagen hydrolysate production.6 Globally, around800 000 metric tons of chromium-free solid waste,a large proportion of which is composed of limedsplit waste, are processed annually. It is interesting tonote the potential possibility of profiting more fromthe limed split waste. The collagen extracted fromlimed calf splits at low temperature by the pepsin-digestion method has been shown to exhibit similarproperties to those of commercial collagen.7,8 Collagenhas many special characteristics, such as biodegrad-ability and weak antigenicity,9 and finds applica-tions in cosmetics,10 biomedical11 and pharmaceuticalindustries.12 Thus, the feasibility of extracting collagenfrom limed split wastes has been investigated.

Because the high content of inorganic substancesaffects collagen extraction, limed split wastes arenot suitable for collagen extraction directly. Certain

∗ Correspondence to: Guo-ying Li, The Key Laboratory of Leather Chemistry and Engineering of Ministry of Education, Sichuan University, Chengdu, PR China,610065E-mail: [email protected](Received 9 November 2007; revised version received 14 January 2008; accepted 14 January 2008)Published online 22 March 2008; DOI: 10.1002/jctb.1912

2008 Society of Chemical Industry. J Chem Technol Biotechnol 0268–2575/2008/$30.00

D Li, W Yang, G-y Li

pretreatments are necessary to remove inorganicsubstances and to increase collagen yields. One ofthe common compounds used for decalcificationis ethylenediamine tetraacetic acid disodium salt(EDTA). This was used as a chelating agent anddoes not disrupt the tissue. Strong acid solutionssuch as nitric acid and hydrochloric acid coulddecrease decalcification time but these harsh chemicalscause tissue damage that is obvious even at lightmicroscopic level.13,14 Microwaves have been usedby some investigators to accelerate decalcification,and reports indicated that there was no differencein appearance between tissue treated with microwavesand tissue processed with routine decalcification.15 Inaddition, various commercial decalcification solutionssuch as RDO (Dupage Kinetic Laboratories Inc.) andDecal (Omega Chemical Corp.) were available, butthey disrupted the tissue to some degree.16

Considering the techno-economic viability, theEDTA- and HCl-pretreatment methods were selectedand carried out in this study, and some physi-cal/chemical properties of the pretreated limed splitwastes and the extracted collagens were analyzed inorder to evaluate the pretreatment processes. Based ona comparative study of pretreatment methods, a pro-cess of native collagen extraction from limed bovinesplit wastes was proposed.

MATERIALS AND METHODSProcessing of limed bovine split wastesMaterial and preparationLimed bovine split wastes were procured from severallocal commercial leather tanneries and analyzed fortheir chemical components. This research was carriedout on a pilot scale and most measurements were madein duplicate or triplicate on independently procuredlimed split wastes. Initially, 50 kg of limed bovinesplit waste were rinsed in a rotary drum with 0.5%non-ionic detergent for 2 h to remove fat and surfacefeculency. The split waste was swelled with sodiumhydroxide at a pH of 12 for 48 h and then delimed with3% ammonium sulfate for about 1 h. The structurallyopened splits were adjusted to pH 8 and bleachedwith 20 g L−1 hydrogen peroxide for 4 h. The bleachedsplits were then washed in a rotary drum for 20 min toremove residual hydrogen peroxide.

PretreatmentComparison of pretreatment methods focused ondecalcifications because the inorganic substances,whose main component was calcium, could affectthe collagen yield by reducing the peptic activity.Decalcifications were carried out using two differentmethods; soaking in EDTA solution and soaking inHCl solution.

Bleached splits were soaked in the rotary drum withtwo volumes of 0.5 mol L−1 EDTA solution at roomtemperature for 24 h to remove the inorganic matterand then washed twice with tap water. The series

of soaking and washing was repeated. The cleaneddecalcified splits were named EDTA-pretreated splits(EPS).

The other method to remove inorganic matter wascarried out with strong acid solution. Bleached splitswere soaked in two volumes of 15% NaCl solutionand adjusted to a final pH of 2 with HCl. The rotarydrum was run for 6 h at room temperature, followedby adjusting the pH of splits to 7 with NaOH solutionand washing them twice with tap water. The cleaneddecalcified splits were named HCl-pretreated splits(HPS).

Extraction of collagenThe pretreated splits were cut into smaller piecesand pulverized with a mill (Fritsch Pulverisette14, Germany). The powders were dispersed in 30volumes of 0.5 mol L−1 acetic acid containing 2%pepsin (1:3000, calculated on the dry weight ofpowders) at 4 ◦C for 48 h. The supernatants of theextracted solutions were collected by centrifugationat 10 000 × g for 15 min at 4 ◦C to remove insolublesubstances, and then salted out using 0.7 mol L−1

NaCl. The resultant precipitate was collected bycentrifugation, dissolved in 0.5 mol L−1 acetic acid,and dialyzed against 0.1 mol L−1 acetic acid at 4 ◦C for3 days. The collagen concentrations were determinedindirectly from the hydroxyproline concentrations,which were analyzed using the method of Bergmanand Loxyler.17

Analytical methodsChemical component analysisThe moisture, protein, fat and ash content of untreatedand pretreated splits were measured. The fat contentwas determined by a Soxhlet extraction method,18

and the protein content by a Kjieldahl method19

Digest System K-437 and Distillation Unit K-399;Buchi, Flawil, Switzerland). The specific conversionfactor from total Kjeldahl nitrogen (TKN) convertedto protein content was assumed to be 6.25.

Organic matter was defined as the amount ofsample material that vaporized during incinerationat 600 ◦C for 6h (i.e. amount of sample weight lossduring ignition), and, conversely, inorganic matter wasdefined as the amount of residual solids followingignition (i.e. fixed solids or mineral ash). ‘Otherorganic matter’ referred to the amount remaining aftersubtracting the weight of protein and fat from totalorganic matter.

Inductively coupled plasma atomic emission spectrometry(ICP-AES)0.05 g ash obtained from untreated and pretreatedsplits were weighed accurately, dissolved in 5 mL6 mol L−1 HCl solution and diluted to 10 mL in avolumetric flask with deionized water. The experimentwas carried out with inductively coupled plasmaatomic emission spectroscope (Optima 2100 DV; PE,

1042 J Chem Technol Biotechnol 83:1041–1048 (2008)DOI: 10.1002/jctb

Collagen from limed bovine split waste

Cincinnati, USA) and the wavelength line selected fordetermining calcium was 317.933 nm.

Histological analysisHistological analysis was carried out on specimensfrom untreated and pretreated splits. Specimens werefixed by immersion in 10% (w/v) buffered formalin for24 h and then serially cut into 20-µm-thick sections oncryostat (Jung Frigocut 2800 N, Leica Inc., Germany).Specimens were stained with heamatoxylin-eosin(H&E)20 and mounted in neutral resin for observationby light microscope. Microphotographs of relevantfields were taken at a magnification of 40.

Isoelectic point (pI) and particle size of collagenMeasurements of zeta potential and particle sizedistribution of collagen were performed using aZeta potential titration apparatus (Zetaweight NanoZS; Malvern Ltd., Malvern, UK). The titrationtemperature was 25 ◦C and the pH intervals were0.5. The pI of the sample was determined at the pHvalue at which the Zeta potential was zero, while theZ-average particle size was calculated based on theparticle size distribution.

Relative viscosity of collagenThe relative viscosity was determined using anUbbelohde viscosimeter (WSN-1; Yiheng, Ningbo,China). The sample was filtrated through a filterfunnel (40–80 µm) and degassed by centrifugation at10 000 × g for 15 min, and adjusted to 0.2 mg mL−1.A 15 mL collagen solution was incubated at 25 ◦C for20 min and the efflux time (t) was then determined.The measurement was carried out three times to obtainthe average efflux time value. The efflux time (t0) ofthe collagen solvent (0.1 mol L−1 acetic acid) was alsodetermined under the same conditions. The relativeviscosity was calculated using the equation ηrel = t/t0.

Determination of primary amino groupsA sample solution (0.250 mL) was mixed with 2.00 mL0.2125 mol L−1 sodium phosphate buffer (pH 8.2)and 2.00 mL 0.10% trinitrobenzene sulfonic acid,followed by incubation in the dark for 60 min at 50 ◦C.The reaction was quenched by 4.00 mL 0.100 molL−1 HCl, and absorbance was read at 340 nm.21 A1.500 mmol L−1 L-glycine solution was used as astandard.

Sodium dodecyl sulphate polyacrylamide gelelectrophoresis (SDS-PAGE)SDS-PAGE was performed according to the methodof Laemmli,22 using the discontinous Tris-HCl/glycinebuffer system with 10% resolving gel and 4% stackinggel. Collagen samples were mixed with a samplebuffer (0.5 mol L−1 Tris-HCl, pH 6.8, containing2% SDS, 25% glycerol, 0.01% bromophenol blue) toreach a final collagen concentration of 1 mg mL−1,and then boiled for 3 min. 20 µL treated sampleswere injected into gel wells and run for approximate

100 min. The gel was stained for 30 min with 0.25%Coomassie Brilliant Blue R-250 and de-stained with7.5% acetic acid/5% methanol solution until the bandswere clear.

Circular dichroism (CD)Collagen samples were adjusted to 0.5 mg mL−1 andscanned at a wavelength range from 185 to 250 nm at25 ◦C. The molar ellipticity [θ] was recorded using acircular dichroism apparatus (J-500C; Jasco, Tokyo,Japan).

Thermal transition of collagenThe transition temperature (Tm) was determined bydifferential scanning calorimetry (DSC) (DSC 200PC;Netzsch, Bavaria, Germany). Approximately 10 mg ofcollagen solution was sealed in an aluminum pan andan equal weight of solvent in another pan was used asreference. The endothermal curve of the sample wasrecorded from 20 to 46 ◦C at a heating rate of 2 Kmin−1 in a nitrogen atmosphere.

RESULTS AND DISCUSSIONCharacteristics of the initial materialDuring the leather-making processing, fresh bovinehides are initially treated with a mechanical operationto get rid of excess flesh, fat, and muscle, and thenlimed with a system of hydrated lime and sodiumsulfide to remove hair. The thickness of limed bovinehides is rendered uniform by a bladed cylinder, andthen the hides are trimmed to remove the offal inperimeter areas. When the limed hides are transportedto the bladed cylinder and trimmed off, limed bovinesplit waste is produced.

The chemical composition of limed bovine splitwaste contrasts with the composition of fresh bovinehides as shown in Table 1. The composition of freshhide varies over a wide range between batches.23

Protein is still the main component of limed splitwastes on a dry weight basis, although the proteinpercentage is decreased due to the pervasion ofwater. The fat content of limed split waste declinessharply following the leather-making procedures offleshing and degreasing. There is a marked increasein ash content in limed split waste. Inorganicmatter is also markedly increased as a result ofthe introduction of lime and sodium sulfide. Inaddition, spots of sulfide occur on the split waste,which need to be scoured off with hydrogen peroxide.Pretreatments are necessary before collagen extractionbecause of the high ash percentage and the highpH level (approximate 12) of limed bovine splitwaste.

Pretreatment of limed bovine split wastesEffect of pretreatment on chemical compositionAs expected, both EPS and HPS had similarcompositions, but they also had some importantunique characteristics (Table 1). Total component

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Table 1. Chemical compositions of fresh bovine hides, limed bovine split waste, EPS and HPS

Fresh bovine hide Limed bovine split wastes EPS HPS

Moisture (%) 60 ∼ 75 81.70 ± 2.50 77.86 ± 3.22 87.27 ± 3.46Protein (%) 30 ∼ 35 14.85 ± 0.53 12.31 ± 0.40 5.88 ± 0.48Fat (%) 2.5 ∼ 3.0 <0.1 <0.1 <0.1Inorganic matter (%) 0.3 ∼ 0.5 2.21 ± 0.02 0.21 ± 0.00 0.07 ± 0.00Other organic matter (%) <2 1.14 ± 0.01 9.52 ± 0.02 6.68 ± 0.01

weight of both EPS and HPS was different from thatof split waste as a result of moisture changes. Althoughthe protein percentages of EPS and HPS were only12.31% and 5.88%, respectively, as a result of theirmoisture changes, the protein content in EPS andHPS were still 55.60% and 46.19%, respectively, ona dry weight basis. The results indicated that proteinwas still the main component of EPS and HPS ona dry weight basis. Ash contents of EPS and HPSwere both obviously lower than that of untreated splitwastes. In addition, fat contents of both EPS and HPShad reduced appreciably, even though they were onlya small quantity before pretreatment.

Calcium hydroxide, the main component ofhydrated lime, had soaked into the interspaces of fibersduring the liming procedure. This reacted with CO2

and transformed to calcium carbonate on the surfaceof split waste because the limed split waste was alwaysexposed to air during the storage period. A smallquantity of calcium oxide existing in the hydrated limewas also adsorbed on the fibers. During the prepara-tion procedures, some of these calcium compoundswere removed, while the majority still remained insidethe spacing or on the surface of fibers. Thus, one ofthe remarkable differences between fresh bovine hidesand limed bovine split waste was the content of inor-ganic matter, whose main component was calciumcompounds (1 to 1.5% on the basis of dry weight ofsplits), such as granulated lime (Ca(OH)2), calciumcarbonate and a small quantity of calcium oxide.

During the process of collagen extraction, a few ofthese calcium compounds slightly dissolve in aceticacid as calcium (II) ion, while others still exist as aconformation of precipitation and crystals. Calcium(II) ion hardly affects the activity of pepsin,24 but thepH rise of the extractive solution resulting from thedissolution of calcium hydroxide obviously inhibitspeptic activity. Granulated lime, calcium carbonateand calcium oxide existing in the form of precipitationmight affect peptic activity by adsorbing pepsinin the same manner as aluminum compounds.25

Furthermore, calcium carbonate crystals will lead toinactivation of pepsin by transforming the secondarystructure of the pepsin, with a decrease in β-turns.26 Accordingly, it is necessary to carry out adecalcification procedure to remove residual calciumcompounds.

The percentage removal of inorganic matter andcalcium compounds were determined to investigatethe different pretreatment methods. The amountsof inorganic matter and calcium (II) contained inuntreated split wastes, delimed split wastes, EPS andHPS are shown in Table 2. The inorganic matterand calcium compounds removal percentages fordelimed split wastes were only 48.69% and 46.65%respectively. The inorganic matter and calciumcompounds removal percentages for EPS (92.40%and 98.89%, respectively) and HPS (95.57% and98.75%, respectively) were much higher than thoseof delimed split wastes. The effects of EDTA- andHCl-pretreatment on the removal percentages ofinorganic matter and calcium compounds is obvious,and no significant differences could be detected inthe removal percentages between EDTA- and HCl-pretreatment. The inorganic matter and calcium (II)contents declined sharply and the inhibitive effect ofinorganic matter on peptic activity disappeared.

Histological analysisIt is known that skin fibers absorb a large amountof moisture under acid and alkaline conditions andthis develops a state called ‘swelling’ with shorterand thicker collagen fibers. Acid swelling results fromthe repulsion of electropositive amino groups, whilealkaline swelling is caused by electronegative carboxylgroups in the same manner. Donnan equilibrium andelectrostatic repulsion theories can be employed toexplain this phenomenon.

Histological analysis of untreated split wastes, EPSand HPS revealed normal dermis without epidermis,which was removed during leather-making procedures(Fig. 1). According to the histological appearance with

Table 2. Inorganic matter residuals and collagen yields of limed bovine split wastes, delimed split wastes, EPS and HPS

Residual

Inorganic matter (g) Ca2+ (g) Collagen yield (%)

Limed bovine split wastes (100 g) 2.210 ± 0.020 1.015 ± 0.008 –Delimed split wastes 0.8537 ± 0.012 0.4121 ± 0.004 4.17 ± 0.06EPS 0.168 ± 0.004 0.011 ± 0.001 10.42 ± 0.07HPS 0.098 ± 0.002 0.013 ± 0.001 41.31 ± 0.12



Figure 1. Effect of pretreatment on histological appearance of limedbovine split waste. Histological appearances of EPS (B) and HPS(C) were compared with that of initial limed bovine split wastes (A).Hematoxylin and eosin staining; 40×.

H&E staining, both EDTA- and HCl-pretreatmentscaused some changes in fibrous structure. In theskin tissue of HPS, large spacing between adjacentcollagen fiber bundles and small bundle size wereobserved, which indicated a high degree of fibrousopening. In contrast, the collagen fibers in EPSwere relatively larger than those in untreated splits,which led to a denser network. According to theelectrostatic repulsion theory, the fiber dispersaldegree of pretreated splits was pH-dependent andwas enhanced by the increased margin between pI

value of skin fibers (7.5 to 7.8) and pH value of theirenvironment. In conclusion, the fibrous structure ofHPS was more open than that of EPS.

Collagen yieldsAs a result of the lower percentage of inorganic matterand the more open fibrous structure, pretreated splitswere available to extract collagen directly. Collagenyields of delimed split wastes, EPS and HPS areshown in Table 2. Little collagen (4.17%) could begained from delimed split wastes with no pretreatment,while collagen could be extracted at 10.42% and41.31% yields on a dry weight basis from EPS andHPS, respectively. The collagen yield of HPS wasobviously higher than that of EPS, and more than1.6 kg native collagen could be produced from 50 kglimed bovine split waste by the HCl-pretreatmentmethod. The percentage removals of inorganic matterwere similar to each other, the higher collagen yieldof HPS being mainly caused by the more openedfibrous structure, which was of advantage for thepenetration of acetic acid and pepsin during theprocess of collagen extraction. In addition, the NaClintroduced in the HCl-pretreatment reduces proteinwastage by avoiding excessive swelling; the commonsalt attracts and ties-up the excess moisture whichwould otherwise cause the fibers to swell.27

Characteristics of collagen from limed bovinesplit wastesPrincipal physical properties of EDTA-pretreated splitcollange (EPC) and HCl-pretreated split collagen (HPC)Table 3 shows great differences in some principalphysical properties of EPC and HPC. HPC had muchfewer primary amino groups, lower acidic isoelectricpoint, larger Z-average particle size and higher relativeviscosity than EPC. The isoelectric point of collagenwas chiefly determined by the proportion of acidicand basic residues. The lower acidic isoelectric pointof HPC (5.38) compared to that of EPC (5.87) wasmainly caused by the decreased number of primaryamino groups. A possible increase in carboxyl groupsas a result of deamination of acid amide groups duringthe pretreatment could also contribute to this lattereffect.28 Both EPC and HPC dissolved in 0.5 mol L−1

acetic acid were electropositive because of theionization of amino groups. HPC, with lower acidicisoelectric point, had weaker electrostatic repulsionbetween fibers as a result of the reduction of net charge.This low mutual repulsion of HPC allowed stronger

Table 3. Principal physicochemical properties of EPC and HPC

EPC HPC

Isoelectric point 5.87 ± 0.03 5.38 ± 0.02Average particle size (d, nm) 1560 ± 26 3050 ± 31Relative viscositya 1.49 ± 0.02 1.72 ± 0.01Primary amino acid (µmol mg−1) 0.127 ± 0.010 0.099 ± 0.004

a Determined at 25 ◦C with 0.2 mg mL−1 samples.



aggregation of collagen fibers than EPC.29 Therefore,the particle size distributions of EPC (153 nm with18.1% intensity and 6180 nm with 80.4% intensity)and HPC (192 nm with 17.2% intensity and 8230 nmwith 82.8% intensity) were different from each otherand the Z-average particle size of HPC was larger thanthat of EPS. Meanwhile, the relative viscosity of HPCdetermined at 25 ◦C with 0.2 mg mL−1 concentrationwas 1.15-fold higher than that of EPC, which wasalso mainly because of the larger particle size and theweaker electrostatic repulsion.

SDS-PAGE analysisFigure 2 shows SDS-PAGE patterns of EPC andHPC on 10% gel, along with CCC as a comparison.It was found that both the EPC and the HPCpatterns consisted of two α bands (about 100 kDa),which were the unfolding polypeptide chains of triplehelixes ([α1 (I)]2 [α2 (I)]s), and one β band (about200 kDa), which was the dimer of the α chains. Thesepatterns presented a molecular mass of 300 kDa andagreed with that of CCC. Because of the reducedintermolecular cross-linkage in EPC and in HPCcaused by the extreme pretreatment conditions, theband of the γ chain was beyond detection. In addition,there was no detection of small components lowerthan 100 kDa. Thus, the result of SDS-PAGE analysisindicated that the polypeptide chains of both EPC andHPC were uninterrupted by the pretreatment process.

Conformation and thermal behavior of EPC and HPCCD spectrums of CCC, EPC and HPC withcollagen solution are given in Fig. 3. A positive peakat 223 nm and a negative peak at 197 nm wereobserved for EPC and HPC as well as CCC. Thesecharacteristic CD spectra were due to the triple helical

Figure 2. SDS-PAGE analysis of molecular weight standards (1),CCC (2), EPC (3) and HPC (4) on 10% gel.

Figure 3. CD spectra of CCC (1), EPC (2) and HPC (3) at 15 ◦C.

conformation of collagen, adopting the polyproline-II-like helical conformation. Compared with CCC,HPC had a decreased molar ellipticity at 223 nmand at 197 nm than EPC. This result indicated thestronger aggregation of HPC because of its weakerelectrostatic repulsion. This trend was similar tothat of collagen treated with chromium complexes,which was interpreted as being due to aggregation ofcollagen molecules owing to coordination of asparticand glutamic acids in the presence of the metal ions.30

Furthermore, the stronger aggregation of HPC was inaccord with its larger Z-average particle size. Thus,the CD spectra indicated that both EPC and HPCmaintained their triple helix conformation, while HPCshowed stronger aggregation of collagen molecules.

Degradation of the collagen triple helical structureinto random coils was employed to interpret the heattransformation of collagen, and the thermal behaviorof collagen solution was examined by DSC. Thethermal transition curve and the Tm of CCC, EPC andHPC are shown in Fig. 4. Tm of EPC (34.7 ◦C) andHPC (34.6 ◦C) was close to that of CCC (35.7 ◦C).It was shown that the thermal behaviors of EPC andHPC were not markedly different from that of CCC.

Economics of the processThe industrialization of wet spun collagen fibers madefrom limed bovine split wastes with this novel HCl-pretreatment method has been successful in SouthYangzi region of China. The process with HCl-pretreatment method was shown to be economicallyfeasible. As an example, a plant operating to process 50tons of limed bovine split waste per day could producemore than 1.6 tons of collagen fiber per day. The costto produce collagen fibers would be about $12 500per ton. Collagen fibers are available commercially forabout $38 500 per ton in China.



Figure 4. Thermal transition curves of CCC (1), EPC (2) and HPC (3),as shown by DSC.

CONCLUSIONThe economic use of material recovered from limedbovine split waste is gaining attention. In order toextract native collagen from the solid waste, the effectsof two pretreatment methods on collagen extractionwere studied.

Pretreated splits were analyzed for chemical compo-sition changes, fibrous structure and collagen yields.The results indicated that both EPS and HPS enabledcollagen extraction but HPS was more economicalbecause of its higher collagen yield. Collagen extractedfrom pretreated splits was characterized, indicatingthat HPC gave a more acidic isoelectric point, mostlydue to its lower content of primary amino groups,and had larger Z-average particle size and higherrelative viscosity than EPC. Electrophoretic analysisrevealed that the polypeptide chains were maintainedintact after the pretreatment process. CD spectra indi-cated that both EPC and HPC maintained thetriplehelix conformation, although stronger aggregation wasdetected in HPC. The thermal behavior of EPC andHPC was not markedly different from that of CCC.

In conclusion, the physicochemical properties ofEPC and HPC were similar to those of CCC. Thus,a process of native collagen extraction from limedbovine split waste using a HCl-pretreatment methodis proposed for its high collagen yield.

ACKNOWLEDGEMENTSThis work was supported financially by theNational Natural Science Foundation of China (NO.20576083). We wish to thank Mr Jun Jin in the Anal-ysis & Testing Center of Sichuan University for hisexcellent technical assistance.

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Extraction of native collagen from limed bovine split wastes through improved pretreatment methods