Food Hydrocolloids 96 (2019) 288–299

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

Improvement of the solubility and emulsifying properties of rice bran T

protein by phosphorylation with sodium trimetaphosphate
Zhenying Hua, Liang Qiub, Yong Suna, Hua Xionga,∗, Yasumitsu Ograc
a
State Key Laboratory of Food Science and Technology, NanChang University, Nanchang, 330047, PR China
b
Jiangxi University of Traditional Chinese Medicine, Nanchang, Jiangxi 330004, PR China
c
Laboratory of Toxicology and Environmental Health, Graduate School of Pharmaceutical Sciences, Chiba University, Chuo, Chiba, 260-8675, Japan

A R T I C LE I N FO A B S T R A C T

Keywords: Rice bran protein (RP) could represent an important component of the protein resources in daily life because a
Rice bran protein large amount of it remains after rice processing. At present work, RP underwent phosphorylation using sodium
Phosphorylation trimetaphosphate under hydrothermal treatment at different pH values (3.0, 5.0, 7.0, 9.0 and 11.0), and the
Emulsion utilization of phosphorylated products in emulsions was also assessed. Phosphorylated RP at pH 9.0 showed the
XPS
highest solubility and emulsifying activity. According to the results of the phosphorus content, zeta potential,
Emulsion stability
infrared spectroscopy and X-ray photoelectron spectroscopy analysis, phosphate groups were introduced to RP,
and the phosphate esters (P=O) were detected. With the analysis of surface hydrophobicity, intrinsic fluores-
cence, and circular dichroism spectra, phosphorylation by hydrothermal treatment changed the secondary and/
or tertiary structure of proteins and increased the solubility and emulsifying activity. Emulsions prepared with
phosphorylated RP at pH 9.0 were stable over a range of environmental conditions: pH 7–9, NaCl < 50 mM, and
temperatures < 90 °C were studied. It shows that RP phosphorylated by sodium trimetaphosphate could be
utilized in the formulation and production of natural emulsion-based products as emulsifier.

1. Introduction characteristics of RP are expected to be modified in order to maximize

Rice bran is the by-product of rice milling and is considered to be a
rich source of protein, fat, carbohydrate and micronutrients, such as
minerals and phenolic acid (Han, Chee, & Cho, 2015). Almost 90% of
the rice bran worldwide is utilized as fodder for livestock, and others
were used for the extraction of rice bran oil (Zullaikah, Melwita, & Ju,
2009). Rice bran seemed to be minimally utilized in the food industry
because of its high fibre content and possible hull contamination
(Hamada, 1997). Accounting for 5–8% of total grain, rice bran contains
11–13% crude protein (Gul, Yousuf, Singh, Singh, & Wani, 2015).
Meanwhile, rice bran protein (RP) is recognized as a suitable protein
source for infants, young children and special groups, which is attrib-
uted to its hypoallergenic nature and reasonable balance of amino acids
(Hou et al., 2017). Thus, RP could represent an important component of
the protein resources in daily life because a large amount of it remains
after rice processing. Additionally, RP shows good physicochemical
characteristic in foaming and emulsifying due to the presence of hy-
drophobic and hydrophilic groups (Fabian & Ju, 2011). Hence, this
finding suggested that RP could be utilized in drinks and baked pro-
ducts (Sharif, Butt, Anjum, & Khan, 2014). The physicochemical
the utilization in the food industry.
To the best of our knowledge, the application of phosphorylation in
protein modification could be an effective and economical way to im-
prove the functional properties of protein and has already been con-
ducted in soybean, peanut, whey, egg white isolated protein, and others
(Cheng et al., 2017; Li, Sun, Ma, Jin, & Sheng, 2018; Sánchez-Reséndiz
et al., 2018; Yu et al., 2015). Based on its chemical versatility, phos-
phate can form mono-, di- and tri-esters with alkyl and aryl hydroxyl
groups as well as acid anhydrides, which are known to occur on nine
amino acids in protein including Ser, Thr, Tyr, Arg, Lys, His, Cys, Asp
and Glu, and are stable in aqueous solution at physiological pH (Hunter,
2012). According to the previous report, the mechanism of modification
of food protein by phosphate is that, the protein side chain groups,
including –OH groups of Ser, Thr and Tyr, ε-NH2 of Lys, the 1 and 3
nitrogen atoms of the His imidazole ring and the nitrogen atom of the
Arg guanidine group, were selectively induced by a large number of
phosphate groups (Fig. 1) (Sung, Chen, Liu, & Su, 1983). With the in-
creasing negative charges introduced by phosphate groups on the pro-
tein molecular surface, the protein will be enhanced with respect to
hydration. This step leads to the improvement in water solubility, water

∗
Corresponding author.
E-mail address: huaxiong100@126.com (H. Xiong).

https://doi.org/10.1016/j.foodhyd.2019.05.037
Received 12 January 2019; Received in revised form 25 April 2019; Accepted 21 May 2019
Available online 22 May 2019
0268-005X/ © 2019 Published by Elsevier Ltd.
Z. Hu, et al.
Fig. 1. Phosphoesterification reaction between protein isolates and sodium trimetaphosphate (STMP) as cited in the work of Sung et al. (1983).
and oil retention capacity, emulsification and foaming properties of the
protein (Moure, Sineiro, Domínguez, & Parajó, 2006). To date, the
study of phosphorylation on RP modification is limited, as well as ap-
plications in the food industry. Sodium tripolyphosphate (STP), sodium
trimetaphosphate (STMP) and POCl3 are regarded as safe food additives
and could represent economical reagents for large-scale production of
modified proteins in the food industry (Kato, Aoki, Kato, Nakamura, &
Matsuda, 1995). Meanwhile, STMP can be more efficient in chemical
reactions compared to the straight chain polyphosphates due to its ring-
shaped molecular structure (Sánchez-Reséndiz et al., 2018). Hence, we
proposed to improve the functional properties of RP by phosphorylation
with STMP.
The proper pH range for the reaction of protein molecules with
STMP spans from 7 to 9 regarding thermal treatment in aqueous solu-
tion (Cheng et al., 2017). However, RP is composed of 37% albumin
(water-soluble), 31% globulin (salt-soluble), 2% prolamin (alcohol-so-
luble) and 27% glutelin (alkali-soluble protein) (Fabian & Ju, 2011).
Thus, it seems that the large proportion of protein fractions (primarily
glutelin) could not react sufficiently with STMP under near-neutral
environments due to its low solubility and dispersibility. It has been
reported that the solubility of rice bran glutelin could be improved by
the phosphorylation under hydrothermal treatment at 55 °C, pH 9.7
(Ma, Na, Cheng, & Wang, 2017). Technically, phosphorylation might
rigidify the RP structure by introducing additional favourable contacts
between RP residues, which would be accompanied by increasing the
exposure of hydrophobic side chains, resulting in decreasing its solu-
bility (Chen, Liu, Wang, Ye, & Shen, 2017). Chen, Liu, Wang, Ye, and
Sheu (2017) conduct phosphorylation to rice protein, of which the
condition was at 35 °C, pH 11.5, and 20.6% of Ser residues were
phosphorylated while no enhancement of solubility was observed, or
even worse. Hence, the improvement of solubility and emulsifying
properties did not only count on the phosphorylation degree, but also
depend on the structure of protein after the modification. In the present
work, we aim to study the effect during phosphorylation under hy-
drothermal treatment on the modification of RP considering the solu-
bility and emulsifying activity. The study was also conducted to eluci-
date the presence and mechanism of phosphorylation during the
chemical modification by the methods of fluorescence, circular di-
chroism (CD), Fourier transform infrared (FT-IR) spectroscopy, scan-
ning electron microscopy (SEM) and X-ray photoelectron spectroscopy
(XPS) analysis. Furthermore, we evaluate the potential utilization of the
modified product within emulsions in terms of stability under different
environmental condition. It is expected that the work could be helpful
to understand the potential utilization of protein modifications by
phosphorylation.
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Food Hydrocolloids 96 (2019) 288–299
2. Material and methods
2.1. Material and chemical reagents
RP was extracted by 0.05 M NaOH solution and isoelectric pre-
cipitation from defatted rice bran (the basic information of rice bran
shown in Table S1), which was purchased from local farmer market,
and the protein purity of RP was 88.9% (determined by Kjeldahl Ni-
trogen, N × 5.95), of which the contamination was starch and minerals.
1-anilio-8naphthalenesulphonate (ANS) was of analytical grade. Whey
protein isolate (WPI; Mingrui Chemicals Company, Zhenzhou, Henan
province, China) and STMP was purchased from Sigma-Aldrich In-
dustrial Co. Ltd. (Shanghai, China). The Reagent C for the determina-
tion of phosphorylation degree, was prepared freshly each time, by
mixing 10 mL of 6 M sulfuric acid with 20 mL of distilled water and
10 mL of 2.5% ammonium molybdate, then adding 10 mL of 10% as-
corbic acid. The soybean oil was bought in the supermarket. All re-
agents used here were of analytical grade or higher.
2.2. Preparation of phosphorylated RP
RP with 2% (w/v) dispersion was added to 0.1 M STMP at different
pH (3.0, 5.0, 7.0, 9.0 and 11.0) with adjustment of 1 M NaOH and/or
HCl. The mixture was continuously stirred at 55 °C for 2 h in water bath.
After the neutralization with NaOH and/or HCl, the samples were
dialyzed to remove free STMP for 72 h against deionized water (sam-
ples/deionized water, 1:100), which was changed every 6 h. After
lyophilizing, the samples were collected, named as P-RP3, P-RP5, P-
RP7, P-RP9 and P-RP11, respectively, and raw RP as the control. Then
the samples were stored at −20 °C for further studies.
2.3. Physicochemical properties of phosphorylated RP
2.3.1. Determination of phosphorylation degree
The phosphorylation degree was represented by the phosphorus
content of sample and determined according to previously report with
minor modification (Xiong, Zhang, & Ma, 2016). 0.5 g of samples was
suspended in perchloric acid and left overnight at room temperature.
Thereafter, the suspension was wet-ashed at 170 °C with additional HCl
until it became transparent. The solution was diluted with deionized
water to 5 mL and filtered through a 0.45 μm syringe filter. 4 mL of
reagent C was added into the digested samples and capped with Par-
afilm and incubated in a 37 °C water bath for 1.5 h. Then cool to room
temperature and read the absorbance in UV/Vis spectrum (TU1810,
Beijing Purkinje General Instrument LTD Co., China) at 820 nm against
the blank. The standard curve for the quantitate was prepared by the
phosphorus standard solution. For the determination of inorganic
phosphorus, 5 mL of 12% trichloroacetic acid was added to the same
Z. Hu, et al.
volume of 10 g/L sample solution, which was centrifuged at 10,000 g
for 15 min. The amount of phosphorus bound to proteins was calculated
by the difference between the total phosphorus and the phosphorus
content in supernatant and presented as mg/g in terms of protein.
2.3.2. Determination of zeta potential
The zeta potential of samples was measured by a Zetasizer Nano-ZS
instrument (Malvern Instruments, Worcestershire, UK). Protein sample
was added into PBS solution (50 mM, pH 7.4) to a final concentration of
0.05% (w/v). After filtration through a 0.45 μm porous membrane, the
diluted samples were induced directly into the chamber of Zetasizer
Nano-ZS particle electrophoresis instrument prior to zeta potential
analysis at 25 °C. Zeta potentials were calculated from the electro-
phoretic mobility data obtained from Smoluchowski’ equation.
Measurements were carried out in triplicate for the time interval of 30 s
for 100 zeta runs.
2.3.3. Protein solubility
Lowry method was conduct here to determine protein solubility
with bovine serum albumin as standard (Lowry, Rosebrough, Farr, &
Randall, 1951; (Xu et al., 2016). In brief, protein sample was diluted in
Tris-HCl (50 mM, pH 7.0) to make 1% aqueous solution (w/v) and kept
stirring magnetically at room temperature for 30 min. After the cen-
trifugation at 10,000×g for 15 min, the protein content of supernatant
was determined by Folin & Ciocalteu's reagent. The result was calcu-
lated by the division of protein content in supernatant and total protein
in samples.
2.3.4. Emulsifying activity and emulsion stability of sample after
phosphorylation
Emulsifying activity and stability were determined by the method
from Zhao et al. (2012) with some modification. In brief, 16 mL of 0.1%
protein aqueous solution (50 mM PBS, pH 7.0) and 4 mL of soybean oil
were mixed and homogenized at 12,000 rpm for 1 min with a high-
shear mixer (IKA-T18, IKA-Werke GmbH&Co., Staufen, Germany). A
50 μL volume of pre-emulsion was dispersed into 5 mL of 0.1% SDS (w/
v) and read at 500 nm for the absorbance with 0.1% SDS solution as
blank by a UV spectrophotometer (TU-1810, Beijing Puxi General In-
strument Co., Beijing, China). The absorbance values measured record
at 0 min (A0) and 10 min (A10) after the formation of pre-emulsion were
used to calculate the emulsifying activity index (EAI) and the emulsion
stability index (ESI):
2 × 2.303
EAI (m2 /g) = × A0 × D
C × (1 − ∅) × 10 4 (1)
10 × A0
ESI (%) =
A0 − A10 (2)
Where A0 and A10 represent the absorbance at 500 nm at 0 min and
10 min, respectively. D was the dilution times and the value was 100
here. C is the protein concentration (g/mL) before homogenizer, and ϕ
is the oil volume fraction (v/v) of the pre-emulsion (ϕ = 0.20).
2.4. Structural characteristics of modified RP by phosphorylation
2.4.1. Surface hydrophobicity and intrinsic fluorescence
Surface hydrophobicity (H0) was measured according to the de-
scription of Zhao, Zhao, Chen, and Xiong (2019) with ANS as fluores-
cence probes. The fluorescence intensity was measured at wavelength
of 390 nm as the excitation and 470 nm as emission by a fluorometer
(F7000, Hitachi Co., Tokyo, Japan). H0 was the initial slope calculated
by a linear regression analysis of fluorescence intensity versus protein
concentration (%, w/v).
Intrinsic fluorescence was gained from a 0.01 mg/mL of protein
sample dispersed in PBS (50 mM, pH 7.0) by a fluorometer. The
fluorescent intensity was recorded as the emission wavelength from 300
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Food Hydrocolloids 96 (2019) 288–299
to 400 nm with an excitation wavelength of 280 nm.
2.4.2. Circular dichroism (CD) measurement
CD profile of samples were obtained from a 0.1 mg/mL of protein
sample dispersed in PBS (50 mM, pH 7.0) by a Bio-Logic MOS-450 CD
spectrometer (French Bio-Logic SAS, Claix, French) with a 1.0 mm path
length quartz cuvette. The samples were scanned from 190 to 250 nm to
gain the far-UV CD spectra with PBS as the reagent blank. The data was
analysed by the online SELCON3 method.
2.4.3. Fourier transform infrared (FT-IR) analysis
The FT-IR information of samples were recorded by an FTIR spec-
trometer (Nicolet Nexus 470, Nicolet Co., USA) with scanning the
whole band of (400-4000 cm−1) at room temperature. In brief, a 2 mg
of samples was mixed with KBr and pressed into pellet. The transmit-
tance intensity was obtained in the wavenumber range of
4000–400 cm−1 at 4 cm−1 resolutions. Infrared spectrogram of sample
was a superposition of 32 times and the KBr spectrum as the back-
ground. All samples prior to determination were baseline corrected,
after background correction.
2.4.4. Scanning electron microscopes (SEM) analysis
The microstructure of sample was determined by SEM (Quanta
200F, FEI, Hillsboro, OR, USA) according to our previous work (Peng
et al., 2010). The samples were sprinkled onto a double-sided tape and
sputter-coated with a 5 nm-thick gold layer.
2.4.5. X-ray photoelectron spectroscopy (XPS) analysis
The surface chemical composition was analysed by a VG Multilab
2000 X-rays at 300 W with the axis of the energy analyzer normal to the
plane of the sample surface. Elemental surface compositions were ob-
tained at a pass energy of 25 eV with a 0.05 eV resolution. All spectra
were recorded by using double anode Al target and 0.05 eV resolution.
2.5. Influence of environmental stress on emulsion stability
The preparation of emulsion and the assessment of emulsion stabi-
lity under environmental stress were performed according to the
method of Xu et al. (2016) with minor modification.
2.5.1. Preparation of emulsion
Protein samples were dispersed into 10 mM PBS (pH 7.0) and stirred
for 3 h to completely hydrate. After centrifugation at 3000×g for
15 min, the supernatant was adjusted to the concentration of 5 mg/mL
determined by Lowry method. Then a 90 mL of solution was mixed with
10 mL of soybean oil and pre-homogenized at 12,000 rpm for 2 min
with a high-shear mixer (IKA-T18, IKA-Werke GmbH&Co., Staufen,
Germany) and emulsified by a Microfluidizer (NCJJ-0.007/200,
Langfang Tongyong Machinery Manufacturing Co., China) at 80 MPa
and repeated 3 times. 0.01% (w/v) of sodium azide was added into the
emulsions to protect from the growth of microbial growth.
2.5.2. pH stability
After adjusting pH at the range of 3–9 by 1M HCl and NaOH solu-
tions. The emulsions were transferred to 15 mL tube and stored 25 °C
for 24 h prior to particle size and zeta potential analysis.
2.5.3. Ionic strength stability
Freshly prepared emulsions were transferred into 15 mL tubes and
modified the ionic strength by adding 6 M NaCl solution to lead various
final NaCl concentrations (0–300 mM). Emulsions were vortexed and
stored for 24 h at 25 °C prior to particle size.
2.5.4. Temperature stability
Emulsion were transferred into 15 mL tubes and incubated in a
water bath at different temperatures (30–90 °C) for 30 min. After
Z. Hu, et al.
cooling to room temperature, samples were vortexed and stored for
24 h at 25 °C prior to particle size.
2.5.5. Storage stability
Emulsion were transferred into 15 mL tubes and incubated at dif-
ferent temperatures (4 °C, 25 °C, 60 °C) for 7 days prior to particle size.
2.6. Statistical analysis
All determination was conduct in triplicate and presented as
mean ± SD. Meanwhile, one-way analysis of variance (ANOVA) with
Duncan test were performed by SPSS statistics 20.0 (SPSS Inc., Chicago,
US). Differences were considered significant at p < 0.05 among the
samples.
3. Results and discussion
3.1. Effect of phosphorylation on physicochemical properties of modified RP
3.1.1. Total phosphorus content
Hydrothermal phosphorylation was conducted in modifying RP
under different pH conditions with STMP, and the total phosphorus
content of modified RP was summarized in Fig. 2A. It has been found
that the highest content of phosphorus in modified RP was achieved
when the reaction was performed at pH 9.0 (9.53 ± 0.522 mg/g),
Fig. 2. Phosphorus content (A), zeta potential (B), protein solubility (C) and the emulsifying properties (EAI and ESI) (D) of phosphorylated RP under treatments at
pH 3.0, 5.0, 7.0, 9.0 and 11.0. Each of the values was determined in triplicate and presented as the mean ± SD, and different lowercase letters indicate significant
differences between each group (p < 0.05).
291
Food Hydrocolloids 96 (2019) 288–299
which was significantly higher than raw RP (0.049 ± 0.006 mg/g)
(p < 0.05). According to the amino acid analysis, most of amino acids
were influenced after phosphorylation, especially Ser (Table S2). It
seems that amino acids of protein side chain groups reacted with
phosphate group by phosphorylation and generated phosphor-amino-
acid residues, leading to the decrease of amino acid contents, such as
Ser (Fig. 1) (Sung et al., 1983). Hydrothermal phosphorylation has been
widely used for protein modification in the food industry and numerous
industrial applications. To the best of our knowledge, the proper pH for
the reaction of protein molecules with STMP is within a range of 7–9
under thermal treatment (Cheng et al., 2017). However, the optimized
pH of reaction differs among various proteins. Ma et al. (2017) sug-
gested that the optimal pH of phosphorylation for rice bran glutelin
treated by STP was 9.7 according to single factor experiments. This
finding was similar to our findings in this study. The phosphorus con-
tents of modified RP reacted above pH 7 were higher than that in the
acidic solution environment (Fig. 2A). The amino or hydroxyl groups in
the side chains of lysine or serine are at least partially deprotonated and
lead to the enhancement of contact with STMP when above pH 8
(Feeney, 1987). When phosphorylated modification was performed at
pH levels higher than 12, deprotonation of OH groups of serine residues
reached equilibrium, and no further improvement of phosphorylation
was observed (Sánchez-Reséndiz et al., 2018). This finding is poten-
tially the reason why the phosphorus content of P-RP11
(8.16 ± 0.476 mg/g) was significantly lower than P-RP9 here
Z. Hu, et al.
Table 1
Secondary structure content of phosphorylated RP compared with native RP by
CD analysis.
Samples α-helix (%) β-sheet (%) β-turn (%) Random coil (%)
RP 20.4 38.6 16.5 24.3
P-RP3 16.2 28.6 24.0 31.2
P-RP5 10.7 34.2 20.8 34.2
P-RP7 23.1 36.8 16.5 23.6
P-RP9 34.5 11.0 23.1 31.3
P-RP11 34.3 11.4 22.9 31.3
(p < 0.05). Nevertheless, some proteins have been reported to contain
percentages of phosphorylation that are higher at pH values above 12
than at pH 11, such as peanut protein (Yu et al., 2015). Peanut protein
was regarded as difficult to modify due to its resistance to denaturation
(Feeney, 1987). Meanwhile, peanut protein was comparatively more
phosphorylated than other proteins because of its higher availability of
serine residues (Sánchez-Reséndiz et al., 2018). It seems that proteins
with different structural properties affect the degree of phosphorylation
in modifications of proteins under the same conditions. Interestingly, it
has been reported that the solubility of rice bran glutelin could be
improved by the phosphorylation under hydrothermal treatment at
55 °C, pH 9.7 (Ma et al., 2017) and the phosphorus contents of phos-
phorylated product were at the range from 7 to 25 mg/g protein, which
is much higher than our results. The different phosphorus contents
between Ma et al. (2017) and our results would be attributed to the
objective material, pH of reaction and reagent dose and chemical
component for phosphorylation. First of all, the RP was gained from the
rice bran and comprises albumin, globulin, prolamin and glutelin,
which Ma et al. only focus on the solo glutelin as the objective to study
the phosphorylation reaction. As we mentioned before, both of the
composition of amino acid, protein structure and properties would in-
fluence the reaction between phosphate and protein molecule. Sec-
ondly, the main purpose for Ma et al. is to optimize the condition of
phosphorylation between rice bran glutelin and sodium tripolypho-
sphate to improve the processing characteristics. The response surface
method was designed according to the principle of Box-Benhnken
central combination design based on the single factor experiments.
Hence, the results would be higher at optimized condition than ours, of
which the purpose is to evaluate the solubility and emulsifying prop-
erties to utilize in the formulation and production of natural emulsion-
based products as emulsifier and reveal the possible mechanism of
phosphorylation in RP. Thirdly, we conduct phosphorylation with 3%
sodium trimetaphosphate, while 9% sodium tripolyphosphate was ap-
plied by Ma et al. The dynamic reaction is different with the dose and
chemical species. Fourthly, the method of determination of phosphor-
ylation degree was different. The details of Ma et al.’s method was that,
‘molybdenum blue colorimetric method was adopted to determine the
phosphorus content in RBG before and after modification. Using
phosphorus standard reserve. Combined phosphorus (mg/g) = (Phos-
phorus content after modification - Phosphorus content before mod-
ification)/sample mass ×100%’. Obviously, the free sodium tripoly-
phosphate, which could still retain with the protein as contamination,
was not taken into consideration by authors. This is quite different with
ours, resulting the higher values of phosphorus probably. Taken to-
gether, it is difficult to discuss the differences between the results of Ma
et al. and our work in terms of phosphorus content.
3.1.2. Zeta potential
Fig. 2B summarized the zeta potential values of modified RPs. The
zeta potential of RP was electronegative because the isoelectric point of
RP (approximately pH 4.5) was lower than the pH value of the buffer
solution (PBS pH 7.0). In addition, the absolute values of the zeta po-
tentials of modified RPs show the same tendency with phosphorus
content of modified RPs. Compared to raw RP, the absolute value of
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Food Hydrocolloids 96 (2019) 288–299
Table 2
Secondary structure content of phosphorylated RP compared with native RP by
FTIR analysis.
Samples α-helix (%) β-sheet (%) β-turn (%) Random coil (%)
RP 21.2 47.9 3.2 27.8
P-RP3 21.2 43.5 11.0 24.2
P-RP5 20.2 43.3 10.8 25.7
P-RP7 20.9 46.8 6.4 26.0
P-RP9 22.8 38.4 14.6 24.3
P-RP11 23.9 36.5 14.7 24.9
zeta potential for phosphorylated RP increased from −4.0 mV (RP) to
−15.12 mV (P-RP3), −16.1 mV (P-RP5), −20.1 mV (P-RP7),
−23.3 mV (P-RP9) and −19.2 mV (P-RP11). This finding could be at-
tributed to the different degree of phosphorylation in RPs, as we
mentioned above (Fig. 2A). Taken together, the greater the abundance
of phosphate is introduced to RP, the higher the absolute value of zeta
potential is increased, since the attachment of phosphate increased the
net negative charge. The value of the zeta potential can be significantly
related to the stability of colloidal dispersions and indicates the degree
of repulsion between similarly charged particles (Wu, Zhao, Yang, &
Chen, 2014). In the emulsion system, the intensity of the electric
charges at the droplet surfaces was improved by the introduced nega-
tively charged phosphate groups with a sufficiently strong electrostatic
repulsion force between the droplets, leading to better steric stabiliza-
tion and prevention of the aggregation and coalescence between dro-
plets (Khan, Mu, Zhang, & Arogundade, 2014; Xiong et al., 2016).
Hence, the assessment of phosphorylated RP with respect to the utili-
zation of the emulsion could be conducted as a next step.
3.1.3. Solubility
The solubility of modified RP products was presented in Fig. 2C. All
sample solubilities were improved after phosphorylated treatment,
especially those of P-RP7 (42.3 ± 2.72%), P-RP9 (58.4 ± 2.61%) and
P-RP11 (35.3 ± 3.44%), as compared to the control (RP,
6.67 ± 1.46%). Even though the solubilities of P-RP3 (12.7 ± 1.10%)
and P-RP5 (15.4 ± 0.62%) were significantly higher than RP
(p < 0.05), the improvement of this property is insufficient for further
utilization. RP shows good physicochemical characteristics with respect
to foaming and emulsifying due to the presence of hydrophobic and
hydrophilic groups (Fabian & Ju, 2011). The hydrophilicity/hydro-
phobicity balance influences the protein solubility, which depends on
the amino acid composition, particularly at the protein surface (Moure
et al., 2006). Under phosphorylated treatment, protein side chain
groups selectively induced a large number of phosphate groups. With
the increasing negative charges introduced by phosphate groups along
the protein molecular surface, the hydration of protein will be en-
hanced and lead to the improvement in water solubility (Moure et al.,
2006; Sung et al., 1983). RP shows the lowest solubility at pI (iso-
electric point, approximately 4.5 for RP), leading to the aggregation of
protein molecules, since hydrophobic interactions between proteins are
far greater than the hydrophilic and hydration repulsion forces pro-
duced by charged residues in this condition. Therefore, the solubility of
RP is poor at acid condition around pI. Fewer amino residues could
react with STMP in acidic conditions near the pI than those in neutral
and alkaline environments. Phosphorus content and solubility of P-RP3
and P-RP5 were lower than those of P-RP7, P-RP9 and P-RP11 (Fig. 2A
and C). Meanwhile, more negative charges were introduced by STMP at
the surface of the protein molecule when the pH value of the reaction is
far from the pI of RP (Fig. 2B). Hydrophilic and hydration repulsion
forces can overcome hydrophobic interactions, enabling phosphory-
lated RP to maintain high solubility (Yu et al., 2015).
3.1.4. Emulsifying activity
As previously mentioned, RP exhibits good physicochemical
Z. Hu, et al. Food Hydrocolloids 96 (2019) 288–299

Fig. 3. Surface hydrophobicity (H0) of phosphorylated RP under pH conditions of 3.0, 5.0, 7.0, 9.0 and 11.0 during treatments (A). Each value was determined in
triplicate and presented as the mean ± SD, and different lowercase letters indicate significant differences between each group (p < 0.05). Also shown are the
intrinsic fluorescence (B) and the FT-IR profile within the wavelength range of 4000–400 cm−1 (C) and the wavelength range of 1700–500 cm−1 (D).

Fig. 4. SEM images for RP (A), P-RP3 (B), P-RP5 (C), P-RP7 (D), P-RP 9 (E) and P-RP 11(F), respectively, with an internal scale of 1 μm.

characteristics with respect to emulsifying due to the presence of hy- higher net negative charge in the surface of RP (Fig. 2B). During the
drophobic and hydrophilic groups (Fabian & Ju, 2011). With the in- preparation of the emulsion, a higher absolute value of the zeta po-
crease of phosphate content introduced to RP molecules, it showed tential of droplets significantly enhances steric stabilization and

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Fig. 5. Surface element content (a) and XPS spectra (536-528 eV) of RP and P-RP (pH 5.0, 7.0, 9.0) and peak fitting spectra of O1s in raw and phosphorylated RP (pH
5.0, 7.0, 9.0) (C–F).
prevents the aggregation and coalescence between droplets (Xiong
et al., 2016). Therefore, the emulsifying activity of RP should be im-
proved after phosphorylation. Fig. 2D shows the emulsifying properties
of raw RP and phosphorylated RPs. The EAI of emulsions from raw RP
and phosphorylated RP at 0 min were 1.70 m2/g (RP), 3.83 m2/g (P-
RP3), 5.45 m2/g (P-RP5), 6.91 m2/g (P-RP7), 13.72 m2/g (P-RP9) and
13.01 m2/g (P-RP11), respectively, indicating that the emulsifying ac-
tivity of RP was obviously improved by phosphorylation, as expected,
and that P-RP9 showed the highest emulsifying activity when compared
to other treatments. The emulsifying activity reflects the rapid ad-
sorption of proteins at the oil/water interfaces during the formation of
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Food Hydrocolloids 96 (2019) 288–299
an emulsion (Benelhadj, Gharsallaoui, Degraeve, Attia, & Ghorbel,
2016). With the induced negative charges of phosphate groups in
protein chain residues, phosphorylated RP could move rapidly to the
oil/water interfaces and improve the adsorption of the protein onto the
oil-water interfacial layer, as well as the dispersion of the oil droplets,
by increasing the electrostatic repulsion force of droplets (Thaiphanit &
Anprung, 2016). However, P-RP3 and P-RP5 showed lower emulsifying
activity when considering EAI values. This may be attributed to the low
solubility and minimal negative charge introduced. The protein could
not be sufficiently hydrated to play an important role as emulsifier.
The ESI of raw and phosphorylated RPs over a time period of 10 min
Z. Hu, et al. Food Hydrocolloids 96 (2019) 288–299

Fig. 6. Influence of temperature on the mean particle size of the P-RP9 emul-
sion and WPI emulsion (A), visual graphs of P-RP9 emulsions (B) and WPI (C)
emulsions, respectively.

were 50.0% (RP), 49.4% (P-RP3), 53.9% (P-RP5), 85.2% (P-RP7),

83.6% (P-RP9) and 89.5% (P-RP11), respectively. The highest ESI was
observed at pH 7.0, 9.0 and 11.0, and no significant difference was Fig. 7. Influence of pH on the mean particle sizes of the P-RP9 emulsion and
found among them (p > 0.05). This finding indicated that ESI im- WPI emulsion (A), visual graphs of P-RP9 emulsions (B) and WPI (C) emulsions,
proved with increasing phosphorylation pH and reached a platform respectively.
after pH 7.0. ESI referred to the ability of emulsions to remain dispersed
without coalescing, flocculating and creaming, and ESI values were
3.2. Mechanism of phosphorylation by STMP in modified RP
found to be positively correlated with protein surface charge and so-
lubility (Karaca, Low, & Nickerson, 2011; Patel & Kilara, 1990).
3.2.1. Surface hydrophobicity and intrinsic fluorescence
Meanwhile, the hydrophilicity/hydrophobicity balance influences the
To confirm whether the hydrothermal treatment with STMP influ-
protein emulsifying activity, as well. After the proteins are absorbed
enced the secondary and tertiary structures of protein, surface hydro-
and move rapidly to oil/water interfaces, the surface hydrophobicity of
phobicity (H0) and intrinsic fluorescence were taken into consideration.
the protein molecule is important to stabilize the oil droplet through
Surface hydrophobicity is a measure of the exposure of hydrophobic
hydrophobic interaction. Even though P-RP11 shows lower solubility
amino acids in the protein chain, which were otherwise buried within
and phosphorus content than that of P-RP9 (Fig. 2A and C), both EAI
the interior (Yerramilli, Longmore, & Ghosh, 2017). ANS was utilized
and ESI of P-RP11 exhibit similarity to P-RP9 (p > 0.05). This result
here as a fluorescent probe, which non-covalently binds to the surface
might be attributed to protein unfolding and increasing exposure of
hydrophobic regions of protein. The H0 values of raw RP and phos-
hydrophobic moieties, leading to the improvement of the emulsifying
phorylated RP under pH treatment were shown in Fig. 2A. Compared to
stability at pH 11.0. With better hydrophilic-lipophilic balance, the oil
raw RP and P-RP5, phosphorylated RP at pH 3.0, i.e., P-RP3, was ex-
droplet surface is easier to anchor by protein (Wan, Wang, Wang, Yuan,
posed to more hydrophobic moieties, an effect which was primarily due
& Yang, 2014). If the hypothesis is correct, the hydrothermal treatment
to aggregation of protein at acidic condition (p < 0.05). Compared to
with STMP should affect the secondary and/or tertiary structure as
raw RP (213), it was found that H0 values of phosphorylated RP im-
well, which could be proven by further analysis.
proved with increasing phosphorylation pH within the range of
5.0–11.0, indicating that protein unfolding leads to the change of
protein tertiary structure upon hydrothermal treatment with STMP.
Fluorescence intensity could play an important role as index of the

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Z. Hu, et al. Food Hydrocolloids 96 (2019) 288–299

extent of exposure of aromatic amino acids to water, which is related to

changes of protein tertiary conformation (Cui, Zhao, Yuan, Zhang, &
Ren, 2013). The emission fluorescence spectroscopy profile of raw and
phosphorylated RPs were shown in Fig. 2B. The fluorescence intensities
of phosphorylated RPs were higher than that of raw RP, except P-RP5,
because of more aromatic groups being exposed at the surface of pro-
tein molecules and increasing emission fluorescence (Xu et al., 2016).
The fluorescence intensity of phosphorylated RP improved with in-
creasing phosphorylation pH within the range of 5.0–11.0. This result
was coincident with previous surface hydrophobicity data (Fig. 2A).
Through hydrothermal treatment with STMP, aromatic groups of RP
were exposed to water, indicating the changes of tertiary conformation
and partial unfolding of protein. However, the maximum emission
wavelength (λmax) of phosphorylated RP shifted from 350 nm to
343 nm. This indicates that the environment of aromatic amino acids of
protein chain residues was changed, and suggest covalent binding to
other groups (Iosin, Toderas, Baldeck, & Astilean, 2009). It has been
reported that λmax was blueshifted when the aromatic amino acid be-
came buried within the interior of the protein, as shown by the decrease
of fluorescence intensity (Chen, Zhao, Sun, Ren, & Cui, 2013). In the
present work, we supposed that aromatic amino acids would be exposed
to water after the hydrothermal treatment with STMP and reacted with
phosphorylated groups at the surface of protein molecules, as we
mentioned before, considering the high fluorescence intensity and
blueshifted λmax in phosphorylated protein here.

3.2.2. Secondary structure content

Fig. 8. Influence of NaCl on the mean particle sizes of the P-RP9 emulsion and
WPI emulsion (A), visual graphs of P-RP9 emulsions (B) and WPI (C) emulsions,
respectively.
A deeper understanding of raw and phosphorylated RP secondary
structure was analysed using the far-UV CD spectrum, which is a gen-
eral technique to predict protein secondary structure which is particu-
larly useful for soluble proteins (Wang, Zhang, Wang, Wang, & Chen,
2015; Xu et al., 2016). Only slight differences were observed in phos-
phorylated RP (P-RP3, P-RP5 and P-PR7) comparing to raw RP (Fig.
S1). It seems that the secondary structure of protein was changed a little
under hydrothermal treatment by phosphorylation at acidic or around
neutral condition. However, the CD profiles of P-RP9 and P-RP11
showed quit different than others with strong negative bands from 200
to 240 nm observed. Generally, α-helical proteins have negative bands
at 222 nm and 208 nm and a positive band at 193 nm (Greenfield,
2006). It indicated that the α-helix content increased after hydro-
thermal treatment by phosphorylation. It has been reported that the β-
sheet structures are relatively stable, whereas the α-helix, β-turn, and
random coil structures are relatively flexible and open, as reported
previously (Yong, Yamaguchi, & Matsumura, 2006). To evaluate the

Fig. 9. Influence of storage time on the mean particle sizes of P-RP9 emulsion and WPI emulsion (A), visual graphs of P-RP9 emulsions (B) and WPI (C) emulsions
with different temperatures after 7 days of storage.

296
Z. Hu, et al.
changes, the contents of secondary structure were calculated by the
online SELCON3 method with CD data. Compared with raw RP, the β-
sheet content in phosphorylated RP decreased from 38.6% to 11.4%
upon hydrothermal treatment with STMP, whereas the contents of α-
helix, β-turn and random coil increased from 20.4% to 34.3%,
16.5%–22.9% and 24.3%–31.3%, respectively, indicating that hydro-
thermal treatment with STMP affected the secondary structure and led
to a more extended form (Table 1). However, the precision of analysis
should be based on the purity of samples (⩾ 95%) and the accurate
molecular weight of protein (Greenfield, 2006). Only imprecise data
about secondary structure contents could be obtained here in terms of
the mixture samples.
Considering the low solubility of raw RP and some phosphorylated
forms of RP, far-UV CD spectra cannot provide sufficient information
regarding the overall changes in proteins, since far-UV CD spectra
merely reflect water-soluble subunits and only the rough data for
mixture protein showed here. FTIR is a powerful technique that pro-
vides comprehensive insights into integrated structures, especially for
solid samples (Wang et al., 2015). Amide Ⅰ bands (1700-1600 cm−1),
according to FTIR analysis, are highly related to the secondary structure
of protein (Hu et al., 2018). Accordingly, the deconvolved spectrum of
the amide Ⅰ band was iteratively fitted with Gaussian band shapes
(Byler & Susi, 1986), and the corresponding secondary structures were
summarized in Table 2. Compared with raw RP, the β-sheet content in
phosphorylated RP decreased from 47.9% to 36.5 by hydrothermal
treatment with STMP, whereas the contents of α-helix and β-turn in-
creased from 21.2% to 23.9% and 3.2%–14.7%, respectively. When
exposed to extreme pH (⩾11) conditions, many globular proteins un-
dergo significant conformational changes, known as the molten globule
(MG) state. The MG state maintains most secondary structures but tends
to unfold and lose some of the tertiary structures (Goto, Calciano, &
Fink, 1990). Generally, β-sheets could be formed in aggregated protein
molecules, whereas turn structures were considered to be a product of
the protein unfolding of any higher ordered structures (Ellepola, Choi,
& Ma, 2005). Even though the data regarding secondary structure
contents showed slightly different results between FTIR and CD ana-
lysis, both of the results tended towards the decrease of relatively stable
structure (β-sheet) and increase of flexible and open structure (such as
α-helix and β-turn) within protein secondary structure. This finding
suggested that phosphorylated RP forms were unfolded and that RP was
disaggregated by hydrothermal treatment with STMP.
3.2.3. Microstructure
Fig. 4 shows scanning electron micrographs (SEM) of raw and
phosphorylated RP: raw RP (A), phosphorylated RP at pH 3.0 (B), 5.0
(C), 7.0 (D), 9.0 (E) and 11.0 (F), respectively. Compared to raw RP,
aggregated protein surfaces were observed in P-RP3, P-RP5 and P-RP7.
This was coincident with our findings with respect to secondary
structure change, since β-sheet content in phosphorylated RP below
neutral pH was shown to be higher than that in the alkaline environ-
ment (Tables 1 and 2). Interestingly, the surface profiles of P-RP9 and
P-RP11 were different from the aggregation pieces formed. Ma et al.
(Ma et al., 2017) showed the same results when phosphorylation was
performed in rice bran glutelin at pH 9.7. This indicated that RP were
unfolded during phosphorylation an d that the changes in protein
conformation differed with various pH conditions.
3.2.4. FT-IR analysis
FT-IR analysis has been widely used to study the protein chains
when the presence of phosphate esters is known or presumed (Sánchez-
Reséndiz et al., 2018). The FT-IR profiles of raw and phosphorylated RP
at the wavelength range of 4000–400 cm−1 were presented in Fig. 3C.
Clearly, the band in the range of 1700–1600 cm−1 was assigned to the
Amide Ⅰ bands, which were quite different between raw and phos-
phorylated RPs. As we discussed with respect to the secondary structure
of phosphorylated RP, the conformation of RP was influenced and
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Food Hydrocolloids 96 (2019) 288–299
caused the changes in secondary structure (Tables 1 and 2). To verify
changes in protein structure due to the phosphorylation in RP, the
details in the major IR-region (1400-800 cm−1) were traced in Fig. 3D.
The bands at ∼1395 cm−1, which were assigned to the stretching vi-
bration of C–N, showed differential absorption, indicating that the
atoms or groups that were bound to C–N of RP changed after phos-
phorylation. Meanwhile, the peaks at approximately 1238 cm−1 were
assigned to the stretching vibration of P=O, while the peaks at ap-
proximately 923 cm−1 were attributed to the stretching vibration of
P–O in phosphorylated RP (Xiong et al., 2016). Taken together, it was
shown that STMP changed the FT-IR profiles of RP due to modifications
within their chemical structures.
3.2.5. XPS analysis of modified RP product
The surface element content (A) and the XPS spectra (536-524 eV)
(B) of raw and phosphorylated RP (pH 5.0, 7.0 and 9.0) and peak fitting
spectra of O1s in raw and phosphorylated RP (pH 5.0, 7.0 and 9.0) (C–F)
were presented in Fig. 5. The surface phosphorus content in RP in-
creased after phosphorylation, and the surface phosphorus atomic
percentages were 0.46% (P-RP5), 0.65% (P-RP7) and 0.6% (P-RP9),
respectively, as compared to the control (0.14% RP). This suggested
that the phosphate group was successfully introduced to RP molecule
surfaces. Meanwhile, it can be observed that the binding energy of O1s
in phosphorylated RP (pH 5.0 and pH 9.0) shifted to ∼532.2 and
∼532.4 eV, respectively, compared with raw RP (∼532.6 eV) ac-
cording to Fig. 4B, suggesting that the chemical environment of oxygen
atoms changed after phosphorylation through a distortion of the elec-
tron clouds of oxygen atoms (Xiong & Ma, 2017). The binding energy
and chemical state of O1s in raw and phosphorylated RP (∼536 eV-
∼524 eV) were summarized in Fig. 5 (C–F) by peak fitting. Two peaks
at ∼532.9 and ∼531.7 eV were assigned to the chemical states of
C–OH and C=O in raw RP (Fig. 5C), respectively. With respect to the
effect of phosphorylation on RP, three peaks were observed in P-RP7
and P-RP9 at ∼539, ∼532.8 and ∼531.7 eV. The peaks around
∼532.8 and ∼531.7 eV were assigned to the chemical states of C–OH
and C=O by comparison to that in raw RP. However, Xiong and Ma
(2017) conjectured that the peak at ∼528.8 eV might be attributed to
the O–P bond which may be formed between ovalbumin and STP. In
addition, it has been reported that the peaks of bridging (P–O–P) and
non-bridging (PO-) oxygen were ∼534 and ∼532 eV, respectively
(Brow, Kirkpatrick, & Turner, 1990). Hence, the third peak at ∼539 eV
might be attributed to the O=P bond, considering the analytical results
of FT-IR and XPS.
3.3. Assessment of emulsifying stability of RP by phosphorylation under
different environmental stress
The physical state of oil-in-water emulsions under various en-
vironmental stresses was studied in this section. We assumed a com-
parison between phosphorylated RP (P-RP5, P-RP7 and P-RP9) with
raw RP in terms of emulsifier. Unfortunately, preliminary experiments
showed that RP could not form emulsions, and that the emulsion sys-
tems separated rapidly into oil and aqueous phases. Furthermore, the
emulsion consisting of P-RP5 and P-RP7 is not stable during storage
over 7 days (data not shown). This finding suggested that the raw RP, P-
RP5 and P-RP7 were unsuitable as emulsifiers with the notable excep-
tion of P-RP9. To assess the emulsion regularly, whey protein isolate
(WPI), which is widely utilized in the food industry as a commercial
emulsifier, was used for the emulsion stability studies as contrast.
3.3.1. Temperature
The effect of temperature on the mean particle diameter and
creaming stability of the emulsions was analysed and shown in Fig. 6.
The initial mean diameters of the droplets coated by P-RP9 and WPI
after homogenization were 981 nm and 731 nm at room temperature
(25 °C). This suggested that phosphorylated RP could be absorbed to the
Z. Hu, et al.
droplet surfaces more rapidly during homogenization, or that they were
more effective at preventing re-coalescence inside the homogenizer (Xu
et al., 2016). Both of the emulsions coated by P-RP9 and WPI showed
relative stability to thermal processing within the range of 25–60 °C,
with a slight change in mean particle diameter. However, a sharp in-
crease in the particle size of the emulsions prepared with P-RP9 was
observed from 985 nm to 1400 nm, in contrast to the mild change in
emulsions coated by WPI from 731 nm to 914 nm. The increase in
droplet aggregation in samples may be attributed to an increase in
exposure of hydrophobic groups during heating, leading to an increase
in electrostatic attraction and/or formation of disulfide bonds and at-
tenuation of the electrostatic repulsion between the droplets (Charoen
et al., 2011; Zhang, Wu, & Yang et al., 2012). Fortunately, visual ob-
servation of the emulsions showed that they were stable with respect to
creaming and that no distinct phase separation occurred.
3.3.2. pH
The influence of pH on the mean particle diameter and visual graphs
of the emulsions were presented in Fig. 7. Both of the emulsions pre-
pared by P-RP9 and WPI showed similar trends in the particle size
versus pH profiles. The mean particle diameter of the emulsion pre-
pared by P-RP9 decreased from 2285 nm to 981 nm, with an increase in
the pH value, and the emulsion prepared by WPI decreased from
1849 nm to 750 nm. Extensive droplet aggregation occurred from pH 3
to 5, and the emulsions were relatively stable from pH 7 to 9. It was also
observed that the emulsions separated into a bottom transparent serum
layer and a top white cream layer from 3 to 5 but were stable in re-
sponse to creaming at pH 7 and 9. The protein emulsifier showed low
emulsifying activity, and relevant emulsions are highly unstable to
aggregation and creaming near the protein isoelectric points, which
occurs at pH values of approximately 3–5 for RP and WPI. Furthermore,
increasing creaming velocity with particle size leads to the creaming
instability at lower pH (McClements, 2015).
3.3.3. Ionic strength
The influence of ionic strength on the particle sizes of the emulsions
was measured (Fig. 8). The WPI emulsion is stabilized through the
addition of NaCl. The emulsion prepared by P-RP9 showed little change
in particle size with increasing salt content in the range from 0 to
50 mM NaCl. In contrast, a sharp increase of droplet sizes occurred in
the range from 100 to 300 mM NaCl with phase separation. Droplet
aggregation can be attributed to the ability of the counter ions in the
salt to screen the electrostatic repulsion between the negatively charged
droplets (Xu, Luo, Liu, & McClements, 2017). When the NaCl con-
centration was above a critical level, the electrostatic repulsion could
no longer overcome the attractive interactions (van der Waals and
hydrophobic) between the droplets, thereby leading to aggregation.
3.3.4. Storage stability
Through a 7 days of storage test period at temperatures of 4 °C, 25 °C
and 60 °C, changes in the sizes of the droplets in the emulsions were
measured and presented in Fig. 9. Compared to the WPI emulsions,
which were stable throughout 7 days of storage at 4 °C and 25 °C, P-RP9
emulsions were shown to be stable systems, with aggregation of dro-
plets occurring from the 3rd day. Even though both of the emulsions
coated by P-RP9 and WPI showed relative stability to thermal proces-
sing within the range of 25–60 °C, with a slight change in mean particle
diameter (Fig. 6), the particle sizes of P-RP9 and WPI emulsions in-
creased to approximately 2000 nm according to the extent of storage.
Slight phase separation was observed in the P-RP9 emulsion on the 7th
day (Fig. 9B). Storage time may alter interfacial properties of emulsions
due to the rearrangement or desorption of the protein molecules at the
droplet surfaces (Huang et al., 2018). It has been reported that the
addition of anionic polysaccharides, such as xanthan gum, could im-
prove the thermal stability of emulsion due to a stronger electrostatic
and steric repulsion between the droplets (Xu et al., 2017). To complete
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Food Hydrocolloids 96 (2019) 288–299
the exploration of phosphorylated proteins in food emulsions, further
research should be conducted.
4. Conclusions
This work demonstrated that the solubility and emulsifying activity
of RP were improved by hydrothermal phosphorylation with STMP,
almost 8.7 times and 8.1 times, respectively, which could represent an
effective emulsifier candidate utilized in the food industry. Through the
phosphorylation, large amounts of phosphate accumulated and were
introduced into the surface of protein molecules, increasing the abso-
lute zeta potential value. Meanwhile, the protein structure unfolded,
showed more flexibility, and was transformed from the insoluble ag-
gregates present in the raw material to soluble components in the
phosphorylated protein under alkaline condition with thermal treat-
ment. In addition, buried hydrophobic groups were exposed to the
outer surface of the molecule and/or reacted with phosphate groups,
leading to the increase of surface hydrophobicity and intrinsic fluor-
escence with blue shift. FT-IR spectra and XPS analysis indicated the
presence of phosphate esters (P=O), confirming the modification of
protein structure. Phosphorylated RP prepared at pH 9.0 showed good
emulsifying activity and stability. However, oil droplets stabilized by P-
RP9 were unstable with respect to aggregation at pH values in the vi-
cinity of the isoelectric point of the modified product and with the
addition of salt and high temperature. Further research is required to
optimize the preparation of phosphorylated RP and improve the sta-
bility of the emulsions.
Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgement
This study was supported by grants from the Research Program of
State Key Laboratory of Food Science and Technology, Nanchang
University (Project No. SKLF-ZZA-201609), the Natural Science
Foundation of Jiangxi Province of China (20171BBF60047), Key project
‘5511’ for Science and Technology Research of Jiangxi Province
(S2018ZDYFE0040) and Major science and technology project of
academy of sciences in Jiangxi (2018-YZD1-05).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.foodhyd.2019.05.037.
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