EDTA-assisted hydrothermal synthesis of cubic SrF2 particles and their catalytic performance for the pyrolysis of 1-chloro-1,1-difluoroethane to vinylidene fluoride

Article abstract of DOI:10.1039/c8ce01546e

Uniform, free-standing and cubic SrF₂ microparticles were successfully fabricated by a facile hydrothermal method with ethylenediaminetetraacetic acid (EDTA) as the chelating agent. The influences of preparation conditions, such as the pH value, amount of EDTA and hydrothermal time, on the formation of SrF₂ crystals were investigated. The formation mechanism of cubic SrF₂ particles was proposed based on the experimental results. Following calcination in air at 500 °C, SrF₂ particles were evaluated as the catalyst for the pyrolysis of 1-chloro-1,1-difluoroethane (HCFC-142b, CH₃CClF₂) to vinylidene fluoride (VDF, CH₂═CF₂) at 350 °C and a space velocity of 600 h^?1. The results indicate that SrF₂ cubes exhibit high catalytic activity with a HCFC-142b conversion of about 70% and a selectivity to VDF of 80-87%. No significant deactivation was observed within the time on stream of 30 h. With the reaction temperature increased to 450 °C, the conversion of HCFC-142b is close to 94%, while the selectivity to VDF remains almost unchanged. Although the SrF₂ catalyst prepared by the conventional precipitation method also shows high conversion, its selectivity to VDF is only around 50-70%. We suggest that the surface acidity and specific surface area play major roles in the catalytic performance. Compared with the temperatures for industrial manufacture of VDF of 650-700 °C, the SrF₂ catalysts provide a promising pathway to produce VDF at much lower temperatures.

Full text of DOI:10.1039/c8ce01546e

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PAPER
Effects of acylation and glycation treatments on
physicochemical and gelation properties of
rapeseed protein isolate†
Cite this: RSC Adv., 2018, 8, 40395
a
b
c
ab
b
*
*
and Rong He
Zhigao Wang, ‡ Cheng Zhang,‡ Tian Zhang, Xingrong Ju
The aim of this study was to improve the gelation property of rapeseed protein isolates (RPI) by means of
acylation and glycation. The results showed that acylation and glycation within RPI occurred at Lys, and
Lys, Met, Ile, Leu and Pro, respectively. Acylation and glycation both increased the surface
hydrophobicity (So) and molecular weight of RPI, and decreased the free sulfhydryl (SH) content of RPI,
while acylation resulted in a lower change of So and SH. The conformational structure of modified RPIs
was changed, and acylated RPI (acylation degree, 38 ꢀ 0.2%) possessed the highest ordered structure
content among the modified RPIs. The thermal stability of the protein was improved after either
acylation or glycation treatments. Furthermore, native RPI with moderate modification (low degree of
acylation, 38 ꢀ 0.2%) showed an overall improvement in the gelation and gel properties as evidenced by
the reduced least gelation concentration and surface roughness, increased water-holding capacity, and
better textural properties.
Received 23rd September 2018
Accepted 20th November 2018
DOI: 10.1039/c8ra07912a
rsc.li/rsc-advances
characteristics of RPI was poorer than that of other proteins,
such as soy protein, which severely affected its applications as
an ingredient in the food industry.3,5
1. Introduction
The cultivation of rapeseed is performed all over China. The
major global demand for rapeseed is for edible oils. However,
once the oil is removed from the seeds, rapeseed meal remains.
Rapeseed meal has been recognized as a rich potential alter-
native source of plant protein containing up to 50% protein on
a dry weight basis. Rapeseed protein isolate (RPI) is usually
produced from rapeseed meal and considered a suitable source
of dietary protein for human consumption due to the high
bioavailability and excellent balance of its essential amino acid
composition.1 However, its use in food industry is limited
mainly due to its inferior functionality compared to other
commercially available protein isolates.2 Although it has been
reported that the RPI had some favorable functional properties,
including good water/oil absorption, as well as foaming and
emulsication abilities,3,4 it was found that the gelation
Chemical modication of proteins has the advantages of
high efficiency and strong controllability, which improves the
function and physicochemical properties of proteins. For
industrialization, chemical modication is easier to implement
in large scale production than physical and enzymatic modi-
cations.6 Acylation has been widely used in food processing as
one of chemical modication methods of proteins. Currently,
succinic anhydride is more commonly used as a safe food
acylating agents.7 The nature of the acylation reaction is the
nucleophilic substitution reaction between the acylating agent
and the free amino or hydroxyl group in the protein molecule,
resulting in changes in the functional properties of proteins.8 It
was reported that gelation, emulsifying properties, and water
solubility of oat protein could be improved by acylation process
when compared to native protein.9,10
The glycation of the protein with reducing sugar by means of
Maillard reaction has been shown to be a promising approach,
which could improve the functional properties of proteins, such
as emulsifying and gelation property, solubility, and thermal
stability.11 Maillard reaction can be achieved under the wet or
dry-heating conditions. Recently, wet-heating method, which
shortened the reaction time of dry-heating method to only
several hours, has been developed to reduce the adverse effects
(insoluble aggregates) of Maillard reaction to ensure the
production of functional protein–polysaccharide conjugates.12
The wet-heating method can change the molecular structure of
proteins, which is conducive to the adsorption and
aSchool of Food Science and Technology, Jiangnan University, Wuxi, 214122 People's
Republic of China. E-mail: xingrongju@163.com; Fax: +86 25 8402 8788; Tel: +86
25 8402 8788
bCollege of Food Science and Engineering, Collaborative Innovation Center for Modern
Grain Circulation and Safety, Key Laboratory of Grains and Oils Quality Control and
Processing, Nanjing University of Finance and Economics, Nanjing, 210003, People's
Republic China. E-mail: rong.he@njue.edu.cn; Fax: +86 25 8402 8788; Tel: +86 25
8402 8788
cAdvanced Pharmaceutics and Drug Delivery Laboratory, Leslie Dan Faculty of
Pharmacy, University of Toronto, 144 College Street, Toronto, Canada, M5S 3M2
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c8ra07912a
‡ These authors contributed equally to this work.
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reorganization of the protein on its interface, hence strongly based on previous method1 with slight modications. Briey,
affecting the interface properties of the protein.13 Several RPI solution (2% w/v, pH 10) was prepared in deionized distilled
proteins, including egg white protein, soy protein, milk protein water with stirring for 30 min, and then butanedioic anhydride
and whey protein, have been demonstrated to achieve improved was added into the RPI solution (5, 10, and 15% w/w, respec-
emulsifying properties, water holding properties and gelation tively, protein basis), to obtain different degree of acylation
properties through glycation with saccharide.4,7,14 However, samples RPI1, RPI2 and RPI3. During the acylation, the pH of
there are few studies on the physicochemical properties and mixture was maintained at 10 by adding 2 M NaOH. The
gelation characteristics of rapeseed protein aer modied by mixture was dialyzed with deionized distilled water for 24 h
acylation and glycation.
(MWCO of 10 kDa) and then freeze-dried.
The objective of this study was to improve the gelation
Preparation of glycated RPI. The glycated RPI was prepared
properties of rapeseed protein by the mean of acylation and by means of the Maillard reaction according to the method of
glycation and enhance its applicability in food industry. The Qu et al. with slight modications.15 Brief, a 10 mg mLꢂ1 RPI
amino acid compositions, surface hydrophobicities, free sulf- solution with pH 10 was prepared and fully mixed for 1 h. The
hydryl, molecular weights and protein conformations of modi- RPI solution was mixed with dextran at 1 : 1 (w/w) ratio at room
ed rapeseed proteins were analyzed. The effect of acylation and temperature for 30 min, and then the solution was adjusted to
glycation on gel properties, water holding capacity, surface pH 10 with 1 M NaOH. The RPI–dextran mixture was then
ꢁ
morphology and thermal properties of the rapeseed protein gels heated at 90 C for 1, 2, and 3 h, respectively, and then imme-
were investigated. Furthermore, the above indicators were diately cooled in an ice-water bath to stop reaction. The reaction
analyzed by principal components.
protein products (RPI4, RPI5, RPI6) were then freeze-dried.
2.4 Surface hydrophobicity (So)
2. Materials and method
The So of protein samples were determined using an aromatic
uorescence probe (ANS, 8-anilino-1-naphthalenesulfonic acid
ammonium salt for uorescence) based on previous method.16
Brief, samples stock solutions were successively diluted to nal
concentrations of 50–250 mg mLꢂ1 using 0.01 M phosphate
buffer (pH 8.0). Then, a 20 mL ANS (8 mM in 0.01 M phosphate
buffer, pH 8.0) was added to 4 mL of each diluted sample
solution. The uorescence intensity of the mixture was tested by
the uorescent microtiter plate reader (SpectraMax Gemini™
XS, Molecular Devices, Inc., USA) at excitation and emission
wavelengths of 390 nm and 470 nm, respectively. The initial
slope of uorescence versus sample concentration plot (calcu-
lated by linear regression analysis) was set as an index of So.
2.1 Materials
The defatted rapeseed meal was provided by COFCO East Ocean
Oils & Grains Industries Co., Ltd (Zhang Jiagang, China).
Butanedioic anhydride, dextran, 5,5-dithiobis(2-nitrobenzoic
acid) (DTNB), 8-anilino-1-naphthalenesulfonic acid ammo-
nium salt for uorescence (ANS), and ethylenediaminetetra-
acetic acid (EDTA) were purchased from Sigma-Aldrich (St.
Louis, MO, USA). 2,4,6-Trinitrobenzene sulfonic acid (TNBS)
was purchased from Beijing Ouhe Technology co., Ltd (Beijing,
China).
2.2 Preparation of rapeseed protein isolates
Rapeseed protein isolates (RPI) were produced from defatted
rapeseed meal according to a previously reported method1 with
slight modications. Briey, defatted rapeseed meal was
dispersed in deionized water (1 : 15, w/v), adjusted to pH 11.0 by
2.5 Determination of free sulydryl
The free sulydryl content was analyzed according to previous
method of Beveridge et al.17 Ellman's reagent was prepared by
dissolving 4 mg DTNB in 1 mL Tris–glycine buffer (pH 8.0).
Then, 1 mL protein samples solution (1 mg mLꢂ1, dissolved in
Tris–glycine buffer that contain 8 M urea) was mixed evenly with
50 mL Ellman's reagent and then incubated for 30 min at room
temperature. The mixture was then centrifuged for 20 min at
8000g. The absorbance of samples was tested by the microtiter
plate reader (SpectraMax Gemini™ XS, Molecular Devices, Inc.,
USA) at 412 nm.
ꢁ
using 1 M NaOH with stirring at 40 C for 2 h, and the slurry
centrifuged at 10 000g for 20 min at 4 ꢁC. The supernatant was
recovered, adjusted to pH 4.5 with 1 M HCl for 1 h, and
centrifuged at 10 000g for 30 min at 4 ꢁC. The recovered
precipitated proteins were then washed with anhydrous ethyl
alcohol, redispersed in deionized water, and adjusted to pH 7.0
with 1 M NaOH. Finally, the protein solution was freeze-dried to
acquire RPI.
2.3 Protein modications
2.6 Amino acid analysis
RPI were modied by two processes: acylation with butanedioic Amino acid composition analysis of protein samples were per-
anhydride (at 5, 10, and 15% w/w, respectively, protein basis) formed based on the method of Hou et al. with slight modi-
and glycation with dextran (at 1 : 1 ratio w/w, protein basis, cation.18 The 0.2 mg protein sample waꢁs added to 10 mL 6 M
heated at 95 ꢁC for 1, 2, and 3 h, respectively). The modied and HCl and reacted under nitrogen at 110 C for 24 h in the vial.
native proteins were then used to investigate the modications The hydrolysate in the vials was cooled down, evaporated to
(acylation and glycosylation) on effects of RPI gel properties.
dryness, and diluted to 50 mL using 0.02 M HCl. 100 mL solution
Preparation of acylated RPI. Carboxyl-propionylated reac- was then further diluted to 1 mL using 0.02 M HCl and ltered
tions were carried out on RPI using butanedioic anhydride (0.22 mm), and then placed in Hitachi Amino Acid Analyzer (L-
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8900, HITACHI, Japan). The analysis conditions were same as temperature in an ice bath followed by storing at 4 ꢁC for
Hou's reported conditions. All results were repeated for three overnight. The RPI samples concentration at which the formed
times.
gel didn't slip when the vial was inverted was taken as the least
gelation concentration (LGC). The properties of formed gels
were then analyzed by textural analyzer according to the method
of He et al. (Stable Micro System TA. XTII plus, Surrey, UK).16
Gels were penetrated to 30% deformation with a P 0.5 (0.5 inch
diameter of gel strength probe) probe at a constant rate of
0.2 mm sꢂ1. The gels were analyzed directly within the vials in
order to get the accurate information about the formed gels,
and hardness, adhesiveness, springiness, cohesiveness, and
chewiness were determined from the typical force–time curves
by soware (Exponent). Hardness was measured by peak force
in the rst compression cycle. Adhesiveness was measured by
maximum negative force generated during upstroke of probe.
Springiness was calculated as the distance of down stroke of the
second compression. Cohesiveness was measured by the ratio
of positive area during the second to that of the rst compres-
sion cycle. Chewiness was then calculated as hardness ꢃ
springiness ꢃ cohesiveness.
2.7 FTIR analysis
FTIR spectrum analysis was carried out according to the
method described by Wang et al.1 A tensor 27 infrared spec-
trophotometer (Bruker Optics, Ettlingen, Germany) was used to
determine IR spectra of samples. The measurements were per-
formed in a transmission mode using an IR cell, with KBr
windows in the frequency region 400–4000 cmꢂ1. Each protein
spectrum was recorded at a resolution of 4 cmꢂ1 and 64 scans.
Aer background correction (KBr spectrum), all spectra were
baseline corrected and obtained. The secondary structural
changes (a-helix at 1650–1659 cmꢂ1,b-sheet at 1618–1640 cmꢂ1
and 1689–1698 cmꢂ1, b-turn at 1660–1688 cmꢂ1, unordered at
1641–1649 cmꢂ1) of protein samples were analyzed by PeakFit
soware (v 4.12 Systat Soware Inc., Point Richmond, CA) based
on amide I (1600 to 1700 cmꢂ1) region. The accuracy of the peak
tting procedure was evaluated by their correlation coefficients
(R2 > 0.99), t standard errors, and residual plots. Protein
samples were analyzed in triplicates.
2.11 Water-holding capacity
The water-holding capacity (WHC) of RPI gels was measured
´
according to the method of Valerie LeungSok Line et al. with
2.8 SDS-PAGE
slight modications.20 The gels were weighted and placed into
1.5 mL centrifugal tubes, and then centrifuged at 9000 rpm for
20 min. The weight of overow water in the tube was measured.
Water-holding capacity was calculated as follows:
The modied and native protein samples were resolved in 12%
SDS-PAGE. In brief, 2 mg sample was added into 1 mL of
deionized distilled water and mixed well. Then, the mixture was
solubilized in a sample buffer (Bio-Rad, USA) and boiled for
10 min. Aer 10 min centrifugation at 10 000 ꢃ g, 20 mL of the
sample were loaded onto a 12% SDS-PAGE and then run at 70 V
for stacking gel and 120 V for separating gel (Mini-protein unit,
Bio-Rad, USA). The gel was stained in 0.1% Coomassie blue (R-
250) solution comprising methanol (90 mL), DDI H2O (90 mL)
and acetic acid (20 mL) for 2 h and de-stained in the same
solution without the dye Coomassie blue.
Wt ꢂ Wr
WHC % ¼
ꢃ 100
(1)
Wt
where Wt is the total weight of gel and Wr is the weight of
overow water aer centrifugation.
2.12 Surface morphology of gel by atomic force microscopy
(AFM)
The surface morphology of RPI gels was analyzed by an AFM
(Bruker Instruments, Madison, WI, USA) according to the
method described by Wang et al. with some modications. The
RPI samples (150 mg mLꢂ1) were prepared and deposited a drop
onto mica, and then placed into a culture dish. The culture dish
was heated at 95 ꢁC in water bath for 30 min, subsequently,
cooled to ambient temperature in an ice bath. Each sample was
dried in air prior to AFM scanning. The surface morphology of
gels was measured using ScanAsyst mode by AFM (probe model:
TAP150). The scan area was 2 ꢃ 2 mm, and the scan frequency
was 0.5. In both cases, the standard cantilever holders for
operation in air were used. The average root mean square (Ra) of
the roughness prole ordinates was calculated by NanoScope
Analysis (Bruker) soware.
2.9 Differential scanning calorimeter (DSC)
The thermal properties of protein samples were analyzed by
a DSC-8000 instrument (Perkin Elmer, Shelton, USA). Nitrogen
was used as a purge gas (20 mL minꢂ1). 1 mg freeze dried
sample and 10 mg deionized distilled water were weighted into
an aluminum pan and hermetically sealed. The aluminum pan
was heated from 30 to 150 ꢁC at a rate of 10 ꢁC minꢂ1. An empty
aluminum pan was used as reference. Denaturation tempera-
ture (Td) was calculated using Universal Analysis soware. All
experiments were performed in triplicate.
2.10 GEL preparation and characteristics
RPI gels were prepared based on previous method described by
Aluko et al. with some modications.19 The native and modied
protein samples were suspended in deionized distilled water at
2.13 Statistics
concentrations of 10–18% (w/v) and placed into a cylindrical All experiments were replicated for at least 3 times (n $ 3).
glass vials with a diameter of 28 mm and height of 57 mm. All Values were expressed as the mean ꢀ standard deviation (SD)
ꢁ
mixtures in the vials were mixed evenly and heated at 95 C in and were analyzed by one-way factor analysis of variance
water bath for 30 min and then immediately cooled to room (ANOVA). Tukey post hoc was applied for the differences
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between group means. SPSS 16.0 was used for all the statistical by 6.0%, 3.0% and 14.9% in its content in RPI1, RPI2 and RPI3,
analyses. P < 0.05 was considered statistically signicant.
respectively. Increasing the degree of acylation of protein led to
the reduced contents of Val, Arg, His and Glu, indicating Val,
Arg, His and Glu could be involved in the acylation reaction.
This might be attributed to the unfolding of protein structure by
the high level of acylation that led to the exposure of reactive
amino acids. It was worth noting that the change of the spatial
structure of protein likely resulted in the exposure of other
buried amino acids,15 which was conrmed by the increased
content of Asp, Tyr and Phe shown in Table 1 in the RPI3 with
high level of acylation degree.
The glycation of protein was mainly achieved by the Maillard
reaction between amino groups of proteins and reducing
sugars.21 Compared to the native RPI, the contents of Lys, Met,
Ile, Leu and Pro in all glycated RPIs were decreased by glycation
treatment, which is likely due to the wet-heating graing reac-
tion that induced the interaction of amino groups of RPI and
dextran, indicating that Lys, Met, Ile, leu and Pro mighty be the
glycation reaction sites of RPI and dextran. The reacted sites
obtained in this study differed slightly from the those of Qu
et al., reported earlier,15 which could be due to the slight
difference in the process conditions, especially in the glycation
method. Additionally, with increasing degree of glycation, Tyr
and Val also were involved, indicating that high level of glyca-
tion could increase the exposure of reactive amino acids. The
contents of His and Arg in all the glycated RPIs samples were
increased, while the total contents of all amino acids did not
signicantly change, which is likely because that the loss of
amines induced by Maillard reaction was offset by the increased
exposure of other amine groups by inducing protein unfolding
3. Results and discussion
3.1 Acylation and glycation of RPI
To obtain the rapeseed protein with improved functional
properties such as gelation, RPI was subjected to acylation and
glycation modications. Before the properties of modied RPI
analysis, the degree of acylation and glycation of samples was
determined rstly, which was shown in Table S1 (ESI).† The
degree of acylation in the three acylated RPIs (RPI1, RPI2, RPI3)
were determined to be 38.0 ꢀ 0.2%, 47.0 ꢀ 0.4%, and 56.0 ꢀ
0.3%, respectively. The graing degree of the three glycated
RPIs (RPI4, RPI5, RPI6) were measured as 7.0 ꢀ 1.8%, 12.0 ꢀ
1.1%, and 17.0 ꢀ 0.9%, respectively.
3.2 Amino acid composition analysis
Analysis of changes in amino acid compositions not only can
determine the basic composition of the modied protein, but
also can reect the extent of modication. The amino acid
composition for native RPI is shown in Table 1, where it was
observed to have high levels of glutamic acid (19.9%), leucine
(9.2%), aspartic acid (8.5%), lysine (6.7%) and glycine (5.9%). In
addition, we found that the contents (57.1–58.6%) of hydro-
philic amino acids in both native RPI and modied RPI were
higher than those (41.4–43.3%) of hydrophobic amino acids.
Acylation refers to the binding of succinyl groups to the free
amino groups on protein. As shown in the Table 1, Lys, the main
reactive amino acid during the acylation process, was reduced
Table 1 Amino acid compositions (%) of native RPI, acylated RPI (RPI1, RPI2, RPI3), and glycated RPI (RPI4, RPI5, RPI6)a
Acylation
RPI1
Glycation
RPI4
Amino acid
RPI
RPI2
RPI3
RPI5
RPI6
Hydrophilic amino acids
Asp
Ser
8.5
5.0
8.5
5.0
8.3
4.9
10.0 (17.6[)
5.0
8.6
5.1
8.5
5.1
8.6
5.1
Glu
Gly
Cys
Tyr
Lys
His
Arg
Total
19.9
5.9
0.1
3.9
6.7
3.6
4.1
57.7
19.9
5.8
0.1
3.9
6.3 (6.0Y)
3.6
19.9
5.8
0.1
3.9
6.5 (3.0Y)
3.6
4.1
57.7
18.8 (5.5Y)
5.9
0.1
4.2 (7.7[)
5.7 (14.9Y)
3.3 (8.3Y)
3.7 (9.8Y)
57.1
20.4
5.9
0.1
3.8
6.1 (9.0Y)
3.9 (8.3[)
4.4 (7.3[)
58.3
20.5
5.8
0.1
20.5
5.9
0.1
3.7 (5.1Y)
3.7 (5.1Y)
6.2 (7.5Y)
4.0 (11.1[)
4.5 (9.8[)
6.1 (9.0Y)
4.0 (11.1[)
4.5 (9.8[)
4.1
57.2
58.6
58.5
Hydrophobic amino acids
Val
Met
Ile
Leu
Thr
Ala
Pro
Phe
Total
5.5
2.3
4.9
9.2
4.9
5.3
4.9
5.6
42.6
5.4
2.3
4.9
9.1
4.9
5.2
4.9
5.6
42.8
5.1 (7.3Y)
4.6 (16.4Y)
2.3
5.0
9.3
5.0
5.4
4.9
6.0 (7.1[)
42.9
5.5
5.3 (3.6Y)
2.1 (8.7Y)
4.6 (6.1Y)
8.8 (4.3Y)
4.9
5.2
4.6 (6.1Y)
5.7
5.3 (3.6Y)
2.1 (8.7Y)
4.6 (6.1Y)
8.9 (3.3Y)
5.0
5.3
4.6 (6.1Y)
5.7
2.3
4.8
9.0
4.9
5.2
4.9
5.6
43.3
2.2 (4.3Y)
4.7 (4.1Y)
8.9 (3.3Y)
5.0
5.3
4.7 (4.1Y)
5.6
41.9
41.4
41.5
a
The values in parenthesis were the change percentage points of amino acid compositions in modied RPI when compared to native RPI.
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Fig. 1 SDS-PAGE pattern of molecular weight marker, acylated RPIs (RPI1, RPI2, RPI3), and glycated RPIs (RPI4, RPI5, RPI6).
by modication.22 In addition, when the conformation around results are similar to those of Yin et al. and Wang et al. who also
the Lys residues changed, it would affect the efficiency of other found that high molecular protein band of kidney bean protein
amino groups reacting with dextran, which in turn inuenced and pea protein tended to dissociate with the increasing degree
the contents of amino groups.21
of the acylation.25,26 In addition, glycation treatment signi-
cantly reduced the intensity of RPI bands and the numbers of
bands in the glycated RPI compared to those of both native RPI
and acylated RPIs. It was observed that characteristic protein
bands of 12S globulin subunit, a-polypeptide (ꢄ30 kDa) and b-
polypeptide (ꢄ20 kDa) of cruciferin, and heavy (ꢄ10 kDa) and
light (ꢄ4.5 kDa) of napin gradually became lighter with
increasing degree of glycation. Moreover, the 12S globulin
subunit and the a-polypeptide and b-polypeptide of cruciferin
seemed to be the most affected in the glycated RPIs, indicating
that globulin subunits were more involved in the glycation
reaction. Thus it was implied that the Maillard reaction conju-
gated dextran to RPI likely by binding to globulin. This nding
was in line with the previous nding from Safoura et al.2
3.3 SDS-PAGE of modied RPI
SDS-PAGE patterns of native RPI and modied RPI were shown
in Fig. 1. The pattern of native PRI showed the characteristic
polypeptide bands including the 12S globulin subunit, a-poly-
peptide (ꢄ30 kDa) and b-polypeptide (ꢄ20 kDa) of cruciferin,
and heavy (ꢄ10 kDa) and light (ꢄ4.5 kDa) chains of napin. This
result was similar to the ndings in the literatures.2,23 It was
reported that acylation could increase the molecular weight of
protein. Compared to the native RPI, by increasing the degree of
acylation, the molecular weight of subunits in the acylated RPIs
was gradually increased, which was likely due to the introduc-
tion of succinyl groups with high molecular weight during the
acylation process.24 Moreover, high level of acylation caused
signicantly dissociation of high molecular of 12S globulin
3.4 Surface hydrophobicity of native RPI and modied RPI
subunit and aggregates between 50 and 100 kDa shown in RPIs Surface hydrophobicity (So), measured by ANS probe, is an
with lower degree of acylation and native RPI, while there were index of the number of hydrophobic groups exposed on the
no signicant changes at bands of the low molecular subunits surface of the protein, which plays an important role in the
(cruciferin and napin) in native and acylated RPIs (Fig. 1). These functional properties of protein. As shown in Fig. 2A, So of RPI
Fig. 2 Surface hydrophobicity (So) (A) and free sulfhydryl content (B) of native RPI, acylated RPIs (RPI1, RPI2, RPI3), and glycated RPIs (RPI4, RPI5,
RPI6). Bars (mean ꢀ SD) with different letters indicated a statistical difference between the mean values (P < 0.05).
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Fig. 3 Fourier spectrum (upper) and Gaussian fitting curves (bottom) in the amide I region of modified RPI (A1–A3 and B1–B3) and native RPI (C).
Secondary structure compositions of the native RPI, acylated RPIs (RPI1, RPI2, RPI3), and glycated RPIs (RPI4, RPI5, RPI6) (D).
was signicantly increased by the acylation and glycation charge of protein and enhancing repulsion among protein
treatments, indicating that native protein possessed the higher subunits, and hence promoting the unfolding of protein
degree of folded structure compared to the modied proteins. structure, leading to the expose of hydrophobic groups.6,9 In this
Although both acylation and glycation treatments improved the study, acylation of RPI signicantly enhanced So of protein
So of RPI, the inuence was signicantly higher in the glycated (especially RPI1, a acylation degree of 38%, Table 1). However,
RPIs than that of acylated RPIs. Additionally, we found that the with the increasing degree of acylation, the So of RPI2 and RPI3
So was partly dependent on glycation, where RPIs with GD of declined to 80% and 60% respectively when compared to the
12.0% and 17.0% (Table 1) have higher So than the RPI with RPI1. This result was consistent with the nding of Zhao et al.
7.0% GD (especially at GD of 12.0%). This phenomenon could who observed that the So of oat protein was decreased by the
be attributed to that the disruption of organized RPI structures high degree of acylation.32 It was likely due to the excess amount
aer covalent binding of sugar molecules by Maillard reaction, of hydrophilic anionic succinyl group introduced into the RPI
resulting in sterically unfolding of the proteins by weakening structure, leading to enhanced electronic repulsion by
intra- and intermolecular interactions.27 It has been reported increasing electronegativity, which prevent ANS probe from
that the So of milk protein, soy protein isolate and b-conglycinin binding to the exposed hydrophobic area, thus resulting in the
increased aer modication by Maillard reaction with saccha- decrease in So.8,33
ride.28–30 Noteworthy, with increasing degree of glycation (from
12.0% GD to 17.0% GD), the So of protein decreased by ꢄ6%,
which is in agreement with W. Li et al.31 who found that the
3.5 Free sulydryl content
higher the degree of glycation, the lower So protein possesses.
Extensive studies reported that acylation could increase the
surface hydrophobicity of protein by reducing the positive
According to Fig. 2B, the amount of free SH groups in modied
proteins were signicantly decreased by the acylation and gly-
cation treatments compared to that of the native RPI. The
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reduction in free SH content following acylation and glycation range of 125.08–130.86 ꢁC is slightly lower than that of native RPI,
are consistent with previous reports for food proteins such as and the DH of acylated and glycated RPI was signicantly higher
whey protein34 and pea protein.26 The reduction in the suly- than that of native RPI, suggesting that more energy was required
dryl groups can be attributed to the formation of disulde for the denaturation of modied proteins and modications
linkages. High extent of acylation could also reduce the number improved the thermal stability of modied RPIs. The aggregation
of accessible free sulydryl groups by blocking the sulydryl of proteins and disruption of hydrophobic bonds were exothermic
groups of detection.26 Glycation led to even lower free SH reactions, which could reduce the enthalpy (DH) measured by
content in RPI compared to acylation treatment, which could be DSC.39 Compared to low degree of acylation (RPI), there was
ascribed to the heat treatment in Maillard reaction promoting a dramatic decrease of DH in the protein within high degree of
the disulde linkages formation in the glycated protein mole- acylation (RPI2 and RPI3). This is likely due to that strong elec-
cules compared to the mild condition (room temperature) used trostatic repulsive force introduced by the high amount of anionic
in acylation treatment.34 The reduction in SH content in RPI succinyl groups disrupted protein molecular interactions, such as
under high temperature was also reported by He et al. 2014.
hydrophobic interactions, resulting in the reduced DH values.40 In
addition, it has been reported that dextran molecules could
enhance the steric spacers between protein molecules, protecting
protein against aggregation by preventing the interactions of
hydrophobic binding sites on its surface,39 which might explain
the increase of DH in the glycated RPIs.
3.6 Protein conformational analysis
The secondary structures of RPIs were analyzed with FTIR in the
amide I (1600–1700 cmꢂ1) region, where the band assignments
were as follows: a helix, b turn, b sheet and random coil. The
quantitation of secondary structure contents was obtained by
the ttings of Gaussian peaks and multiple peaks, which was
summarized in Fig. 3A–C. As shown in Fig. 3D, compared to
native RPI, the content of a helix and b turn increased (espe-
cially in RPI2 and RPI3) in the acylated RPIs, the content of
b sheet decreased, and random coil was nearly unchanged with
increasing extent of acylation. These results indicated that the
structure of protein was signicantly changed aer the succinic
groups were conjugated, and high level of acylation was prone
to induce the transformation of b sheet to a helix and b turn.
Similar ndings have been reported in secondary structure of
kidney bean protein.35 It was possible that acylation induced
strong electrostatic repulsion interactions in combination with
steric hindrance within RPI due to the conjugation of bulky
anionic succinyl groups.36 The glycated RPIs showed an increase
in content of a helix, b turn and random coil, while the b sheet
content decreased (Fig. 3D). The alteration of secondary struc-
ture by glycation might be ascribed to multiple factors: (1) the
conjugation of saccharide to RPI likely led to changes in spatial
structure and unfolding of protein molecules, inducing random
coil and b turn while reducing b sheet (generally located in the
interior of the protein molecules);37 (2) the introduction of
extensive hydroxyl groups from dextran could result in
enhanced intermolecular interaction among the proteins, and
therefore an increase in a-helix structure.38 It is well known that
structure and functionality were closely related. Since the
content of ordered structure (a helix and b sheet) of RPI1 was
highest in the modied RPI (up to 56%), RPI1 likely possessed
the better protein properties among the modied RPIs.
3.8 Gelation and RPI gel properties
The gel properties of native RPI, acylated RPI, and glycated RPI
are summarized in Table 2. As shown in Table 2, LGC (least
gelation concentration) of RPI signicantly (P < 0.05) decreased
from 15% to 11% and 12% by slight acylation and glycation
treatments, respectively. However, the high level of glycation-
treated RPI resulted in an increase of LGC from 15% to 17%.
These ndings were consistent with data analysis of protein
structure unfolding during acylation and glycation treatments
(Fig. 2), which showed a signicant increase in the So of modied
RPI. The unfolded proteins were then able to interact through
hydrophobic bonding to increase strength of gel networks and
reduce the amount of proteins required to form gels, i.e.,
decreased LGC.41 Previous study found that hydrophobic inter-
action was a key factor during the formation of gel networks.42
3.7 DSC properties
In this study, DSC was conducted to evaluate the thermal prop-
erties of RPI samples. In the DSC spectra, the denaturation
temperature and the denaturation enthalpy (DH) can be deter-
mined by the temperature and peak area corresponding to the
maximum peak. As shown in Fig. 4, the denaturation temperature
Fig. 4 DSC curves of rapeseed protein, acylated rapeseed protein, and
glycosylated rapeseed protein. Insets show the denaturation temper-
ꢁ
of native RPI is 132.47 C. The denaturation temperature of acyl-
ation and glycation treated rapeseed protein (except RPI 3) in the ature, Td (ꢁC) and enthalpy, DH (J gꢂ1) values.
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gel textural properties may be attributed to suitable proportion
within the RPI aer less extensive acylation process. The chewi-
ness, which is expressed as the energy required to chew a solid
prior to swallowing, were, 23.165 mJ and 15.491 mJ in RPI1 and
RPI4, respectively, which further conrmed the capability of
acylation treatment to induce formation of stronger RPI gels.
3.9 Water holding capacity of the gels
Water holding capacity is an important indicator of the interac-
tion between protein and water.43 The water holding capacities of
native RPI, and RPIs with different modication treatments were
summarized in Fig. 5. The acylated RPI gels gained signicantly
better water holding capacity than the native RPI gel (P < 0.05).
However, there was no signicant difference among RPI1, RPI2
and RPI3, suggesting that the most robust network structure of
gel to hold water could be formed under low degree of acylation.
The acylation treatment has been demonstrated to improve the
solubility of rapeseed protein, as well as the water holding
capacity of the protein gel.44 The uniform spatial structure might
also contribute to the increase of the gel holding capacity.45 Aer
modication by glycation, the water holding capacity of RPI5 and
RPI6 was higher than that of RPI4 (Fig. 5), which might be due to
the high extent of glycation that introduced large amount of
hydrophilic polysaccharides, enhancing the affinity among
proteins and water molecules.46
Fig. 5 Water holding capacity (WHC) of native RPI, and RPIs with
different modification treatments. Bars (mean ꢀ SD) with different
letters indicated a statistical difference between the mean values (P <
0.05). Test was completed at the 15% protein concentration, while the
RPI6 was completed at 17% protein concentration.
However, the RPI5 and RPI6 (high extent of glycation protein)
didn't reduce amount of proteins for forming gel even if they
possessed of higher So than that of native RPI. This could be
attributed to that the steric spacers between glycated protein
molecules formed from the dextran coating, protected proteins
against interaction by decreasing hydrophobic binding sites on
its surface.39 As shown in Table 2, observed rheological properties
of RPI gels were strongly related to types and extent of modi- 3.10 Surface morphology of the gels
cations. The hardness, springiness, cohesiveness and chewiness
Surface morphology is an important factor in understanding the
of the acylated RPI gel (RPI1) and the glycated RPI gels (RPI4 and
RPI5) were signicantly (P < 0.05) higher than those of the native
RPI gel. The adhesiveness of all acylated RPI gels was lower than
the native RPI gel and the glycated RPI gels. Gels prepared from
RPI of low degree of acylation was shown to be relatively harder
than gels obtained from the glycated RPI, or even
transglutaminase-treated canola protein reported by Pinterits
et al.,5 suggesting that superiority of acylation treatment when
compared with enzymatic and glycation modications. A certain
degree of acylation modication not only increased the So
(Fig. 2A), but also decreased the free SH content (Fig. 2B), and
improved ordered secondary structure (Fig. 3). He et al. reported
that the proportion of hydrophobic interactions, electrostatic
interactions, disulde bonds, and structural conformation can
strongly inuence protein gel properties.41 The observed better
characteristics of heat induced gels. The surface morphology of
RPI gels was observed using AFM by 2D images, cross-section
images, 3D images, and Ra values (Fig. 6). The degree of rough-
ness of the gel surface could be visually observed from the 2D and
3D images. In addition, the straight lines on the 2D images are
used to generate the cross-section images.47 Based on the 2D/3D
images and cross-section images, the roughness of the gel
surface decreased in the order of acylated RPI gels, glycated RPI
gels and native RPI gel. Ra is a more accurate and quantitative
indicator of the average roughness and was calculated from the
height of all locations throughout the area. The Ra value decreased
from 4.41 nm in the gel of original RPI to 1.10, 2.22 and 2.10 nm in
the gels of acylated RPI1, RPI2 and RPI3, respectively. However, as
the degree of glycation increases, glycation reduced Ra value to
2.95, 3.15, and 3.52 nm in the gels of RPI4, RPI 5 and RPI6,
Table 2 The gelation properties of native RPI, acylated RPI, and glycated RPIa
Sample
LGC (%)
Hardnessb (g)
Adhesivenessb
Springinessb
Cohesivenessb
Chewinessb (mJ)
RPI
15a
11b
14c
15a
12d
15a
17e
18.019 ꢀ 1.314a
45.304 ꢀ 0.568b
17.901 ꢀ 1.021ac
16.250 ꢀ 1.451c
27.747 ꢀ 0.879d
20.562 ꢀ 1.976a
—
ꢂ6.916 ꢀ 0.745a
ꢂ9.117 ꢀ 0.324b
ꢂ16.109 ꢀ 0.765c
ꢂ15.023 ꢀ 1.212c
ꢂ4.023 ꢀ 0.314d
ꢂ0.638 ꢀ 0.543e
—
0.686 ꢀ 0.003a
0.858 ꢀ 0.005b
0.651 ꢀ 0.004c
0.621 ꢀ 0.004d
0.830 ꢀ 0.006e
0.774 ꢀ 0.004f
—
0.553 ꢀ 0.004a
0.596 ꢀ 0.004b
0.442 ꢀ 0.002c
0.578 ꢀ 0.003d
0.672 ꢀ 0.005e
0.603 ꢀ 0.005f
—
6.830 ꢀ 1.023a
23.165 ꢀ 0.786b
5.147 ꢀ 0.653c
5.827 ꢀ 0.896a
15.491 ꢀ 1.360e
9.597 ꢀ 1.431f
—
RPI1
RPI2
RPI3
RPI4
RPI5
RPI6
a
Results were presented as mean ꢀ SD. Different letters in the same column indicate a statistical difference between the mean values (P < 0.05).
LGC ¼ least gelation concentration. b Test was completed at the 15% protein concentration.
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Fig. 6 AFM images of native RPI gel, acylated RPI (RPI1, RPI2, RPI3) gels, and glycated RPI (RPI4, RPI5, RPI6) gels (2 ꢃ 2 mm2 scan size). The images
displayed on the left were the 2D image of different RPI gels. The image cross-sections corresponded to the section cut by the black straight line
on the 2D image. This corresponded to the gel largest surface roughness. The average roughness values (Ra) displayed on the right were
presented as mean ꢀ SD with different letters, indicating statistical differences between the mean values (P < 0.05).
respectively. The acylated RPI gels had a lower Ra than that of the in Fig. 7 further conrm the results obtained by AFM. In conclu-
glycated protein gels, implying that interaction based on low level sion, slight modication with native RPI can reduce the roughness
of acylation RPI could produce a smoother and atter protein gel of the RPI gels and change the gels surface morphology to a more
network than glycated RPI. The pictures of the RPI gels are shown continuous and uniform gel network structure.
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Fig. 7 The photos of native RPI gel, acylated RPI gels (RPI, RPI2, RPI3), and glycated RPI gels (RPI4, RPI5, RPI6). Gels were prepared at 15% (w/v) at
95 ꢁC in water bath for 30 min, while RPI6 was prepared at 17% (w/v) at 95 ꢁC in water bath for 30 min.
Fig. 8 The principal component analysis of native RPI and modified RPIs formed by PC1 and PC2: factor loading (A) and factor scores (B).
Secondary structure is the percentage of ordered a-helix and b sheet contents.
differentiations among samples with different modications
based on the placement of each sample. The RPI1 sample resided
in the rst quadrant, but RPI4 and RPI5 samples were clustered
in the fourth quadrant. This indicated that acylated RPI with low
acylation degree were negatively correlated based on PC2, but
positively correlated based on PC1. In addition, we found that the
RPI1 was negatively correlated with RPI, RPI2 and RPI3 based on
the PC1, whereas it was positively correlated with RPI and RPI 2
based on PC2. Thus, according to the PCA modied RPI with low
acylation degree tended to facilitate the protein gel properties
with regards to hardness, springiness, denaturation enthalpies,
secondary structure, SH and WHC.
3.11 Principal component analysis of RPI samples
Principal component analysis (PCA) was conducted to investigate
the associations among the protein gel properties (SH, So, WHC,
secondary structure, hardness, springiness, denaturation
enthalpies, cohesiveness, Ra) of native RPI, acylated RPIs and
glycated RPIs samples.48 The factor loading plot and factor scores
plot were shown in Fig. 8. As shown in the Fig. 8A, the rst
component (PC1), which showed 43.9% of the variance, was
positively correlated to cohesiveness, hardness, springiness,
denaturation enthalpies and Ra, but was negatively correlated to
the SH, WHC and secondary structure. The secondary compo-
nent (PC2), which showed 24.8% of the variance, was positively
correlated with SH, WHC, secondary structure, hardness,
springiness and denaturation enthalpies. However, So, cohe-
siveness, and Ra showed negative correlations with PC2 (Fig. 8A).
The two principal components explained 68.7% of the total
variation. The score plots shown in Fig. 8B enabled
4. Conclusion
In this study, the effect of acylation and glycation on the phys-
icochemical and gelation properties of RPI was investigated.
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Amino acid composition analysis and SDS-PAGE demonstrated
that acylated and glycated RPIs were likely formed by the
conjugation of succinyl groups and dextran to Lys, and Lys, Met,
Ile, leu and Pro, respectively. Both modications increased the
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Conflicts of interest
22 X. Guo and Y. L. Xiong, LWT–Food Sci. Technol., 2013, 51,
397–404.
No conict of interest exits in the submission.
23 M. Aider and C. Barbana, Trends Food Sci. Technol., 2011, 22,
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27 T. J. Wooster and M. A. Augustin, Food Hydrocolloids, 2007,
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Acknowledgements
Funding for this work was provided by the National Natural
Science Foundation of China (31501522, 31571767), Nanjing
Agricultural Science and Technology Research project (Project
No. 201505056) and a project funded by the Priority Academic
Program Development of Jiangsu Higher Education Institutions
(PAPD).
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