Food Hydrocolloids 132 (2022) 107838

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

Effect of downstream processing on the structure and rheological properties

of xanthan gum generated by fermentation of Melaleuca alternifolia
residue hydrolysate
Zhi-Xuan Li a, 1, Jia-Yu Chen a, 1, Yi Wu a, Zhong-Ying Huang a, Shu-Ting Wu a, Yun Chen a, b,
Jing Gao a, b, Yong Hu a, b, **, Chao Huang a, b, *
a
School of Food Science, Guangdong Pharmaceutical University, Zhongshan, 528458, People’s Republic of China
b
GDPU-HKU Zhongshan Biomedical Innovation Platform, Zhongshan, 528437, People’s Republic of China

A R T I C L E I N F O A B S T R A C T

Keywords: Low-cost Melaleuca alternifolia residue hydrolysate has great potential as a promising substrate for xanthan gum
Xanthan gum production but further downstream processing in addition to traditional alcohol-precipitation is necessary to
Downstream processing obtain purified xanthan gum from this substrate. In this study, the effect of downstream processing on the
Structure analysis
structure and rheological properties of xanthan gum generated by fermentation of Melaleuca alternifolia residue
Rheological characterization
hydrolysate was evaluated for the first time. Compared with acid-precipitation and alkali-precipitation, dialysis
Melaleuca alternifolia residue hydrolysate
Fermentation was much more suitable for xanthan gum purification as demonstrated by the energy dispersive spectrometer
(EDS) and thermogravimetric analysis (TGA) results, and the xanthan gum with relatively high purity could be
obtained after alcohol-precipitation and dialysis. Dialysis after alcohol-precipitation showed little influence on
the functional groups of xanthan gum but the microstructure of xanthan gum changed greatly in that a fila­
mentous structure was formed after this treatment. The fermentation performance could be evaluated accurately
based on the suitable downstream processing. The purified xanthan gum had similar rheological properties
(pseudoplasticity and solid-like/gel-like behavior) to the typical xanthan gum according to its flow curves and
linear viscoelastic analysis. However, some rheological properties (existence of Newtonian plateau in flow
curves, and crossover of storage modulus (G′ ) and loss modulus (G′′ ) in frequency sweep test) of the purified
xanthan gum solution were different from that of the typical xanthan gum. Overall, this study can offer some
important information for efficient and effective purification of xanthan gum with a good rheological perfor­
mance from low-cost Melaleuca alternifolia residue hydrolysate.

1. Introduction
Xanthan gum, a microbial polysaccharide mainly produced by Xan­
thomonas campestris, has many attractive characteristics such as special
rheological properties, good water solubility and compatibility to
various salts, good stability to heat, acid, and alkali, and therefore it has
many applications in food, petroleum, tissue engineering, and pharma­
ceutical industry (Andrew, 1977; Huang, Zhong, & Yang, 2020; Kumar,
Rao, & Han, 2018; Riaz, Iqbal, Jiang, & Chen, 2021; Sandford, Cottrell,
& Pettitt, 1984). In particular, xanthan gum is an important food addi­
tive that has been widely applied as the thickener, stabilizer, emulsifier,
etc. (Blok et al., 2021; Du et al., 2022; Matsuyama et al., 2021). To make
xanthan gum production more suitable for industrialization, exploiting
low-cost substrates for fermentation is always an important research
topic. To date, many low-cost feedstocks such as kitchen waste (Li et al.,
2016), glycerol (Wang, Wu, Zhu, & Zhan, 2017), hydrolyzed starch
(Niknezhad, Asadollahi, Zamani, & Biria, 2016), etc. have been proven
to be suitable for xanthan gum production.
For a long time, essential oils have attracted much attention and
profitable market for their important functions such as antioxidant ac­
tivity, antibacterial and antifungal activity, food packaging and pre­
servative, anxiolytic effect, etc. (Falleh, BenJemaa, Saada, & Ksouri,

* Corresponding author. No. 9 Changmingshui Avenue, Wuguishan Street, Zhongshan, 528458, People’s Republic of China.
** Corresponding author. No. 9 Changmingshui Avenue, Wuguishan Street, Zhongshan, 528458, People’s Republic of China.
E-mail addresses: lidhyong@126.com (Y. Hu), huangc@gdpu.edu.cn (C. Huang).
1
Both authors contributed equally to this work.

https://doi.org/10.1016/j.foodhyd.2022.107838
Received 3 January 2022; Received in revised form 1 April 2022; Accepted 27 May 2022
Available online 31 May 2022
0268-005X/© 2022 Elsevier Ltd. All rights reserved.
Z.-X. Li et al.
2020; Ju et al., 2019; Tariq et al., 2019; Zhang & Yao, 2019). Among
various plants, Melaleuca alternifolia (Australian tea tree) plays an
important role in essential oils production for its many advantages such
as high essential oils extraction yield, ease of cultivation, and broad
activities of its essential oils (Davies, Larkman, Marriott, & Khan, 2016;
Pereira, de Sant’Anna, Silva, Pinheiro, & de Castro-Prado, 2014; Shep­
herd, Wood, Raymond, Ablett, & Rose, 2015). Many solid residues are
generated after essential oils extraction, e.g., about 2–3 kg Melaleuca
alternifolia can generate about 20–30 mL essential oils, and the
remaining solid residues are considered as wastes. Obviously, it is wise
and necessary to explore some high value-added utilization of the bio­
resource of plant residues after essential oils extraction (Saha & Basak,
2020).
In this work, the hydrolysate of Melaleuca alternifolia residue after
essential oils extraction was used as the substrate for xanthan gum
production by Xanthomonas campestris. Generally, xanthan gum in the
fermentation broth can be simply recovered by alcohol-precipitation
with high efficiency and easy operation (Li et al., 2016; Nejadman­
souri et al., 2020; Palaniraj & Jayaraman, 2011). However, it is worth
noting that various alcohol insoluble impurities exist in the Melaleuca
alternifolia residue hydrolysate as found in our pre-research (unpub­
lished), and therefore merely alcohol-precipitation is not enough for
obtaining xanthan gum from this substrate. Obviously, the structure and
rheological properties of xanthan gum will be affected greatly due to the
low purity.
In this situation, exploration of suitable downstream processing
(separation and purification) is very important for xanthan gum pro­
duction from Melaleuca alternifolia residue hydrolysate. On the one
hand, the suitable downstream processing can be found by investigating
the effect of different downstream processing on the performance of
purified xanthan gum. On the other hand, based on the suitable down­
stream processing, the accurate evaluation on the efficiency of this
bioconversion could be performed to show its potential for large-scale
application. Considering the above discussion, various downstream
processing steps were applied to separate and purify xanthan gum from
Melaleuca alternifolia residue hydrolysate, and the effect of downstream
processing on the structure and rheological properties of xanthan gum
were evaluated systematically for the first time in this study. Overall,
this study can offer some important information for efficient and effec­
tive purification of xanthan gum with a good rheological performance
from low-cost Melaleuca alternifolia residue hydrolysate.
2. Methods and materials
2.1. Fermentation substrate preparation
Melaleuca alternifolia residue after essential oils extraction by steam
distillation was smashed (particle size≤0.45 mm) and dried before
usage. Then, the residue was hydrolyzed by 2% (w/v) sulphuric acid at
121 ◦ C for 1 h, and the hydrolysate was recovered by vacuum filtration.
Then, the hydrolysate was neutralized by calcium oxide to pH around
9–11 and recovered by vacuum filtration. After that, the pH of the hy­
drolysate was adjusted to around 5–6 by sulphuric acid and recovered by
vacuum filtration. Subsequently 1% (w/v) activated carbon was added
into the hydrolysate and stirred in 80 ◦ C water bath for 1 h, and then the
hydrolysate was recovered by vacuum filtration. After the above treat­
ments, the Melaleuca alternifolia residue hydrolysate was used for xan­
than gum production. The sugars concentration of this substrate was
15.7 g/L.
2.2. Xanthan gum production
Xanthomonas campestris HC003 (Laboratory of Food Science,
Guangdong Pharmaceutical University) was used for xanthan gum
production in this study. Firstly, the pre-culture was performed on pre-
cultivation medium (g/L, glucose 5, xylose 5, yeast extract 3, peptone
2
Food Hydrocolloids 132 (2022) 107838
10, NaCl 5, initial pH 7.0) at 30 ◦ C and 200 rpm for 48 h. Subsequently
10% (v/v) seed culture was translated into 30 mL fermentation medium
(Melaleuca alternifolia residue hydrolysate, pH 7.0) in 250 mL conical
flask, and the fermentation was carried out at 30 ◦ C and 200 rpm. The
fermentation broth was taken out periodically for the measurement of
biomass, xanthan gum yield, and residual sugars concentration. All
fermentation was carried out in duplicate and the results were expressed
as the averages.
2.3. Biomass and residual sugars concentration determination, and
alcohol-precipitation of crude xanthan gum
The cells of X. campestris were separated from the fermentation broth
by centrifugation at 10610×g for 10 min. After that, the precipitate was
washed by deionised water and recentrifuged at 10610×g for 10 min.
Then, the biomass was determined by the dry cell weight after lyophi­
lization. The residual sugars concentration in the cell-free supernatant of
fermentation broth after centrifugation was measured by the DNS
method (Miller, 1959). To obtain xanthan gum, the cell-free supernatant
was added with three volumes (v/v) of ethanol and maintained at
− 20 ◦ C for 1 h and then centrifuged at 10610×g for 15 min. The pre­
cipitation of crude xanthan gum, including both pure xanthan gum and
other alcohol insoluble impurities present in the Melaleuca alternifolia
residue hydrolysate, was then further dried by lyophilization.
2.4. Further xanthan gum separation and purification
After alcohol-precipitation and drying, the crude xanthan gum was
further separated and purified by three different methods. The first
method was acid-precipitation. In detail, the dry alcohol-precipitate
(crude xanthan gum) was suspended in hydrochloric acid solution (pH
2.0) and shaken at 800 rpm at room temperature for 0.5 h to remove acid
soluble impurities. Subsequently the acid insoluble precipitate (purified
xanthan gum) was recovered by centrifugation at 10610×g for 15 min
and then lyophilized. The second method was alkali-precipitation. In
detail, the dry alcohol-precipitate (crude xanthan gum) was suspended
in 0.5 M sodium hydroxide solution and shaken at 800 rpm at room
temperature for 0.5 h to remove alkali soluble impurities. Subsequently
the alkali insoluble precipitate (purified xanthan gum) was recovered by
centrifugation at 10610×g for 15 min and then lyophilized. The third
method was dialysis. In detail, the crude xanthan gum was dissolved in
deionised water and then dialyzed with an 8000 Da dialysis membrane
against deionised water for 2 d to remove the small molecule impurities.
The remaining xanthan gum solution was then lyophilized to obtained
dry purified xanthan gum. For each treatment, the polysaccharide
(xanthan gum) content was measured by the phenol-sulphuric acid
method (DuBois, Gilles, Hamilton, Rebers, & Smith, 1956). After further
separation and purification, the xanthan gum yield was expressed as the
amount of dry purified xanthan gum in per liter fermentation broth.
2.5. Structure and rheological characterization
The morphology of xanthan gum samples was observed by a field
emission scanning electron microscope (SEM, JSM-7610F PLUS, JEOL,
Japan) operated at a voltage of 15 kV after spray-gold treatment. The
elemental analysis of xanthan gum samples was carried out by an energy
dispersive spectrometer (EDS, Ultim Max 40, Oxford Instruments, UK).
The functional groups of xanthan gum samples were measured by a
Fourier transform infrared spectroscopy (FT-IR, Thermo Scientific
Nicolet IS5, Thermo Fisher Scientific, USA) in a wavenumber ranging
from 500 to 4000 cm− 1. The thermogravimetric analysis (TGA) was
carried out by a TA SDT 650 (TA Instruments, USA) heated from room
temperature to 600 ◦ C with a heating rate of 10 C/min under nitrogen
◦
atmosphere (flow rate of 100 mL/min).
The rheological characterization of xanthan gum solutions was
measured by an MCR-102 rheometer (Anton Paar, Austria). The steady-
Z.-X. Li et al.
Fig. 1. Polysaccharide (xanthan gum) content and residual content of xanthan
gum products after different treatments. (a) Crude xanthan gum; (b) xanthan
gum after acid-precipitation; (c) xanthan gum after alkali-precipitation (d)
xanthan gum after dialysis.
state viscosity curves were obtained at a temperature ranging from 25 to
45 ◦ C with a shear rate ramp from 0.01 to 1000 1/s. A strain sweep test
was applied to measure the linear viscoelastic properties at a frequency
of 10 rad/s in a strain range of 0.01–100%. A frequency sweep test was
used to determine the dynamic viscoelastic properties at a constant
strain in the linear viscoelastic region (LVE) obtained from the strain
sweep test with frequencies from 0.1 to 100 rad/s. At the same time, a
complex viscosity measurement was carried out in LVE. All the rheo­
logical characterization was carried out in triplicate and the results were
expressed as the averages.
Fig. 2. SEM micrographs of xanthan gum products after different treatments. (A) Crude xanthan gum; (B) xanthan gum after acid-precipitation; (C) xanthan gum
after alkali-precipitation (D) xanthan gum after dialysis.
3
Food Hydrocolloids 132 (2022) 107838
3. Results and discussion
3.1. Effect of downstream processing on the purity of xanthan gum
After fermentation, xanthan gum generated by X. campestris needs to
be recovered from the fermentation broth. Although the technologies of
xanthan gum separation and purification have been studied for many
decades, alcohol-precipitation is usually applied in most situations for
its advantages such as high efficiency, and ease of operation (Borges,
Moreira, Vendruscolo, & Ayub, 2008; Li et al., 2016; Nejadmansouri
et al., 2020; Palaniraj et al., 2011; Yoo & Harcum, 1999; Zhang & Chen,
2010).
However, it is worth noting that alcohol-precipitation might not be
completely suitable for xanthan gum separation and purification from
the fermentation substrate that also contains various alcohol insoluble
impurities because both xanthan gum and alcohol insoluble impurities
will be precipitated after adding alcohol into the fermentation broth. As
for the Melaleuca alternifolia residue hydrolysate after detoxification
without adding microorganisms for fermentation, various alcohol
insoluble compounds were generated after adding three volumes (v/v)
of ethanol (the ratio of ethanol addition was the same as the alcohol-
precipitation of crude xanthan gum from fermentation broth), suggest­
ing that merely using alcohol-precipitation for xanthan gum separation
and purification might not be enough, and more procedures were
necessary for further xanthan gum separation and purification from
alcohol insoluble impurities. In addition to alcohol-precipitation, acid-
precipitation, alkali-precipitation, and dialysis also have the potential
for xanthan gum separation and purification (Bilanovic, Shelef, & Green,
1994; Smith, 1984; Towle, 1977). Therefore, to evaluate their possibility
for further xanthan gum separation and purification, all these three
procedures were applied to treat the crude xanthan gum (contained both
xanthan gum and alcohol insoluble impurities) after
alcohol-precipitation.
To evaluate the performance of further xanthan gum separation and
purification, the effect of different treatments on polysaccharide
Z.-X. Li et al.
Fig. 3. EDS analysis of xanthan gum products after different treatments. (A) Crude xanthan gum; (B) xanthan gum after acid-precipitation; (C) xanthan gum after
alkali-precipitation (D) xanthan gum after dialysis.
(xanthan gum) content of xanthan gum products should be measured
firstly and it is the most important consideration for choosing the suit­
able method. As shown in Fig. 1, without further treatment, the poly­
saccharide content of crude xanthan gum after initial alcohol-
precipitation was very low (48.8%). Obviously, this crude xanthan
gum was not suitable as the final product and further separation and
purification was necessary. After different treatments (acid-precipita­
tion, alkali-precipitation, and dialysis), the polysaccharide content
(88.2%) after dialysis was much higher than those (66.2% and 62.7%,
respectively) after acid-precipitation, and alkali-precipitation, showing
that dialysis was the better choice for further xanthan gum separation
and purification. Overall, after dialysis, the high polysaccharide content
(close to 90%) of purified xanthan gum made it much more suitable to be
the final product.
To be an ideal method for xanthan gum separation and purification,
it should keep xanthan gum and remove impurities as much as possible.
Therefore, in addition to polysaccharide content, the residual content of
crude xanthan gum products after its further separation and purification
was also measured. As shown in Fig. 1, the residual content after alkali-
precipitation was higher than that after acid-precipitation, indicating
that the amount of acid soluble impurities was more than that of alkali
soluble impurities. Compared with acid-precipitation, the residual
content after dialysis was even lower, suggesting both acid soluble and
alkali soluble impurities could be removed by dialysis.
4
Food Hydrocolloids 132 (2022) 107838
3.2. Effect of downstream processing on the structure of xanthan gum
3.2.1. SEM analysis
The morphology of xanthan gum has been extensively reported in
many previous studies that the native xanthan gum usually has an
irregular sheet-like or granular structure (Ahmad & Hasan, 2017;
Kumar, Deepak, Sharma, Srivastava, & Kumar, 2017; Lei et al., 2021;
Nejadmansouri et al., 2020; Wang, Xiang, Li, Zhang, & Bai, 2021). It is
worth noting that many modifications including the physical and
chemical methods will alter the morphological structure of xanthan gum
and therefore ameliorate the properties of xanthan gum (Bhatia, Ahuja,
& Mehta, 2015; He et al., 2022; Wang et al., 2021). Besides improving
the xanthan gum purity, like these physical and chemical methods,
further downstream processing might also change the morphological
structure of xanthan gum.
The SEM morphologies of xanthan gum after different downstream
processing are shown in Fig. 2. As it depicted, the microstructure of
crude xanthan gum was irregular granules and many tiny particles
(possible impurities) covered the surface of these granules (Fig. 2A).
After acid-precipitation, the irregular granules of purified xanthan gum
as shown in the SEM image were much smaller than those of crude
xanthan gum (Fig. 2B), possibly due to the removal of acid soluble im­
purities. While for alkali-precipitation, some porous microstructure with
rougher surface possibly caused by the removal of alkali soluble impu­
rities was observed, but the irregular granules of purified xanthan gum
did not become smaller (Fig. 2C). Overall, the morphological structure
Z.-X. Li et al. Food Hydrocolloids 132 (2022) 107838

(Fig. 3A). Among these other elements, the contents of S (7.29%), Ca

(7.17%), and K (3.69%) were relatively high. The elements S and Ca
mainly come from the existence of CaSO4 generated after the neutrali­
zation of the Melaleuca alternifolia residue hydrolysate, while the
element K might come from the hydrolysis of the Melaleuca alternifolia
residue. After acid-precipitation, the contents of K, Na, Mg, Si, and Cl
reduced totally but the contents of S and Ca increased (Fig. 3B) possibly
because HCl cannot react with CaSO4. As for alkali-precipitation,
although the S content reduced greatly, the contents of O and Na
increased greatly due to the adding of NaOH, and the alkali-
precipitation showed little effect on reducing Ca content (Fig. 3C).
Thus, alkali-precipitation seems not suitable for xanthan gum further
separation and purification. As shown in Fig. 3D, dialysis showed a good
performance on the removal of the impurities that the content of all the
elements including S, Ca, K, Na, Mg, Si, and Cl became much lower than
those of crude xanthan gum. The EDS results also proved that dialysis
was suitable for further xanthan gum separation and purification.

3.2.3. FT-IR analysis

The effect of different downstream processing (acid-precipitation,
Fig. 4. FT-IR spectrum of xanthan gum products after different treatments. (a)
Crude xanthan gum; (b) xanthan gum after acid-precipitation; (c) xanthan gum
alkali-precipitation, and dialysis) on the functional groups of xanthan
after alkali-precipitation (d) xanthan gum after dialysis. gum was measured by FT-IR in the wavenumber of 4000–500 cm− 1. As
shown in Fig. 4, the characteristic peaks of xanthan gum which were
reported in many previous studies (Kang et al., 2019; Liu et al., 2021;
of xanthan gum after both acid-precipitation and alkali-precipitation
Nejadmansouri et al., 2020; Sujithra, Deepika, Akshaya, & Ponnusami,
was irregular granules, which was similar to the crude xanthan gum,
2019) could be observed in the FT-IR image of the xanthan gum samples
suggesting that both treatments had merely a small influence on the
after different treatments. In detail, the peaks appeared from 3405 to
morphological structure of xanthan gum. However, the acid-
3449 cm− 1 corresponded to the hydroxyl (-OH) stretching, the peaks of
precipitation and alkali-precipitation might chemically modify (e.g.,
2921–2929 cm− 1 corresponded to the C–H stretching from aldehyde,
deacetylation, depyruvation, etc.) xanthan gum as systematically
CH2 or CH3 groups, the peaks of 1618–1621 cm− 1 corresponded to the
introduced in the recent review article (Riaz et al., 2021), and thus more – O), the peaks of 1405–1413 cm− 1
stretching of the carbonyl group (C–
attention should be paid to their effect on the molecular architecture of
corresponded to the C–H deflection angle, and the peaks of 1062–1116
xanthan gum.
cm− 1 corresponded to the stretching of C–O and acetal groups. It is
Interestingly, compared with acid-precipitation and alkali-
worth noting that the peaks that appeared from 1726 to 1729 cm− 1
precipitation, the dialysis treatment had a much greater influence on
corresponding to acetyl C– – O stretching vibration (Wang, Zhu, Yang, &
the morphological structure of xanthan gum. As shown in Fig. 2D, the
Lu, 2013) could be found in the FT-IR spectra of crude xanthan gum,
microstructure of purified xanthan gum after dialysis changed from the
xanthan gum after acid-precipitation, and xanthan gum after dialysis,
irregular granules to the intertwined filaments with smooth surfaces. In
but disappeared in the FT-IR spectrum of xanthan gum after
the previous study, some special rheological properties such as crossover
alkali-precipitation. In the previous study, alkali (NaOH) treatment was
of storage modulus (G′ ) and loss modulus (G′′ ), higher dynamic viscosity
applied for xanthan gum modification (deacetylation) and the FT-IR
than steady-state shear viscosity (η<|η*|, departure from Cox-Merz
results showed that the peak value of acetyl C– – O stretching vibration
rule), etc. could be found for the semiflexible wormlike xanthan gum
absorption decreased gradually as the NaOH concentration was higher,
sample, which was different from the rheological properties of rodlike
and even disappeared due to the full deacetylation of xanthan gum
xanthan gum sample (Lee & Brant, 2002b). Recently, it was found that
(Wang et al., 2013). Similarly, the peak disappearance from 1726 to
industry-scale microfluidization treatment, an innovative superfine
1729 cm− 1 in the FT-IR spectrum of xanthan gum after
pulverization technology, could form the similar filamentous structure
alkali-precipitation was possibly caused by the deacetylation of xanthan
of xanthan gum as shown in Fig. 2D, and this structure was related to the
gum. Although strong acid treatment with high temperature and long
changing rheological properties of xanthan gum from non-Newtonian
period can hydrolyze xanthan gum and then the composition of xanthan
flow to Newtonian flow (He et al., 2022). As a result, the downstream
gum can be analyzed by high-performance liquid chromatography (Tait,
processing of dialysis after alcohol-precipitation had potential to modify
Sutherland, & Clarke-Sturman, 1990), weak/mild acidic environment
xanthan gum and alter its rheological properties. On the other hand, the
(e.g., decreasing pH caused by cold plasma treatment) might not change
smooth surfaces of intertwined filaments as shown in Fig. 2D demon­
the basic infrared spectra of xanthan gum (Bulbul, Bhushette, Zambare,
strated that most impurities existing in crude xanthan gum have been
Deshmukh, & Annapure, 2019). This can partly explain the little influ­
removed after dialysis. According to the above results, dialysis after
ence of acid-precipitation with relatively mild conditions (diluted hy­
alcohol-precipitation can not only separate and purify xanthan gum
drochloric acid treatment at room temperature for a short period) on the
from the Melaleuca alternifolia residue hydrolysate, but also modify the
FT-IR spectra of xanthan gum in the present study. Compared with
microstructure of xanthan gum, therefore it is an attractive downstream
alkali-precipitation, dialysis mainly removed the small molecule impu­
processing for xanthan gum production from the Melaleuca alternifolia
rities, and thus the peaks that appeared from 1726 to 1729 cm− 1 could
residue hydrolysate.
be observed in the FT-IR spectrum of xanthan gum after dialysis (Pinto,
Furlan, & Vendruscolo, 2011). Overall, although the downstream pro­
3.2.2. EDS analysis
cessing of dialysis after alcohol-precipitation had a great influence on
To further evaluate the performance of different downstream pro­
the microstructure of xanthan gum, it affected little on the functional
cessing, the element composition of crude and purified xanthan gum was
groups of xanthan gum.
analyzed by EDS (Fig. 3). Without further separation and purification, in
addition to its main elements (C and O), the crude xanthan gum con­
3.2.4. TGA analysis
tained various other elements such as S, Ca, K, Na, Mg, Si, and Cl
The thermal degradation behaviors of xanthan gum after different

5
Z.-X. Li et al. Food Hydrocolloids 132 (2022) 107838

Fig. 5. TGA and DTG spectra of xanthan gum products after different treatments. (a) Crude xanthan gum; (b) xanthan gum after acid-precipitation; (c) xanthan gum
after alkali-precipitation (d) xanthan gum after dialysis.

downstream processing were evaluated (Fig. 5). Typically, there are

three stages in the TGA curves of xanthan gum (Bulbul et al., 2019). In
this study, this “three stages” thermal degradation could also be
observed for the crude xanthan gum after alcohol-precipitation. For the
first stage (room temperature to 180 ◦ C), the weight loss was mainly due
to dehydration and degradation of some volatile compounds (impu­
rities). For the second stage (180–385 ◦ C), a fast weight loss happened
caused by the pyrolytic depolymerization. The maximum degradation
rate of crude xanthan gum was 282.49 ◦ C. Finally, after the third stage
(385–600 ◦ C), the remaining percentages of crude xanthan gum sample
was 53.21%, indicating that many impurities existed in the crude xan­
than gum. For the xanthan gum obtained after acid-precipitation, more
stages rather “three stages” thermal degradation was observed. It was
reported that polysaccharides would be degraded after acid treatment
(Trygg, Beltrame, & Yang, 2019). Similarly, it was possible that xanthan
gum was ruptured after acid-precipitation, and therefore its TGA curve
was more complex and contained various stages. For the xanthan gum
obtained after alkali-precipitation, the “three stages” thermal degrada­
tion was not obvious in the TGA curve, and a gradual thermal degra­
dation was observed. In the previous study, acetyl groups had a great Fig. 6. Evolution of biomass, xanthan gum yield, and residual sugars concen­
influence on the thermal degradation behavior of xanthan gum that tration during the fermentation process on Melaleuca alternifolia residue
acetylation on xanthan gum chain could improve its thermal stability hydrolysate.
(Endo, Setoyama, Yamamoto, & Kadokawa, 2015) while breaking of
acetyl groups would make the decomposition temperature of xanthan gum in the following study.
gum to be lower (Sara, Yahoum, Lefnaoui, Abdelkader, &
Moulai-Mostefa, 2020). As introduced above, alkali-precipitation
caused deacetylation of xanthan gum (Wang et al., 2013), and there­ 3.3. Evaluation on fermentation performance based on the suitable
fore reduced the thermal stability of xanthan gum, and decreased the downstream processing for xanthan gum production
temperature of its depolymerization. It is worth noting that the
remaining percentages after thermal degradation of xanthan gum after Although the cost of Melaleuca alternifolia residue hydrolysate is low
acid-precipitation and alkali-precipitation (53.29% and 49.76%, enough to be a fermentation substrate for xanthan gum production, but
respectively) were still close to that of crude xanthan gum, suggesting its potential negative effect on the fermentation should not be ignored.
that many impurities still existed in the xanthan gum. For the xanthan On the one hand, antimicrobial activity is an important application of
gum obtained after dialysis, the “three stages” thermal degradation was Melaleuca alternifolia essential oils (Battisti, Caon, & Machado de Cam­
even more obvious than that of crude xanthan gum. During the first pos, 2021). Although most Melaleuca alternifolia essential oils have been
stage, the weight loss mainly happened from room temperature to extracted after steam distillation, some antimicrobial compounds might
100 ◦ C due to dehydration. For the second stage (pyrolytic depolymer­ still exist in the Melaleuca alternifolia residue. On the other hand, after
ization of xanthan gum), the maximum xanthan gum degradation rate acid hydrolysis of lignocellulosic biomass like the Melaleuca alternifolia
was greater (301.33 ◦ C vs. 282.49 ◦ C). And finally, the remaining per­ residue in this study, various complex and toxic components such as
centage of xanthan gum sample was much lower (22.94% vs. 53.21%). furan and aromatic compounds will also be generated in the lignocel­
These results indicated that many impurities have been removed after lulosic hydrolysates, which will inhibit the growth and products syn­
dialysis. Again, the TGA results showed that dialysis was a promising thesis of microorganisms (Palmqvist & Hahn-Hägerdal, 2000). Thus, it is
method for further separation and purification of xanthan gum. necessary to evaluate the fermentation performance of bioconversion
Considering the above results and discussion, dialysis showed much from Melaleuca alternifolia residue hydrolysate to xanthan gum. How­
better performance for xanthan gum separation and purification than ever, because the crude xanthan gum contains many impurities after
acid-precipitation and alkali-precipitation. Additionally, dialysis is a traditional alcohol-precipitation from Melaleuca alternifolia residue hy­
green technology without the potential pollution from acid and alkali. drolysate. In this situation, it is difficult to learn the fermentation per­
As a result, dialysis after alcohol-precipitation was chosen as the formance of xanthan gum production due to the difficulty in accurate
downstream processing for the separation and purification of xanthan measurement of xanthan gum yield.
As introduced in the previous sections, xanthan gum with high purity

6
Z.-X. Li et al. Food Hydrocolloids 132 (2022) 107838

Table 1
Comparison of xanthan gum yield on carbon sources consumption.
Carbon sources Xanthan gum yield on References
carbon sources
consumption (%)
Glucose 56 Mohsin et al. (2021)
Xylose 53 Mohsin et al. (2021)
Cheese whey 42 Niknezhad, Asadollahi, Zamani,
(lactose) Biria, and Doostmohammadi
(2015)
Crude glycerol 27.5 Wang et al. (2016)
Sugarcane molasses 22.4 Khosravi-Darani et al. (2009)
Kitchen waste 67.07 Li et al. (2016)
Melaleuca 44.2 This study
alternifolia
residue
hydrolysate
can be obtained with the downstream processing of dialysis after
alcohol-precipitation. Based on this technology, the xanthan gum yield
can be measured accurately, and therefore the fermentation process of
xanthan gum production is evaluated in this part (Fig. 6). In this study,
simply treated by calcium oxide neutralization and activated carbon
adsorption, the Melaleuca alternifolia residue hydrolysate was used for
xanthan gum production. As shown in Fig. 6, there was almost no lag
phase existing during this bioprocess that during the first 12 h of
fermentation, the synthesis of xanthan gum was quickly, suggesting that
the fermentation environment of the Melaleuca alternifolia residue hy­
drolysate showed little inhibition on the biosynthesis of xanthan gum by
X. campestris. Similarly, the sugars utilization was very fast during the
first 12 h of fermentation, showing that X. campestris had adapted the
fermentation environment of the Melaleuca alternifolia residue hydro­
lysate quickly. The bioprocess of xanthan gum production has also been
evaluated in some previous studies with different fermentation sub­
strates and in many cases, the xanthan gum synthesis was usually later
than that of cell growth of X. campestris (Li et al., 2016; Wang, Wu, Zhu,
& Zhan, 2016). Interestingly, in this study, the cell growth and xanthan
gum synthesis of X. campestris were simultaneous from the first 12 h of
fermentation as the consumption of fermentable sugars. After 12 h of
fermentation, the biomass and xanthan gum yield continued to increase
and the xanthan gum synthesis became even faster during 12–24 h of
fermentation. After 24 h of fermentation, the cell growth and xanthan
gum synthesis of X. campestris were slower and the biomass (1.7–2.2
g/L) and xanthan gum yield (4.8–5.0 g/L) were stable after 36 h and 48
h of fermentation, respectively due to the exhaustion of fermentable
sugars in the medium. Overall, the xanthan gum yield was 5.0 g/L after
48 h of fermentation, and the xanthan gum yield on sugars consumption
was 44.2% (g/g). The performance of this bioprocess was compared
with other xanthan gum productions from various substrates. As shown
in Table 1, both the typical lignocellulose-derived fermentable sugars
(glucose, xylose) (Zhou, Liu, & Zhao, 2021) and low-cost substrates
(cheese whey, crude glycerol, sugarcane molasses, and kitchen waste)
can be used as media for xanthan gum production. Although the xanthan
gum yield on carbon sources (sugars) consumption in this study was
lower than that of xanthan gum production from glucose, xylose, and
kitchen waste, it was higher than that of xanthan gum production from
cheese whey, crude glycerol, and sugarcane molasses. By the compari­
son and considering the low cost of Melaleuca alternifolia residue hy­
drolysate and the relatively high xanthan gum yield on sugars
consumption, this bioprocess is efficient and promising for large-scale
application.
3.4. Effect of downstream processing on the rheological properties of
xanthan gum
3.4.1. Flow curves
As a classical microbial polysaccharide that has been widely applied
as a thickener and stabilizer in the food industry, xanthan gum is famous
for its rheological properties in its solution. Generally, xanthan gum
solution has high viscosity in low shear and low concentration, and high
stability in the environment of heat, acid, and alkali (Faria et al., 2011;
Tian et al., 2015; Wu et al., 2021). As the pseudoplastic behavior is an
important characteristic of xanthan gum solution, the flow curves of the
xanthan gum solution (1%) were evaluated firstly at 25 ◦ C for both the
crude xanthan gum without further purification and the xanthan gum
purified by dialysis after alcohol-precipitation (Fig. 7A). As shown in
Fig. 7A, both the solutions of crude xanthan gum and purified xanthan
gum showed an obvious pseudoplasticity. Namely, the xanthan gum
solutions had high viscosity at low shear rates possibly caused by
polymer entanglements and form a partial self-associated conformation;
however, as the shear rate increased, the weak structure of polymer
entanglements was changed to orienting polymer chains, and therefore
an obvious shear-thinning behavior (the viscosity of xanthan gum so­
lution decreased with the higher shear rate) was observed (Jang, Zhang,
Chon, & Choi, 2015; Xu, Xu, Liu, Chen, & Gong, 2013). Generally, the
characteristic of shear-thinning in the solution of polysaccharides is
beneficial as dysphagia diets (Wei, Guo, Li, Ma, & Zhang, 2021). It is
worth noting that the viscosity of the solution of crude xanthan gum was
much lower than that of xanthan gum purified by dialysis after
alcohol-precipitation especially in low shear rate (Fig. 7A), indicating
that the impurities in the crude xanthan gum affected the viscosity of its
solution greatly.
Typically, a Newtonian plateau exists for the solutions of many food
gums/hydrocolloids such as guar gum and tara gum under low shear
rate (e.g., <1 s− 1) as shown in the flow curves (Kongjaroen, Meth­
acanon, & Gamonpilas, 2022). In contrast, this Newtonian plateau is
hardly observed in the solution of xanthan gum (He et al., 2022) and
even in the solutions of hybrid containing xanthan gum with the same
shear rate (Kongjaroen et al., 2022), and usually, the Newtonian plateau
can be only found for the xanthan gum solution under extremely low
shear rate (e.g., <10− 2 s− 1) (Yahoum, Moulai-Mostefa, & Le Cerf, 2016).

Fig. 7. (A) Flow curves of solutions (1%) of (a) purified xanthan gum and (b) crude xanthan gum; (B) Flow curves of solutions (1%) of purified xanthan gum under
different temperatures.

7
Z.-X. Li et al.
Table 2
The rheological parameters obtained by Cross model for the solutions of both
crude and purified xanthan gum.
T/oC η0 /mPa⋅s η∞ /mPa⋅s k n R2
Crude xanthan gum 25 803 2.092 0.483 0.672 0.998
Purified xanthan 25 23298 8.714 3.710 0.769 0.992
gum 35 12002 6.742 2.314 0.754 0.994
45 6785 3.357 1.448 0.733 0.998
As shown in Fig. 7A, a Newtonian plateau existed for the solution of
crude xanthan gum under a low shear rate (1 s− 1), which was similar to
the laws of the solutions of guar gum and tara gum (Kongjaroen et al.,
2022), indicating that the impurities in the crude xanthan gum changed
the laws of flow curves of its solution. Interestingly, a Newtonian plateau
was also found for the solution of purified xanthan gum with a relatively
high shear rate (10− 1 s− 1) when compared with the previous study
(Yahoum et al., 2016). Generally, no Newtonian plateau was observed
for xanthan gum solution under this shear rate. The relationship be­
tween the structure and rheological properties of xanthan gum have
systematically learned in many previous studies, and the structure of
xanthan gum can be reflected from its rheological properties (Ross-­
Murphy, 1995; Shatwell, Sutherland, Ross-Murphy, & Dea, 1990).
Usually, xanthan gum has a typical weak-gel network in a salt solution
(Ross-Murphy, 1995), but this situation might be different in a solution
without salt. In this study, the xanthan gum solution was prepared by
deionised water (in absence of salt), and the Newtonian plateau existing
in the flow curves indicated that the purified xanthan gum had an
entanglement network (Shatwell et al., 1990). Obviously, the alteration
of rheological properties was due to the variation of xanthan gum
structure, and this could also be found from the research on the visco­
elasticity (discussed in section 3.4.2). It is worth noting that some
modifications such as microfluidization treatment and high-pressure
homogenization can change xanthan gum solution from
non-Newtonian flow to Newtonian flow (Eren, Santos, & Campanella,
2015; He et al., 2022). Obviously, the downstream processing of dialysis
after alcohol-precipitation could fulfill a similar effect as these advanced
modification technologies.
To date, various models have been developed to fit the rheological
results, and the Cross model was used widely to fit the flow curves of
non-Newtonian fluids (Cross, 1965; Eren et al., 2015; Kongjaroen et al.,
2022; Xu et al., 2013; Yahoum et al., 2016). To depict the rheological
laws of crude and purified xanthan gum more systematically, the
rheological result of flow curves was further fitted to the classical Cross
model (Cross, 1965; Pelletier, Viebke, Meadows, & Williams, 2001;
Yahoum et al., 2016):
η0 − η∞
η= + η∞
1 + kγ̇n
where η0 is the zero-shear viscosity, η∞ is the infinite-shear viscosity, γ̇ is
the shear rate, k is the consistency factor, and n is the flow index.
The results of flow curves exhibited a good fit to the Cross model with
the correlation coefficients (R2) totally higher than 0.99 and the rheo­
logical parameters acquired from the Cross model are summarized in
Table 2. As shown in Table 2, the η0 of the solution of purified xanthan
gum was much higher than that of the solution of crude xanthan gum
(23298 mPa s vs. 803 mPa s), again showing that the viscosity of xan­
than gum was affected greatly by the impurities in the crude xanthan
gum. As discussed in the previous sections, the impurities in the xanthan
gum reduced the purity of xanthan gum, and thus the actual xanthan
gum concentration was lower in the solution (1%) of crude xanthan gum
than that in the solution (1%) of purified xanthan gum. This can partly
explain the different η0 of the solutions of purified xanthan gum and
crude xanthan. Moreover, as the crude xanthan gum might contain
various salts according to the EDS analysis (Fig. 3A), the existence of
these salts in the xanthan gum solution also decreased the viscosity by
8
Food Hydrocolloids 132 (2022) 107838
Fig. 8. Strain sweep dependency of storage modulus (G′ ) and loss modulus (G′′ )
of solution (1%) of (a) purified xanthan gum and (b) crude xanthan gum.
the charge shielding effect (Alquraishi & Alsewailem, 2012). In addition
to the flow curves measured at 25 ◦ C, the flow curves of solutions of
purified xanthan gum were also evaluated at higher temperatures (35
and 45 ◦ C) and fitted by the Cross model (Fig. 7B and Table 1). The
temperature had an obvious influence on the pseudoplasticity of xan­
than gum solutions (Fig. 7). Typically, increasing temperature will cause
order-disorder transition of xanthan gum from an ordered state (helix)
to a disordered conformation (random coil) which involves the changes
of polymer network and dissociation of the side chains from the back­
bone (Bercea & Morariu, 2020; Pelletier et al., 2001). Thus, the viscosity
and pseudoplasticity of xanthan gum solution decrease with the higher
temperature. As shown in Table 1, all the n values in Table 1 were less
than 1, showing that all the samples tested were pseudoplastic fluids. It
is worth noting that the values of η0 , η∞ , k, and n of the solutions of
purified xanthan gum became lower as the temperature increased, again
showing that the viscosity and pseudoplasticity of xanthan gum solution
decreased with the higher temperature.
3.4.2. Viscoelasticity and complex viscosity
Besides flow curves, viscoelasticity is also an important rheological
property of xanthan gum solution that usually exhibits gel-like behavior
(Milas, Rinaudo, Knipper, & Schuppiser, 1990). The linear viscoelastic
behavior of the solutions (1%) of both crude and purified xanthan gum
was learned by a strain sweep measurement firstly. As shown in Fig. 8,
both the storage modulus (G′ ) and the loss modulus (G′′ ) changed little
with the variation of strain sweep. Interestingly, the downstream pro­
cessing of dialysis after alcohol-precipitation exhibited a great influence
on the G′ and G′′ of xanthan gum solutions that the G′ and the G′′ of
solution of purified xanthan gum were much higher than that of solution
of crude xanthan gum. Moreover, the G′′ was higher than G′ within the
strain range tested for the solution of crude xanthan gum, suggesting
that the solution of crude xanthan gum exhibited a typical viscous-like
behavior. Obviously, this linear viscoelastic behavior is quite different
from the typical xanthan gum solution which usually exhibits a
solid-like/gel-like behavior (Eren et al., 2015; Sara et al., 2020; Wei
et al., 2021). In contrast, after downstream processing of dialysis after
alcohol-precipitation, the G′ was totally higher than G′′ within the strain
range tested for the solution of purified xanthan gum, showing that the
elastic property was much more predominant than the viscous one
within the linear viscoelastic region (LVE). As a result, suitable down­
stream processing was necessary to obtain purified xanthan gum whose
solution has a solid-like/gel-like behavior.
For deeper learning the rheological property of the solution of
Z.-X. Li et al.
Fig. 9. (A) Frequency sweep dependency of storage modulus (G′ ) and loss modulus (G′′ ) of solution (1%) of purified xanthan gum; (B) Dependency of steady-state
shear viscosity (η) and complex viscosity (|η*|) of solution (1%) of purified xanthan gum versus shear rate and frequency respectively.
purified xanthan gum, the dynamic viscoelastic behavior of the xanthan
gum solution (1%) was measured in a constant strain in the LVE. As
shown in Fig. 9A, the slope of storage modulus (G′ ) was higher than that
of loss modulus (G′′ ). With low frequency, G′′ was higher than G′ , and as
the frequency became higher, G′ was higher than G′′ with high fre­
quency. Therefore, two regions were separated by the crossover point,
and the critical frequency (ωc) and relaxation time (tR = 2π/ωc) were
0.734 rad/s, and 8.560 s, respectively. For the region with the frequency
lower than the crossover point, the xanthan gum solution had viscous-
like behaviors (G’’> G′ ) while for the region with the frequency
higher than the crossover point, the xanthan gum solution had solid-
like/gel-like behavior (G’> G′′ ). The relationship between the dynamic
viscoelastic properties and structure of xanthan gum has been elucidated
in many previous studies (Lee & Brant, 2002a; Lee et al., 2002b).
Different from our results, no crossover of G′′ and G′ was observed for
rodlike xanthan gum sample at various salt concentrations (Lee et al.,
2002a). Moreover, it was reported in many studies that G′ was always
higher than G′′ for a native xanthan gum over the entire frequency range
in dynamic viscoelasticity measurement (Eren et al., 2015; Sara et al.,
2020; Yahoum et al., 2016). To obtain the similar rheological property
of dynamic viscoelasticity like this study (existence of the critical fre­
quency), suitable physical or chemical modifications (e.g., high-pressure
homogenization, carboxymethylation, and alkylation) were carried out
in these studies (Eren et al., 2015; Sara et al., 2020; Yahoum et al.,
2016). In contrast to the rodlike xanthan gum sample, the crossover of
G′′ and G’ could be observed for the semiflexible wormlike xanthan gum
sample (Lee et al., 2002b), and this dynamic viscoelastic property was
similar to our results. The dynamic viscoelastic properties indicated that
in addition to purification, the structure of xanthan gum could also be
modified by the downstream processing of dialysis after
alcohol-precipitation, and the structure alteration might result in the
change of rheological properties of xanthan gum solution.
Besides the frequency sweep test, the complex viscosity (dynamic
viscosity) of xanthan gum solution was also analyzed. Fig. 9B depicts the
relationship between steady-state shear viscosity (η) and complex vis­
cosity (|η*|) versus shear rate and frequency respectively. As can be
observed, |η*| was higher than η with the shear rates and frequencies
tested, and the deviation between |η*| and η became greater as the shear
rates and frequencies were higher. Obviously, the solution of purified
xanthan gum did not obey the Cox-Merz rules, where η≈|η*| at a given
shear rate and frequency (Cox & Merz, 1958). The deviation between |η
*| and η (η<|η*|) was mainly caused by the elastic gel-like structure
which is broken under steady-state shear but affected little under
oscillatory measurements (Martín-Alfonso, Cuadri, Berta, & Stading,
2018). Thus, the departure from Cox-Merz rule confirmed that the pu­
rified xanthan gum sample had a weak-gel structure, which was in good
accordance with other xanthan gum solution reported in the previous
study (Abson et al., 2014). As introduced previously, this special rheo­
logical property (η<|η*|, departure from Cox-Merz rule) could be
9
Food Hydrocolloids 132 (2022) 107838
observed for the semiflexible wormlike xanthan gum sample rather than
the rodlike xanthan gum sample (Lee et al., 2002b), again showing that
the downstream processing of dialysis after alcohol-precipitation could
modify the structure of xanthan gum and thus alter its rheological
properties.
4. Conclusions
To fulfill and learn the bioconversion from low-cost Melaleuca
alternifolia residue after essential oils extraction to high value-added
food additive xanthan gum, exploration of suitable downstream pro­
cessing is necessary. After systematical comparison, dialysis after
alcohol-precipitation was found to be suitable for xanthan gum sepa­
ration and purification, and based on this method, the bioconversion
process could be evaluated accurately. The effect of downstream pro­
cessing on the structure and rheological properties of xanthan gum was
systematically studied with a series of characterization including SEM,
EDS, FT-IR, TGA, and rheological analysis. In addition to xanthan gum
separation and purification, downstream processing of dialysis after
alcohol-precipitation also modified the structure of xanthan gum and
therefore affect the rheological properties of xanthan gum. Overall, the
purified xanthan gum with a good rheological performance could be
obtained efficiently and effectively from the low-cost Melaleuca alter­
nifolia residue hydrolysate, and therefore this substrate is attractive for
industrial xanthan gum production. Future studies should focus on the
scale-up of the downstream processing especially for exploring prom­
ising (efficient, cost-effective, reusable, and durable) material, equip­
ment, and technology for large-scale dialysis considering its relatively
high cost. Moreover, the application of xanthan gum obtained from the
low-cost Melaleuca alternifolia residue hydrolysate should be learned to
promote its industrialization.
CRediT authorship contribution statement
Zhi-Xuan Li: Conceptualization, Methodology, Validation, Formal
analysis, Investigation, Data curation, Writing – original draft, Writing –
review & editing. Jia-Yu Chen: Conceptualization, Validation, Formal
analysis, Investigation, Data curation, Writing – original draft, Visuali­
zation. Yi Wu: Investigation, Formal analysis, Visualization. Zhong-
Ying Huang: Investigation, Formal analysis. Shu-Ting Wu: Validation,
Investigation. Yun Chen: Supervision, Funding acquisition. Jing Gao:
Supervision, Funding acquisition. Yong Hu: Writing – review & editing,
Supervision, Project administration, Funding acquisition. Chao Huang:
Writing – original draft, Writing – review & editing, Methodology, Su­
pervision, Resources, Project administration, Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial
Z.-X. Li et al. Food Hydrocolloids 132 (2022) 107838

interests or personal relationships that could have appeared to influence Kang, Y., Li, P., Zeng, X., Chen, X., Xie, Y., Zeng, Y., et al. (2019). Biosynthesis, structure
and antioxidant activities of xanthan gum from Xanthomonas campestris with
the work reported in this paper.
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Research Foundation (2019A1515011843, 2019A1515010640, and extensional rheology of thickened liquids from commercial gum-based
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Project of Guangdong Province (2016TQ03N881), National Natural gum graft copolymer and its application for controlled release of highly water
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