AN ECO-FRIENDLY TANNING SYSTEM FOR LEATHER MANUFACTURE

M.Nirenjana*, T.N. Archana*, Anubhav Malhotra*, Swarna V Kanth#, B Madhan#, J. Raghava

Rao#, Balachandran Unni Nair#, S. Sadulla#, T Ramasami#
* - Department of Leather Technology, Allagappa College of Technology, Anna University, Chennai - 25
#-
Central Leather Research institute, Adyar, Chennai-20
E-mail: nirenltit21@yahoo.co.in
Key words: Alginic acid, crosslinking, collagen

Abstract
Polysaccharides are abundant in nature and are profuse, non-toxic, biodegradable natural polymers
possessing high degree of functionalization. However to fulfil the demand for tailored application profiles,
native biopolymers are often modified. Alginic acid is one such polysaccharide that has been modified and
widely used in food ,phamaceutical, and medical industries. Alginic acid has been modified to dialdehyde
alginic acid and used as a tanning agent for the stabilization of collagen. Shrinkage temperature
measurements, collagenase activity, scanning electron microscopy, infrared spectroscopy and differential
scanning colorimetry techniques have been investigated.

1.Introduction most commercial tanning practices, as they result

Leather is one of the unique material which
posses characteristics like breathability,
viscoelasticity which arises from the naturally
woven fibrous skin matrix. These characteristics
of leather is well suited in several applications.
In leather processing the natural material skin is
being stabilized against degradation. The unit
process involved in the stabilization of skin into
leather is called tanning. There are various
chemical agents used for tanning. Currently the
tanning industry is dominated by the use of
chromium salts.
Hides and skins predominantly contain collagen,
the most abundant protein in animals. There are
19 different types of collagen [1], of which type I
collagen is the main component of skin, tendon,
bone and other tissues. This protein is present as
chains wound in triple helical form [2], which
are organized into fibrils of great strength and
stability [3]. The structure of collagen is
stabilized by inter- and intra-chain hydrogen
bonds [4] and by water mediated hydrogen
bonds [5,6].
In olden days collagen stabilization was brought
about by plant polyphenols, through multiple
hydrogen bonds [7,8]. Some of the oligomers of
chromium were known to interact with collagen
[9,10], bringing about irreversible matrix
changes, thus imparting higher stability [11]. The
networks formed between collagen and tanning
agents are covalent bonds, complex bonds
Coulombic forces, H-bonds and hydrophobic
interactions depending on the tanning system.
Chrome tanning is a primary tanning system in
in leathers for different end uses with good
physical and organoleptic properties. In recent
times because of the ecological concerns the
leather industry is looking for an alternative
tanning system. It faces serious disadvantages
due to the constraint of its discharge norms of
2ppm[12]. The currently practiced chrome
tanning procedures results in only 60-65% of the
chromium being offered to the leather and a
substantial amount of chrome is discharged into
the effluent [13]. As of now there is no safe
method of disposal method available for the
chrome effluent or the chrome tanned leather
products, which is a major concern. Hence
researchers throughout the world are looking for
alternative tanning systems, which would result
in leathers with good hydrothermal stability,
strength characteristics, organoleptic properties
and very importantly eco-friendly. A cleaner and
sustainable tanning agent/system that can result
in leather with good functional properties is the
current need of the tanning industry.
Biomaterials, biodegradable polymers are
important biomaterials, which are abundant in
occurrence possessing a high degree of
functionalisation. Alginic acid is one such
material, which has already established its
applications in food, pharmaceutical, and
medical industries (Kennedy, Griffith, Atkins,
1984; Mc Neely and Pettitt, 1973). Molecular
design of these materials plays an important role
in determining their suitability in such
applications. Alginic acid is a linear, binary
copolymer of 1,4-linked -L-guluronic acid and
-D-mannuronic acid [14], usually isolated from
a brown algae but is also present in some species
of bacteria [15,16]. Along the polymer chain the
two residues are arranged in an irregular
blockwise pattern [17-19]. There are three types
of blocks: homopolymeric sequence of
mannurronate (MM blocks) and guluronate
residues (GG blocks) and a region where the two
residues alternate (MG blocks). The relative
proportions of these block types are affected by
several factors [20-22] such as the botanical
source, plant maturity, collection site and
seasonal variations.
In solution, alginates behave like flexible coils
[24]. However on interaction with metal ions,
they form ordered structure as evidenced by their
X-ray diffraction patterns [25]. Attempts have
been made to covalently crosslink sodium
alginate with gelatin and athylenediamine using
water-soluble carbodiimide. Recently it is
reported that although higher molecular weight
alginate is non-biodegradable, its dialdehyde
derivative is biodegradable [26]. Alginic acid is
the only polysaccharide, which naturally
contains carboxyl groups in each constituent
residue, and possesses various abilities for
functional materials. To fulfill the demand for
tailored end use applications the native
biopolymers are often need to be modified.
Tanning with aldehydes is known to mankind for
a very long time and aldehydes such as
formaldehyde, glyoxal and glutaraldehyde have
been used for stabilization of skin[27-30]. In the
present study alginic acid is converted to
dialdehyde alginic acid and the crosslinking
ability of this modified biopolymer is studied.
Structural formula of alginic acid [23]
Dialdehyde alginic acid has been proved to be
biodegradable and toxologically accepted
chemical [26]. Hence dialdehyde alginic acid has
the dual advantage of being a tanning material
and the leather products after usage will not have
disposal problems, as they can be made
biodegradable under certain conditions. In this
context, the dialdehyde starch has immense
potential to be used as a tanning agent in leather
processing as it is abundant and one of the
cheapest raw material with large number of
aldehyde groups in a single molecule.
This paper presents the studies on the feasibility
of using dialdehyde alginic acid as a tanning
agent for stabilization of collagen.
2. Materials and Methods
2.1. Materials
Type IA collagenase was obtained from SIGMA
ALDRICH, U.S. The Alginic acid and other
chemicals used for the process were obtained
from S.D fine chemicals.
2.2. Methods
2.2.1.Periodate oxidation of alginic acid
20 g Alginic acid and calculated amount of
sodim metaperiodate were dispersed in 100ml of
distilled water and stirred in the dark
magnetically at 25C for 6 hrs to obtain
dialdehyde Alginic acid of different degree of
oxidation. The degree of oxidation was followed
by determining the concentration of periodate
left unconsumed by iodometry after 6 hrs [31],
and the degree of oxidation was found to be
98%.
2.2.2 Fourier transform infrared (FT-IR)
spectra
FT-IR spectra of Alginic acid and dialdehyde
Alginic acid were acquired on a Impact 400 FT-
IR instrument using a potassium bromide (KBr)
disc containing 1% finely ground sample.
2.2.3 Differential scanning calorimetry(DSC)
studies
Thermal properties of Alginic acid and
dialdehyde Alginic acid were analyzed using
Dupont 2910 Differential Scanning Colorimeter.
All measurements in the instrument were
conducted under a nitrogen atmosphere. The
samples were scanned at 5C/min . The
temperature at the midpoint of the change in
slope of the DSC change was taken as the
structural change in Alginic acid to dialdehyde
Alginic acid with respect to thermal heat.
2.2.4 Pretreatment of collagen
Collagen matrix of goat skin has been used for
crosslinking experiments. The raw skin has been
converted to acidified collagen (pickled pelt) at
pH 3.0 after adopting a sequence of
operations/processes that is conventionally
carried out in the pretanning operation during
leather manufacture [32].
2.2.5 Crosslinking of collagen with dialdehyde
alginic acid
Ten gram portions of acidified collagen matrix
have been brought to a pH of 8 by treating the
collagen with 8% sodium chloride and 80%
water in a bottle shaker at 8 rpm for 30 minutes
at room temperature, 2% sodium formate as 10%
solution has been added and the shaker has been
run for 30 minutes and then 3% sodium
bicarbonate as 10% solution was added in 3
feeds of 10 minutes in the running shaker.
Dialdehyde alginic acid at concentration of 10%
has been used for crosslinking treatment to the
marked bottle shaker containing collagen matrix
at pH 8 and was run for 24 hours at 300C. At the
end of the crosslinking period, the waste solution
was collected and the uptake of dialdehyde
alginic acid was calculated. The crosslinked
collagen samples were thoroughly washed and
aged for 24 hours. Hydrothermal stability and
Scanning Electron Microscopy experiments were
carried out. The samples were further taken for
collagenase activity.
2.2.6 Determination of hydrothermal stability
The temperature at which the collagenous fiber
shrinks to one third of its original length is noted
as the shrinkage temperature of the fibre. The
measurement of the shrinkage temperature
determines the thermal stability of collagenous
matrices. The shrinkage temperature is also
termed as hydrothermal stability as the
measurement is carried out in the presence of
water. The hydrothermal stability of native and
dialdehyde alginic acid treated collagen was
measured using a Theis shrinkage meter by the
standard method [37].
2.2.7 Collagenase Hydrolysis of the stabilized
collagen
The enzymatic degradation of collagen adjusted
to pH 7.0(native) and collagen stabilized with
dialdehyde alginic acid by collagenase was
analyzed by estimating the amount of
hydroxyproline released in the solution after
hydrolysis. The native and stabilized collagen
was treated with bacterial collagenase (Type IA)
from Clostridium histolyticum. Collagenase
treatment was carried out in 0.04 M CaCl2
solution buffered at pH 7.2 with 0.05 M tris HCl.
The collagen: enzyme ratio was maintained at
40:1. The samples were incubated at a
temperature of 37oC. Samples were collected at
different time interval upto 96 hrs and stored in
freezer. The cleavage of native and stabilized
collagen was monitored by the release of soluble
form of hydroxyproline from insoluble collagen
[38]. Aliquots of 750 l of supernatant of
samples collected at different time interval were
withdrawn after centrifuging at 10,000 rpm for
10 min. The collagenase hydrolysate was
hydrolyzed in sealed hydrolysis tubes with 6 N
HCl for 16 hrs. The hydrolysates were
evaporated to dryness in a porcelain dish over a
water bath to remove excess acid. The residue
free of acid was made up to a known volume and
the percentage (%) of hydroxyproline was
determined using the method of Woessner [39].
This method of determining hydroxyproline
involves the oxidation of hydroxyproline to
pyrrole-2-carboxylic acid, which complexes with
p-dimethylaminobenzaldehyde exhibiting
maximum absorbance at 557 nm.
Hydroxyproline = (concentration in g/weight of
sample) x dilution factor. Soluble collagen =
Hydroxyproline 7.4. % Collagen degradation =
(Weight of soluble collagen/Initial collagenous
weight) x 100. (Initial collagenous weight has
been adjusted after taking into account the
amount of Dialdehyde alginic acid fixed)
2.2.8 Scanning Electron microscopic (SEM)
studies
Dialdehyde alginic acid crosslinked sample
were cut from the official sampling position [40]
after the treatment process. The sample were
fixed by soaking them with buffered formalin for
18 h. Samples were then dehydrated gradually
using acetone and methanol as per standard
procedure [41]. Samples were then cut into
specimens with uniform thickness. The
specimens were then coated with gold using a
JEOL JFC-1100E ion-sputtering device. A JEOL
JSM-5300 scanning electron microscope was
used for the analysis. The micrographs for the
cross section were obtained by operating the
SEM at an accelerating voltage of 20 kV with
different lower and higher magnification levels.
3. Results & Discussions
There are two hydroxyls attached to carbons at
2,3 position and one carboxyl in 6 position in
each dehydrated glucose unit. There are many
reactive groups available in the alginic acid
molecule that can react with different chemical
reagents. Periodate oxidation of alginic acid
selectively cleaves the C2-C3 bond between the
two adjacent hydroxyl groups and the 1,2--diol
group in glucose is converted into a dialdehyde.
The advantage of periodic acid lies in the
specificity of its oxidation. It forms aldehydes
within the polysaccharide molecule but it does acid reduces, which again is an indication of the
not continue the oxidation of the polymers to low oxidation of alginic acid to alginic dialdehyde.
molecular weight water-soluble forms. The
extent of oxidation can be readily controlled, and
a complete range of aldehyde derivatives of a
starch is made available as the oxidation level
varies between 0 and 100%. At 100% oxidation
hydroxyl groups of each glucose residue in the
Alginic acid is converted to its corresponding
dialdehyde structure; at 50% oxidation half of
the glucose residues of the alginic acid are
converted.

3.1 Structural properties of alginic acid and

dialdehyde alginic acid
The FT-IR spectrum of alginic acidis shown in
Fig. 1a. In the fingerprint region of the spectrum
of alginic acid three characteristic peaks appear
between 937 and 1111 cm-1; these peaks are
attributed to C-O bond stretching. The band at
948 cm-1 is assigned to 14 linkage. The peak
at 1038 cm-1 is characteristic of the
anhydroglucose ring O-C stretch. The peaks at
808 cm-1 and 787 cm-1 can be assigned to
mannuronic and guluronic acids. The band at
815 cm-1 is due to the polyguluronate structure
and the band at 880 presents the characteristic -
mannuronic residues. Another characteristic peak
occurs at 1630 cm-1, which could be a feature of
tightly bound water present in starch. The
spectrum also displays absorption peak at 1743
cm-1corresponding to the stretching band of the
free carboxyl anions. An extremely broad band
due to the hydrogen bonded hydroxyl group (O-
H) appears at 3403 cm-1 that is attributed to the
complex vibrational stretches associated with
free inter and intra molecular bound hydroxyl
groups which make up the gross structure of
Alginic acid. The sharp band at 2926 cm-1 is
characteristic of the C-H stretches associated
with the ring methane hydrogen atoms.
Comparison of the spectrum of alginic acid with
alginic acid dialdehyde (Fig. 1b) clearly indicates
the deviations around 1600 1700 cm1, which
could be due to the introduction of additional
carbonyl stretching (C=O) of aldehydic
functional group. The occurrence of sharp band
at 2926 cm-1 in the spectrum is associated with
the C-H stretching with the dialdehyde alginic
acid substituents. The strong band at 3404 cm-1
(hydroxyl groups) of dialdehyde alginic acidis of
decreased intensity as the oxidation of alginic
acid to dialdehyde alginic acid reduces the
number of hydroxyl groups as compare to alginic
b
Fig. 1. FT-IT of a) Alginic acid and b) Alginic
acid dialdehyde
3.2 Thermal properties of alginic acid and
dialdehyde alginic acid
The thermal stability of the alginic acid and
alginic acid dialdehyde were studied using DSC.
The exothermal and the endothermal changes of
Alginic acid and oxidized alginic acid at the
increase of constant temperature of 5oC were
measured by DSC. Fig. 2a shows the results of
native alginic acid. The peak at 124.18 C is due
to the thermal transition of Alginic acid. The
DSC of the dialdehyde alginic acid shown in Fig.
2b indicates a peak at 135.53C, which is due to
the thermal transition of introduction of
dialdehyde functionality. The DSC curve of
dialdehyde alginic acid and Alginic acid indicate
different thermal degradation characteristics
between the native and the oxidized Alginic acid.
The dialdehyde alginic acid appears to be more
stable since the inception of degradation is at
higher temperature. This greater thermal
stability of the dialdehyde alginic acid molecule
may be due to the introduction of aldehyde
functionality by lowering the amount of
hydroxyl groups after oxidation of Alginic acid
molecules. Thus the thermal stability is increased
as a large proportion of aldehyde functionality
prevails in the large Alginic acid molecule.
a collagen. Hence the treatment of dialdehyde
b acid treated skin are given in Table 1. The
Fig. 2. DSC of a) Alginic acid and b) Alginic
acid dialdehyde
3.3 Interaction of collagen with dialdehyde
alginic acid
The aldehydes are known to interact with the
amino groups of the protein [46]. The amino
groups are generally protonated at lower pH and
hence aldehyde interaction with proteins is
preferable at pHs above iso electric point (IEP)
of the protein. The IEP of acid pretreated (alkali
followed by acid) collagen is 4.7 and above this
pH collagen will remain negatively charged and
amino sites are available for the interaction of
aldehydes. Earlier studies indicate pH 8 is
conducive for the interaction of aldehydes with
collagen [43]. Also, periodate oxy starch, like
periodate oxycellulose, has been shown [44,45]
to be extremely sensitive to degradation by
alkali, by virtue of the presence of dialdehyde
groups. Hydrolysis may take place either at the
1,4-bonds or at the C2-O bonds, giving
depolymerized and decomposition products,
which may take part in the reaction in the
alkaline range [46]. Hence reaction pH 8 can
also favor such hydrolysis process which can
result in the reduction of molecular weight and
size and can favor the penetration of dialdehyde
alginic acid into the collagen matrix. It has also
been established that a maximum offer of 10% of
aldehydes is sufficient for the stabilization of
alginic acid with collagen had been carried out at
10% offer, pH 8 for a period of 24 hrs. The
aldehydic functionality in the dialdehyde alginic
acid can covalently bind with amino groups of
the collagen and the hydroxyl groups of the
dialdehyde alginic acid can involve in hydrogen
bonding interaction with the functional groups
available in collagen matrix.
3.4 Hydrothermal stability of the stabilized
collagen
The shrinkage temperatures measured for the
acidified(pickled) skin and the dialdehyde alginic
shrinkage temperature for the acidified collagen
fibres has been found to be 560C. Shrinkage
temperature is the temperature at which the skin
(collagen) matrix shrinks to one third of its
original length. The temperature at which the
shrinkage of collagen matrix occurs is highly
specific and occurs due to destabilization of the
protein. It is also termed as hydrothermal
stability. The hydrothermal stability of native
skin is around 600C, which is higher compared to
the acidified skin, because acidification alters the
IEP of the protein and the electrostatic
interaction due to salt bridges within the collagen
matrix is comparatively stronger at IEP and
hence the matrix can offer higher resistance to
shrinking. The long-range ionic interactions and
other non-covalent interactions are the cause for
ordering of the collagen molecules the fibre
matrix. Hence the basic forces, which are
responsible for the high denaturation temperature
of this protein, could be attributed to such long-
range interactions. Any further increase in the
ordering of the collagen will also increase the
stability of the matrix against hydrothermal
stress. Increase in resistance against
hydrothermal stress is one of the important
aspects in the stabilization of collagen matrix. It
is found that treatment of collagen with
dialdehyde alginic acid has resulted in increase
of the hydrothermal stability of the collage
matrix considerably. As seen from the Table 1,
the hydrothermal stability of the collagen matrix
treated with dialdehyde alginic acid is found to
be 800C, which is 240C more than is observed for
acidified collagen. The interaction of dialdehyde
alginic acid with collagen has further increased
the long range ordering of collagen, which in
turn is responsible for the increase in the
hydrothermal stability of the collagen matrix.

Table 1: Hydrothermal stability of acidified and
dialdehyde alginic acid treated goat skin
Process Shrinkage
temperature(C)
Acidified skin 56
Dialdehyde alginic 80
acid treated skin
3.5 Enzymatic Stability of dialdehyde alginic
acid treated Collagen:
Collagen is resistant to all enzymes except
collagenase. Triple helical structure of the
collagen renders stability against degradation to
several proteinases other than collagenases [47].
The stability of the dialdehyde alginic acid
treated collagen matrix against enzymatic
degradation has been studied by analyzing the
rate of hydrolysis of collagen on treatment with
bacterial collagenase. Bacterial collagenase
preferentially cleaves X-Gly (X is most
frequently a neutral amino acid) bond of the -
Gly-Pro-X-Gly-Pro-X- sequence in the non polar
regions of the collagen molecule [48]. Bacterial
collagenases from Clostridium histolyticum
cleave collagen at multiple sites [49], whereas,
mammalian collagenases from human-fibroblast
cleave collagen only at a single site (leu-Ileu)
breaking it into a 3/4th and 1/4th fragment [50].
Hence a treated collagen matrix exhibiting
stability against the activity of bacterial
collagenase can exhibit high resistance to
degradation by any class of enzymes. Collagen
matrix treated with dialdehyde alginic acid has
been subjected to hydrolysis by collagenase and
the resulting hydrolysates have been analyzed for
hydroxyproline released into solution. The
hydroxyproline content is a direct measure of the
amount of collagen solubilised. The amount of
hydroxyproline released in the supernatant for
the native and dialdehyde alginic acid treated
collagen matrix samples subjected to collagenase
treatment for a period of 72 hrs is given in Table
2. From the table it is observed that skin treated
with alginic acid dialdehyde offers resistance to
degradation by collagenase. The stabilization
effect of the collagen fibers treated with
dialdehyde alginic acid, where marked decrease
in the degradation is observed as against the
untreated collagenous matrix. Native collagen
matrix has undergone extensive hydrolysis with
the treatment of collagenase. The native collagen
exhibited 98% hydrolysis of collagen in the
above time period whereas collagen matrix
treated with alginic acid dialdehyde exhibited
only 40% degradation. The interaction of
dialdehyde alginic acid with collagen has
brought in changes in the active sites of collagen
because of which collagenase is not able to
express it activity of breaking down the
collagenous matrix.
Table2: % Collagen degradation (based on
release of hydroxy proline) from native and
alginic acid dialdehyde treated collagen after 72
hrs of collagenase hydrolysis
% Collagen
Degradation
Native Collagen 98
Collagen treated with 40
alginic acid dialdehyde
3.6 Scanning Electron microscopic (SEM)
analysis of the native and stabilized collagen:
Evaluation of opening up of fiber bundles by
implicit approach was done using Scanning
Electron Microscope. Scanning electron
micrographs of collagen matrix stabilized using
the polyaldehyde showing the cross section at a
magnification of x 600 is given in Fig. 3. The
SEM micrograph of dialdehyde alginic acid
treated collagen fibres as seen from Fig. 3
appears to be coated. The coating could be due to
the presence of dialdehyde alginic acid around
the collageanous fibres. Normally sample from
acidified collagen displays a very fine opening
up of fiber bundles because in pretreatment of
collagen with alkali followed by acid treatment,
fiber opening is developed as an osmotic-driven
splitting of fiber bundles and the hydrostatic
pressure built inside the matrix keeps the fibers
apart from each other.
Fig. 3. Scanning electron micrograph of the cross
section (600X) alginic acid dialdehyde treated
collagen fibres

4. Conclusion
The presented work clearly indicates that the
biopolymer alginic acid in oxidized form as
dialdehyde alginic acid can be used in the
stabilization of another biopolymer collagen. The
treatment of collagen matrix with dialdehyde
alginic acid has improved the thermal and
enzymatic stability of collagen. Substantial
increase in hydrothermal stability is observed on
treatment with dialdehyde alginic acid . The18.
dialdehyde alginic acid treatment renders
collagenous matrix resistant to enzymatic
hydrolysis. The stability against heat and
enzymatic hydrolysis are the main aspects to be20.
considered for the permanent stabilization of
collagen which is achieved in the process called
tanning. The dialdehyde alginic acid, a modified
polymer of alginic acid appears to be an
effective alternative material that can be used in
the process of stabilization of collagen.
24.
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