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

23

PHYSICOCHEMICAL PROPERTIES OF COLLAGEN,

GELATIN AND COLLAGEN HYDROLYSATE DERIVED
FROM BOVINE LIMED SPLIT WASTES
ZHONGKAI ZHANG, GUOYING LI* and BI SHI
The Key Laboratory of Leather Chemistry and Engineering of Ministry of Education, Sichuan University, Chengdu, P. R.
China, 610065

Abstract
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
membrane.
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
industries.

Introduction In this work, the different physicochemical properties of

collagen, gelatin and collagen hydrolysate derived from
Nowadays, approximately 170 million pieces of all
bovine limed split wastes by different preparative processes
kinds of raw hides are converted into leather in China and,
will be compared in order to better utilize them as potential
in the leather making processes, about 1.45 million tons of
biomaterials or additives.
leather wastes - including about 0.45 million tons of limed

Experimental methods
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 Extraction of pepsin-digested collagen
hydrolysate.
Bovine limed split waste pieces were fleshed and then
Collagen is an important biomaterial in medical
delimed with 2% NH4Cl and 0.5% HCl for 60 min. The
applications due to its special characteristics, such as
delimed pieces were neutralized to pH6.0-7.0 with 0.5%
biodegradability and weak antigenecity.1 Thus collagen, as HCl followed by rinsing with distilled water. Then they
a new type of biomaterial, has been used in drug delivery were cut into smaller pieces and pulverized with a mill
systems2 and tissue engineering.3 In addition, there are (Fritsch Pulverisette 14, Germany). The powders were
some intrinsic relationships between collagen and many extracted with 30 volumes of 0.5 M acetic acid containing
diseases such as rheumatoid arthritis4 and systemic 1% pepsin (1:10 000, calculated on the dry weight of limed
sclerosis.5 Gelatin can be made into roll film and drug split wastes) at 4°C for 48 hrs. The supernatants of the
capsules and can also be used as a biomaterial in extracted solutions were collected by centrifugation at 10
biomedical applications, such as in drug delivery systems.6 000g for 15 min at 4°C, and then salted out by adding NaCl
which are very different from the traditional capsule. to a final concentration of 3 M followed by centrifugation
Collagen hydrolysate is a polypeptide composite made by under the same conditions. The precipitate was dissolved in
further hydrolysis of denatured collagen. It has been used 0.5 M acetic acid and salted out in 0.7 M NaCl solution.
in cosmetics and as a food additive.7-9 Many attempts have The precipitate was again dissolved in 0.5 M acetic acid,
been made to utilize the limed split wastes in ways other and then dialyzed against 0.1 M acetic acid. Collagen
than utilization as the source of commercial gelatins. It had concentration was determined indirectly from the
been shown that the collagen extracted from calf limed hydroxyproline concentration, which was analyzed by the
splits at a low temperature by the pepsin-digestion method method of Bergman and Loxyley.13
had properties similar to those of the commercial
collagen.10-11 In addition, collagen polypeptide had also Preparation of gelatin and collagen hydrolysate
been prepared from the limed hide offal in a two-step Bovine limed split wastes were relimed with 3-4% lime
protease (EA and EA 537) treatment process at 45-60°C.12 and 0.5% NaOH for two weeks. The relimed split wastes
*Corresponding author: E-mail: [email protected]

23
were washed and neutralized to pH5.5-6 with 1.5% HCl. wavelength range from 190 to 250nm at 25°C and its molar
Gelatin (type B) was extracted about five times from 70 to ellipticity[θ] was recorded using a circular dichroism
90°C at a 5°C interval. Collagen hydrolysate was prepared apparatus (Jasco J-500C, Japan).
by hydrolysis of gelatin using 0.02% (w/v) trypsin (1:250)
at 40°C for 4 hrs. The solutions were dried using a spray Denaturation temperature determined by viscosity method
dryer (Buchi B-191, Switzerland). The concentrations of Collagen (0.02mg/ml) was prepared in the same method
the samples were determined by the Biuret method.14 as used for the CD sample. Gelatin and collagen
hydrolysate solutions were adjusted to 8 and 10mg/ml
Sodium dodecyl sulphate-polyacrylamide gel respectively. All the samples were filtered through a filter
electrophoresis (SDS-PAGE) funnel (40-80µm) and degassed by centrifugation. The
Samples were mixed with 0.5 M Tris-HCl buffer (1% denaturation temperature was determined from the
SDS, 10% glycerol and 0.01% bromophenol blue, pH 6.8) viscosity changes using an Ubbelohde viscosimeter. 15ml
and then boiled for 5 min. 15µl treated samples were solution was incubated for 15 min at the given temperature
injected into 10% gel wells and run for approx. 120 min. from 25 to 50°C, and then the efflux time (t) was recorded.
The gel was stained with 0.25% Coomassie Brilliant Blue The efflux times (t0) of 0.1 M acetic acid solution (collagen
R-250 for 45 min and de-stained with 7.5% acetic acid/5% solvent) and water (solvent for gelatin and collagen
methanol solution until the bands were clear. hydrolysate) were also determined under the same
conditions. The reduced viscosity (ηsp/c), where ηsp is the
Isoelectric points of samples specific viscosity and is calculated by (t-t0)/t0 and c is the
Collagen, gelatin and collagen hydrolysate solutions concentration of the samples, was plotted against the
(0.05% w/v) were titrated with 0.25 M NaOH or 0.25 M temperature. The denaturation temperature was determined
HCl and the Zeta potentials at a given pH were recorded by as the mid-point temperature where viscosity changes
a Zeta potential titration apparatus (Malven Zetaweight reached 50%.
Nano ZS, UK). The titration temperature was 25°C and the
decreasing pH intervals were 0.5 pH. The change of Zeta Preparation of collagen and gelatin membranes
potential was plotted against the change of pH for the Collagen and gelatin were dried to form membranes at
samples. The isoelectric points of the samples were 30°C in silica gel desiccators. The collagen hydrolysate
determined at the pH value where the Zeta potential was membrane dried under the above conditions was too fragile
zero. to be used in practice. So only the collagen and the gelatin
membranes were chosen for assay.
Fibril formation experiment
Samples were dialyzed against phosphate buffer (0.1 M Physical properties of membranes
Na2HPO4, 0.05 M citric acid, pH7.2) in the presence of The denaturation temperatures of the collagen and the
NaCl (final concentration adjusted to 0.15 M) at 4°C. The gelatin membranes were determined by a differential
concentrations of the samples were adjusted to 0.5mg/ml scanning calorimetry apparatus (DSC) (Netzsch DSC
using the same buffer solution. The samples were incubated 200PC, Germany). The membranes were conditioned in a
to 35°C to initiate fibril formation, and the turbidities were humidistat containing saturated NH4NO3 solution at 20°C
recorded at 313nm using a UV spectrometer (Perkin Elmer for 24 hours before test. Approx. 2mg samples were sealed
Lambda 25, USA). The resulting collagen fibril was in aluminum pans and the empty pan was used as a control.
lyophilized in a freeze dryer (Labconco Freeze Dryer The endothermal curves of the samples were recorded from
FreeZone 6 Liter, USA) and its morphology was observed 10 to 80°C at a heating rate of 5°C/min in a nitrogen
by scanning electron microscopy (SEM) (JEOL JSM- atmosphere. After cooling with liquor nitrogen, secondary
5900LV, Japan). The SEM specimen was prepared by the heating was run under the same conditions.
method described by Li.10 The morphologies of the membranes were examined by
SEM as in the fibril formation experiment. In addition,
Resistance to trypsin degestion their tensile strength and elongation at the break were also
Collagen was dialyzed against the same phosphate determined by a strength tester (Gaotie GT-AI-7000S,
buffer as used for fibril formation without the NaCl at 4°C China).

Results and discussions

for 72 hours. The gelatin and collagen hydrolysate were
directly dissolved in deionised water. All the samples were
digested with 0.04% (w/v) trypsin (1:250) and the degree
of hydrolysis was examined by determining the increasing
amounts of primary amines in the samples. The samples SDS-PAGE analysis
taken at the given time were added 1% (w/v) SDS and The electrophoresis patterns of the samples are shown in
boiled for 5 min in order to inactivate the enzyme. The Fig. 1. Collagen displayed one β band (200 kDa) and two
primary amines were determined using the α bands (100 kDa for α1 and α2), which were the unfolding
trinitrobenzensulfonic acid (TNBS) method as described polypeptide chains of the triple helix ([α1(I)]2[α2(I)]). The
by Adler-Nissen.15 molecular weight of type I collagen was about 300 kDa. In
contrast, the molecular weights of gelatin and collagen
Circular dichroism (CD) hydrolysate were less than 300 and 50 kDa respectively
Collagen was dialyzed against 0.1 M acetic acid solution and their molecular weight distributions were very wide.
using a dialysis tube at 4°C for 72 hrs. The other samples So the components of gelatin and collagen hydrolysate
were dissolved in distilled water. All the samples were were more complex than those of collagen. Different
centrifuged at 10 000g for 15 min and adjusted to 0.5 preparative processes lead to different molecular weight
mg/ml before CD analysis. The solution was scanned at the distribution. Collagen was extracted in the acid solution

24
containing pepsin, which only attacked the non-triple
helical domain of native collagen, whereas gelatin and
collagen hydrolysate were prepared under severe
conditions (above the denaturation temperature). Most of
the triple helices of gelatin and collagen hydrolysate had
been destroyed and parts of their peptide bonds were also
broken out. Therefore, there were wide distributions and

Wavelength (nm)
Figure 2. CD spectra of collagen, gelatin and collagen hydrolysate.

an important role in the clarification of cloudy alcoholic

beverages by aggregation of the yeast and other insoluble
particles.19
The triple helix of native collagen can transform to the
Figure 1. SDS-PAGE analysis of molecukar weight standards (lane 1),
collagen (lane 2) gelatin (lane3) and collagen hydrolysate (lane 4) on
random coil configuration when it is heated above the
10% gel. denaturation temperature. In the denaturation process, the
physical properties such as viscosity, solubility and optical
activity changed due to the collapse of the triple helical
lower molecular weights for gelatin and collagen
structure. CD spectra and viscosity change are widely used
hydrolysate. to determine the denaturation temperature of native
collagen. The denaturation temperatures determined by the
Isoelectric points of samples
two methods are compared to each other when the protein
Isoelectric point is an important parameter of proteins, concentration and the solvent are same.20 The curves of the
which is related to the proportion of acid amino residues reduced viscosity plotted against the temperature are given
and base amino residues in protein. Gelatin and collagen in Fig. 3. The reduced viscosity of collagen decreased at
hydrolysate were composites of different molecular weight 30°C and reached a plateau at 42.5°C. The denaturation
polypeptides, and thus, the values of isoelectric points were temperature was approx. 37.5°C. The helix-coil transition
the values of the system which included the various of collagen involves the breakage of hydrogen bonds
polypeptides and buffer. The isoelectric points of collagen, between the adjacent polypeptide chains of collagen
gelatin and collagen hydrolysate were 8.26, 4.88 and 4.56, molecules in the denaturation process. The intact trimers
respectively. The isoelectric point of collagen was in the
(γ) of collagen turn into individual chains (α) or dimers (β)
basic range due to the acidic extraction, which kept side
in the transition, causing the decrease of viscosity at the
amide residues intact. In contrast, the isoelectric points of
same time. In the case of gelatin and collagen hydrolysate,
gelatin and collagen hydrolysate were in the acid range due
to the higher density of carboxyl groups caused by the the reduced viscosity changed slightly with the increase of
hydrolysis of side amide groups of samples under the temperature, without showing the helix-coil transition. It
strong base and high temperature preparative conditions.

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.
CD spectra of the samples are shown in Figure 2. Collagen
had a positive maximum peak at 221nm and a negative
minimum peak at 192nm, suggesting a typical triple helical
conformation,17 whereas the positive peaks in gelatin and
collagen hydrolysate disappeared, showing the random
coils configuration. However, the triple helix can partly re-
form for gelatin when the temperature of the sample falls
below the gelation temperature.18 The different secondary
structures of the samples can greatly affect some special
properties of the samples. As examples, the triple helical Figure 3. Thermal denaturation curves for collagen, gelatin and collagen
hydrolysate determined by viscosity method.
and rod like structure of collagen as a clarifying agent play

25
was mainly because there were only a few or possibly no
triple helices remaining to be decomposed in the heating
process.
The denaturation temperature of collagen is similar to
the body temperature and the environmental temperature,
and thus the denaturation temperature of mammalian
animal collagen is generally higher than that of marine
animal collagen. Therefore, the application area of
mammalian animal collagen is more extensive than the
marine animal collagen in respect of the thermal stability.

Fibril formation experiment

Native collagen can aggregate into collagen fibrils under Figure 5. SEM image of reconstructed collagen fibrils.
physiological conditions. In the process of fibril formation Bar = 5 µm
in vitro, it can be separated into three steps including Samples digested with trypsin
temperature-dependent initiation, temperature-independent
growth and temperature-dependent growth according to the The collagenous domain is hardly digested by enzymes
study of Gelman.21 The fibril formation profiles are shown other than collagenase due to its stable triple helix but the
in Fig. 4. Collagen solution became turbid with increasing denatured products such as gelatin and collagen
time, showing fibril formation in the solution. In addition, hydrolysate are easily attacked by proteinases. Trypsin was
the SEM image of the resulting collagen fibrils presented used to test the samples for their resistance to enzyme
obvious fibril morphology (Fig. 5). However, the optical digestion. The content of primary amines can be used as an
intensities of gelatin and collagen hydrolysate were stable, index for the degree of hydrolysis of samples. The trypsin
suggesting no fibrillogenesis. The collagen fibrillogenesis digestion curves are shown in Fig. 6. The level of primary
involved different collagen types, different extraction amines of collagen increased very little in 6 hrs at 30°C
methods and self-assembly conditions (buffer, temperature, (below denaturation temperature), showing little
proteins concentration and so on). Different types of hydrolysis. By contrast, the amount of primary amines in
collagens with different triple helical structures can gelatin increased greatly especially in the first 2 hrs.
influence the time course and extent of fibril formation.22 Collagen hydrolysate was prepared by the hydrolysis of
Different preparative processes of the same type collagen gelatin using trypsin. Thus, there were only a few sites
have also different fibrilogenesis behavior and conditions.11 remaining for trypsin to futher attack under these mild
Collagen was extracted in acidic solution containing pepsin conditions as a consideration of the denaturation of
which attacked only the non-triple helical domain of native collagen. Collagen was more resistant to proteinase
collagen. Gelatin was prepared under basic and high hydrolysis than the denatured products (gelatin and
temperature conditions, which hydrolysed the amide collagen hydrolysate) due to its specific triple helical
groups and decomposed the triple helices. Collagen structure.
hydrolysate is a denatured hydrolysis product with small
molecular weight and without the triple helical structure.
Therefore, the structures of gelatin and collagen
hydrolysate were disordered and their molecular weight
distributions were very wide, which made fibrillogenesis
impossible. In addition, self-assembly of collagen
monomers into fibrils involves hydrophobic and
electrostatic interactions between collagen chains. The
preparative process reduced the isoelectic points of gelatin
and collagen hydrolysate due to the increase of negative
charges, which also made it difficult for the molecules to
undergo interaction due to the repellency of negative
charges. Thus these products lost the ability of fibril Figure 6. Trypsin hydrolysis curves for collagen, gelatin and collagen
formation. hydrolysate at 30°C.

Physical properties of membranes

The thermo-physical properties of the collagen and the
gelatin membranes were examined by DSC. Fig. 7 shows
the thermograms of the collagen and the gelatin membrane.
The peak temperature of the collagen membrane was about
59°C similar to that of the gelatin membrane (62.5°C), but
its peak area (enthalpy) was markedly larger than that of
the gelatin membrane in the first scan. In the second run,
the endothermal peaks of the collagen and the gelatin
membrane both disappeared and only exhibited a weak step
baseline, showing the glass transition. The thermal
behaviors of collagen and gelatin are related to the moisture
Figure 4. Turbidity - time curves for collagen, gelatin and collagen content and the thermal history (preparative conditions).
hydrolysate incubated at 35°C.

26
The collagen membrane presented a larger peak area than There were obvious fibril networks with a rough
that of the gelatin membrane in the denaturation process membranous structure for the collagen membrane. In
due to the greater presence of collagen triple helices. In contrast, the gelatin membrane without fibril networks
addition, the DSC traces in the first scan corresponded to appeared only as a smooth structure on the surface.
the helix-coil transitions during the heating of the samples, In addition, the tensile strengths and elongations
while those in the second scan corresponded to the at the break for the collagen and the gelatin
characteristics of completely amorphous biopolymer due to membrane were 58 ± 5N/mm2, 7 ± 0.5% and 36 ± 4
the elimination of the triple helical structure. Therefore, N/mm2, 3 ± 0.5%, respectively. The differences of the
there were no endothermal peaks in the second scan. mechanical properties were caused by the different
structures of the membranes. There were more rigid native
triple helices and fibril networks in the collagen membrane
than in the gelatin membrane, which played important roles
in elevating the mechanical properties of the collagen
membrane.

Conclusions
It was found that there were many different
characteristics of collagen, gelatin and collagen
hydrolysate from the different preparative processes,
although they were all derived from bovine limed split
wastes. Only collagen possessed the triple helical
conformation. It exhibited many properties distinct from
B gelatin and collagen hydrolysate, such as high molecular
weight and narrow molecular distribution, basic isoelectric
point, triple helix to coil translation in the process of
denaturation, high resistance to protease, and ability of
fibril formation.
Gelatin and collagen hydrolysate were the denatured
products of native collagen and adopted a random coil
conformation, showing different properties, e.g. relative
lower molecular weights and wide molecular distributions,
lower isoelectric points, without conformation translation,
Figure 7. Thermograms of the collagen membrane A and the gelatin loss of ability of fibril formation, and being easily attacked
membrane B determined by DSC.
by proteinase. In addition, the collagen membrane had a
larger endothermal peak area, a special network structure
and better physical/ mechanical properties compared with
those of the gelatin 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. Therefore, the extraction of collagen from
bovine limed split wastes is a high value route for the
utilization of leather wastes.

Acknowledgement
This work was supported financially by the National
Natural Science Foundation of China (No. 20576073).
(Received September 2005)

References
1. Lee, C. H., Singla, A. and Lee, Y., Biomedical applications of
collagen. Int. J. Pharm., 2001, 221, 1-22.
2. Friess, W., Collagen - biomaterial for drug delivery. Eur. J. Pharm.
Biopharm., 1998, 45, 113-136.
3. Pachence, J. M., Collagen-Based Devices for Soft Tissue Repair.
J. Biomed. Res. (Applied Biomaterials), 1996, 33, 35-40.
4. Holmdahl, R., Bockermann, R., Backlund, J. et al., The molecular
pathogenesis of collagen-induced arthritis in mice - a model for
rheumatoid arthritis. Ageing Research Reviews, 2002, 1, 135-147.
5. Tamby, M. C., Chanseaud, Y., Guillevin, L. et al., New insights into
Figure 8. Surface morphologies of the collagen membrane A and the the pathogenesis of systemic sclerosis. Autoimmunity Reviews, 2003,
gelatin membrane B observed by SEM. Bars 5 µm. 2, 152-157.
6. Einersona, N. J., Stevensa, K. R. and Kao, W. J., Synthesis and
The surface morphologies of the collagen and the gelatin physicochemical analysis of gelatin-based hydrogels for drug carrier
membrane were greatly different from each other (Fig. 8). matrices. Biomaterials , 2002, 24, 509-523.

27
7. Langmaier, F., Mladek, M., Kolomaznik, K. et al., Isolation of elastin 15. Adler-Nissen, J., Determination of the degree of hydrolysis of food
and collagen polypeptides from long cattle tendons as raw material protein hydrolysates by trinitrobenzenesulfonic acid. J. Argric. Food
for the cosmetic industry. Int. J. Cosmet. Sci., 2002, 24, 273-279. Chem., 1979, 27, 1256-1262.
8. Langmaier, F., Mladek, M., Kolomaznik, K. et al., Collagenous 16. Linsenmayer, T. F., Collagen. In: Cell Biology of extracellar matrix,
hydrolysates sources of proteins. Int. J. Cosmet. Sci., 2001, 23, 193-199. Hay, E.D., ed., 2, 8-9, Plenum, New York, 1991.
9. Morimura, S., Nagata,, H., Uemura, Y. et al., Development of an 17. Engel, J., Folding and unfolding of collagen triple helices. Advances
effective process for utilization of collagen from livestock and fish in Meat Research, 1987, 4, 145-161.
waste. Proc. Biochem., 2002, 37,1403-1412 . 18. Gomez-Guillen, M. C., Turnay, J., Fernandez-Diaz, M. D. et al.,
10. Li, G. Y., Fukunaga, S., Takenouchi, K. et al., Physicochemical Structural and physical properties of gelatin extracted from different
properties of collagen isolated from calf limed splits. J. Amer. Leather marine species: a comparative study. Food Hydrocolloids, 2002, 16,
Chem. Ass., 2003, 98, 224-229. 25-34.
11. Li, G. Y., Fukunaga, S., Takenouchi, K. et al., Physioligical and cell 19. Hichman, D., Simes, T. J., Miles, C. A. et al., Isinglass/collagen:
biological properties in vitro of collagen isolated from calf limed denaturation and functionality. J. Biotechnol., 2000, 79, 245-257.
splits. J. Soc. Leather Technol. Chem., 2003, 88, 66-71. 20. Ogawa, M., Portier, R. J., Moody, M. W. et al., Biochemical
12. Li, Y. C., Zhu, D. Y., Jin, L. Q. et al., Preparation and analysis of properties of bone and scale collagens isolated from the subtropical
collagen polypeptide from hides by enzymes. J. Soc. Leather Technol. fish black drum (Pogomia cromis) and sheepsheas seabream
Chem., 2005, 89, 103-106. (Archosargus probatocephalus). Food Chem., 2004, 88, 495-501.
13. Bergman, I. and Loxley, R., Two improved and simplified methods 21. Gelman, R. A., Williams, B. R. and Piez, K. A., Collagen fibril
for the spectrophotometric determination of hydroxyproline. Anal. formation. J. Biol. Chem., 1979, 254, 180-189.
Chem., 1965, 35:1961-1965. 22. Birk, D. E. and Silver, F. H., Collagen fibrillogenesis in
14. Gornall, A. G., Bardawill, C. J. and David, M. M., Determination of
vitro:comparison of type I, II and III. Arch. Biochem. Biophys., 1984,
Serum proteins by means of the Biuret reaction. J. Biol. Chem., 1949,
235, 178-185.
177, 751-766.

28