Food Hydrocolloids 44 (2015) 129e135

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Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Cross-linking xanthan and other compounds with glycerol

Dragoljub Bilanovic a, *, Jeanna Starosvetsky b, Robert H. Armon b
a
Center for Environmental, Earth, and Space Studies, Bemidji State University, Bemidji, MN 56601, USA
b
Faculty of Civil and Environmental Engineering, Department of Water and Agricultural Engineering, Technion e Israel Institute of Technology, Haifa 32000,
Israel

a r t i c l e i n f o a b s t r a c t

Article history: In a waterless or near-waterless environment glycerol's hydroxyl groups react with xanthan's functional
Received 12 April 2014 groups to make glycerol cross-linked xanthan (GCX) since not enough water is present to inhibit
Accepted 12 September 2014 glycerol-xanthan reactions. The water, formed during cross-linking, in fact catalyzes the unwinding of
Available online 28 September 2014
xanthan's double-helix thus making functional groups of its side chains and of its backbone more
accessible for cross-linking. Functional groups of xanthan's side chains and those in its backbone are
Keywords:
cross-linked with glycerol monomers and oligomers. Glycerol monomers and its oligomers cross-link
Bio-polymer
xanthan when glycerol to xanthan weight ratio is smaller than 27.6. Hardness increases with an in-
Cross-linking
Glycerol
crease of xanthan to glycerol ratio; GCX made with 50% wt xanthan is a hard solid material almost 40
Hydrocolloid times harder than GCX gel made with 5% wt xanthan. A gram of GCX absorbs more than 39 g of water. In
Polysaccharide a waterless or near-waterless environment glycerol cross-links xanthan and other bio-polymers. Mate-
Xanthan rials made by glycerol cross-linking of bio-polymers can be used as hydrogels, absorbents, coatings,
carriers in controlled delivery of chemicals, films, membranes and are of interest for those and other
applications in agriculture, food, pharmaceutical and other industries. Using glycerol to cross-link bio-
polymers and other compounds will also help decrease the pressure on the water resources and mini-
mize pollution of the environment.
© 2014 Elsevier Ltd. All rights reserved.

1. Introduction
Hydrocolloids are major functional agents used in the food in-
dustry (Varela & Fiszman, 2011). Xanthan, the most successful
microbial hydrocolloid approved for human consumption (FDA,
2014), is used as an emulsifier, friction and water mobility
reducer, stabilizer, thickener in food, pharmaceutical, gas and oil,
cosmetic, and other industries. Xanthan fermentations are con-
ducted in submerged mode mostly on glucose containing media
and in solid/semisolid mode on different alternative media (Ben
Salah et al., 2010; Bilanovic, Chang, Isobaev, & Welle, 2011;
Bilanovic, Shelef, & Green, 1994, Kurbanoglu & Kurbanoglu, 2007;
Stredansky & Conti, 1999; Yoo & Harcum, 1999).
Xanthan, an anionic polyelectrolyte, forms a “weak gel” when
added at concentration
0.3% wt (Morris, Nishinari, & Rinaudo,
2012) but does not gel by conventional methods (Iijima,
Shinozaki, Hatakeyama, Takahashi, & Hatakeyama, 2007).
* Corresponding author. Tel.: þ1 218 755 2801; fax: þ1 218 755 4107.
E-mail addresses: dbilanovic@bemidjistate.edu (D. Bilanovic), starjean@tx.
technion.ac.il (J. Starosvetsky), cvrrobi@tx.technion.ac.il (R.H. Armon).
Xanthan forms cryogel (Giannouli & Morris, 2003) but also some
mixed gels with different polymers (Agoub, Smith, Giannouli,
Richardson, & Morris, 2007; Argin-Soysal, Kofinas, & Lo, 2009;
Fitzpatrick, Meadows, Ratcliffe, & Williams, 2013).
Glucose makes xanthan's backbone to which short side chains
are attached to every second glucose. The side chains contain: two
D-mannose, one D-glucuronic acid and variable amount of acetate
(degree of substitution 50e70%) and of pyruvate (degree of sub-
stitution up to 40%). Xanthan molecules form a double-helix which
is stabilized by hydrogen bonds, electrostatic interactions and steric
effects. Xanthan's transition from ordered to disordered confor-
mation is reversible and can be induced by changing temperature
or ionic strength; the transition is also dependent on the degree of
ionization of carboxyl and acetyl residues (Born, Langendorff, &
Boulenguer, 2004).
Glycerol is also approved for human consumption; currently
there is oversupply of technical grade glycerol originating from the
biodiesel plants. Glycerol hydroxyl group react with the fatty acid
carboxyl group to form ester bond with one water molecule
released per each ester bond formed (Nelson & Cox, 2005).
We hypothesized that in a waterless or near-waterless envi-
ronment glycerol's hydroxyl groups would react with xanthan's

http://dx.doi.org/10.1016/j.foodhyd.2014.09.024
0268-005X/© 2014 Elsevier Ltd. All rights reserved.
130 D. Bilanovic et al. / Food Hydrocolloids 44 (2015) 129e135
functional groups to make glycerol cross-linked xanthan (GCX). We
thought that glycerol-xanthan cross-linking should happen in a
waterless or near-waterless environment because both xanthan
and glycerol are hydrophilic so elimination of excess water would
eliminate inhibiting side reactions of water with both glycerol and
xanthan.
If glycerol would cross-link xanthan in waterless or near-
waterless environment then it would also cross-link other poly-
mers with reactive and accessible functional groups. Xanthan's side
chains contain up to three groups (i.e. one acetyl and two carboxyl)
which are likely to cross-link with glycerol following dissociation of
xanthan double helix into two single helices. Testing xanthan and
bio-polymers with carboxyl groups (i.e. casein, fibrin, and pectin),
and without carboxyl but with other reactive functional groups (i.e.
chitosan, cellulose, DNA, and starch), could help us understand if
glycerol would cross-link all bio-polymers or only those incorpo-
rating particular functional groups.
The objectives of this work were to conduct a feasibility study of
glycerol cross-linking of xanthan and to assess whether bio-
polymers other than xanthan cross-link with glycerol.
An extensive literature review shows no journal publications or
patents on the subjects; the review indicates the idea of glycerol
cross-linking of bio-polymers in either waterless or near-waterless
environments to be novel and deserves further studies.
If developed the glycerol cross-linked xanthan, and other glyc-
erol cross-linked polymers, could be rather interesting materials for
numerous applications in food, pharmaceutical, and many other
industries.
2. Materials
Xanthan fermented locally (Bilanovic, Malloy, & Remeta, 2010)
and commercial xanthan (Spectrum Chemicals; pyruvic acid 1.5%
minimum) were used. Viscosity of 2% aqueous solution at 20  C was
4108 ± 360 mPas (n ¼ 4) fermented xanthan and 4310 ± 270 mPas
(n ¼ 4) commercial xanthan (viscometer Brookfield DV-E).
Analytical grade glycerol and potato starch were obtained from
J.T. Baker. Casein, fibrin and citrus pectin were from Sigma. Fish
sperm DNA was from Wards. Chitosan was from CTI, cellulose
powder CF1 from Whatman. Food dyes “Blue 1” and “Red 40” were
supermarket purchased.
3. Methods and materials
3.1. Glycerol cross-linked xanthan e procedure
To dissociate xanthan's double helices and to make its functional
groups accessible for glycerol cross-linking we heated glycerol-
xanthan mixtures to T
105  C; both open and closed containers
were used. Experimental samples were made by mixing a known
amount of xanthan (0e50% wt) with a known amount of glycerol
(100e50% wt). To cross-link xanthan with glycerol a weighed
amount of the mixture of known composition was placed into
24 mL glass vial and heated in microwave oven (DAEWOO-CRS)
with or without screw cap attached. Alternatively a weighed
amount of the mixture of known composition was placed in an Al-
weighing dish or into open glass container and heated in a drying
oven (GCA-CO). Sample temperature was determined immediately
after heating with infrared thermometer (CE XIRT-BTA).
3.1.1. Cross-linking other polymers with glycerol
Experimental samples were made by mixing a known amount of
bio-polymer (0e50% wt) with a known amount of glycerol
(100e50% wt). The procedure described in 3.1 above was used to
test glycerol cross-linking of seven other bio-polymers, three with
carboxyl groups (i.e. casein, fibrin, and pectin), and four without
carboxyl groups (i.e. chitosan, cellulose, DNA, and starch).
3.2. Water release in preparation of the glycerol cross-linked bio-
polymers
The amount of water released during cross-linking was deter-
mined gravimetrically by weighing samples before and after
heating (Section 3.1 and 3.1.1.); the averages of three replicas for
each sample were reported.
3.2.1. Water absorption by the glycerol cross-linked xanthan
To test water absorption a known amount of GCX sample was
placed into a 24 mL glass vial to which a known volume of ddH2O
was added; closed vials were left undisturbed for 24 h when the
weight of free water was measured (Ohaus GA200D balance).
Swelling degree (SD) values were calculated using the following
equation:
SDð%Þ ¼ ðWs  Wd Þ  ð100=Wd Þ
where Ws and Wd are the weight of swollen GCX sample and that of
dry GCX sample respectively; the averages of three replicas for each
sample were reported.
3.2.2. Food dye absorption and release
GCX samples were tested for the release of food dyes into water
and absorption of the dyes from water. To test dye release a known
mass of the sample containing a known amount of food dye was
placed into 24 mL glass vial to which a known volume of ddH2O
was added; closed vials were put on a rocker for 24 h. Beckman DU
650 spectrophotometer was used to measure concentration of dye
at 629 nm (Blue 1) and at 503 nm (Red 40). To test the dye uptake a
known mass of the sample containing no food dye was placed into
24 mL glass vial to which a known volume of dye solution was
added; closed vials were put on a rocker for 24 h. Concentration of
dye in H2O was measured as described above.
3.3. FTIR spectra
IR spectra of samples were recorded on Nicolet 6700 FT-IR
spectrometer with OMNIC software over the range 4000 to
650 cm1 using 0.09 cm1 resolution. KBr disc method was used to
get glycerol spectra; glycerol was mixed with dry KBr powder then
pressed in hydrostatic press for 10 min. Spectra of compressed
xanthan as well as spectra of GCX samples were recorded with
“Smart iTR e ZnSe Plate”; xanthan powder or GCX sample were
pressed for 10 min in hydrostatic press; recorded spectra were
average of three scans.
3.4. Hardness
Cross-linked samples were casted in a “drum” or rectangular
cuboid molds of roughly equal size (L 3.0 ± 0.2 cm, W 3.0 ± 0.2 cm,
H 1.5 ± 0.2 cm) following the above procedure (Section 3.1 and
3.1.1.). Durometers (models 306L and 411 e PTC Instruments)
were used to measure hardness following ASTM Standard D2240;
the reported results were average of five determinations.
3.5. Statistical analysis
Design Expert Software V7.16 (State Ease Inc.) was used to plan
the experiments and analyze the results.
 D. Bilanovic et al. / Food Hydrocolloids 44 (2015) 129e135 131

4. Results and discussion oligomers and release a molecule of water per each bond formed
(Medeiros, Leite, & Lago, 2012). Functional groups of xanthan's
Upon heating to GCX mixtures made with 50% wt xanthan backbone and of its side chains are likely to be cross-linked with
released on average 4.3 times less water than GCX mixtures made both glycerol monomers and its oligomers at all ratios of glycerol to
with 7.5%wt xanthan (XE in Fig. 1). If the water released from the xanthan used in this work (i.e. GCX made 50% wt and less xanthan).
GCX samples would be proportional to the mass of xanthan in the A portion of water formed during the cross-linking stays in the bulk
GCX samples the samples made with 50% wt xanthan should of GCX material and, in part because of its high temperature, cat-
release 6.67 times more water than the samples made with 7.5% wt alyzes the unwinding of xanthan's chains thus making functional
xanthan. We estimated that meager amounts of water would be groups of its backbone and of its side chains more accessible for
released if glycerol cross-linking of xanthan would exclusively go cross-linking.
via functional groups existing in xanthan's side chains (XC in Fig 1). IR spectra of glycerol (G), xanthan (X) and glycerol cross-linked
At all xanthan concentrations tested, measured water releases are xanthan (GCX) are given in Fig. 2. OeH group stretching bands are
two to three orders of magnitude larger than the estimated water in range 3600e2900 cm1; those due to asymmetric COO
release (XE vs XC in Fig 1). This implies glycerol reacts with func- stretching are in range 1640e1590 cm1. Symmetric COO
tional groups of xanthan's backbone and with those in its side stretching bands are in range 1440e1400 cm1 while CeOeC
chains. This finding also implies that glycerol oligomers, formed via stretching bands cover range from 1050 to 950 cm1 (NIST, 2014
condensation of glycerol monomers, could cross-link functional and Fig 2).
groups of xanthan's backbone and those in its side chains.
If the amount of water released was calculated per mole of
added glycerol, GCX samples made with 50% wt xanthan released
roughly 2.8 times more water than those made with 7.5% wt xan-
than (GE in Fig. 1). If the amount of water released would be pro-
portional to the amount of glycerol used to make GCX samples
those made with 50% wt xanthan should release roughly 1.8 times
less water than GCX samples made with 7.5% wt xanthan (GE in Fig
1). Fig. 1 shows the calculated amount of water which would be
released when all three OH groups of glycerol participated in the
cross-linking (GC in Fig 1). The curves depicting experimental (GE)
and calculated (GC) water release cross at 35% wt xanthan (GE vs
GC in Fig 1) to indicate that the amount of water released from
these GCX samples equals the amount of water which could come
from the added glycerol. The sample made with 50% wt xanthan
released 1.64 times more water than there could come from the
added glycerol. Samples containing less than 35% wt xanthan
released much less water than could come from added glycerol
only (GC i GE in Fig 1). Xanthan is more acidic than the compounds
which it is made off (Causse et al. 2013). Glycerol molecules
condense in both acidic and in basic environments to form glycerol

Fig. 1. Water released from glycerol cross-liked xanthan. Remarks: Known amount
(16.083 ± 0.442 g) of the glycerol-xanthan mixtures were oven heated to 140  C for 1
hour then dried overnight at 105  C. XE amount of water released (mole H2O/mole
xanthan monomer added), averages of three replicas, SD  0.655. XC calculated
amount of H2O which would be released provided two moles of H2O are released per Fig. 2. IR spectra of glycerol (G), xanthan (X), and glycerol cross-linked xanthan (GCX).
mole of xanthan monomer added, assuming the monomer's molecular weight equals Remarks: GCX samples made of 25 % wt xanthan and 75 % wt glycerol were placed in
933. GE amount of water released (mole H2O/mole glycerol added), averages of three closed glass vials and heated twice for 13 seconds in microwave oven; temperature
replicas, SD  0.006. GC calculated amount of H2O which would be released provided 3 after heating was on average 110 ± 2  C. Averages of three consecutive scans are
moles of H2O are formed per mole of glycerol added. presented (Section 3.3).
132 D. Bilanovic et al. / Food Hydrocolloids 44 (2015) 129e135

The areas under specific bands, which reflect the relative was released per gram of biopolymer-glycerol mixtures (H2O in
amounts of corresponding functional groups, differed by the Table 1); this further point towards (at least partial) cross-linking of
samples. the bio-polymers tested with glycerol in waterless environments.
The area under GCX's OeH bands is much larger than the area Attempts were made to test GCX matrices on absorption and
under glycerol's OeH bands and the area under xanthan's OeH release of food dyes; GCX matrix made with 15% wt xanthan
bands (OeH region in Fig. 2). Larger area under GCX's OH bands released “Red 40” somewhat faster than “Blue 1” (RR and RB in
point toward glycerol cross-linking of xanthan. We think that a Fig. 3); absorption of “Red 40” from aqueous solution was faster
smaller portion of H2O generated in formation of xanthan-glycerol when compared to “Blue 1” (UR and UB in Fig 3). GCX matrix
bonds was released from GCX samples while a large portion of released within first 6.5 h of experiment 52 ± 3 percent (n ¼ 3) of
generated water was instantaneously absorbed in the bulk of GCX “Red 40” (RR in Fig 3); 69 ± 2.5% of “Red 40” was release from GCX
sample (Figs. 1 and 2 and Table 1). matrix after 24 h. Some 44 ± 2.4 percent (n ¼ 3) of “Blue 1” con-
Areas under “CeH” bands are roughly equal for glycerol and GCX tained in GCX matrix was released from the matrix after 6.5 h;
and much smaller for xanthan (CeH in Fig 2); areas under xan- 62 ± 3.1% of “Blue 1” was released from the matrix after 24 h (RB in
than's C]O bands are roughly equal to these of GCX's C]O bands. Fig 3). Glycerol cross-linked xanthan is seemingly a good material
In contrast the area of GCX CeOeC bands is almost three times as for construction of slow-release matrices.
large as that of xanthan's CeOeC bands suggesting glycerol's Almost all “Blue 1”(99 ± 0.6%; n ¼ 3) was absorbed from the
monomers and oligomers cross-link xanthan. Overall the IR spectra solution by GCX matrix after only 6.5 h of contact; GCX matrix
for G, X and GCX strongly point towards the chemical cross-linking absorbed more than 85% of “Red 40” in the same time frame (UR
of glycerol and xanthan. and UB in Fig 3). Matrices made of glycerol cross-linked xanthan
In a waterless environment glycerol cross-links xanthan; this appear to be good absorbents for application in food, pharmaceu-
implies glycerol should cross-link other bio-polymers as well; some tical and other industries.
compounds we tested for glycerol cross-linking are listed in Hardness increased as the percentage of a bio-polymer in the
Table 1; functional groups of these bio-polymers are given in glycerol-polymer mixture increased; for example GCX made with
Table 2. Pectin and casein (50% wt) cross-linked with glycerol 50% wt xanthan was almost 16 times harder than GCX made with
(50% wt) yield hard solid materials the hardness of which is com- 15% wt xanthan (Xanthan in Table 1, Fig 1 in “Supplementary
parable to that of GCX made with 50%wt xanthan (Xanthan, Pectin Information”) and almost 40 times harder than GCX made with
and Casein in Table 1 and Figs. 1e4 and 8 “Supplementary 5% wt xanthan (XG in Fig 4). Hardness of glycerol cross-linked
Information”). pectin made with 15% wt pectin is on average 35% lesser than
Glycerol cross-linked starch (Starch in Table 1 and Fig. 7 in hardness of glycerol cross-linked pectin containing 50% wt pectin
“Supplementary Information”) was a rather weak gel but it (PC in Fig 4, Pectin in Table 1, Fig. 8 in “Supplementary
released 214 percent more H2O than cellulose (Cellulose in Table 1) Information”). The same is true for glycerol cross-linked casein
which also appear to yield even weaker gel than starch. Glycerol samples maid with 50% wt casein which are on average 26 times
cross-linking of DNA yields a reddish-brown solution at 15% wt harder than samples maid with 15% wt casein (CG in Fig 4, Casein in
DNA and a very viscous gel-like material at 50% wt DNA (DNA in Table 1, Fig 4 in “Supplementary Information”).
Table 1 and Fig. 5 in “Supplementary Information”). If left to dry the Swelling Degree (SD) also seemed to increase as the share of
reddish-black solution turns into a hard film. It seems that all the xanthan increased; GCX samples made with 25% wt xanthan
compounds tested react with glycerol to some extent by forming showed an average SD of 1730 ± 105% while those made with
three kinds of solids (hard, granular, porous), gels, films, and stable 50% wt xanthan showed an average SD of 3948 ± 26%.
suspensions/solutions (Table 1 and Figs. 1e8 in “Supplementary GCX materials exhibited a different environmental stability;
Information”). All tested biopolymer-glycerol mixtures released some of them were left on lab-bench in May of 2011 and appear
water upon heating; anywhere from 0.45 to 3.95 mmol of water unchanged until this day, microbes degraded others within a few
week time.
Table 1 At a low concentration (1.0 g/L) xanthan double helix disso-
Glycerol cross-linked bio-polymers. ciates into two single helices when heated to about 80  C; at a
Bio-polymer AHBP15; AHBP50; H2O Remark concentration higher than 10 g/L dissociation is incomplete; xan-
n¼5 n¼3 (g/gM) than undergoes an irreversible denaturation accompanied by
Xanthan 0.46 ± 0.06 7.15 ± 0.86 0.0531 ± 0.0029 G15, S50
dissociation of its double helix into two single helices when heated
Pectin 3.5 ± 0.27 5.45 ± 0.66 0.0151 þ 0.0014 GL15, S50 above 95  C (Capron, Brigand, & Muller, 1998; Matsuda, Biyajima, &
Starch 0.2 ± 0.22 0.48 ± 0.11 0.0188 ± 0.0019 GL15, GL50 Sato, 2009).
Casein 0.2 ± 0.42 5.25 ± 0.41 0.0185 ± 0.0011 GL15, S50 Glycerol readily reacts with carboxyl group of acetic acid to yield
DNA CND CND 0.0661 ± 0.0051 L15; VWG50
mono-, di- or tri-acetate. We think that glycerol monomers and
Fibrin CND CND 0.0128 ± 0.0012 PS15, PS50
Chitosan CND CND 0.0129 þ 0.0019 WGr15, DGr50 oligomers cross-links all carboxyl groups available at xanthan's
Cellulose CND CND 0.0088 þ 0.0007 WDLM 15&50 helices; glycerol monomers and oligomers probably cross-link,
Remarks: AHBP15 and AHBP50 are average hardness (Newton) of material made of
though to less extent, other functional groups existing on xan-
15% wt bio-polymer (BP) and 85% wt glycerol (G) and material made with 50% wt BP than helix. Xanthan's side chains contain three carboxyl groups (i.e.
and 50%wt G. H2O (g/gM) is the amount of water released (g) per gram of material glucuronic acid, pyruvic and an acetyl residues); the groups
made of 15% wt BP and 85% wt G after four cycles of consecutive heating in mi- possibly react with glycerol to yield glycerol cross-linked xanthan.
crowave (i.e. two heating cycles of 13 s each and two heating cycles of 26 s each to
eCH3 and eCH2 groups frame xanthan's acetyl residues making
make the total heating time equal 78 s). GL15 and GL50 stand for gel like material
made of 15% wt BP and 85% wt G and gel like material containing 50% wt BP and them less reactive than other two carboxyl groups; in addition
50% wt G. L15 stands for liquid material made of 15% wt BP and 85% wt G. PS15 and RHN- and eCH3 groups frame acetyl residues on chitosan (Rinaudo,
PS50 stand for porous solid material made with 15% w and 50% wt BP. S15 and S50 2006) which apparently does not react with glycerol.
stand for solid material made of 15% wt BP and 85% wt G and solid material con- To unwind xanthan's double helices and to make its functional
taining 50% wt BP and 50% wt G. VWG50 very weak gel containing 50% wt BP and
50% wt G. WGr15 wet granular material containing 15% wt BP. DGr50 dry granular
groups accessible for glycerol cross-linking we varied quality and
material containing 50% wt BP. WDLM 15&50 wet dough like material containing 15 duration of energy input by heating glycerol-xanthan mixtures to
or 50% wt BP. CND could not be determined or below detection limit. T
104  C in close and open containers (Section 3.1). Gels, solids
 D. Bilanovic et al. / Food Hydrocolloids 44 (2015) 129e135 133

Table 2
Bio-polymers with various functional groups tested on glycerol cross-linking.

Functional group Bio-polymer

Xanthan Casein Chitosan Cellulose DNA Fibrin Pectin Starch

ReCOOH 1 1 0 0 0 1 1 0
ReOH 1 1 1 1 1 1 1 1
ReOeR 1 1 1 1 1 1 1 1
ReCOeR 1 1 1 0 0 1 1 0
ReH 1 1 1 1 1 1 1 1
ReNH2 0 1 1 0 1 1 0 0
RePO4 0 1 0 0 1 0 0 0

Remarks: 1 ¼ functional group integral part of bio-polymer molecule; 0 ¼ functional group is not integral part of bio-polymer.

and porous materials were produced (Table 1 and Figs. 1e3 in
“Supplementary Information”) by glycerol cross-linking of the
exposed carboxyl-groups. Glycerol could cross-link carboxyl
groups: a) present at the same side chain of xanthan helix, b)
existing at two adjunct side chains of a single xanthan helix, and c)
existing at side chains of two or more xanthan's helices; three
simple schemes of glycerol-xanthan cross-linking are given in
Fig. 5.Glycerol could react with itself to form di-, tri- and longer
oligo-mers which could also cross-link xanthan's helices.
Seven other polymers, three with carboxyl groups (i.e. casein,
fibrin, and pectin), and four without carboxyl groups but containing
reactive functional groups like eNH2 and ePO4 (i.e. chitosan, cel-
lulose, DNA, and starch), were tested glycerol cross-linking. This
was also performed to estimate whether functional groups other
than carboxyl participate in glycerol cross-linking (Tables 1 and 2
and Figs. 1e8 in “Supplementary Information”).
Upon heating to T
108  C glycerol cross-links casein's and
pectin's carboxyl groups to form, much like with xanthan, strong
gels and solid materials (Tables 1 and 2, Figs 4 and 8 in
“Supplementary Information”). Glycerol also reacts with fibrin's
carboxyl groups to form erodible porous materials (Table 1 and
Fig. 6 in Supplementary Information). It appears that stronger gels
and solids cannot be made with fibrin because the concentration of
carboxyl groups in fibrin is not high enough; it also appears that
other functional groups which make fibrin are not involved in
cross-linking with glycerol.
There are no carboxyl groups in chitosan, cellulose, DNA and
starch. Upon heating with glycerol, chitosan and cellulose do not
form either gels or solids. Glycerol forms a very week gel with DNA
(Tables 1 and 2, Fig 5 in “Supplementary Information”) indicating a
probable involvement of PO4 group. Starch forms week gels with
glycerol; the gels are stronger than glycerol-DNA gels but quite
weak in comparison to glycerol-xanthan, glycerol-casein and
glycerol-pectin gels (Tables 1 and 2, Fig 7 in “Supplementary
Information”). Starch contains CeH, CeOH and CeOeC groups
but there are no carboxyl groups in starch which signals that under
right experimental conditions these groups could participate in
glycerol cross-linking.
Polymer geometry, nature of monomer linkage, chain length,
spatial arrangement of monomers, accessibility of functional
groups, and other macromolecular characteristics affect glycerol
cross-linking of polymers. For example CeH and CeOeC groups are
present in both cellulose and in starch and are less reactive than
carboxyl groups present in other polymers tested. Cellulosic CeH
and CeOeC groups, existing in tightly packed b(1 / 4)-glucose
chains, do not cross-link with glycerol. In contrast starch's CeH and
CeOeC groups, existing in loosely packed a(1/)-glucose chains,
appear to cross-link with glycerol (Tables 1 and 2 and Fig 7 in
“Supplementary Information”). Strong gels and solid materials
made in glycerol-casein, glycerol-pectin, and glycerol-xanthan
cross-linking much like weak materials made in glycerol-fibrin

Fig 3. Uptake and release of food dyes by glycerol cross-linked xanthan (GCX). Re-
marks: RR is release of “Red 40” where 100% equals 1.321 ± 0.014 mg Red 40/L; RB is
release of “Blue 1” where 100% equals 0.289 ± 0.014 mg Blue 1/L, UR is absorption of
“Red 40” with 100% equals 10.031 ± 0.085 mg Red 40/L, UB is absorption of “Blue 1”
with 100% equals 2.501 ± 0.042 mg Blue 1/L (averages of three replicas). GCX matrices
were prepared by placing a known amount of XG mixture containing 15% wt xanthan
in closed glass vials which were heated twice for 13 seconds in microwave oven to
temperature of 109 ± 3 T; matrices for uptake experiments were prepared without
food dyes; matrices for release experiments were prepared with a known amount of
food dye.
Fig. 4. Hardness of glycerol cross-linked bio-polymers. Remarks: Samples of glycerol
(G) crosslinked xanthan (XG), pectin (PG), starch (SG), and casein (CG) were placed in a
“drum” and rectangular cuboid molds of roughly equal size (L 3.0 ± 0.2 cm, W
3.0 ± 0.2 cm, H 1.5 ± 0.2 cm) and heated in microwave oven for 26 seconds to a final
temperature of 109 ± 4.0  C (Section 3.1 and 3.1.1.) Hardness was determined on five
samples following ASTM Standard D2240 (Section 3.4).
134 D. Bilanovic et al. / Food Hydrocolloids 44 (2015) 129e135
Fig. 5. Glycerol cross-linking of xanthan e proposed schematics. Remarks: Not to scale. Dashed lines are used for glycerol to differentiate between xanthan and glycerol molecules.
Glycerol monomers and oligomers cross-link xanthan; oligomers are not shown for simplicity.
cross-linking indicate that glycerol-polymer cross-linking could be
tailored to yield materials of different characteristics by varying
quantity and quality of polymer's reactive accessible groups.
Xanthan's acetyl content, degree of xanthan polymerization,
and other xanthan's characteristics are likely to a play role in
glycerol cross-linking e effects of these are yet to be studied. The
study in progress shows hardness of materials made by glycerol
cross-linking of high pyruvate xanthan (>1.5% pyruvate) is at least
twice higher than the hardness of the materials made with low
pyruvate xanthan (<0.5%).
4.1. Conclusions
We have shown glycerol cross-links xanthan and other com-
pounds in a waterless environment. A portion of water formed in
cross-linking stays in GCX catalyzing the unwinding of xanthan's
chains thus making functional groups of its backbone and of its side
chains more accessible for cross-linking. Functional groups of the
xanthan's side chains and probably those in its backbone are cross-
linked with glycerol. Glycerol monomers and its oligomers cross-
link xanthan when glycerol to xanthan weight ratio is smaller
than 27.6; very weak gel-like materials form above this ratio indi-
cating incomplete cross-linking since concentration of xanthan's
functional group is not high enough. Glycerol cross-linked mate-
rials of different characteristics are simple to prepare by varying the
following: the ratio of glycerol to polymer, the quality and duration
of energy input (i.e. dry oven heating, heating in microwave oven
using closed or open containers), and by changing concentration of
functional groups in a polymer molecule. Materials made by glyc-
erol cross-linking of bio-polymers can be used as hydrogels, ab-
sorbents, coatings, carriers in controlled delivery of chemicals,
films, membranes and are of interest for those and other applica-
tions in agriculture, food, pharmaceutical and other industries.
Using glycerol in bio-polymer cross-linking and other cross-linking
reactions would also help decrease the pressure on the water re-
sources and minimize pollution of the environment.
Acknowledgments
This research work was in part funded through Minnesota Space
Grant (Sub-award No. A001584007). We thank Visnja Remeta-
Bilanovic for making photos presented in this work.
 D. Bilanovic et al. / Food Hydrocolloids 44 (2015) 129e135
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.foodhyd.2014.09.024.
References
Agoub, A. A., Smith, A. M., Giannouli, P., Richardson, R. K., & Morris, E. R. (2007).
“Melt-in-the-mouth” gels from mixtures of xanthan and konjac glucomannan
under acidic conditions: a rheological and calorimetric study of the mechanism
of synergistic gelation. Carbohydrate Polymers, 69(4), 713e724.
Argin-Soysal, S., Kofinas, P., & Lo, M. Y. (2009). Effect of complexation conditions on
xanthan-chitosan polyelectrolyte complex gels. Food Hydrocolloids, 23,
202e209.
Ben Salah, R., Chaari, K., Besbes, S., Ktari, N., Blecker, C., Deroanne, C., et al. (2010).
Optimization of xanthan gum production by palm date (Phoenix dactylifera L.)
juice by-products using response surface methodology. Food Chemistry, 121(2),
627e633.
Bilanovic, D., Chang, F.-H., Isobaev, P., & Welle, P. (2011). Lactic acid and xanthan
fermentations on an alternative potato residues media e carbon source costs.
Biomass and Bioenergy, 35(7), 2683e2689.
Bilanovic, D. D., Malloy, S. H. & Remeta, P. (2010). Solid or semi-solid state
fermentation of xanthan on potato or potato waste. U.S. Patent 7,727,747 B2.
Assignee: Bemidji State University Foundation.
Bilanovic, D., Shelef, G., & Green, M. (1994). Xanthan fermentation of citrus waste.
Bioresource Technology, 48(2), 169e172.
Born, K., Langendorff, V., & Boulenguer, P. (2004). Xanthan. In E. J. Vandamme, S. De
Baets, & A. Steinbüchel (Eds.), Polysaccharides I: Polysaccharides from pro-
karyotes: Vol. 5. Biopolymers (pp. 259e269). Wiley-VCH.
Capron, I., Brigand, G., & Muller, G. (1998). Thermal denaturation and renaturation
of a fermentation broth of xanthan: rheological consequences. International
Journal of Biological Macromolecules, 23, 215e225.
Causse, B., Spadini, L., Martins, J. M. F., Lenoir, L., Heyraud, A., & Delolme, C. (2013).
Xanthan exopolysaccharide: acidebase reactivity related to structure and
135
conformation. A model for understanding the reactivity of degraded and
colloidal soil organic matter. Chemical Geology, 359, 150e158.
FDA (2014). http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.
cfm?CFRPart¼172 Accessed: January 2014.
Fitzpatrick, P., Meadows, J., Ratcliffe, I., & Williams, P. A. (2013). Control of the
properties of xanthan/glucomannan mixed gels by varying xanthan fine
structure. Carbohydrate Polymers, 92(2), 1018e1025.
Giannouli, P., & Morris, E. R. (2003). Cryogelation of xanthan. Food Hydrocolloids, 17,
495e501.
Iijima, M., Shinozaki, M., Hatakeyama, T., Takahashi, M., & Hatakeyama, H. (2007).
AFM studies on gelation mechanism of xanthan gum hydrogels. Carbohydrate
Polymers, 68, 701e707.
Kurbanoglu, E. B., & Kurbanoglu, N. I. (2007). Ram horn hydrolysate as enhancer of
xanthan production in batch culture of Xanthomonas campestris EBK-4 isolate.
Process Biochemistry, 42(7), 1146e1149.
Matsuda, Y., Biyajima, Y., & Sato, T. (2009). Thermal denaturation, renaturation and
aggregation of a double-helical polysaccharide xanthan in aqueous solution.
Polymer Journal, 41(7), 526e532.
Medeiros, A. M., Leite, M. M. C., & Lago, M. R. (2012). Use of glycerol by-product of
biodiesel to produce an efficient dust suppressant. Chemical Engineering Journal,
180, 364e369.
Morris, E. R., Nishinari, K., & Rinaudo, M. (2012). Gelation of gellan e a review. Food
Hydrocolloids, 28, 373e411.
Nelson, L. D., & Cox, M. M. (2005). Lehninger e Principles of biochemistry (4th ed.).
(pp. 343e355). New York: W.H. Freeman and Company.
NIST. (2014). Chemistry WebBook. http://webbook.nist.gov/cgi/cbook.cgi?
ID¼C64197&Units¼SI&Type¼IR-SPEC&Index¼2#IR-SPEC Accessed January
2014.
Rinaudo, M. (2006). Chitin and chitosan: properties and applications. Progress in
Polymer Science, 31, 603e632.
Stredansky, M., & Conti, E. (1999). Xanthan production by solid state fermentation.
Process Biochemistry, 34(6e7), 581e587.
Varela, P., & Fiszman, S. M. (2011). Hydrocolloids in fried foods. A review. Food
Hydrocolloids, 25, 1801e1812.
Yoo, D. S., & Harcum, W. S. (1999). Xanthan gum production from waste sugar beet
pulp. Bioresource Technology, 70(1), 105e109.