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Journal of the Society of Leather Technologists and Chemists, Vol. 90 p.



The Key Laboratory of Leather Chemistry and Engineering of Ministry of Education, Sichuan University, Chengdu, P. R.
China, 610065
Collagen, gelatin and collagen hydrolysate were prepared from bovine limed split wastes by different preparative
processes. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the
molecular weight distribution of collagen was very narrow (about 200 and 100kDa for β and α chains
respectively) compared with those of gelatin (less than 300kDa and wide distribution) and collagen hydrolysate
(less than 50kDa and wide distribution ).
The isoelectric points of collagen, gelatin and collagen hydrolysate were 8.26, 4.88 and 4.54 respectively
determined by Zeta potential titration. Circular dichroism (CD) spectra revealed that there were two peaks, a
positive peak around 221nm and a negative peak around 192nm for collagen, which are the characteristics of
collagen triple helix. However, gelatin and collagen hydrolysate lacked any positive peaks around 220nm,
suggesting random coils. The denaturation temperature of collagen was about 37.5°C determined by the viscosity
method, the helix-coil transitions for gelatin and collagen hydrolysate were not present in the heating process.
Collagen reaggregated to fibrils at 35°C monitored at 313nm. In contrast, gelatin and collagen hydrolysate lost
the ability of fibril formation. Collagen was more resistant to trypsin hydrolysis compared with gelatin and
collagen hydrolysate. In addition, the collagen membrane exhibited superior features such as higher enthalpy,
greater network structure and better physical/mechanical properties compared with those of the gelatin
Therefore, collagen isolated from limed split wastes can be a high value product due to its special characteristics
and has many potential future applications in biomaterials, functional additives, cosmetics and pharmaceutical


In this work, the different physicochemical properties of
collagen, gelatin and collagen hydrolysate derived from
bovine limed split wastes by different preparative processes
will be compared in order to better utilize them as potential
biomaterials or additives.

Nowadays, approximately 170 million pieces of all
kinds of raw hides are converted into leather in China and,
in the leather making processes, about 1.45 million tons of
leather wastes - including about 0.45 million tons of limed
split wastes are simultaneously produced. Limed split
wastes are less contaminated by chemicals than tanned
leather or finished leather wastes, and can be a source of
high value products such as collagen, gelatin and collagen
Collagen is an important biomaterial in medical
applications due to its special characteristics, such as
biodegradability and weak antigenecity.1 Thus collagen, as
a new type of biomaterial, has been used in drug delivery
systems2 and tissue engineering.3 In addition, there are
some intrinsic relationships between collagen and many
diseases such as rheumatoid arthritis4 and systemic
sclerosis.5 Gelatin can be made into roll film and drug
capsules and can also be used as a biomaterial in
biomedical applications, such as in drug delivery systems.6
which are very different from the traditional capsule.
Collagen hydrolysate is a polypeptide composite made by
further hydrolysis of denatured collagen. It has been used
in cosmetics and as a food additive.7-9 Many attempts have
been made to utilize the limed split wastes in ways other
than utilization as the source of commercial gelatins. It had
been shown that the collagen extracted from calf limed
splits at a low temperature by the pepsin-digestion method
had properties similar to those of the commercial
collagen.10-11 In addition, collagen polypeptide had also
been prepared from the limed hide offal in a two-step
protease (EA and EA 537) treatment process at 45-60°C.12

Experimental methods
Extraction of pepsin-digested collagen
Bovine limed split waste pieces were fleshed and then
delimed with 2% NH4Cl and 0.5% HCl for 60 min. The
delimed pieces were neutralized to pH6.0-7.0 with 0.5%
HCl followed by rinsing with distilled water. Then they
were cut into smaller pieces and pulverized with a mill
(Fritsch Pulverisette 14, Germany). The powders were
extracted with 30 volumes of 0.5 M acetic acid containing
1% pepsin (1:10 000, calculated on the dry weight of limed
split wastes) at 4°C for 48 hrs. The supernatants of the
extracted solutions were collected by centrifugation at 10
000g for 15 min at 4°C, and then salted out by adding NaCl
to a final concentration of 3 M followed by centrifugation
under the same conditions. The precipitate was dissolved in
0.5 M acetic acid and salted out in 0.7 M NaCl solution.
The precipitate was again dissolved in 0.5 M acetic acid,
and then dialyzed against 0.1 M acetic acid. Collagen
concentration was determined indirectly from the
hydroxyproline concentration, which was analyzed by the
method of Bergman and Loxyley.13
Preparation of gelatin and collagen hydrolysate
Bovine limed split wastes were relimed with 3-4% lime
and 0.5% NaOH for two weeks. The relimed split wastes

*Corresponding author: E-mail:


1 M acetic acid solution (collagen solvent) and water (solvent for gelatin and collagen hydrolysate) were also determined under the same conditions. The isoelectric points of the samples were determined at the pH value where the Zeta potential was zero. Approx. After cooling with liquor nitrogen. The samples taken at the given time were added 1% (w/v) SDS and boiled for 5 min in order to inactivate the enzyme.15 Results and discussions SDS-PAGE analysis The electrophoresis patterns of the samples are shown in Fig. All the samples were centrifuged at 10 000g for 15 min and adjusted to 0. and the turbidities were recorded at 313nm using a UV spectrometer (Perkin Elmer Lambda 25.5% HCl. So the components of gelatin and collagen hydrolysate were more complex than those of collagen. secondary heating was run under the same conditions. The concentrations of the samples were determined by the Biuret method. The samples were incubated to 35°C to initiate fibril formation. 2mg samples were sealed in aluminum pans and the empty pan was used as a control.15 M) at 4°C.10 Physical properties of membranes The denaturation temperatures of the collagen and the gelatin membranes were determined by a differential scanning calorimetry apparatus (DSC) (Netzsch DSC 200PC.04% (w/v) trypsin (1:250) and the degree of hydrolysis was examined by determining the increasing amounts of primary amines in the samples. USA). the molecular weights of gelatin and collagen hydrolysate were less than 300 and 50 kDa respectively and their molecular weight distributions were very wide. Collagen was extracted in the acid solution Circular dichroism (CD) Collagen was dialyzed against 0.01% bromophenol blue. Denaturation temperature determined by viscosity method Collagen (0. which were the unfolding polypeptide chains of the triple helix ([α1(I)]2[α2(I)]).2) in the presence of NaCl (final concentration adjusted to 0. The solution was scanned at the 24 . Different preparative processes lead to different molecular weight distribution.1 M acetic acid solution using a dialysis tube at 4°C for 72 hrs.5 mg/ml before CD analysis. pH7. The reduced viscosity (ηsp/c).25 M NaOH or 0.5% acetic acid/5% methanol solution until the bands were clear. The collagen hydrolysate membrane dried under the above conditions was too fragile to be used in practice.8) and then boiled for 5 min. Collagen hydrolysate was prepared by hydrolysis of gelatin using 0.02% (w/v) trypsin (1:250) at 40°C for 4 hrs. Switzerland). where ηsp is the specific viscosity and is calculated by (t-t0)/t0 and c is the concentration of the samples.5 pH. China). The membranes were conditioned in a humidistat containing saturated NH4NO3 solution at 20°C for 24 hours before test. Resistance to trypsin degestion Collagen was dialyzed against the same phosphate buffer as used for fibril formation without the NaCl at 4°C for 72 hours. pH 6. The denaturation temperature was determined as the mid-point temperature where viscosity changes reached 50%. So only the collagen and the gelatin membranes were chosen for assay. Germany). Collagen displayed one β band (200 kDa) and two α bands (100 kDa for α1 and α2). their tensile strength and elongation at the break were also determined by a strength tester (Gaotie GT-AI-7000S. Gelatin and collagen hydrolysate solutions were adjusted to 8 and 10mg/ml respectively. 10% glycerol and 0. 15ml solution was incubated for 15 min at the given temperature from 25 to 50°C. The SEM specimen was prepared by the method described by Li.1 M Na2HPO4. USA) and its morphology was observed by scanning electron microscopy (SEM) (JEOL JSM5900LV. All the samples were digested with 0.5mg/ml using the same buffer solution. The efflux times (t0) of 0. The solutions were dried using a spray dryer (Buchi B-191. Japan). The gelatin and collagen hydrolysate were directly dissolved in deionised water.05 M citric acid. 15µl treated samples were injected into 10% gel wells and run for approx. The titration temperature was 25°C and the decreasing pH intervals were 0.were washed and neutralized to pH5. The change of Zeta potential was plotted against the change of pH for the samples. The concentrations of the samples were adjusted to 0. UK). The resulting collagen fibril was lyophilized in a freeze dryer (Labconco Freeze Dryer FreeZone 6 Liter.5-6 with 1. Isoelectric points of samples Collagen. gelatin and collagen hydrolysate solutions (0. The molecular weight of type I collagen was about 300 kDa. 1. was plotted against the temperature.02mg/ml) was prepared in the same method as used for the CD sample. 0. 120 min. The other samples were dissolved in distilled water. The gel was stained with 0.05% w/v) were titrated with 0. The endothermal curves of the samples were recorded from 10 to 80°C at a heating rate of 5°C/min in a nitrogen atmosphere. The morphologies of the membranes were examined by SEM as in the fibril formation experiment. In contrast. The denaturation temperature was determined from the viscosity changes using an Ubbelohde viscosimeter. Preparation of collagen and gelatin membranes Collagen and gelatin were dried to form membranes at 30°C in silica gel desiccators. Gelatin (type B) was extracted about five times from 70 to 90°C at a 5°C interval. Japan). All the samples were filtered through a filter funnel (40-80µm) and degassed by centrifugation. and then the efflux time (t) was recorded. In addition.14 wavelength range from 190 to 250nm at 25°C and its molar ellipticity[θ] was recorded using a circular dichroism apparatus (Jasco J-500C.25 M HCl and the Zeta potentials at a given pH were recorded by a Zeta potential titration apparatus (Malven Zetaweight Nano ZS.5 M Tris-HCl buffer (1% SDS. Fibril formation experiment Samples were dialyzed against phosphate buffer (0.25% Coomassie Brilliant Blue R-250 for 45 min and de-stained with 7. The primary amines were determined using the trinitrobenzensulfonic acid (TNBS) method as described by Adler-Nissen. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) Samples were mixed with 0.

which is related to the proportion of acid amino residues and base amino residues in protein.20 The curves of the reduced viscosity plotted against the temperature are given in Fig. 4. Collagen had a positive maximum peak at 221nm and a negative minimum peak at 192nm. The isoelectric point of collagen was in the basic range due to the acidic extraction. CD spectra of collagen.26.88 and 4. suggesting a typical triple helical conformation. and thus. the isoelectric points of gelatin and collagen hydrolysate were in the acid range due to the higher density of carboxyl groups caused by the hydrolysis of side amide groups of samples under the strong base and high temperature preparative conditions. However. causing the decrease of viscosity at the same time. an important role in the clarification of cloudy alcoholic beverages by aggregation of the yeast and other insoluble particles. gelatin and collagen hydrolysate determined by viscosity method. It Figure 1. The denaturation temperature was approx. the values of isoelectric points were the values of the system which included the various polypeptides and buffer. respectively. the physical properties such as viscosity. 25 . there were wide distributions and Wavelength (nm) Figure 2. lower molecular weights for gelatin and collagen hydrolysate. CD spectra and viscosity change are widely used to determine the denaturation temperature of native collagen. As examples. without showing the helix-coil transition. 3. the reduced viscosity changed slightly with the increase of temperature. CD spectra of the samples are shown in Figure 2.17 whereas the positive peaks in gelatin and collagen hydrolysate disappeared. Isoelectric points of samples Isoelectric point is an important parameter of proteins. the triple helical and rod like structure of collagen as a clarifying agent play Figure 3. which only attacked the non-triple helical domain of native collagen. which kept side amide residues intact. collagen (lane 2) gelatin (lane3) and collagen hydrolysate (lane 4) on 10% gel. the triple helix can partly reform for gelatin when the temperature of the sample falls below the gelation temperature. gelatin and collagen hydrolysate were 8. The reduced viscosity of collagen decreased at 30°C and reached a plateau at 42. SDS-PAGE analysis of molecukar weight standards (lane 1). whereas gelatin and collagen hydrolysate were prepared under severe conditions (above the denaturation temperature).19 The triple helix of native collagen can transform to the random coil configuration when it is heated above the denaturation temperature. The helix-coil transition of collagen involves the breakage of hydrogen bonds between the adjacent polypeptide chains of collagen molecules in the denaturation process. 37. Thermal denaturation curves for collagen.5°C.containing pepsin. Triple helical conformation and helix-coil transition Collagen is a sort of optically active protein and adopts the polyproline II-like helical conformation16 with a negative minimum absorption band around 190nm and a weak positive maximum absorption band at 210-230nm. The denaturation temperatures determined by the two methods are compared to each other when the protein concentration and the solvent are same. solubility and optical activity changed due to the collapse of the triple helical structure.18 The different secondary structures of the samples can greatly affect some special properties of the samples. showing the random coils configuration.5°C. Most of the triple helices of gelatin and collagen hydrolysate had been destroyed and parts of their peptide bonds were also broken out. In contrast. Therefore. In the denaturation process.56. gelatin and collagen hydrolysate. The intact trimers (γ) of collagen turn into individual chains (α) or dimers (β) in the transition. Gelatin and collagen hydrolysate were composites of different molecular weight polypeptides. In the case of gelatin and collagen hydrolysate. The isoelectric points of collagen.

and thus the denaturation temperature of mammalian animal collagen is generally higher than that of marine animal collagen. proteins concentration and so on). Trypsin was used to test the samples for their resistance to enzyme digestion. which made fibrillogenesis impossible. The level of primary amines of collagen increased very little in 6 hrs at 30°C (below denaturation temperature). the amount of primary amines in gelatin increased greatly especially in the first 2 hrs. 6. Collagen hydrolysate is a denatured hydrolysis product with small molecular weight and without the triple helical structure. The peak temperature of the collagen membrane was about 59°C similar to that of the gelatin membrane (62. 7 shows the thermograms of the collagen and the gelatin membrane. 5). Turbidity . Physical properties of membranes The thermo-physical properties of the collagen and the gelatin membranes were examined by DSC. gelatin and collagen hydrolysate incubated at 35°C. gelatin and collagen hydrolysate at 30°C.21 The fibril formation profiles are shown in Fig.5°C). The trypsin digestion curves are shown in Fig. In the second run. 4. However. different extraction methods and self-assembly conditions (buffer. self-assembly of collagen monomers into fibrils involves hydrophobic and electrostatic interactions between collagen chains. the endothermal peaks of the collagen and the gelatin membrane both disappeared and only exhibited a weak step baseline. 26 . the optical intensities of gelatin and collagen hydrolysate were stable.22 Different preparative processes of the same type collagen have also different fibrilogenesis behavior and conditions.was mainly because there were only a few or possibly no triple helices remaining to be decomposed in the heating process. Thus these products lost the ability of fibril formation. the SEM image of the resulting collagen fibrils presented obvious fibril morphology (Fig. Thus. Figure 4. Fig. there were only a few sites remaining for trypsin to futher attack under these mild conditions as a consideration of the denaturation of collagen. In addition. Collagen hydrolysate was prepared by the hydrolysis of gelatin using trypsin. By contrast. showing little hydrolysis. temperature-independent growth and temperature-dependent growth according to the study of Gelman. The preparative process reduced the isoelectic points of gelatin and collagen hydrolysate due to the increase of negative charges. The collagen fibrillogenesis involved different collagen types. Trypsin hydrolysis curves for collagen. but its peak area (enthalpy) was markedly larger than that of the gelatin membrane in the first scan. which hydrolysed the amide groups and decomposed the triple helices. the structures of gelatin and collagen hydrolysate were disordered and their molecular weight distributions were very wide. SEM image of reconstructed collagen fibrils.time curves for collagen. The denaturation temperature of collagen is similar to the body temperature and the environmental temperature. Bar = 5 µm Samples digested with trypsin The collagenous domain is hardly digested by enzymes other than collagenase due to its stable triple helix but the denatured products such as gelatin and collagen hydrolysate are easily attacked by proteinases. The content of primary amines can be used as an index for the degree of hydrolysis of samples. Gelatin was prepared under basic and high temperature conditions. showing the glass transition. which also made it difficult for the molecules to undergo interaction due to the repellency of negative charges. Therefore. Different types of collagens with different triple helical structures can influence the time course and extent of fibril formation. showing fibril formation in the solution. Therefore. In addition. Fibril formation experiment Native collagen can aggregate into collagen fibrils under physiological conditions. Figure 5. suggesting no fibrillogenesis. temperature.11 Collagen was extracted in acidic solution containing pepsin which attacked only the non-triple helical domain of native collagen. Figure 6. the application area of mammalian animal collagen is more extensive than the marine animal collagen in respect of the thermal stability. it can be separated into three steps including temperature-dependent initiation. In the process of fibril formation in vitro. Collagen solution became turbid with increasing time. The thermal behaviors of collagen and gelatin are related to the moisture content and the thermal history (preparative conditions). Collagen was more resistant to proteinase hydrolysis than the denatured products (gelatin and collagen hydrolysate) due to its specific triple helical structure.

while those in the second scan corresponded to the characteristics of completely amorphous biopolymer due to the elimination of the triple helical structure. 113-136.a model for rheumatoid arthritis. Chanseaud. 2. Gelatin and collagen hydrolysate were the denatured products of native collagen and adopted a random coil conformation. which played important roles in elevating the mechanical properties of the collagen membrane. The results suggest that collagen derived from bovine limed split wastes has a greater potential biomaterial utilization than gelatin and collagen hydrolysate due to its special physicochemical properties. Pharm. lower isoelectric points.g.The collagen membrane presented a larger peak area than that of the gelatin membrane in the denaturation process due to the greater presence of collagen triple helices. Synthesis and physicochemical analysis of gelatin-based hydrogels for drug carrier matrices. 3 ± 0. e. In addition. and ability of fibril formation. R. 3.biomaterial for drug delivery. Holmdahl.5% and 36 ± 4 N/mm2. the gelatin membrane without fibril networks appeared only as a smooth structure on the surface. Conclusions It was found that there were many different characteristics of collagen. 1998. the collagen membrane had a larger endothermal peak area. Bars 5 µm. the DSC traces in the first scan corresponded to the helix-coil transitions during the heating of the samples. Thermograms of the collagen membrane A and the gelatin membrane B determined by DSC.. C. 4. Surface morphologies of the collagen membrane A and the gelatin membrane B observed by SEM. and Kao. Figure 8... 27 . Eur. Only collagen possessed the triple helical conformation. H. New insights into the pathogenesis of systemic sclerosis. 35-40. Biomaterials . 45. In addition. R. et al. Tamby. Therefore. basic isoelectric point. Therefore. Backlund. 2002. J. W.. Collagen . relative lower molecular weights and wide molecular distributions. Acknowledgement This work was supported financially by the National Natural Science Foundation of China (No. such as high molecular weight and narrow molecular distribution. There were obvious fibril networks with a rough membranous structure for the collagen membrane. Guillevin. Biomedical applications of collagen. J. M. triple helix to coil translation in the process of denaturation.. Y. Friess. Y. a special network structure and better physical/ mechanical properties compared with those of the gelatin membrane. The molecular pathogenesis of collagen-induced arthritis in mice . et al. It exhibited many properties distinct from gelatin and collagen hydrolysate. Ageing Research Reviews. L. and Lee. B Figure 7. 7 ± 0. J. respectively. 509-523. Biopharm. (Applied Biomaterials). Pharm. N.5%. The surface morphologies of the collagen and the gelatin membrane were greatly different from each other (Fig. 2003. 5. 135-147. R. the tensile strengths and elongations at the break for the collagen and the gelatin membrane were 58 ± 5N/mm2. Collagen-Based Devices for Soft Tissue Repair. 2002. 1.. J. J. the extraction of collagen from bovine limed split wastes is a high value route for the utilization of leather wastes. 221. 24. Singla.. loss of ability of fibril formation... 2. M. Autoimmunity Reviews. 33. Einersona. There were more rigid native triple helices and fibril networks in the collagen membrane than in the gelatin membrane.. In contrast. 6. Stevensa. The differences of the mechanical properties were caused by the different structures of the membranes. 20576073). K. without conformation translation. and being easily attacked by proteinase. Res. although they were all derived from bovine limed split wastes. 1996. J. J.. In addition. showing different properties.. there were no endothermal peaks in the second scan. C. 2001. 152-157. A. 8). Pachence. high resistance to protease. gelatin and collagen hydrolysate from the different preparative processes. W... Lee. Bockermann. (Received September 2005) References 1. Int. Biomed. 1-22.

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