International Journal of Food Science and Technology 2020 1

Original article
Mechanisms of breakdown of Haematococcus pluvialis cell wall
by ionic liquids, hydrochloric acid and multi-enzyme treatment

Zhang Ye,1 Xing-He Tan,1 Zhi-Wei Liu,1* Rana Muhammad Aadil,2 Yi-Cheng Tan1 &
Muhammad Inam-ur-Raheem2
1 College of Food Science and Technology, Hunan Agricultural University, Changsha 410128, China
2 National Institute of Food Science and Technology, University of Agriculture, Faisalabad 38000, Pakistan
(Received 31 January 2020; Accepted in revised form 24 March 2020)

Summary The robust cell wall structure of Haematococcus pluvialis (H. pluvialis) consists of polysaccharides and
tough non-hydrolysable sporopollenins, which makes it difficult to extract superpotent antioxidant, astax-
anthin from these cells. Therefore, breakdown of cell wall is a key step in the overall process of astaxan-
thin recovery. In this study, the mechanism of three well-established chemical techniques for cell
disruption of H. pluvialis cysts [ionic liquids (IL), hydrochloric acid (HCl) and multiple enzymes (multi-
enzyme, ME)] on deconstruction of the cyst cell wall of H. pluvialis was explored and characterised by
Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), nuclear magnetic resonance
(NMR) and gas chromatography–mass spectrometry (GC-MS) analyses. The results demonstrated that
the three cell wall breakdown techniques exhibited high extraction efficacies for the recovery of astaxan-
thin from H. pluvialis [IL (86.71
2.06%), HCl (80.52
2.28%) and ME (71.08
2.49%)]. However,
their performances on disrupting the trilayered cell walls of H. pluvialis were significantly different, which
were confirmed by distinct morphologies of the treated cell walls visualised by scanning electron micro-
scopy (SEM) and transmission electron microscopy (TEM). Meanwhile, the results of FTIR confirmed
that, to some extent, cellulose, hemicellulose and lignin in the cell walls were hydrolysed by HCl, IL and
ME treatments. However, ME exhibited a less hydrolytic effect on lignin than HCl and IL. Moreover,
XRD and NMR analyses implied that the amorphous region of cell wall was susceptible to hydrolysis/
breakdown by the three techniques.
Keywords Astaxanthin extractability, disruption mechanism, FTIR, Haematococcus pluvialis, structural analysis, X-ray diffraction.

Introduction skin diseases and cancer prevention (Lee et al., 2017;

Luthria et al., 2018).
Haematococcus pluvialis (H. pluvialis) is a unicellular
The robust structure of the trilaminar cell wall of
biflagellate microalga that forms a mature red-coloured,
H. pluvialis makes this microalga remarkably resis-
non-motile resting cell, namely an aplanospore (cyst)
tant to physical and chemical cell disruptions and
under unfavourable environmental conditions. Astaxan-
complicates the extraction process of astaxanthin
thin is abundantly accumulated in the aplanospores as a
from these cyst cells. Cell disruption techniques
mixture of monoesters and diesters, reaching up to 5%
such as ionic liquids (IL), hydrochloric acid (HCl),
of dry weight of algae (Khoo et al., 2019). As a lipid-sol-
ultrasound, high-pressure homogenisation and dif-
uble keto-carotenoid with a strong antioxidation effect,
ferent multi-enzyme (ME) treatments have been pre-
astaxanthin exhibits a stronger antioxidant potential
viously applied to facilitate the extraction of
than b-carotene and vitamin E due to its hydroxyl and
astaxanthin (Greenly & Tester, 2015). Among these
carbonyl functional groups (Liu et al., 2016; Fakhri
techniques, chemical cell wall breakdown techniques
et al., 2018). Astaxanthin is widely used in medicine,
(IL, HCl and ME) exhibit a great potential for cell
health products, cosmetics and aquaculture because of its
wall disruption and high-efficiency astaxanthin
enormous therapeutic benefits, such as immune adjust-
extraction. Enzyme-assisted extraction has been
ment; anti-inflammatory and antiobesity properties; and
regarded as a potentially feasible means for H. plu-
*Correspondent: E-mail: zwliu@hunau.edu.cn vialis extraction, and the results suggested that the

doi:10.1111/ijfs.14582
© 2020 Institute of Food, Science and Technology (IFSTTF)
The peer review history for this article is available at https://publons.com/publon/10.1111/ijfs.14582
2 Cell-wall breakdown mechanism of H. pluvialis Z. Ye et al.
extraction could reach above 70% (Zhao et al.,
2019). Moreover, HCl treatment is also shown to
be an effective cell disruption technique, which can
breakdown the cell wall in a relatively shorter time
(<60 min). Sarada et al. (2006) stated that the
astaxanthin extraction rate could reach more than
80% by HCl treatment (2 h). Furthermore, IL have
been reported as green, high-efficiency solvents; Orr
et al. (2016) proved that IL can completely hydrol-
yse wet chlorella at room temperature to obtain
higher lipid recovery rates. Effects of IL on break-
down of H. pluvialis cell wall structure were investi-
gated by Liu et al. (2019), who found that the cell
walls of H. pluvialis were efficiently disrupted by
1-butyl-3-methylimidazolium chloride ([Bmim] Cl) at
a mild temperature (50 °C), with an 85% extraction
rate of astaxanthin from H. pluvialis. Although the
cell wall breakdown of H. pluvialis for astaxanthin
recovery has been studied extensively, investigations
related to breakdown and changes in the cell wall
structure at the molecular level have rarely been
conducted.
Thus, the aim of this study was to explore the
mechanisms of breakdown of H. pluvialis cell wall
related to the changes occurring in characteristic
absorption bands and crystallinity of cell wall, using
three chemical pretreatment techniques (HCl, IL and
ME). To achieve these objectives, different high-
impact techniques, such as scanning electron micro-
scopy (SEM), transmission electron microscopy (TEM),
Fourier transform infrared spectroscopy (FTIR), X-ray
diffraction (XRD) and nuclear magnetic resonance
(NMR), were used. Finally, gas chromatography–mass
spectrometry (GC-MS) was also employed to investi-
gate the monosaccharide composition in the super-
natants obtained after cell wall treatment by these
techniques.
Materials and methods
Intact H. pluvialis cysts were purchased from Baiou
Biogenic Co. Ltd. (Yunnan, China) (astaxanthin con-
tent: 3.5%). The sample was stored in a dry, air-tight
aluminium pack at 18 °C to prevent degradation.
1-Butyl-3-Methylimidazolium chloride ([Bmim] Cl,
97%), 1-Butyl-3-methyl pyridinium chloride ([Bmpy]
Cl, 98%), triethylmethylammonium chloride ([Tmam]
Cl, 98%) and standard astaxanthin were obtained
from Aladdin Chemistry Co. Ltd. (Shanghai, China).
Cellulase (400 U mg1) and pectinase (500 U mg1)
were obtained from Ruiyong Biogenic Co. Ltd.
(Shanghai, China). Monosaccharides were purchased
from Sigma-Aldrich. All the organic solvents and
chemicals were of analytical grade and purchased from
Sinopharm Chemical Reagent Co. Ltd. (Shanghai,
China).
International Journal of Food Science and Technology 2020
IL and HCl pretreatments
IL and HCl pretreatments were performed using the
method described by Liu et al. (2018).
Enzymatic pretreatment
Intact H. pluvialis cysts (10 mg) were incubated with a
mixture of cellulase, pectinase and complex enzymes,
such as [cellulase: pectinase = 1:1 (U/U)], [cellulase:
pectinase = 2:1 (U/U)] and [cellulase: pectinase = 1:2
(U/U)] in 0.2 mol L1 acetic acid–Na acetate buffer
(pH 5) in a 50 °C water bath for 6 h. To stop the
reaction, the enzyme mixture was inactivated by incu-
bation in a water bath (80 °C for 5 min), and the cells
were subsequently collected by centrifuging at 2925 g
for 10 min. The cells were further extracted twice with
4 mL methanol for 1 h at 50 °C, and the organic sol-
vent thus obtained was collected and stored at 18 °C
until further analysis.
HPLC analysis
Astaxanthin content in the cell extracts was deter-
mined using the method described by Liu et al. (2018).
Cell wall preparation
Cell walls of H. pluvialis were obtained by grinding
and leaching the cells with methanol repeatedly. To
remove proteins, the obtained cell walls were mixed
with distilled water (1:100, m/m) and incubated in a
45 °C water bath for 30 min. The mixture was then
centrifuged at 1785 g for 5 min to recover the cell
walls as a pellet. These operations were repeated sev-
eral times until the absorption of the supernatant at
280 nm was lower than 0.05. The collected cell walls
were lyophilised and stored at 4 °C for further
studies.
SEM and TEM
SEM (Zeiss EVO18, Germany) was used to image
H. pluvialis cells after astaxanthin extraction. The sam-
ples were prepared using the method described by
Wang et al. (2016).
Determination of cell wall monosaccharides by GC-MS
The cell walls of H. pluvialis were treated with HCl,
IL and ME using the procedures described above.
After the reactions, supernatants were collected by
centrifugation at 2925 g for 5 min for analysis of
monosaccharide composition. Each supernatant
(2 mL) was mixed with 4 mL trifluoroacetic acid (3 M)
in a 10 mL screw tube, incubated for 2 h and then
© 2020 Institute of Food, Science and Technology (IFSTTF)

evaporated to dryness. Next, sodium carbonate (20%,
78 lL) was added, and the tubes were incubated at
30 °C for 60 min using a dry block heater. Later,
1 mL of 2 M sodium borohydride was added, and
incubation was continued for 1.5 h at room tempera-
ture. Acetic acid was added to remove excess sodium
borohydride. The solution was passed through a
cation column and eluted with 6 mL distilled water,
after which the elute was evaporated to dryness at
60 °C. Pyridine (2 mL) and n-propylamine (2 mL)
were added to the residue, and the mixture was incu-
bated for 30 min at 55 °C, cooled down and then
evaporated to dryness at 60 °C. Finally, 1 mL of pyri-
dine and 1 mL of acetic anhydride were added to the
residue, and the solution was reacted at 105 °C for
1 h. The reaction was quenched using distilled water,
and the extracted liquid was passed through a small
column of anhydrous sodium sulphate. The monosac-
charides in the eluate were analysed by gas chromatog-
raphy–mass spectrometry (QP 2010 PLUS, Shimadzu,
Japan) using an RXI-5 SIL MS (30 9 0.25 9
0.25 nm) column. The column temperature was first
set at 120 °C, increased to 250 °C at intervals of
3 °C min1 and finally kept at 250 °C for 5 min.
Other parameters were as follows: detector tempera-
ture, 250 °C min1; carrier gas, helium; flow rate,
1 mL min1. The standards were used in the following
order: rhamnose, fucose, arabinose, xylose, mannose,
glucose, galactose, glucuronic acid and galacturonic
acid.
FTIR measurement
The change in the structure of cell walls before and
after treatment by the three different methods was
analysed by FTIR (Affinity-1, Shimadzu, Japan) using
the method described by Zhang et al. (2017).
XRD measurement
The cell walls pretreated with HCl, ionic liquid and
enzyme, and untreated cell walls were analysed by
XRD (7000 S/L, Shimadzu, Japan). A peak deconvo-
lution method was used to analyse the XRD data
(Ahvenainen et al., 2016)
Cr ¼ Fc =ðFa þ Fc Þ  100%:
13
C nuclear magnetic resonance (NMR) spectroscopy
measurements
Cell wall samples (25 mg) were mixed with 0.5 mL
deuteroxide in a vial, sealed and heated in an oil bath
at 55 °C for 4 h. Next, they were scanned by NMR
spectroscopy (AVANCE NEO, Bruker, Switzerland),
and the spectra were recorded at 298 K. 13C NMR
© 2020 Institute of Food, Science and Technology (IFSTTF)
Cell-wall breakdown mechanism of H. pluvialis Z. Ye et al. 3
spectra (100.62 MHz) were obtained from 12 000 to
72 000 scans, depending on the sample concentration,
in 64 K data points with a recycle delay of 2 s. The
chemical shift was recorded by using an external hex-
amethyl benzene methyl resin, expressed in ppm.
Statistical analysis
Each experiment was repeated three times, and the
results are expressed as mean
standard deviation.
The statistical analyses were performed using Origi-
nLab 8 software (OriginLab. Inc. Northampton, MA,
USA). The results obtained were evaluated using a
one-way ANOVA (Duncan post hoc method), and the
level of significance adopted was P < 0.05.
Results and discussion
Comparison of cell wall disruption efficiency
The recovery of astaxanthin from H. pluvialis after the
three cell wall disruption techniques and subsequent
extraction employing different organic solvents is pre-
sented in Figure S1. In control cells, the astaxanthin
extractability using methanol, ethanol and acetone
alone was negligible (<6%), which indicates that the
thick trilaminar cell wall renders H. pluvialis less per-
meable and extremely resistant to penetration by
organic solvents, thus making astaxanthin extraction
difficult. Hence, better cell wall disruption techniques
are required for extracting astaxanthin from H. pluvi-
alis (Kim et al., 2016). In our studies, among the
applied techniques, IL ([Bmim] Cl) exhibited a high
potential for cell disruption and astaxanthin extrac-
tion, with a yield of 86.71
2.06%, and these results
agree with previous studies (Liu et al., 2018; Liu et al.,
2019). In a study, imidazolium-based IL played a more
critical role in dissolving some components of the
intact H. pluvialis cell wall than other kinds of IL
(Desai et al., 2016; Choi et al., 2019). In addition, IL
showed excellent recyclability, and a large number of
studies have proved that it can be reused in several
cycles without a reduction in efficiency (Orr et al.,
2016; Liu et al., 2019).
For HCl treatment, the extractability of astaxanthin
ð1Þ was evaluated using different HCl concentrations (2, 4
and 6 M) for pretreatment followed by acetone extrac-
tion. The results showed that the HCl pretreatment
method was an efficient method for cell disruption of
H. pluvialis (Figure S1). The cell walls were signifi-
cantly broken down by the various concentrations of
HCl, facilitating astaxanthin extraction. The highest
astaxanthin extraction (80.52
2.28%) was achieved
at 4 M HCl concentration. Further increase in the con-
centration of HCl led to a decrease in astaxanthin
extractability. The possible reason for this
International Journal of Food Science and Technology 2020
4 Cell-wall breakdown mechanism of H. pluvialis Z. Ye et al.
phenomenon may be attributed to the excessive acid
concentration (Bustos-Garza et al., 2013). Our results
were in accordance with previous findings by Sarada
et al. (2006). As shown in Figure S1, the enzymatic
extraction method is less efficient than the other two
methods. The highest astaxanthin extractability
(71.08
2.49%) was attained after enzymatic pretreat-
ment [pectinase and cellulose mixed at a ratio of 1:1(U/
U)] with an enzymatic dosage of 7000 U/mL. As shown
in Figure S2, ME treatment showed a higher potential
for cell disruption than single-enzyme treatment.
Among the different formulae used, the best ME for-
mula was the enzyme complex of cellulose and pectin
(1:1). These results agreed well with the study of Gerken
et al. (2013) who found that appropriate combinations
of various enzymes have a greater potential to digest the
cell walls and weaken their structure, thereby facilitating
the utilisation of algae or algal biomass.
Mechanism of cell disruption by IL, ME and HCl
The breakdown mechanism of the cell walls using
these three techniques was studied further. The mor-
phology and microstructure of the cell wall of H. plu-
vialis were visualised by SEM and TEM (Figures S3
and S4). Compared to the intact cysts with smooth
and integrated morphology of untreated cells (Fig-
ure S3a), significant changes were observed after the
cells were treated with the IL. The surface structure
seems to be disintegrated and shrunken, generating a
honeycomb-like morphology (Figure S3b). These
results are in agreement with our previous findings
(Liu et al., 2018), which indicated that the morphology
of the encysted cell surface of H. pluvialis became
rough and wrinkly, and some cavities were generated
on the cell wall surface after pretreatment with [Bmim]
Cl and methanol extraction. Unlike an inner collapse
of a large cell wall surface area after IL treatment, the
surface of ME-treated cell wall was less affected, and
discontinuous and tiny holes were generated (Fig-
ure S3c). Regarding cells pretreated with 4 M HCl
(Figure S3), the cell wall was lysed and broken down
completely, thus losing its integrity. Thus, SEM analy-
sis demonstrated that the mechanisms of breakdown
of the cell wall of H. pluvialis related to these three
techniques were different from each other. It is well
known that the cell wall of aplanospores of H. pluvi-
alis consists of an elaborate three-layered cell wall (tril-
aminar sheath, secondary wall and tertiary wall)
(Desai et al., 2016). Most notably, IL have a less effect
on the outmost trilaminar sheath which is composed
of a sporopollenin-like hydrolysis-resistant polymer
known as algaenan. However, they are capable of
breaking down the homogeneous middle layer arrange-
ment of polysaccharides (secondary wall) and the
heterogeneous inner layer arrangement of
International Journal of Food Science and Technology 2020
polysaccharides (tertiary wall). ME treatment has the
capacity to hydrolyse all the three layers of the cell
wall. However, the hydrolysis of the cell wall was
selective and non-homogeneous, resulting in the cre-
ation of discontinuous cavities in the cell wall. To
uncover the intercellular structure change of H. pluvi-
alis, further studies conducted by TEM are shown in
(Figure S4e); the cell walls after pretreatment with IL
still exhibited an intact trilayered structure, which
forms an outer layer adjacent to the inner layer of
astaxanthin. After pretreatment with IL and subse-
quent extraction by organic solvent, it was clearly
observed that the cell wall of H. pluvialis had lost its
integrity and the whole cell was distorted, which was
consistent with the morphology of H. pluvialis as
observed by SEM. After ME and solvent extraction
treatments, the tiny pores gradually became larger and
a loss of cell wall boundaries was observed, which
facilitated the extraction of hydrophobic intracellular
astaxanthin by solvents (Figure S4f).
The underlying mechanism by which [Bmim] Cl
degraded the cell wall might be attributed to the fact
that the cell wall constituents (such as cellulose) were
dissolved by IL. Pinkert et al. (2009) reported that cel-
lulose was highly soluble in [Bmim] Cl, and it could be
possible due to Cl anions, which show a good capa-
bility for hydrogen bonding. It is believed that IL
along with Cl anions are the most effective solvents
for dissolving cellulose and that these anions interact
with the hydroxyl groups of cellulose molecules to pro-
mote cellulose dissolution. Remsing et al. (2006) also
confirmed that the Cl anions of [Bmim] Cl interact
with the hydroxyl groups of cellulose and disturbs the
hydrogen bonds of different types of cellulose, leading
to their dissolution. The action of the enzyme on cellu-
lose is a heterogeneous catalytic reaction. First, the
crystalline cellulose is hydrolysed by the enzyme to
form amorphous cellulose and soluble oligosaccha-
rides. A further reaction resulted in glucose generation,
which ultimately leads to cell wall degradation (Zhao
et al., 2012; Datta et al., 2017). With respect to HCl
treatment, hydrolysis of glycosidic bonds of cellulose
in the cell wall is the main reason for cell wall break-
down. The degradation of cellulose primarily occurs in
the amorphous cellulose (Adel et al., 2011). A report
has suggested that acid hydrolysis has a great potential
in degrading cellulosic walls of sugarcane, sorghum
and corn into glucose for further production of biofu-
els, such as bioethanol (Harun & Danquah, 2011).
FTIR analysis
To determine chemical changes in the cell walls, FTIR
spectra of the untreated, and IL- ME-, and HCl-trea-
ted samples were investigated (Figure S5). All the three
techniques showed different abilities of hydrolysing the
© 2020 Institute of Food, Science and Technology (IFSTTF)

H. pluvialis cell walls. The intensity of the absorption
peaks at 798 cm1, expressed as a C-H/C-O-C stretch,
was enhanced after treatment with the three techniques.
The most significant changes were observed in the HCl-
treated sample. Compared to untreated sample, the
absorption peaks at 860 cm1 (a characteristic absorp-
tion peak of lignin) gradually weakened and ultimately
disappeared after IL and HCl treatments, but not after
ME treatment, illustrating that ME did not have the abil-
ity to hydrolyse lignin in the cell walls. The absorption
peaks appearing at 1022–1179 cm1 are characteristic
peaks related to cellulose and hemicellulose. The peak at
1022 cm1 represents the stretching vibration of C = O/
C-C/C-C-O (Djikanovic et al., 2016). The narrow bands
at 1079 and 1154 cm1 belong to the glycoside ether
bond C-O-C stretching vibration absorption peaks (Blok-
ker et al., 1998). Compared to that of the untreated sam-
ple, the characteristic peak of the treated cell walls at
1022 cm1 was clearly weakened, and the peak intensity
was no longer sharp after IL, ME and HCl treatments.
This implies that IL, ME and HCl treatments break
down the hydroxyl/ether and C = O bonds. However,
the ability to hydrolyse these bonds by HCl was greater
than that of IL and ME treatments. The peaks at 1154
and 1179 cm1 were shifted and even disappeared after
HCl treatment. This phenomenon may be ascribed to the
generation of new materials as the decomposition of cel-
lulose or hemicellulose progressed under HCl treatment
(Pascoal Neto et al., 1995).
The characteristic peaks at 1630–1650 cm1 represent
the C = C stretching vibration of ester bonds in car-
bonyl or carboxyl groups as a bridge to link the hemi-
cellulose and lignin (Gao et al., 2011; Scholz et al.,
2014). The relative intensity of the characteristic peaks
of the treated samples at 1630–1650 cm1 was signifi-
cantly decreased as compared to that of control. The
results indicated that lignin in the cell wall was hydrol-
ysed and the intensity of the absorption peak after ME
treatment was higher than after treatment with the
other two methods. These results confirmed the charac-
teristic absorption peak at 860 cm1, which indicated
that ME treatment had a lesser effect on lignin. The
dominant peaks at 2800–3300 cm1 are ascribed to the
CH groups in cellulose and lignin. The peaks at 2852
and 2920 cm1 indicated C-H asymmetric and symmet-
ric stretching vibrations of methyl/methylene (Haider
et al., 2017). Our results thus suggest that some of the
cellulose, hemicellulose and lignin in the cell wall were
hydrolysed after the different treatment methods.
X-ray diffraction
An XRD diffractogram was carried out to investigate
the crystallinity of the cellulose in the samples. As
depicted in Figure S6, the diffractogram of the samples
show peaks at 2h = 22.48–22.64°, which represent the
© 2020 Institute of Food, Science and Technology (IFSTTF)
Cell-wall breakdown mechanism of H. pluvialis Z. Ye et al. 5
structure of cellulose. Treatment with the three methods
did not alter the main crystalline structure of the cell
wall (Zhang et al., 2014). However, the intensity of the
main diffraction peaks in the crystalline region of cellu-
lose changed. These phenomena implied that the crys-
tallinity was increased, which was confirmed by
calculating the crystallinity in all the samples (Luduena
et al., 2011). As shown in Table 1, the crystallinity
index of untreated, and HCl, IL and ME treatments
were 12.93
0.13, 37.52
0.18, 21.56
0.27 and
24.28
0.21%, respectively. The crystallinity of the
treated samples was significantly higher than that of the
untreated sample. The increase in crystallinity might be
due to the fact that the partially crystalline and amor-
phous regions were rearranged in the crystalline zone,
as HCl, IL and ME directly reacted with the amor-
phous zone. The crystallinity after HCl treatment was
higher than after treatment with the other two methods,
which may be due to the fact that the amorphous
region was more sensitive to HCl treatment. Our results
agreed with those of Li et al. (2015), who reported that
the crystallinity of cellulose increased by 10.9 % after
acid hydrolysis. Regarding IL treatment, the crystalline
regions of the cellulose were unaffected, but amorphous
parts were partially destroyed; this phenomenon may be
responsible for IL treatment to effectively hydrolyse the
hemicellulose and lignin, leading to an increase in the
relative cellulose content, thus resulting in an increase
in the cellulose crystallinity (Zhang et al., 2015).
13
C NMR analysis
NMR spectroscopy is a useful method to analyse
changes in the cellulose structure during degradation.
In Figure S7, a total of five C atom chemical shift val-
ues were observed for the untreated samples (60.32,
70.99, 78.99, 101.32 and 171.56 ppm). The signal peak
at 55–65 ppm is a characteristic peak of cellulose C6,
that at 64–76 ppm of cellulose C2, C3 and C5, and at
76–88 ppm of cellulose C4, which consists of two
parts. One is the signal peak at 85.5 ppm that repre-
sents the crystal part, and the other is the signal peak
at 79 ppm, which represents semi-crystalline or amor-
phous cellulose (Liiti€a et al., 2003). The signal peak at
95–105 ppm represents cellulose C1 (Earl & Van-
derHart, 1981), and the peak at 170–176 ppm is
derived from C6 of the methyl structure (Xu et al.,
2006). Compared to those of the untreated sample,
most of the 13C of the compounds of cell walls shifted
to the high field after treatment with HCl, IL and ME.
In addition, many new chemical shifts appeared, and it
was observed that most of the chemical shifts in amor-
phous regions had disappeared, implying that the
hydrolysis sites in the cell wall treated with HCl, IL
and ME were amorphous. These results were similar
to the results of XRD, which demonstrated that the
International Journal of Food Science and Technology 2020
6 Cell-wall breakdown mechanism of H. pluvialis Z. Ye et al.

Table 1 XRD analysis of samples
Sample 2h Cr %
Untreated sample 22.48 12.93
IL treatment 22.44 21.56
ME treatment 22.33 24.28
HCl treatment 22.47 37.52
Values with different letters in the same column (a–d) are significantly
different (P < 0.05) from each other.
changes mainly occurred in the amorphous area. In
addition, many saturated alkanes were produced after
treatment by the three methods; for example, the
chemical shift at 28–32 ppm was a methyl group of
the hemicellulose structure.
Monosaccharide composition in supernatants
The monosaccharide composition in the supernatant
after cell wall treatment with HCl, IL and ME is shown
in Figure S8. The results indicated that the monosaccha-
ride composition in the supernatants obtained by the
three methods was different from each other. Glucose
and mannose were detected in the supernatant of HCl-
treated cell walls, which accounted for 80.8
3.34 and
19.2
1.49%, respectively. Regarding IL treatment,
only glucose was detected, whereas in the supernatant of
ME, glucose, mannose, arabinose and xylose were pre-
sent. The contents of glucose, xylose, arabinose and
mannose were 87.0
2.28, 3.1
0.36, 2.8
0.41 and
7.1
91%, respectively (Table 2). In all treatment tech-
niques, glucose was the most abundant monosaccharide.
These results were consistent with a previous study by
Scholz et al. (2014), who estimated the monosaccharide
composition of Nannochloropsis gaditana cell walls after
treatment with a different type of enzymatic digestive
Table 2 Monosaccharide composition in supernatants after
treatment of cell walls with ME, HCl, and IL
solution. Consistent with our results, they also found
that 98%–99% of the monosaccharide was glucose.
These data demonstrate that hydrolysis and breakdown

0.13% d of the cell wall of H. pluvialis are due to diverse phe-

0.27% c nomena.

0.21% b

0.18% a
Conclusions
In this study, the mechanisms of cell wall breakdown
by three pretreatments were investigated, and the
results indicated that the means by which the barriers
of the cell wall of H. pluvialis were broken down using
these treatments were quite different from each other,
which was reflected from the results of SEM, TEM,
FTIR, XRD and NMR. FTIR demonstrated that cel-
lulose, hemicellulose and lignin were hydrolysed by
HCl, IL and ME treatments to some extent. However,
ME treatment had a lesser effect on lignin than the
other two treatments. Analyses by XRD and NMR
implied that the amorphous region of cellulose of cell
wall was susceptible to hydrolysed/breakdown by the
three techniques. Although these three strategies have
a similar efficiency to recover astaxanthin from H. plu-
vialis, differences in the biological activity of extracted
astaxanthin by these techniques, such as in vivo/vitro
antioxidant activities, and antiproliferative activities
should be investigated for further knowledge in this
field. In addition, the biological safety of astaxanthin
extracted by IL treatment should be evaluated before
being applied in cosmetics, medicine, health products.
Acknowledgments
This research was supported by the Natural Science
Foundation of Hunan Province (2019JJ50266).
Ethical approval
Ethics approval was not required for this research.

Different supernatants Conflict of interest

Monosaccharides b (HCl) c (IL) d (ME) No.
Rhamnose ND ND ND
Author elects to not share data: Research data are
Fucose ND ND ND not shared.
Arabinose ND ND 2.8
0.41%
Xylose ND ND 3.1
0.36%
Author Contribution
Mannose 19.2
1.49% ND 7.1
0.91%
Glucose 80.8
3.34% 100% 87
2.28% Zhang Ye: Formal analysis (equal); Methodology (equal);
Galactose ND ND ND Writing-original draft (equal). Zhi-Wei Liu: Conceptual-
Glucuronic acid ND ND ND ization (equal); Data curation (equal); Formal analysis
Galacturonic acid ND ND ND
(equal); Investigation (equal); Project administration
The data represent relative content obtained by peak area ratio. b, c, d (equal); Supervision (equal); Writing-review & editing
were correlated to the results of monosaccharide composition for dif- (equal). Xinghe Tan: Conceptualization (equal); Method-
ferent pretreatment presented in Figure S8. ology (equal); Software (equal); Writing-review & editing
ND, Not determined. (equal). Rana Muhammad Aadil: Methodology (equal);

International Journal of Food Science and Technology 2020 © 2020 Institute of Food, Science and Technology (IFSTTF)

Project administration (equal); Writing-review & editing
(equal). Yi-Cheng Tan: Conceptualization (equal); For-
mal analysis (equal); Software (equal); Writing-review &
editing (equal). Muhammad Inam-ur-Raheem: Writing-re-
view & editing (equal).
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Supporting Information
Additional Supporting Information may be found in
the online version of this article:
Figure S1. Effect of each pretreatment technique on
astaxanthin extractability of H. pluvialis.
International Journal of Food Science and Technology 2020
Figure S2. Effect of various enzymes on astaxanthin
extractability of H. pluvialis.
Figure S3. SEM photographs of an H. pluvialis cell.
Figure S4. TEM photographs of an H. pluvialis cell.
Figure S5. FTIR spectra of different pretreated sam-
ples: (a) HCl treatment, (b) IL treatment, (c) ME
treatment, (d) untreated.
Figure S6. X-ray diffractograms of different pretreat-
ment samples: (a) HCl treatment, (b) IL, treatment, (c)
ME treatment, (d) Untreated.
Figure S7. 13C NMR spectrum of different pre-
treated samples: (a) HCl treatment, (b) IL treatment,
(c) ME treatment, (d) untreated.
Figure S8. Monosaccharide composition in super-
natants after treatment of cell walls with HCl, IL, and
ME: (a) monosaccharide standard, (b) HCl treatment,
(c) IL treatment, (d) ME treatment.
© 2020 Institute of Food, Science and Technology (IFSTTF)