FOOD AND BIOPRODUCTS PROCESSING 9 9 ( 2 0 1 6 ) 2 4 4 -2 5 1

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Food and Bioproducts Processing IChemE CHEMICAL

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Extraction, purification and characterisation of CrossMark

dermatan sulphate from bovine collagen waste
liquor
Simone A. Osborne0’*, Robyn A. Daniela, Wei Chena, Peter Stockwell0,
Kerri Tyrrell0, Kirthi Desilv a b, Robert B. Seymour 0
a CSIRO, Agriculture, Queensland Bioscience Precinct, 306 Carmody Road, St Lucia, Queensland 4067, Australia
b CSIRO, Food and Nutrition, 671 Sneydes Road, Werribee, Victoria 3030, Australia

ARTICLE INFO ABSTRACT

Article history: The aim of this study was to extract, purify and characterise glycosaminoglycans present in
Received 29 March 2016 bovine collagen waste liquor. Processing of bovine hides supplies collagen and gelatine to
Received in revised form 17 May food, cosmetic and pharmaceutical industries generating abundant and unutilised waste
2016 streams that contain bioactive polysaccharides known as glycosaminoglycans. Some of
Accepted 18 May 2016 these molecules, like heparin and dermatan sulphate, have known anti-thrombotic and
Available online 24 May 2016 anti-coagulant activities and are prescribed therapeutics. The predominant glycosamino-
glycan in bovine collagen waste liquor, dermatan sulphate, was extracted and purified
Keywords: to greater than 93% w/w using only serial filtration and anion exchange chromatography.
anti-thrombotic Bioactivity was measured using an in vitro assay revealing anti-thrombotic activity equiva­
bovine hide lent to commercial dermatan sulphate sourced from porcine mucosa. Fluorophore-assisted
collagen waste liquor carbohydrate electrophoresis revealed a composition rich in the predominant mammalian
dermatan sulphate disaccharide, uronic acid-»N-acetyl-D-galactosamine-4-0-sulphate. Depolymerization and
processing waste fractionation enriched for low molecular weight fragments with significantly improved anti­
thrombotic activity. This study identifies an alternative source of anti-thrombotic dermatan
sulphate that can be easily extracted without proteolysis or solvent precipitation that can
be further processed into low molecular weight fractions with improved anti-thrombotic
activity.
© 2016 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

1. Introduction m anufacture of leather w here the degradation or removal

Hides, one of the m ost valuable by-products from anim als
(Jayathilakan et al., 2012), are used in the production of
food grade collagen and gelatine. Gelatine is usually derived
from collagen in bovine hide th a t is extracted industrially
using alkaline or acid treatm ents. As well as being rich
in collagen, bovine hide (or skin) contains glycosaminogly­
cans (GAGs) th a t are predom inantly derm atan sulphate (DS)
(Osborne et al., 2008). Bovine hides are also used in the
of GAGs from hide during processing helps to open the
fibrous structure of skin favourably affecting leather q u al­
ity. The liberation of GAGs, including DS, into processing
w aste stream s during hide preparation h as been used to in d i­
cate the effectiveness of processing and the potential quality
of the derived leather products (Choudhury et al., 2006).
Thus, w aste from the processing of bovine hide contains DS
th a t could be a significant new source of biologically active
DS.

Abbreviations: ADi-OS, UA—GalNAc; ADi-4S, UA^GalNAc4S03; ADi-6S, UA-»GalNAc6S03; ADi-UA2S, IdoA2S03-»GalNAc; ADi-diSB,
IdoA2S03->GalNAc4S03; ADi-diSD, IdoA2S03->GalNAc6S03; ADi-diSE, UA->GalNAc4,6S03; ADi-triS, IdoA2S03-*GalNAc4,6S03.
* Corresponding author.
E-mail address: Simone.Osborne@csiro.au (S.A. Osborne).
http://dx.doi.Org/10.1016/j.fbp.2016.05.008
0960-3085/© 2016 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
 FOOD AND BIOPRODUCTS PROCESSING
D erm atan sulphate, a sulphated polysaccharide belonging
to the sam e family of GAGs as heparin, has attracted signif­
icant attention in the p ast 20 years as an anti-throm botic
drug candidate (Kretz et al., 1996; Nenci, 2002). Unlike h ep ­
arin, the m ost widely used anti-throm botic drug, DS inhibits
throm bin associated w ith clots, acts non-system ically w ith
less side-effects by selectively inhibiting the action of throm ­
bin through heparin cofactor II (HCII) and is also reported to
have profibrinolytic effect (Lauricella et al., 2013). One of the
m ore recent applications of DS is in the preparation of m edi­
cal devices and artificial tissues (Raghava Rao and Nair, 2010).
This application, in addition to the anti-throm botic activ­
ity from DS, highlights the potential to add value to bovine
hide processing w aste through the recovery of DS. D erm atan
sulphate w ith anti-throm botic properties has already been
identified in a range of bovine tissues (Osborne et al., 2008),
m arine organism s (Higashi et al., 2015; Pavao et al., 1998; Volpi
and Maccari, 2009) and other m am m als (Halldorsdottir et al.,
2006; M aimone and Tollefsen, 1990; Ofosu et al., 1987; Osborne
et al., 2008).
Reagent grade DS is com mercially sourced from porcine
intestinal m ucosa th a t is com prised of m ultiple GAGs includ­
ing d erm atan sulphate, heparin, heparan sulphate and
chondroitin sulphate (Tufano et al., 2010). C urrent extraction
m ethods com prise proteolysis to disrupt th e tissue m atrix and
ion exchange and precipitation w ith organic solvents or salts
to isolate derm atan sulphate from the mix of native GAGs
(Osborne et al., 2008; Volpi, 1994). The purification of one type
of GAG from such a mix presents a level of difficulty th a t
m ay be reduced by a source less com plex in com position.
For example, GAG extracts from porcine intestinal m ucosa
are com prised of only 35% DS (Vittoria et al., 1988), w hereas
GAG extracts from bovine (Osborne et al., 2008; Pearson and
Gibson, 1982) and porcine skin are predom inantly DS w ith only
trace am ounts of other GAGs present. As such, w aste from the
processing of bovine hide th a t is predom inantly com prised
of DS represents a new source of DS th a t is readily available,
potentially anti-throm botic and possibly easier to extract.
Other glycosaminoglycans are sourced from different
processing w aste stream s; chondroitin sulphate has been
retrieved from different m arine processing w aste (Grimes
and Lateef, 2013; Vazquez et al., 2013; Zhang et al., 2014),
hyaluronic acid has been extracted and purified from fish eye­
ball (Murado et al., 2012) and heparin is com m only sourced
from porcine m ucosa and bovine lung. In the utilisation
of bioactive glycosaminoglycans from bovine collagen w aste
liquor, a process for th e extraction and purification of DS is
described. Thus, a m ethod involving serial filtration and anion
exchange chrom atography is presented conferring bioactive
DS w ith anti-throm botic activity. Furtherm ore, a m ethod to
depolymerize and separate DS into lower m olecular w eight
fractions w ith significantly improved anti-throm botic activity
is also described and is relevant for further developm ent of DS
as a therapeutic.
2. Materials and methods
2.1. Materials
U nsaturated chondro/derm ato/hyaluro-disaccharides (ADi-
0S, ADi-4S, ADi-6S, ADi-UA2S, ADi-diSB, ADi-diSD, ADi-diSE
and ADi-triS) and chondroitin ACII lyase (Arthrobacter
aurescens) were purchased from Seikagaku Corporation
99 (2OI 6) 2 4 4 -2 5 1 245
(Tokyo City, Japan). Chondroitin sulphate A (bovine trachea),
chondroitin sulphate B (porcine in testin al mucosa), chon­
droitin sulphate C (shark cartilage), heparin (porcine intestinal
m ucosa, >180 USP heparin units mg~a), hep aran sulphate
(bovine kidney), dextran sulphates (8kDa and lOkDa),
2-am inoacridinone (AMAC), sodium cyanoborohydride, 1,3-
diam inopropane, toluidine blue O, 1,9-dim ethyl-m ethylene
blue, acetic acid (glacial), dim ethyl sulphoxide and chon­
droitin ABC lyase (Proteus vulgaris) were from Sigma-Aldrich
(St Louis, Missouri). Q Sepharose Big Beads and agarose NA
were from Amersham/GE H ealthcare Life Sciences (Little Chal-
font, England). Cetyl trim ethyl am m onium brom ide (CTAB)
was from Ajax Chemicals (Sydney, Australia). Heparin cofactor
II (human) and throm bin (alpha, hum an) were from USA Bio­
logical (Swampscott, W eightachusetts). Chromzym TH was
from Roche Diagnostics (Indianapolis, Indiana). N,N,N’,N’-
tetra-m ethyl-ethylenediam ine, am m onium persulphate and
40% acrylam ide/bis solution 37.5:1 (2.6% C) were from Bio-Rad
(Hercules, California). The Blyscan™ Sulphated Glycosamino-
glycan Assay was from Biocolor Ltd (Newtonabbey, N orthern
Ireland). All other reagents were of analytical grade and were
obtained from local suppliers.
2.2. Preparation of native bouine DS (B-DS)
A w aste stream from the alkaline extraction of food grade col­
lagen from bovine hides w as collected and stored a t 4 °C for no
longer th a n 48 h. The GAG concentration w as estim ated in the
w aste stream using the Blyscan™ Sulphated Glycosaminogly-
can Assay (according to the m anufacturer’s instructions). The
w aste liquid w as th en h eated and held a t 53 °C to allow precip­
itation of solids before being clarified using serial filtration and
diafiltration techniques until all sedim ent was removed. The
filtrate pH was adjusted to 7.0 using concentrated HCl before
Q. Sepharose (Big Beads) anion exchange chrom atography was
u ndertaken (at pH 7.0) using 2 M NaCl to elute the bound GAGs.
The eluant w as de-salted using ultrafiltration prior to freeze
drying of the final m aterial. The GAG concentration w as also
estim ated in the freeze-dried m aterial using th e Blyscan™
Sulphated Glycosaminoglycan Assay.
2.3. Visualization of B-DS using agarose gel
electrophoresis
To identify the glycosaminoglycan species present in the
w aste stream , 30 (rg GAG m aterial w as digested w ith 10
mU chondroitinase ACII/ABC lyase in 50 mM Tris-HCl pH
8.0/60 mM sodium acetate/0.02% (w/v) BSA (final volum e 45 |xl)
at 37 °C for 16-18 h. The GAGs were th en visualized using
diam inopropane agarose gel electrophoresis, w ith o u t and
w ith lyase treatm ent, as previously described w ith some
modifications (Osborne et al., 2008). Briefly, 10 |xg chondroitin
sulphate A, B, and C com mercial standards (Sigma) and 15 |xl
(equivalent to 10 |xg) ABC/ACII lyase digested m aterial were
applied to a 0.5% agarose/50 mM diam inopropane gel (adhered
to GelBond Film, Cambrex Bio Science Rockland Inc, Rock­
land, Maine) in 50 mM diam inopropane for 90 m in at 80 V. The
sam ples were fixed for 16-18 h in 0.1% (w/v) cetyl trim ethyl
am m onium brom ide before the agarose gel w as dried, stained
w ith 0.1% toluidine blue/50% (v/v) ethanol/1% (v/v) acetic
acid solution, destained using 50% (v/v) ethanol/1% (v/v)
acetic acid, and scanned on a desktop scanner (hp scanjet
3670).
246 FOOD AND BIOPRODUCTS PROCESSING 9 9 ( 2 0 I 6 )
2.4. Preparation of low molecular w eight DS (L-DS)
The freeze-dried B-DS w as depolym erized as described (Volpi
et al., 1992), w ith m odifications. This controlled m ethod of
DS depolym erization is based on the generation of hydroxyl
radicals by m eans o f m etal ions and hydrogen peroxide.
Briefly, 1 g DS (as determ ined using the Blyscan™ Sulphated
G lycosam inoglycan Assay) w as dissolved in 10 m l sterile
deionized w ater w ith 40 m g copper acetate m onohydrate
added w h ilst stirring at 55 °C. The pH w as adjusted to 7.5 and
m aintained throughout the reaction period using 1M NaOH.
Depolym erization w as facilitated by the addition of 9% (v/v)
H2 O2 delivered at a rate o f 2 ml h -1 . Final incubation tim e w as
50 min.
2.5. Separation of L-DS using anion exchange
chromatography
The L-DS material w as fractionated using anion exchange
chromatography w ith Q Sepharose Big Beads. One gram of
depolym erized m aterial w as fractionated at pH 7.0 using
a linear 0-1.5 M NaCl gradient in 50 mM sodium citrate.
Fractions were collected every 5 m in, for a total 150 min,
using a 2 m l m in-1 flow rate. Fractions containing DS were
confirmed by interaction w ith 1,9-dim ethylm ethylene blue
dye (40 mM glycine/40m M NaCl, 10 mM HCl/60|xM 1,9-
dim ethylm ethylene blue) m easured at 525 nm . The GAG
containing fractions were de-salted using Chem icon Tlibe-O-
DIALYZERs w ith a 1 kDa m olecular w eight cut-off.
2.6. Estimation of molecular w eight by HPLC
The average m olecular w eigh t o f the GAGs w as estim ated
using HPLC. A TSK-GEL G4000SW (7.5 m m x 30cm ) and a
G2000SW (7.5 m m x 30 cm) were used in series. The HPLC
system from Shim adzu con sisted of a Model SIL-lOAi auto
injector, a SCL-10A vp System Controller and a SPD-M10A
diode array detector. The m obile phase w as com posed of
125 mM Na 2 SC>4 and 2mM NaH 2 P0 4 adjusted to pH 6.0 w ith
0.1 M NaOH. Flow rate w as 1.0 ml m in -1 . Glycosam inoglycan
elution w as detected at 206 nm and confirmed via fraction
interaction w ith dim ethylm ethylene blue dye. Then, 200 to
400 (xg of each sam ple w as solubilised in w ater and the m o lec­
ular w eight calculated as an average o f three determ inations
using a standard curve plotted w ith com m ercial chondroitin
sulphate A, chondroitin sulphate B (DS), chondroitin sulphate
C, and dextran sulphates (with an average m olecular w eight
of 10 kDa and 8 kDa).
2.7. Anti-thrombotic assay: heparin cofactor
II-mediated thrombin inhibition by DS preparations
Anti-throm botic activity w as m easured through heparin
cofactor II (HCII) in a 96-w ell plate kinetic assay as previously
described (Dupouy et al., 1988) w ith m odifications. Briefly,
3 (jil 680 nM HCII, 21 jxl 0.02 M Tris-HCl pH 7.4/0.15 M NaCl/PEG
l m g m l-1 and 3(xl 0.125-0.25m g m l-1 o f all DS sam ples (DS
concentration determ ined using the Blyscan™ Sulphated Gly­
cosam inoglycan assay) or 3 fxl 0.15625-2.5 (xg m l-1 heparin
standard were m ixed and incubated at room temperature
for 2 m in before 3 |xl o f 150 nM throm bin w as added. This
solution w as gently m ixed for 1 m in prior to the addition of
150 (xl o f 100 |xM ChromozymTH. The assay w as incubated
at 37 °C for 40 m in w ith the absorbance m easured at 405 nm
2 4 4 -2 5 1
(Spectra m ax PLUS 384) at 2 m in intervals. All DS sam ples
were assayed w ithout and w ith chondroitin ABC lyase diges­
tion in replicates o f six. HCII-mediated throm bin inhibition
by the DS sam ples w as calculated using a heparin standard
curve (0.15625-2.5 (xgml-1 or 0.028125-0.45 USP heparin units
m l-1 ) and expressed as USP heparin units m g-1 DS required to
inhibit throm botic activity by 50%. Each sam ple w as expressed
as the m e a n ± SE.
2.8. Determination of disaccharide composition within
DS preparations using fluorophore-assisted carbohydrate
electrophoresis (FACE)
The disaccharide com position o f all the DS preparations w as
determ ined using FACE as previously described (Osborne et al.,
2008).
2.9. Statistical an alysis
Statistical analyses to m easure differences b etw een the
m olecular w eight size and anti-throm botic activity o f B-DS,
L-DS and L-DS fractions were conducted using a one-w ay
ANOVA follow ed by p ost hoc com parisons using D u n n ett’s test
(with B-DS as the control). Statistical correlations to m easure
the degree to w hich anti-throm botic activity and disaccharide
com position were related were perform ed using nonparam et-
ric Spearman correlations (due to the data not passing the
D’A gostino-Pearson norm ality test). All statistical calculations
were perform ed using GraphPad Prism 5 Software for W in­
dow s (www.graphpad.com ).
3. Results
3.1. Preparation o/B -D S, L-DS and L-DS fractions
To m easure the potential yield o f DS in the bovine col­
lagen w aste liquor prior to processing, total GAGs were
estim ated in the w aste stream to be 0.185 ± 0.01 gL_1 (data
not show n) using the Blyscan™ Sulphated G lycosam ino­
glycan Assay. Following extraction o f total GAGs via serial
filtration, anion exchange chromatography using Q Sepharose
Big Beads, ultrafiltration and freeze drying, the GAG con ­
ten t o f the final dried preparation w as estim ated to be
96.2 ±2.9% (data not shown). To identify the sp ecies of GAG
in the w aste stream, the extracted material w as digested
w ith chondroitin ACII lyase and chondroitin ABC lyase, and
visualized using agarose gel electrophoresis (Osborne et al.,
2008). Fig. 1 show s that the m aterial extracted from the
w aste stream w as com pletely digested by ABC lyase and
unaffected by ACII lyase, indicating it w as likely to be DS.
A sim ilar pattern w as observed follow ing ABC and ACII
lyase digestion o f com m ercially available DS. A com parison
o f the migration o f the extracted material w ith com m er­
cial chondroitin sulphate A and C, DS, heparin and heparan
sulphate provided further evidence that the material w as
DS.
High Performance Liquid Chromatography (HPLC) w as
used to estim ate the average m olecular w eigh t o f the
extracted B-DS to be 26.7 ± 0 .2 kDa. Thble 1 show s the
estim ated m olecular w eigh t o f B-DS coincided w ith an
average m olecular w eigh t o f 30 kDa for com m ercial DS.
The bovine hide DS w as th en depolym erized under con­
trolled oxidative conditions (Volpi et al., 1992) to pro­
duce low m olecular w eigh t DS oligosaccharides ranging
 FOOD AND BIOPRODUCTS PROCESSING 9 9 (2 0 I 6 ) 244-251 247

Table 1 - Average molecular weight ± SE (kDa) and

anti-thrombotic activity t SE (USP heparin units mg 1
DS) of commercial DS bovine hide dermatan sulphate
(B-DS), low molecular weight DS (L-DS) and L-DS
fractions 5-16 (F5-F16).
Source of DS Anti-thrombotic activity Average molecular
(USP heparin units mg-1 weight (kDa)
ACM - + - - + - CSA CSB CSC Hep HS
ABC + + Standards
DS)
Commercial Bovine hide DS 3.8±0.38 30
B-DS 4.7 ±0.42 26.6 ±0.2
Fig. 1 - Agarose gel electrophoresis of GAGs extracted from
L-DS 4.3 ±0.38 9.3 ±1.4*
bovine hide processing waste stream compared to F5 0.03 ±0.038* 6.9 ±0.5*
commercial GAG standards, before and after digestion with F6 0.024 ±0.042* 7.5 ±0.7*
chondroitin ABC lyase (ABC) and chondroitin ACII lyase F7 0.029 ±0.041* 8.0 ±0.7*
(ACII). 10 M-g amounts of the bovine hide derived GAGs and F8 0.10 ±0.042* 8.4 ±0.7*
commercial chondroitin sulphate B (CSB) before (-) and F9 0.28 ±0.067* 9.0 ±0.6*
after (+) digestion with chondroitin ABC and ACII lyase F10 1.7 ±0.11* 9.6 ±0.1*
F ll 1.02 ±0.12* 10.0 ±1.3*
along with 10 p,g chondroitin sulphate A (CSA), chondroitin
F12 5.1 ±0.29 9.6 ±0.9*
sulphate B (CSB), chondroitin sulphate C (CSC), heparin F13 6.07 ±0.46* 7.7 ±0.8*
(Hep) and heparan sulphate (HS) were applied to 0.35% F14 6.0 ±0.41* 7.8 ±0.8*
agarose/50 mM diaminopropane gels and run at 80 V for F15 6.5 ±0.45* 7.7 ±0.8*
90 min. The GAGs were fixed using 0.1% (w/v) F16 6.6 ±0.51* 7.7 ±0.8*
cetyl-trimethyl-ammonium bromide solution and stained
‘Denotes mean values in columns that are significantly different to
using 0.1% toluidine blue (w/v)/50% (v/v) ethanol/1% acetic
B-DS (P<0.05) using a one-way ANOVA followed by post hoc com­
acid (v/v) solution. parisons using Dunnett's test.

3.2. In vitro heparin cofactor II-mediated thrombin

in size from 7 to lOkDa (Table 1). To separate the
oligosaccharides on the basis of charge and potentially
isolate oligosaccharides with enhanced anti-thrombotic activ­
ity, the depolymerized DS was fractionated using anion
exchange chromatography (Q Sepharose Big Beads). Fig. 2
shows that fractions containing DS were identified by inter­
action with dimethylmethylene blue (DMMB) dye. The elution
profile revealed twelve fractions (numbered 5-16) that con­
tained DS above background levels.
inhibition by DS samples
Following preparation of all DS samples an in uitro kinetic
assay (Dupouy et al., 1988) with the chromogenic substrate
chromozymTH was used to measure HCII-mediated thrombin
inhibition. Prior to this assay, the concentration of sulphated
GAGs in all DS samples (B-DS, L-DS and L-DS fractions)
was estimated using the Blyscan™ Sulphated Glycosamino-
glycan Assay and diluted to 0.125-0.25 m gm h1 to ensure

Fig. 2 - Anion exchange chromatography of depolymerized DS using Q Sepharose Big Beads. Elution profile of
depolymerized DS fractionated using a 1.5 M NaCl gradient at pH 7.0. Fractions were collected every 5 min (for 150 min)
using a 2 ml min-1 flow rate. Interaction with DMMB dye confirmed the presence of DS. Fractions 5-16 were collected for
activity and structural analysis.
248 FOOD AND BIOPRODUCTS PROCESSING 9 9 ( 2 0 X 6 )
that comparable amounts of each DS sample were assayed.
HCII-mediated thrombin inhibition by the DS samples was cal­
culated using a heparin standard curve and expressed as USP
heparin units mg-1 DS required for inhibition of thrombotic
activity by 50% (Table 1).
The bovine hide DS (B-DS) inhibited thrombin in the pres­
ence of HCII comparable to commercial DS and equivalent
to 4.7 USP heparin units mg-1 DS (Table 1), as did L-DS at
4.3 USP heparin units mg-1 and L-DS fraction 12 (F12) at 5.1
USP heparin units mg^1. The DS oligosaccharides in L-DS frac­
tions 5-11 (F5-F11) exhibited the lowest inhibition of thrombin
(significantly less than commercial DS P<0.05) ranging from
0.024-1.7 USP heparin units mg-1. However, the highest inhi­
bition of thrombin, and almost 2-fold higher than commercial
DS, was potentiated by the DS oligosaccharides in L-DS frac­
tions 13-16 (F13-F16) that ranged in activity from 6.0-6.6 USP
heparin units mg-1. To control for the presence of contam inat­
ing GAGs, such as heparin or heparan sulphate, the DS sample
extracts were digested with ABC lyase and then assayed for
anti-thrombotic activity. No significant anti-thrombotic activ­
ity was observed following digestion (data not shown).
3.3. Determination o f disaccharide composition in DS
samples using FACE
Disaccharide composition in all of the DS samples was
determined using FACE (Osborne et al., 2008). The DS sam ­
ples were completely digested with chondroitin ABC lyase
producing disaccharides that, along with the commercial
unsaturated chondro/dermato/hyaluro-disaccharides, were
labelled using 2-aminoacridone. Fig. 3 shows the labelled
disaccharides that were separated using 35% acrylamide
gel electrophoresis and visualized under UV light. The
disaccharide composition was measured as the relative
percentage of each detectable disaccharide and expressed
as the mean of triplicate m easurem ents ± standard error
(SE) in Table 2. The majority of the disaccharides in the DS
prepared directly from the bovine hide processing waste were
4-0-mono-sulphated (71.7%, UA-*GalNAc4S03), with 17.5%
non-sulphated (UA^-GalNAc) and 5.5% 6-O-monosulphated
(UA^>-GalNAc6S03). Importantly for the inhibition of throm ­
bin through HCII, 4.7% were IdoA2S03->-GalNAc4S03. Trace
levels (<4%) of IdoA2S03->GalNAc6S03 were also detected.
The mono-sulphated disaccharide IdoA2S03->GalNAc, the
di-sulphated disaccharide, UA^>GalNAc4,6S03, and the
tri-sulphated disaccharide IdoA2S03-*-GalNAc4,6S03, were
below detectable levels.
In Table 2, the disaccharide composition in the L-DS sample
showed a pattern similar to native DS, however L-DS contained
a significantly higher percentage of non-sulphated disaccha­
rides (P< 0.05) accompanied by a non-significant decrease in
4-0 and 6-O-mono-sulphated disaccharides. The loss of sul­
phate groups from mono-sulphated disaccharides has been
observed previously in the depolymerisation of DS and could
be due to removal of sulphate groups by hydroxyl radicals
generated during the depolymerisation process by means of
copper ions and hydrogen peroxide (Ofman et al., 1997).
Fractionation of L-DS appeared to exclude non-sulphated
disaccharides as all L-DS fractions displayed a composi­
tion similar to, or with less non-sulphated disaccharides
(F5) than the DS extracted from the bovine hide processing
waste. Fractionation also produced L-DS oligosaccharides with
increased percentages of sulphated disaccharides, particu­
larly in L-DS fractions 13-16 where there were significant
2 4 4 -2 5 1
increases in the amount of di-sulphated disaccharides
(IdoA2S03-*-GalNAc4S03 and IdoA2S03->-GalNAc6S03) and
significant decreases in the amount of 4-O-mono-sulphated
disaccharides.
4. Discussion
In an attem pt to extract, purify and characterise the gly-
cosaminoglycans present in bovine collagen waste liquor,
anti-thrombotic DS was prepared from this waste stream
using only serial filtration and anion exchange chromatogra­
phy. Commercial DS has predominantly been sourced from
porcine intestinal mucosa, however several studies reveal
porcine (Halldorsdottir et al., 2006; Maimone and Tollefsen,
1990) and numerous bovine tissues (Ofosu et al., 1987; Osborne
et al., 2008) as alternative sources of anti-thrombotic DS.
Reported extraction methods often employ proteolytic m eth­
ods followed by chromatography and/or precipitation with
ethanol/acetone; in this study however, bovine collagen waste
liquor represents a relatively pure source of DS requiring only
filtration and anion exchange to produce an anti-thrombotic
product with purity greater than 93%.
Comparison of the prepared bovine collagen waste liquor
DS (B-DS) with commercial DS using chondroitinase lyase
digestions confirmed the presence of DS (Fig. 1). An in vitro
kinetic assay (Dupouy et al, 1988) with the chromogenic
substrate chromozymTH allowed HCII/DS-mediated throm­
bin inhibition to be measured from B-DS. The ability of
DS to inhibit thrombin activity was compared to a heparin
standard curve and was found to be comparable to com­
mercial DS. To control for the presence of contaminating
GAGs, such as heparin or heparan sulphate, the DS sample
extracts were digested with ABC lyase and then assayed for
anti-thrombotic activity. No significant anti-thrombotic activ­
ity was observed following digestion (data not shown). These
results indicated that DS present in bovine hide processing
waste has anti-thrombotic activity comparable to commer­
cial DS, identifying an additional source of bioactive DS
that is abundant and potentially easier to extract from and
purify.
Poor bioavailability is an issue when considering DS as
an oral therapeutic. Studies estimate bioavailability following
oral administration to hum an volunteers to be low, varying
between 1 and 10% (Dawes et al., 1991; Volpi, 1996). Applica­
tion of low molecular weight DS fragments has the potential
to improve oral bioavailability. However, to be efficacious as
an anti-thrombotic drug, biological activity m ust at least be
retained in low molecular weight DS material. In uiuo studies
involving intravenous or subcutaneous administration of low
molecular weight DS to animals show depolymerized DS to
be as effective as native DS at preventing thrombus formation
in rat (Fabiana Alberto et al., 2008) and rabbit (Du et al., 2007)
models of thrombosis.
Low molecular weight DS (L-DS) was prepared from B-
DS under controlled oxidative conditions (Volpi et al., 1992).
Depolymerization produced oligosaccharides ranging in size
from 7 to lOkDa (Table 1). FACE analysis showed a disac­
charide composition similar to B-DS (Table 1), however, the
depolymerized DS contained a significantly higher percent­
age of non-sulphated disaccharides (P < 0.05). This difference
in disaccharide composition did not appear to affect the ability
of the depolymerized DS to facilitate HCII-mediated thrombin
inhibition in vitro (Table 1), as anti-thrombotic activity from
 FOOD AND BIOPRODUCTS PROCESSING 9 9 (20 I 6) 244-251 249

- W8 ■

DS L-DS F5 F6 Std F7 F8 F9 F10 F ll F12 F13 F14 Std F15 F16

Fig. 3 - Agarose gel electrophoresis showing disaccharide analysis of chondroitin ABC lyase digested bovine hide dermatan
sulphate pS), depolymerized bovine hide DS (L-DS) and fractionated depolymerized DS from bovine hide (F5-F16).
Combined disaccharide standards (Std) top to bottom: (1) ADi-OS (2) ADi-6S, (3) ADi-4S, (4) ADi-UA2S (5) ADi-diSE (6)
ADi-diSD and (7/8) denotes unresolved (7) ADi-diSB and (8) ADi-triS 7/8 disaccharide standards. - * ■ Denotes excess from the
2-aminoacridone labelling reaction.

the depolymerized DS (4.3 USP heparin units mg-1 DS) was
comparable to both the native and commercial DS.
To isolate oligosaccharides with enhanced anti-thrombotic
activity, the depolymerized DS was fractionated using
ion exchange chromatography. The elution profile (Fig. 2)
revealed twelve fractions (numbered 5-16) that contained
DS above background levels. These fractions were ana­
lysed for average molecular weight using HPLC (Table 1),
disaccharide composition using FACE mapping (Fig. 3 and
Table 2) and anti-thrombotic activity using the i n uitro
assay (Table 1). Molecular weight of the DS oligosac­
charides ranged from 6.9-10 kDa. Characterization of the
oligosaccharides using FACE analysis revealed significant
differences in disaccharide composition. The relative per­
centages of the 4-0 and 6-0-mono-sulphated disaccharides
decreased as fraction number increased (Table 1). Conversely,
the relative percentages of the di-sulphated disaccha­
rides, iduronic acid-2-0-sulphate^N-acetyl-D-galactosamine
4-O-sulphate and iduronic acid-2-0-sulphate->N-acetyl-D-
galactosamine 6-O-sulphate, increased as fraction number
increased.
No trends were observed in the relative percentages of
the non-sulphated disaccharide. Analysis of anti-thrombotic
activity revealed that fractions 5-11 were significantly lower
in activity (P < 0.05) compared to the commercial DS standard
(and the native bovine DS) ranging from 0.031-1.02 USP
heparin units m g"1 DS. Fraction 12 was comparable to
the commercial standard (and native DS), whilst frac­
tions 13-16 were up to 2-fold higher in anti-thrombotic
activity ranging from 6.07-6.60 USP heparin units mg-1
DS.
The increase in di-sulphated disaccharides, particu­
larly in the relative percentage of iduronic acid-2-O-
sulphate->N-acetyl-D-galactosamine 4-O-sulphate, positively
correlated with anti-thrombotic activity (r = 0.9789; P < 0.0001).
The am ount of iduronic acid-2-O-sulphate-^N-acetyl-D-
galactosamine 6-O-sulphate also positively correlated with
anti-thrombotic activity (r = 0.9366; P< 0.0001). No other pos­
itive correlations were observed between the remaining
disaccharides and anti-thrombotic activity, however signif­
icant negative correlations were observed between anti­
thrombotic activity and 4-0 and 6-O-mono-sulphated disac­
charides disaccharide composition (r= -0.9371; P< 0.0001 and
r = -0.8112; P<0.01 respectively). The structural characteris­
tics of DS required for HCII-mediated thrombin inhibition
are repeating di-sulphated disaccharides of either iduronic
acid-2-0-sulphate->N-acetyl-D-galactosamine 4-O-sulphate
or uronic acid^-N-acetyl-D-galactosamine 4,6-O-sulphate.
(Fabiana Alberto et al., 2008; Halldorsdottir et al., 2006;
Linhardt et al., 1994; Maimone and Tollefsen, 1990; Mascellani
et al., 1993; Mascellani et al., 1994; Volpi, 1994). By fraction­
ating the depolymerized DS, oligosaccharides containing the
high affinity HCII binding site, iduronic acid-2-O-sulphate-^N-
acetyl-D-galactosamine 4-O-sulphate, were isolated.

Table 2 - Disaccharide composition, expressed as a mean relative percentage ± SE, of bovine hide dermatan sulphate
(B-DS), low molecular weight DS (L-DS) and L-DS fractions (F5-F16).
DS A -d isacch arides

Source o f DS A-di-nonS A -di-m ono 6S A -di-m ono 4S A -di-m ono 2S A -di-di 4,6S A -di-di 2,6S A -di-di 2,4S A -di-tri 2,4,6S

B-DS 1 7 .5 ± 3 .4 5.5 ± 0 .9 5 71.7 ± 1 .2 4 0 0 <4% 4.7 ± 0 .9 4 0

L-DS 25.3 ±1.1* 3.54± 0.22 65.7 ± 0.81 0 0 0 5 .5 ± 0 .1 7 0
F5 8.7 ±0.6* 9.4 ±0.7* 81.8 ±0.9* 0 0 0 0 0
F6 11.0 ± 0 .4 7.6 ± 0 .5 81.4±0.6* 0 0 0 0 0
F7 1 9 .9 ± 3 .3 6.7 ± 0 .9 7 3 .4 ± 2 .4 0 0 0 0 0
F8 1 7 .1 ± 0 .7 7 .1 ± 0 .9 73.7 ± 3 .3 0 0 <4% <4% 0
F9 16.0 ± 0 .8 7.2 ± 0 .7 71.6 ± 2 .1 0 0 <4% <4% 0
F10 1 5 .8 ± 1.6 7.3 ± 0 .9 70.8 ± 1 .7 0 0 <4% <4% 0
F ll 16.6 ± 2 .5 6.9 ± 0 .4 67.8 ± 1 .4 0 0 <4% 5 .8 ± 1.6 0
F12 17.4 ± 0 .5 5.8 ± 0 .9 63.4 ± 1.9 0 0 4.7 ± 0 .3 8.7 ± 0 .7 0
F13 1 1 .4 ± 0 .9 5.9 ± 0 .7 63.5 ± 3 0 0 4.9 ± 0 .3 14.3 ± 1.3* 0
F14 12.9 ± 1 .4 4 .8 ± 0 .9 54.0 ±3.9* 0 0 5.8 ±0.4* 22.4 ±2.4* 0
F15 1 5 .6 ± 0 .8 4.4 ± 0 .9 52.0 ±3.0* 0 0 3.6 ± 1 .9 24.3 ±1.9* 0
F16 14.4 ± 0 .7 3.70 ± 1 .2 46.7 ±1.6* 0 0 6.9 ±2.0* 28.3 ±2.5* 0

’D en o tes m e a n v alu es in co lu m n s th a t are significantly d ifferen t to B-DS (P <0.05) u sin g a one-w ay ANOVA follow ed by p o st hoc c o m p ariso n s
u sin g D u n n e tt’s test.
250 FOOD AND BIOPRODUCTS PROCESSING
5. Conclusion
Bovine collagen w a ste liq u o r is a large v o lu m e w a ste stre a m
th a t is c u rren tly o f little or no value. Purification of DS
from su ch a w aste stre a m could ad d sign ifican t value to
h id e p ro cessin g in d u s trie s w h ilst pro v id in g q u a n titie s o f DS
req u ired for th e p re p a ra tio n o f m ed ical devices a n d artificial
tissu es, an d to fu rth e r in v estig ate it as a n a n ti-th ro m b o tic
m olecule. W h ilst fu rth e r re se a rc h n e e d s to be u n d e rta k e n to
provide sufficient d a ta to ju stify its u se as a n a n ti-th ro m b o tic
th e ra p e u tic , DS derived from b ovine co llagen w a ste liq u o r
could be u sed as a n a lte rn a tiv e to h e p a rin (Syed a n d Reilly,
2009). In c o m p ariso n to th e c u rre n t m e th o d s o f DS e x tra c ­
tio n from porcine an d b ovine tissu e s, p re p a ra tio n o f DS from
bovine hid e p ro cessin g w a ste is p o te n tia lly m o re efficient
as th e DS h a s alread y b e e n lib erated from th e e x tracellu lar
m atrix. F u rth erm o re, bovine h id e is a very p u re so u rce of DS
w ith th e m ajo rity of th e e x tra c te d GAGs b ein g DS. T his m ay
avoid issu es w ith c o n ta m in a n t GAGs a s se e n in so m e c o m ­
m ercial h e p a rin p re p a ra tio n s (Pan e t al., 2010).
T his stu d y d e m o n s tra te s b ovine collagen w a ste liquor
as a source o f bioactive DS th a t can m e d ia te th e in h ib i­
tio n o f th ro m b in th ro u g h HCII co m p arab le to co m m ercial
DS. T he e x tracted DS c a n be fu rth e r m odified to p roduce
low er m o lecu lar w eig h t fractio n s w ith significantly e n h a n c e d
a n ti-th ro m b o tic activity th a t m ay h e lp to a d d re ss th e issu e s
o f bioavailability asso c ia te d w ith n ativ e (or h ig h m o lecu lar
w eight) DS. In sum m ary, a n e w source o f DS h a s b e e n id e n ­
tified w ith a sim ple m e th o d o f p ro c e ssin g th a t p ro d u c e s a
bioactive m olecule w ith g re a te r th a n 93% p u rity a n d p o te n tia l
th e ra p e u tic applicatio n s.
Author contributions
S.A.O. developed th e FACE m e th o d w ith in th e lab o rato ry
a n d co n trib u ted to th e p la n n in g o f e x p e rim e n ts a n d w rit­
ing of th e m a n u sc rip t. R.A.D. e sta b lish e d th e a n ti-th ro m b in
(H CII/throm bin) a ssay w ith in th e laboratory. W.C. p erfo rm ed
th e FACE m a p p in g a n d FIPLC a n aly sis o f th e DS sam p le s, an d
c o n trib u ted to th e w ritin g of th e m a n u sc rip t. P.R.S. depoly-
m erized th e DS m a te ria l a n d co n trib u te d to w ritin g o f th e
m a n u sc rip t. K.T. p erfo rm e d th e a n ti-th ro m b in a ssa y a n d c o n ­
trib u te d to th e w ritin g o f th e m a n u sc rip t. K.D. d eveloped an d
p erfo rm ed th e large-scale e x tra c tio n o f DS from th e w a ste
stre a m , a n d co n trib u te d to th e w ritin g of th e m a n u sc rip t. R.B.S
c o n trib u ted to th e p la n n in g o f e x p e rim e n ts a n d w ritin g o f th e
m an u scrip t.
Acknowledgements
T his w ork w as su p p o rte d by CSIRO A griculture (form erly Live­
stock Industries) a n d CSIRO Food a n d N utrition.
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