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Relationship between the emulsifying properties and formation time of rice bran protein fibrils
Journal Pre-proof Relationship between the emulsifying properties and formation time of rice bran protein fibrils Shuxian Pang, Ping Shao, Qingjie Sun, Chuanfen Pu, Wenting Tang PII:
S0023-6438(19)31327-1
DOI:
https://doi.org/10.1016/j.lwt.2019.108985
Reference:
YFSTL 108985
To appear in:
LWT - Food Science and Technology
Received Date: 22 September 2019 Revised Date:
4 December 2019
Accepted Date: 21 December 2019
Please cite this article as: Pang, S., Shao, P., Sun, Q., Pu, C., Tang, W., Relationship between the emulsifying properties and formation time of rice bran protein fibrils, LWT - Food Science and Technology (2020), doi: https://doi.org/10.1016/j.lwt.2019.108985. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
Pang shuxian: Conceptualization, Writing - Original Draft, Data Curation. Shaoping: Supervision. Sun qingjie: Project administration. Pu chuanfen: Methodology. Tang wenting: Writing - Review & Editing, Funding acquisition.
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Relationship between the Emulsifying Properties and Formation Time of Rice Bran
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Protein Fibrils
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Shuxian Pang a, Ping Shao b, Qingjie Sun a, Chuanfen Pu a,*,Wenting Tang a,*
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a School of Food Science and Engineering, Qingdao Agricultural University,
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Qingdao 266109, China
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b Department of Food Science and Technology, Zhejiang University of Technology,
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Zhejiang, Hangzhou 310014, China
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*Corresponding author.
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Tel.: +8615863002210; +8613854269282
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E-mail: [email protected] (W. T. Tang); [email protected] (C. F. Pu)
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Abstract:
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In this study, oil-in-water emulsions stabilized with rice bran protein fibrils
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(RBPFs) were prepared, and the effect of the fibrils’ formation time on the structural
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and emulsion characteristics, including droplet size, zeta-potential, and surface
27
morphology, were analyzed. As the heating time increased to 420 min, the β-sheet
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structure content and surface hydrophobicity of the RBPFs increased. As the heating
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time continued to heat up to 600 min, a downward trend occurred. The formation time
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also affected the mean contour length of the fibrils, and the scale began to decrease at
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540 min. The emulsion prepared by heating for 480 min showed the best emulsifying
32
activity, and the 420-min fibril emulsion showed the best emulsion stability and the
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highest interfacial protein content. The rheological results showed that as the heating
34
time increased (0–480 min), the storage modulus increased, and the loss modulus and
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the apparent viscosity were reduced. The emulsions in the range of 0–100 s-1 shear
36
rate did not have gel properties and shear thinning. The results of this study will help
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to provide a new perspective to develop a stable food-grade emulsion in the future.
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Keywords ： Rice bran protein fibrils; RBPFs emulsion; Rheological properties;
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Formation time
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2
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1. Introduction
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Oil-in-water (O/W) emulsions are widely used in food, nutrition, and medicine as a
44
means of delivering lipophilic flavorings, antioxidants, functional lipids, and various
45
other biologically active compounds (Ge et al., 2017). However, since the emulsion
46
consists of water and a water-immiscible non-polar liquid (oil phase), the colloidal
47
dispersion is thermodynamically unstable colloidal dispersion, with the oil phase
48
dispersed within an aqueous phase. Therefore, interfacial stabilizers, including protein,
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starch and other polysaccharides, small molecule polymers, amphiphilic lipids, etc.,
50
have been used to stabilize the emulsion system. Among these stabilizers, natural food
51
proteins have favorable interfacial film-forming properties when adsorbed to the
52
oil-water interface, which reduce the interfacial tension. To date, an emulsion system
53
is considered an important component in food processing (Feng, Cai, Wang, Li, & Liu,
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2018).
55
Recently, fibrils derived from globulin have received much attention and are used
56
in materials science such as food and biopharmaceuticals (Serfert et al., 2014). At the
57
pH away from the isoelectric point (pI, commonly at pH ≈ 2.0), thermal denaturation
58
of the protein can lead to an aggregation of proteins into micron-length (1―10 µm),
59
nano-diameter (1―10 nm) fibrils and multi-stranded twisted structures rich in
60
β-sheets perpendicular to the fibril axis (Mohammadian, & Madadlou, 2018). Acid
61
heat treatment usually plays a role in unlocking protein molecules and exposing 3
62
internal hydrophobic areas. The formation of linear fibrils is mainly controlled by the
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balance of intermolecular forces, including the attraction (mainly hydrophobic bonds)
64
between pyrolytic folding molecules and repulsive forces. With high-aspect ratio
65
anisotropic properties, fibrils have specific interfacial behaviors due to capillary
66
forces. Most food proteins have the ability to form such fibrils, such as
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beta-lactoglobulin, whey protein isolate, and soy protein isolate (Gao et al., 2017).
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Studies have pointed out (Serfert et al., 2014) that as anisotropic particles, protein
69
fibrils have better emulsifying effects than rigid-bound spherical particles due to a
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larger aspect ratio. In the interfacial shear rheology analysis, fibrils exhibited a high
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degree of elasticity at the O/W interface. Recently, protein fibrils have been widely
72
used to prepare emulsions. Serfert et al. (2014) prepared anti-oxidized fish oil
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emulsions with β-lactoglobulin fibrils, which have better barrier properties than the
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original protein, resulting in lower permeability, effectively improving the oxidative
75
stability of fish oil. Gao et al. (2014) studied the preparation of stable O/W Pickering
76
emulsions of β-lactoglobulin fibrils at different fibril concentrations and pH. When
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the concentration of fibrils in O/W emulsion is above 5 mg/ml, the emulsion has
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long-term stability. Although stability is acceptable, an excessive concentration of
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fibrils can lead to larger emulsion droplet sizes.
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Rice bran protein (RBP) is composed of albumin, globulin, gluten, and prolamin.
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RBP is recognized as a high-quality vegetable protein with low hypoallergenic
82
activity. Zhang, Huang, Liu & Wei (2014) prepared fibrils by heating RBP under pH 4
83
2.0 at 90 ℃, and found that adding fibrils could significantly promote
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solution-thickening and gel-hardening behavior. Li, Zhang & Li (2014) studied the
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effect of ionic strength on the formation and characteristics of RBPFs, and pointed out
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that the presence of cellulose can change the structural properties of the RBP gel
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system, which is suitable for natural nano-scale gel materials. However, the
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emulsification characteristics of RBPFs are not clear.
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The objective of this study was to evaluate the effect of formation times on the
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structural properties and molecular flexibility of RBPFs. Then the emulsion properties
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of the RBPFs obtained under different times were evaluated. The physicochemical
92
properties of fish oil emulsion stabilized by RBPFs prepared under different times
93
were also compared, such as rheological properties, morphology, particle size
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potential, interface protein content.
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2. Materials and methods
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2.1. Materials
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Rice bran protein with 0.98 g/mL purity was obtained from Xi'an Connor Chemical
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Co., China. Fish oil obtained from Shanxi Guanchen Biotech Co., Ltd (Xi’an, China).
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8-anilino-1-naphthalenesulfonic acid (ANS), Thioflavin T (Th T), trypsin was
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purchased from Sigma-Aldrich (St. Louis, Mo, U.S.A). Trichloroacetic acid (TCA)
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was obtained from Sangon Biotech Co., Ltd (Shanghai, China). All other reagents
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used were analytical grades.
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2.2. Methods 5
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2.2.1. Preparation of RBP
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RBP was dissolved in distilled water (0.06 g/mL) and magnetically stirred for 24 h
106
to obtain RBP dispersion. This RBP dispersion was adjusted to pH 2.0 with 1 mol/L
107
HCl (ionic strength, 150 mmol/L) and centrifuged at 3000 ×g for 30 min in order to
108
remove insoluble materials. The supernatant was taken at 90 °C for a specific time (60,
109
180, 360, 420, 480, 540 and 600 min). Immediately after the end of the sample
110
heating, the samples were placed in an ice bath and stored under 4 °C for further
111
study.
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2.3. RBAFs evaluation
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2.3.1. Th T fluorescence analysis
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The fluorescence analysis was performed according to Zhang et al.'s method (2014)
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with some modifications. The Th T stock solution was prepared by dispersing 8 mg of
116
Th T into 10 mL of phosphate buffer solution (PBS, pH 7, 0.05 mol/L). Before the test,
117
the Th T stock solution was diluted 50 times as a working solution. Then, 20 µL of the
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RBPF sample was mixed with 3-mL Th T working solution, shaken evenly, and
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allowed to stand for 1 min before the fluorescence analysis using a fluorescence
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spectrophotometer (F2700, Hitachi, Japan). The excitation wavelength was set to 460
121
nm, the emission wavelength was set to 470―600 nm, the excitation and emission
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slits were both 5 nm, the voltage was 700 mV, and the scan rate was 500 nm/min. The
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value of F0/F as a function of heating time was determined, in which F0 was the
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maximum fluorescence intensity of unheated protein at 496 nm, and F was the 6
125
fluorescence intensity of protein fibrils at 496 nm for a different heating time.
126
2.3.2. Surface hydrophobicity(H0)
127
H0 was measured by 1-anililo-naphthalene-8-sulfonate (ANS) fluorescent probe,
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according to the method of Mantovani, Guilherme, Netto, & Cunha (2018) with a few
129
modifications. ANS was dispersed in PBS buffer (pH=7, 0.1 mol/L) to prepare a
130
working solution (0.01 mol/L) with the RBPF sample, mixed at a ratio of 15:1; then
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the sample mixture was shaken for 1 min and determined. The excitation wavelength
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was 370 nm, the emission wavelength scope was 400-600 nm, the emission and
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excitation slits were set to 2.5 nm, the operating voltage was 250 mV, and the
134
scanning rate was 500 nm/min.
135
2.3.3. Mean contour length and zeta- potential analysis
136
The mean contour length and zeta-potential of the sample were measured by
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dynamic light scattering (DLS) using Zetasizer Nano-ZS (Malvern Instruments, UK)
138
instrument at 25℃. The sample was diluted appropriately to meet the requirement of
139
the equipment.
140
2.3.4. Transmission electron microscope (TEM)
141
The RBPF sample was dripped onto copper grids coated with amorphous carbon
142
film and dyed with phosphotungstic acid (0.02 g/mL). After natural air drying, the
143
sample was observed using a transmission electron microscope (TEM, JEM-2200 FS,
144
JEOL Ltd., Japan) with 80 kV acceleration voltage, the levels of magnification were
145
1500 times. 7
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2.3.5. Attenuated total reflectance Fourier Transform infrared spectroscopy
147
(ATR/FTIR)
148
An ATR infrared spectrum of the samples was taken using a Nicolet iS10 (Thermo
149
Scientific, USA) instrument. RBPFs were freeze-dried and measured. The scanning
150
was performed 32 times in the wave number range of 4000 cm - 1 to 400 cm -1. Images
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and data were analyzed using OMNIC 8.0 software (Thermo Fisher Scientific，USA)
152
and Peakfit software.
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2.3.6. Molecular flexibility
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This measure was conducted according to the method of Li et al. (2019) with some
155
modifications. The trypsin solution (1 mg/ml) was prepared using Tris-HCl buffer
156
(0.05 mol/L, pH = 8.0). Then, 4 ml of RBPFs (1 mg/ml) was mixed with 250 µL of
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the enzyme solution at 37 °C for 5 min, and the reaction was terminated with 4 ml of
158
5 mg/mL trichloroacetic acid (TCA). The supernatant was collected by centrifugation
159
at 4000 rpm for 20 min. Molecular flexibility was measured at 280 nm and expressed
160
by its absorbance.
161
2.4. Preparation of RBPFs emulsion
162
An aqueous phase was prepared by dispersing 0.04 g/mL RBPFs in aqueous buffer
163
solution (10 mmol/L phosphate, pH 7.0). Coarse fish O/W emulsions containing 1/10
164
organic phase and 9/10 aqueous phase were formed using a high-shear mixer (D50,
165
WIGDENNS) for 5 min at 10,000 rpm. Then, ultrasonic treatment (5 min) was carried
166
out using a 6-mm probe in the ice bath with a sequence of 1 s of sonication and 1 s of 8
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rest (power: 540 W, frequency: 25 kHz). The prepared RBPFs emulsion was kept
168
under 4 °C.
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2.5. Emulsifying activity index (EAI) and emulsifying stability (ES)
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Emulsification activity (EAI) and emulsion stability (EAI) were determined
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according to Tang et al.’s method (2005). The emulsion sample was centrifuged at
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3500 r/min for 10 min, and the supernatant was taken for 100 µL mixed with 10 ml
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sodium dodecyl sulfate buffer (SDS, 1 mg/mL). The absorbance of the emulsion
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sample was measured at 500 nm using an ultraviolet spectrophotometer (TU-1810,
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PERSEE, China). EAI and ES were calculated by the following equations (Tang et al.,
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2005):
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(1)
178 179
(2)
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Where c is the concentration of protein in the sample solution, g/mL;
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EAImin and EAImax are the EAI values of the emulsion after 10 min and 0 min;
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φ is the fraction of the oil phase, which is 1/10 in this experiment;
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EAI is the largest EAI after emulsion formation;
184
L is the cuvette path, 0.01 m.
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2.6. Emulsion characterization
186 187
Based on the results of the RBPFs, we selected the samples heated for 60 min, 180 min, 360 min, 420 min and 480 min to prepare the emulsion. 9
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2.6.1. Emulsion droplet size and zeta-potential analysis
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The physical stability of emulsion was quantified by measuring the average
190
droplet diameter and zeta-potential of a freshly prepared emulsion by a Zetasizer
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Nano-ZS (Malvern Instruments, UK) instrument. Emulsion samples were
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moderately diluted with ultrapure water to avoid multiple scattering. All
193
measurements were carried out at 25℃.
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2.6.2. Adsorption rate of protein (AP) and interfacial protein concentration (Γ) of
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RBPFs emulsion
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According to the method of Ye (2008), some modifications have been made. The
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fresh emulsion was centrifuged at 13000 r/min for 15 min, and the top layer of the
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centrifuge tube was removed. Determination of protein content (Cf) was performed by
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the Bradford (Redmilegordon, Armenise, White, Hirsch, & Goulding, 2013) method.
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Adsorption rate of protein:
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Interfacial protein concentration:
(3) (4)
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Where, C0 was total protein content in the emulsion; ϕ was the oil phase ratio. In
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this paper, the volume concentration of oil phase was 1/10. D3,2 was the average
204
radius of emulsion surface.
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2.6.3. Optical microscopy observation
206
One mL of Th T buffer was added to the RBPFs solution, shaken, and mixed. After
207
1 min, the new RBPFs emulsion was prepared according to section 2.4. The whole
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process of production was kept from the light. The emulsion sample was dripped onto 10
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the slide with a volume of 1 mL, and the appearance of the emulsions were observed
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by an optical microscope (BX 35, Olympus, Japan) under a bright and fluorescence
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field with a 40×objective lens.
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2.6.4. Rheology test
213
The rheological properties of the RBPFs emulsion were evaluated by a MCR320
214
rheometer (Anton Paar, Austria), using a plate configuration and PP50 with 1-mm gap
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at 25 °C. Freshly prepared emulsions were used for all tests.
216
2.6.4.1. Static rheological measurement
217
Viscosity was evaluated in the linear viscoelastic region (LVER) at 25℃ with a
218
shear rate from 0.1―100 s-1. Viscoelastic data were fitted by the Ostwald de Waale
219
model (Zhang, Tan, Abbas, Eric, Xia, & Zhang, 2015):
220
(5)
221
Where, η was apparent viscosity, Pa·s; γ was shear rate, S-1; K was the consistency
222
index, Pa sn; and n was the index that provides information about the flow behavior
223
affected by shear rate.
224
2.6.4.2. Dynamic rheological measurement
225
Frequency scanning was carried out from 0.1 to 100 Hz at 25℃. The relationship
226
between storage modulus (G') and loss modulus (G'') was measured.
227
2.6.5. Raman spectra analysis
228
The Raman spectrum was recorded by Raman spectrometer (Thermo Fisher
229
Scientific, USA) and excited by a near-infrared laser at 785 nm according to Niu et 11
230
al’s method (2016) with a modification. The laser output power was 334 mV and the
231
data acquisition time was set to 40 s to obtain the best peak intensity. The scan range
232
for each test was 300 to 3050 cm-1. Images and data were analyzed using OMNIC 8.0
233
software (Thermo Fisher Scientific，USA).
234
2.6.6. Differential scanning calorimetry (DSC)
235
DSC-Q1000 differential scanning calorimetry (TA Instrument, USA) was used to
236
identify the thermal transitions in the samples during heating according to Zhang, Pu,
237
Tang, Wang & Sun (2019)’s method with some modifications. The lyophilized sample
238
(4-7 mg) was placed in a crucible and sealed, the temperature range was 25-300 ℃,
239
and the heating rate was 10 ℃/min. Sample was tested under nitrogen flow, reaction
240
gas set to 50-60 mL/min, dry gas was set to 200 mL/min.
241
2.7. Data analysis
242
Data analysis and mapping were performed using Origin 8 software. The data were
243
expressed as mean ± standard deviation (Means ± SD), and the significant level was p
244
< 0.05. All tests were performed in triplicate. The correlation analysis was analyzed
245
by SPSS 17.0 software and expressed as Pearson correlation coefficient.
246
3. Results and discussion
247
3.1. Th T fluorescence spectral analysis of fibrils
248
Th T is a cationic benzothiazole dye that can bind in parallel to the β-sheet
249
structure. This reaction results in a significant increase in the maximum fluorescence
250
intensity of Th T. Based on this principle, this fluorescent probe was used to study the 12
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formation and the number of amyloid fibrils in order to study the reaction. Figure 1A
252
shows the Th T fluorescence intensity of RBPFs at specific heating times. As the
253
heating time increased from 60 min to 420 min, the fluorescence intensity reached its
254
highest point, and then heated from 480 min to 600 min, the β-sheet content in RBP
255
began to decrease and entered the decline period of fibrils growth. This may be due to
256
the loss of the β-sheet structure due to excessive heating. Figure 1B shows the
257
fluorescence intensity changes of F0/F. When heated to 420 min, the fluorescence
258
intensity increased sharply, possibly due to the increase of β-sheets and the
259
enhancement of hydrophobic interactions. The subsequent sharp decline may be due
260
to the fibrosis reaction, the protein has reached a certain degree of hydrolysis, no more
261
hydrophobic groups as the building unit continue to self-assemble into fibrils.
262
3.2. Surface hydrophobicity (H0)
263
H0 is one of the most important factors affecting the interface properties of
264
proteins. 1-anililo-naphthalene-8-sulfonate (ANS) was used as the fluorescent probe
265
to determine the H0 values. Increased surface hydrophobicity improved fibrillation of
266
rice bran protein (Moayedzadeh, Madadlou, & Khosrowshahi, 2015). According to
267
Fig. 1C, the protein fibrils heated for 420 min showed higher fluorescence intensity
268
than the other times, indicating that the exposed hydrophobic groups varied with
269
heating time, that acid-heat treatment exposed a large number of hydrophobic groups,
270
and that the RBPFs exhibited an increase in hydrophobicity before heating for 420
271
min. After heating for 480 min, H0 of the RBPFs began to decline. This phenomenon 13
272
may be due to the fact that proteins are no longer hydrolyzed to form a large number
273
of beta-folded structures, but aggregate to form nuclei, which are linked to the
274
construction units and increase the length of protein fibrils.
275
3.3. Mean contour length, zeta-potential and flexibility of RBPFs
276
The distribution of RBPFs mean contour length was affected by the heat-induced
277
environment (Feng et al., 2018). Figures 2A―B show the mean contour length and
278
distribution of fibrils at different heating times. A typical bimodal shape appeared in
279
RBPFs heated for 420 min, resulting in a dramatic increase in fibrils. The mean
280
contour length of the fibrils gradually increased with the increase of heating time from
281
60 min to 420 min, until the fibrils heated for 480 min showed a downward trend.
282
This indicates that heat treatment promotes the fibrillation of RBP to form RBPFs
283
with larger size, and excessive heating time may cause the further fibrillation.
284
Zeta-potential is widely used to characterize the electrostatic interaction between
285
charged particles. Figure 2C shows the zeta-potential distribution of RBPFs, Figure
286
2C shows the zeta-potential distribution of RBPFs. When heating for a short time (60
287
min), the zeta-potential becomes less negative, and then becomes more negative
288
(180-360 min) as the heating time increases, and finally the zeta-potential becomes
289
less negative as heating continues (420-600 min). This shows that when heated for
290
360 min, the absolute value of zeta-potential was the largest, the electrostatic
291
interaction between protein molecules was the strongest, and the protein solution
292
tends to be stable. 14
293
Protein flexibility is an important aspect that affects its emulsification properties.
294
The flexibility of proteins is a controlling factor, which plays an important role in the
295
surface activity of proteins (Li et al., 2019; Razumovsky, & Damodaran, 1999).The
296
molecular flexibility of proteins is not only affected by covalent interactions but also
297
by non-covalent interactions such as van der Waals forces, electrostatic bonds, and
298
hydrophobic interactions (Li et al., 2019). Heat treatment may destroy some of the
299
above bonds and expand the flexible structure of RBPFs, thus increasing flexibility
300
with the increase of reaction time. The molecular flexibility of the protein slowly
301
increased before heating for 180 min, and quickly increased to the maximum value
302
(0.054) at 420 min (Fig.2D).
303
with RBP, the flexibility of RBPF increased significantly with prolonged heating time
304
(P <0.05), which indicates that the structure of RBP molecule was unfolded (Li et al.,
305
2019). Thermal denaturation can destroy non-covalent effects, so heat treatment of the
306
protein can improve its emulsifying properties (Damodaran, 2005).
307
3.4. TEM image of RBPFs
Then it gradually decreased after 420 min. Compared
308
The TEM image (Fig. 3) shows the profile length of the RBP fibrils, showing that
309
the prepared fibrils are semi-flexible under these conditions. It can also be seen that
310
the unheated protein (Fig. 3A) is a spherical particle, which gradually changes to the
311
shape of fibrils as the heating time is further increased. In addition, the DLS results
312
are smaller than the TEM size, probably because the DLS was hydrated, while the
313
TEM sample was measured after drying. 15
314
3.5. ATR/FTIR analysis
315
ATR/FTIR was widely used to analyze the secondary structure of various samples.
316
The characteristic bands in the protein ATR/FTIR spectra mainly include amide I
317
(1600―1700 cm-1) and amide II (1500―1600 cm-1) (Jung, Gunes, & Mezzenga, 2010)
318
combined with the original map (Fig. 4) peak position obtained by deconvolution and
319
secondary structure identification: α- helix, 1648―1660 cm-1; β-folding, 1626―1640
320
cm-1; β-turns, 1662―1684 cm-1; irregular curl, 1640―1650 cm-1. Table 1 shows that
321
as the heating time increases, the β-sheet structure of the protein also increases,
322
reaching the highest at 360 min, and then remaining stable. Gosal, Clark, Pudney &
323
Ross (2002) studied the fibrillation of bovine serum albumin and also found that the
324
number of β-sheets increased after heat-induced protein formation of amyloid fibrosis.
325
The main component of RBP was β-sheets structure, accounting for 26% of the total
326
secondary structure. The main secondary structure of RBP after the heat treatment
327
was still β-sheets structure, and the content of β-sheets structure of RBPFs was higher
328
than that of RBP, the RBPFs of 360 min was the highest reaching 30%. However, the
329
content of β-sheets structure of RBPFs 360 min was no longer increasing, which
330
indicates that the further heat treatment of RBP induced the loss of β-sheets structure.
331
3.6. EAI and ES analysis
332
EAI is an indicator of the emulsification efficiency of an emulsifier. The
333
emulsification activity of a protein is measured by the emulsification efficiency of a
334
protein molecule during homogenization. EAI represents the ability of a protein to 16
335
participate in emulsion formation under external force. ES is the ability of emulsion
336
droplets to maintain stable (Li et al., 2019). The higher the EAI, the higher the
337
emulsification activity. The emulsification activity of a protein is related to the
338
structure of its own molecule. At the same time, according to Table 2, in these six
339
emulsion samples, the RBPFs emulsion heated for 420 min had the best stability, so it
340
was believed that increasing the heating time within a certain range could improve
341
emulsion stability. Tang et al. (2013) found that after protein adsorption to the
342
oil-water interface, structural rearrangement generally occurs, which affects the
343
emulsification properties of proteins.
344
For the above reason, the relationship between the hydrophobicity and molecular
345
flexibility of RBPFs and the EAI and ES between RBPFs emulsions was investigated.
346
According to Table 3, the correlation coefficient between H0 and flexibility was 0.881
347
(p<0.05), the correlation coefficient of ES was 0.899, and the correlation coefficient
348
of EAI was 0.887. The correlation coefficient between flexibility and EAI was 0.962
349
(p<0.01), and the correlation coefficient of ES was 0.685. Hydrophobicity H0 is
350
positively correlated with flexibility, EAI and ES. This indicates that the trends in
351
hydrophobicity and molecular flexibility are consistent. Surface hydrophobicity and
352
flexibility are important factors affecting emulsification performance. In the previous
353
studies, researchers focused on the relationship between H0 and protein emulsification
354
properties and found a strong correlation (Li et al., 2019). Prak, Nakatani, Katsube,
355
Adachi, Maruyama & Utsumi (2005) found the emulsifying ability of protein is not 17
356
exactly the same as the emulsifying ability of surface hydrophobicity. Flexible protein
357
molecules tend to denature at the interface, while rigid protein molecules are less
358
susceptible. Studies by Kato, Komatsu, Fujimoto & Kobayashi (1985) have shown
359
that surface hydrophobicity is ultimately an important factor in achieving good
360
foaming and emulsifying properties due to the high surface hydrophobicity imparted
361
by the mild effector.
362
3.7. Emulsion characterization
363
3.7.1. Average droplet size and zeta-potential
364
The droplet size of the emulsion is one of the most important qualitative
365
properties of emulsions (Ge et al., 2017). According to our preliminary experiments
366
(Fig. S1), 0.01―0.05 g/mL of the fish oil concentration was selected, and the results
367
showed that the 0.04 g/mL protein concentration emulsion was the most stable. Liu &
368
Tang (2014) found that increasing the concentration (0.005―0.06 g/mL) of protein
369
particles can reduce the droplet size and increase the stability of the emulsion. As
370
result, the 0.04 g/mL RBPFs solution was chosen to prepared the emulsion. Figure 5A
371
shows that the average droplet size of the RBPFs emulsion did not change
372
significantly within 0-360 min. While the average droplet size of the emulsion
373
stabilized by 420 min RBPFs rose sharply, which was probably due to the more
374
formation of fibrils, causing the increase of the length of RBPFs those were located at
375
the O/W interface of the emulsion. In contrast, the concentration of protein fibrils was
376
not enough to cover the oil-water interface. For example, the length of the protein 18
377
fibril may not be enough to coat the fish oil droplets, the interfacial tension between
378
the O/W interface is great, and the oil droplets combine to make the particle size
379
larger.
380
Zeta-potential is an important indicator for evaluating the stability of the system.
381
Generally, the absolute value of the zeta-potential is high, the protein molecules are
382
repelled, and the solution tends to be stable. Figure 5B shows the zeta-potential
383
distribution of the RBPFs emulsion droplets, from +31 mV to +35 mV, and the
384
emulsion stabilized by RBPFs was positive. Zeta―potential did not show significant
385
changes indicating that the heating time has little effect on the RBPFs emulsion
386
zeta-potential. It might be due to the length of the heating time has little effect on the
387
zeta-potential of the RBPFs stabilized-emulsion, the overall emulsion potential does
388
not change much. Compared with protein fibrils, the zeta-potential of the emulsion
389
was converted into a positive charge, probably because the fibrils are negatively
390
charged, and the adsorption on the droplets is not enough to change the positive
391
charge of the droplets.
392
3.7.2. AP and Γ of RBPFs emulsion between O/W interface
393
Some studies (Kato et al., 1985) have pointed out that proteins with softer
394
structures are more likely to undergo structural transformation and thus adsorb at the
395
interface. According to the interface adsorption principle of polyelectrolytes, the
396
protein structure will be expanded and denatured during the interface adsorption
397
process. 19
398
It can be seen from Fig. 5C―D that as the heating time increases to 420 min, the
399
AP and Γ of the protein are continuously increasing, and only the emulsion sample
400
heated for 480 min shows a downward trend. This may be due to increased
401
hydrophobicity of the protein, which is consistent with the results of molecular
402
flexibility in Fig. 2. In the protein adsorption process, because the molecular weight of
403
the protein is larger, the adsorption rate at the interface is much lower than that of the
404
small molecule surfactant. After adsorption to the interface, the hydrophobic amino
405
acid of the protein tends to stretch toward the oil-containing direction and rearrange.
406
Therefore, compared with small molecule surfactants, protein emulsifiers have
407
weaker emulsifying ability, but the prepared emulsions have higher physical and
408
chemical stability.
409
3.7.3. Rheology properties of fish oil emulsion stabilized with RBPFs
410
Figure 6 shows the rheological characteristics of the fish oil emulsion stabilized
411
with RBPFs. Within the angular frequency range (0―100 s-1), the fact that the storage
412
modulus G' was less than the loss modulus G'' indicates that in this frequency range,
413
the emulsion does not exhibit gel properties, but exists in a liquid state in which
414
fluidity is good. As the heating time increases, the G' increases gradually, and the G''
415
decreases. The data in the figure were fitted by the Ostwald de Waale model (the
416
relevant parameters are shown in the insert table), and the K-value is lower, indicating
417
lower viscosity, and the n-value is higher, indicating that the emulsion is closer to a
418
Newtonian fluid (Wang, Li, Wang, & Adhikari, 2011). The table shows that the 20
419
K-value was the smallest for the emulsion heated for 420 min, indicating that the
420
viscosity
421
the emulsion exhibits shear thickening, n=1 means an ideal viscosity flow and n<1
422
suggests shear thinning occurs (Wei, & Gao, 2015). As seen in Figure 6 within the set
423
shear rate range (0-100 s-1), the viscosity of all samples gradually decreases, and shear
424
thinning occurs. The reason for this phenomenon is that oil droplets gather together,
425
the weak interaction force that maintains stable flocculation under high-speed shear
426
was destroyed, and shearing occurs thinning phenomenon. In protein emulsions, when
427
the protein concentration reaches a certain value, RBPFs that are not adsorbed to the
428
oil-water interface are present. Due to the presence of unabsorbed and amphiphilic
429
protein fibrils in the continuous phase, the fibrils tend to approach each other due to
430
secondary forces such as hydrophobic interactions, apparently increasing the viscosity
431
of the emulsion. And this thickening enhances the stability of the emulsion (Voutsinas
432
et al., 1983).
433
3.7.4. Morphology
was
the
lowest.
The
value
n
＞
1
represents
that
434
Figure 8 shows the morphology of six emulsion samples under an optical
435
microscope and a fluorescence microscope. According to the results of the
436
fluorescence microscope, it can be seen that the emulsion droplets gradually increase
437
as the heating time increases. And the unheated protein emulsion is darker, which may
438
be caused by the combination of the Th T dye and the β-sheet in the protein, which is
439
the same as that of the fluorescence spectrophotometer. 21
440
3.7.5. Raman spectroscopic analysis.
441
In order to examine the interaction between RBPFs and lipids, the structural
442
properties of RBPFs emulsion were determined using Raman spectroscopy. The ratio
443
of relative intensity ratio I2850 / I2880 and I2935 / I2880 reflects the interaction between
444
lipid chains and the state of order or disorder of acyl chains (Ruiz, Carmona, Jiménez,
445
& Herrero, 2013). It can be visualized from Table 4 that the emulsion of 420 min
446
RBPFs shows the lowest intensity ratios. The disorder of the acyl chain indicates that
447
more proteins were inserted between the acyl chains of the oil. (Herrero, Ruiz,
448
Pintado, Carmona, & Jiménez, 2018). Lower I2850/ I2880 and I2930/ I2880 intensity ratios
449
were also related to hydrophobic interactions including the CH groups of the lipid
450
with protein (Herrero et al., 2018). In addition, combined with the results of infrared
451
spectroscopy analysis, I2850 / I2880 was negatively correlated with the number of
452
β-sheets. Ruiz et al. (2013) also reported that the intensity ratio of the emulsion (I2850 /
453
I2900) was lower than that of the separated fatty phase and pointed out that most of the
454
hydrocarbon chains of milk fat globules and / or triglycerides are closely packed. This
455
hydrocarbon chain accumulation was very important for the stability of the emulsion.
456
3.7.6. DSC analysis
457
DSC were determined the thermal behavior of RBPFs stabilized emulsions with
458
different heating times. The emulsion stabilized by unheated RBP showed an initial
459
endothermic peak at 185 °C and a maximum exothermic peak at 146 °C. The
460
endothermic peak was caused by dehydration associated with the hydrophilic group of 22
461
the polymer, while the exothermic peak was associated with degradation of the
462
polyelectrolyte due to dehydration and depolymerization reactions (Li et al.,
463
2018).The exothermic peak of the RBPFs stabilized emulsion after heating was
464
smaller and broader compared with those of RBP emulsion (Fig. 9). This can be
465
explained as the hydrophobic interaction between RBPFs and fish oil (Sarmento,
466
Ferreira, Veiga, & Ribeiro, 2006).
467
The unheated RBP emulsion began to denature at about 85 °C, the RBPFs emulsion
468
denature temperature began to gradually increase, and the 420 min RBPFs emulsion
469
denature temperature reached about 140 °C, indicating that the corresponding
470
emulsion was the most stable among all the samples. In addition, the emulsion
471
stabilized by RBPFs heated for 180 min has a sharp exothermic peak near 176 °C,
472
suggesting the polymer degradation (Paula, Sombra, Cavalcante, Abreu, & Paula,
473
2011). This has been confirmed that RBPFs heated for 180 min will cause emulsion
474
instability in section 3.6
475
4. Conclusion
476
This study better understands the effect of different heating times on the
477
physicochemical properties of an RBPF-forming emulsion. When RBP were heated
478
for 420 min, the mean contour length and hydrophobicity of the fibrils were highest
479
and the corresponding RBPFs were exhibited favorable emulsifying properties. And
480
the fibril-stabilized O/W emulsion heated for 420 min had better emulsifying
481
properties, and the good hydrophobicity and flexibility also improved emulsifying 23
482
activity of the RBPFs emulsion. This study provides a theoretical basis for
483
constructing protein fibrils suitable for stabilizing emulsions and these properties
484
increase the potential of RBP to prepare emulsions as an effective delivery system for
485
nutraceuticals in functional foods.
486
Acknowledgements
487
The authors thank Key Research and Development Program of Shandong Province of
488
China (No. 2019GNC106051), National Nature Science Foundation of China (No.
489
31501578), University Science and Technology Project of Shandong Province of
490
China (No. J16LE22), Doctoral Science Foundation of Shandong Province of China
491
(No. BS2015SW019), Advanced Talents Foundation of Qingdao Agricultural
492
University (No. 6631115030), and Special Funds for Taishan Scholars Project of
493
Shandong Province of China (ts201712058) for financial support.
494
24
495
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FIGURE CAPTIONS Fig.1. Characterisation of unheated RBP and RBPFs. A, Th T fluorescence intensity of unheated RBP and RBPFs; B, the emission fluorescence intensity ratios F0/F at 496 nm, F0 is the maximum fluorescence intensity of unheated protein at 496 nm, and F is the fluorescence intensity of protein fibril at 496 nm for different heating time; C, the surface hydrophobicity (H0) of RBPFs Fig. 2. Average droplet size distribution (A), mean hydrodynamic diameter (B), zeta- potential (C) and flexibility (D) of RBPFs. Different letters: a, b, c in different columns represent significant differences when P < 0.05. Fig. 3. TEM images of RBPFs, A, B, C D and E were unheated RBP, RBPFs heated for 60 min, 180 min, 360 min and 420 min, respectively. Fig. 4. ATR/FTIR of unheated RBP and RBPFs heated for 60 min, 180 min, 360 min, 420 min, 480 min, 540 min and 600 min, respectively. Fig. 5. Average droplet size (A), zeta-potential (B) and adsorption rate of protein (AP%, C), interfacial protein concentration (Г, D) of the emulsions stabilized by different RBPFs. Different letters: a, b in different columns represent significant differences when P < 0.05. Fig. 6. Storage modulus (A), loss modulus (B) and rheological properties (C) of RBPFs emulsion, the inserted table is the rheological parameters obtained from fitting using power law model for RBPFs emulsion.
32
Fig. 7. Visual appearance of the emulsions stabilized by different RBPFs, A, B, C, D and E were the emulsions stabilized by unheated RBP, RBPFs heated for 60 min, 180 min, 360 min and 420 min, respectively. Fig. 8. Optical microscope and fluorescence microscope images of the emulsions stabilized by different RBPFs, A, B, C, G, H, and I was the bright field, D, E, F, J, K, and L was the fluorescence field.
These were emulsions stabilized by unheated
RBP, RBPFs heated for 60 min, 180 min, 360 min and 420 min, respectively. Fig.9. DSC profiles of RBPFs emulsions stabilized by unheated RBP, RBPFs heated for 60 min, 180 min, 360 min and 420 min, respectively.
33
Table 1 Analysis of protein secondary structure base on attenuated total Fourier Transform infrared spectroscopy (ATR/FTIR). Heat time (min)
α- helix (%)
β-folding (%)
β-turns (%)
Irregular curl (%)
0
22±0.38b
26±0.19a
16±0.42a
26±0.19a
60
21±0.66a
26±0.50a
17±0.54b
23±1.28b
180
21±0.03a
27±0.36b
18±1.55b
25±1.48b
360
22±0.16b
30±0.22c
16±0.14a
26±0.16a
420
21±0.23a
26±0.25a
18±0.18b
25±0.39b
480
22±0.34b
26±0.31a
16±0.25a
26±0.43a
540
23±0.24c
26±0.02a
16±0.34a
26±0.19a
600
21±0.06a
26±0.05a
16±1.75ab
26±0.23a
The data are expressed as ‘mean + standard deviation’. Different letters: a, b, c, d, e in the same line represent significant differences when P < 0.05.
34
Table 2 Emulsifying activity index (EAI) and emulsifying stability (ES) of emulsions stabilized by different RBPFs. Heat time (min)
EAI (m²/g)
ES×10-2
0
1.13±0.02e
1.18±0.06a
60
0.44±0.06c
2.71±0.04e
180
0.43±0.01b
2.47±0.01d
360
0.29±0.01a
2.37±0.01c
420
0.49±0.04d
2.87±0.06f
480
1.20±0.03f
1.92±0.08b
The data are expressed as ‘mean + standard deviation’. Different letters: a, b, c, d, e in the same line represent significant differences when P < 0.05.
35
Table 3 Correlation analysis between flexibility with surface hydrophobicity (H0), ES and EAI H0 and flexibility Flexibility and ES Correlation
0.881*
0.685
Flexibility and
H0 and ES
H0 and EAI
0.899
0.887
EAI 0.962**
coefficient * 0.01
36
Table 4 The relative intensity ratios of I2850/I2880 and I2935/I2880 of RBPFs emulsions Parameters 0 min
60 min
180 min
360 min
420 min
480 min
I2850/I2880
1.00±0.009b
0.97±0.002b
0.96±0.004b
0.95±0.006a
0.94±0.009a
0.97±0.007b
I2935/I2880
1.01±0.006d
1.00±0.005d
0.97±0.01b
1.02±0.004e
0.94±0.002a
0.99±0.002c
The data are expressed as ‘mean + standard deviation’. Different letters: a, b, c, d, e in the same line represent significant differences when P < 0.05.
37
Fig.1
38
Fig. 2
39
Fig. 3
40
Fig. 4
41
Fig. 5
42
Fig. 6
43
Fig. 7
44
Fig. 8
45
Fig.9
46
Highlights
-
Suitable heating time exerted effect on structural properties of rice bran protein
fibrils (RBPFs). -
The molecular flexibility of RBPFs was influenced by formation time.
-
Appropriate formation time induced to RBPFs favorable physic chemical and
emulsifying properties. -
RBPFs structure was closely related to their emulsification properties.
S0023-6438(19)31327-1
DOI:
https://doi.org/10.1016/j.lwt.2019.108985
Reference:
YFSTL 108985
To appear in:
LWT - Food Science and Technology
Received Date: 22 September 2019 Revised Date:
4 December 2019
Accepted Date: 21 December 2019
Please cite this article as: Pang, S., Shao, P., Sun, Q., Pu, C., Tang, W., Relationship between the emulsifying properties and formation time of rice bran protein fibrils, LWT - Food Science and Technology (2020), doi: https://doi.org/10.1016/j.lwt.2019.108985. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
Pang shuxian: Conceptualization, Writing - Original Draft, Data Curation. Shaoping: Supervision. Sun qingjie: Project administration. Pu chuanfen: Methodology. Tang wenting: Writing - Review & Editing, Funding acquisition.
1
Relationship between the Emulsifying Properties and Formation Time of Rice Bran
2
Protein Fibrils
3
Shuxian Pang a, Ping Shao b, Qingjie Sun a, Chuanfen Pu a,*,Wenting Tang a,*
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a School of Food Science and Engineering, Qingdao Agricultural University,
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Qingdao 266109, China
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b Department of Food Science and Technology, Zhejiang University of Technology,
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Zhejiang, Hangzhou 310014, China
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*Corresponding author.
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Tel.: +8615863002210; +8613854269282
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E-mail: [email protected] (W. T. Tang); [email protected] (C. F. Pu)
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22 23
Abstract:
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In this study, oil-in-water emulsions stabilized with rice bran protein fibrils
25
(RBPFs) were prepared, and the effect of the fibrils’ formation time on the structural
26
and emulsion characteristics, including droplet size, zeta-potential, and surface
27
morphology, were analyzed. As the heating time increased to 420 min, the β-sheet
28
structure content and surface hydrophobicity of the RBPFs increased. As the heating
29
time continued to heat up to 600 min, a downward trend occurred. The formation time
30
also affected the mean contour length of the fibrils, and the scale began to decrease at
31
540 min. The emulsion prepared by heating for 480 min showed the best emulsifying
32
activity, and the 420-min fibril emulsion showed the best emulsion stability and the
33
highest interfacial protein content. The rheological results showed that as the heating
34
time increased (0–480 min), the storage modulus increased, and the loss modulus and
35
the apparent viscosity were reduced. The emulsions in the range of 0–100 s-1 shear
36
rate did not have gel properties and shear thinning. The results of this study will help
37
to provide a new perspective to develop a stable food-grade emulsion in the future.
38
Keywords ： Rice bran protein fibrils; RBPFs emulsion; Rheological properties;
39
Formation time
40
2
41 42
1. Introduction
43
Oil-in-water (O/W) emulsions are widely used in food, nutrition, and medicine as a
44
means of delivering lipophilic flavorings, antioxidants, functional lipids, and various
45
other biologically active compounds (Ge et al., 2017). However, since the emulsion
46
consists of water and a water-immiscible non-polar liquid (oil phase), the colloidal
47
dispersion is thermodynamically unstable colloidal dispersion, with the oil phase
48
dispersed within an aqueous phase. Therefore, interfacial stabilizers, including protein,
49
starch and other polysaccharides, small molecule polymers, amphiphilic lipids, etc.,
50
have been used to stabilize the emulsion system. Among these stabilizers, natural food
51
proteins have favorable interfacial film-forming properties when adsorbed to the
52
oil-water interface, which reduce the interfacial tension. To date, an emulsion system
53
is considered an important component in food processing (Feng, Cai, Wang, Li, & Liu,
54
2018).
55
Recently, fibrils derived from globulin have received much attention and are used
56
in materials science such as food and biopharmaceuticals (Serfert et al., 2014). At the
57
pH away from the isoelectric point (pI, commonly at pH ≈ 2.0), thermal denaturation
58
of the protein can lead to an aggregation of proteins into micron-length (1―10 µm),
59
nano-diameter (1―10 nm) fibrils and multi-stranded twisted structures rich in
60
β-sheets perpendicular to the fibril axis (Mohammadian, & Madadlou, 2018). Acid
61
heat treatment usually plays a role in unlocking protein molecules and exposing 3
62
internal hydrophobic areas. The formation of linear fibrils is mainly controlled by the
63
balance of intermolecular forces, including the attraction (mainly hydrophobic bonds)
64
between pyrolytic folding molecules and repulsive forces. With high-aspect ratio
65
anisotropic properties, fibrils have specific interfacial behaviors due to capillary
66
forces. Most food proteins have the ability to form such fibrils, such as
67
beta-lactoglobulin, whey protein isolate, and soy protein isolate (Gao et al., 2017).
68
Studies have pointed out (Serfert et al., 2014) that as anisotropic particles, protein
69
fibrils have better emulsifying effects than rigid-bound spherical particles due to a
70
larger aspect ratio. In the interfacial shear rheology analysis, fibrils exhibited a high
71
degree of elasticity at the O/W interface. Recently, protein fibrils have been widely
72
used to prepare emulsions. Serfert et al. (2014) prepared anti-oxidized fish oil
73
emulsions with β-lactoglobulin fibrils, which have better barrier properties than the
74
original protein, resulting in lower permeability, effectively improving the oxidative
75
stability of fish oil. Gao et al. (2014) studied the preparation of stable O/W Pickering
76
emulsions of β-lactoglobulin fibrils at different fibril concentrations and pH. When
77
the concentration of fibrils in O/W emulsion is above 5 mg/ml, the emulsion has
78
long-term stability. Although stability is acceptable, an excessive concentration of
79
fibrils can lead to larger emulsion droplet sizes.
80
Rice bran protein (RBP) is composed of albumin, globulin, gluten, and prolamin.
81
RBP is recognized as a high-quality vegetable protein with low hypoallergenic
82
activity. Zhang, Huang, Liu & Wei (2014) prepared fibrils by heating RBP under pH 4
83
2.0 at 90 ℃, and found that adding fibrils could significantly promote
84
solution-thickening and gel-hardening behavior. Li, Zhang & Li (2014) studied the
85
effect of ionic strength on the formation and characteristics of RBPFs, and pointed out
86
that the presence of cellulose can change the structural properties of the RBP gel
87
system, which is suitable for natural nano-scale gel materials. However, the
88
emulsification characteristics of RBPFs are not clear.
89
The objective of this study was to evaluate the effect of formation times on the
90
structural properties and molecular flexibility of RBPFs. Then the emulsion properties
91
of the RBPFs obtained under different times were evaluated. The physicochemical
92
properties of fish oil emulsion stabilized by RBPFs prepared under different times
93
were also compared, such as rheological properties, morphology, particle size
94
potential, interface protein content.
95
2. Materials and methods
96
2.1. Materials
97
Rice bran protein with 0.98 g/mL purity was obtained from Xi'an Connor Chemical
98
Co., China. Fish oil obtained from Shanxi Guanchen Biotech Co., Ltd (Xi’an, China).
99
8-anilino-1-naphthalenesulfonic acid (ANS), Thioflavin T (Th T), trypsin was
100
purchased from Sigma-Aldrich (St. Louis, Mo, U.S.A). Trichloroacetic acid (TCA)
101
was obtained from Sangon Biotech Co., Ltd (Shanghai, China). All other reagents
102
used were analytical grades.
103
2.2. Methods 5
104
2.2.1. Preparation of RBP
105
RBP was dissolved in distilled water (0.06 g/mL) and magnetically stirred for 24 h
106
to obtain RBP dispersion. This RBP dispersion was adjusted to pH 2.0 with 1 mol/L
107
HCl (ionic strength, 150 mmol/L) and centrifuged at 3000 ×g for 30 min in order to
108
remove insoluble materials. The supernatant was taken at 90 °C for a specific time (60,
109
180, 360, 420, 480, 540 and 600 min). Immediately after the end of the sample
110
heating, the samples were placed in an ice bath and stored under 4 °C for further
111
study.
112
2.3. RBAFs evaluation
113
2.3.1. Th T fluorescence analysis
114
The fluorescence analysis was performed according to Zhang et al.'s method (2014)
115
with some modifications. The Th T stock solution was prepared by dispersing 8 mg of
116
Th T into 10 mL of phosphate buffer solution (PBS, pH 7, 0.05 mol/L). Before the test,
117
the Th T stock solution was diluted 50 times as a working solution. Then, 20 µL of the
118
RBPF sample was mixed with 3-mL Th T working solution, shaken evenly, and
119
allowed to stand for 1 min before the fluorescence analysis using a fluorescence
120
spectrophotometer (F2700, Hitachi, Japan). The excitation wavelength was set to 460
121
nm, the emission wavelength was set to 470―600 nm, the excitation and emission
122
slits were both 5 nm, the voltage was 700 mV, and the scan rate was 500 nm/min. The
123
value of F0/F as a function of heating time was determined, in which F0 was the
124
maximum fluorescence intensity of unheated protein at 496 nm, and F was the 6
125
fluorescence intensity of protein fibrils at 496 nm for a different heating time.
126
2.3.2. Surface hydrophobicity(H0)
127
H0 was measured by 1-anililo-naphthalene-8-sulfonate (ANS) fluorescent probe,
128
according to the method of Mantovani, Guilherme, Netto, & Cunha (2018) with a few
129
modifications. ANS was dispersed in PBS buffer (pH=7, 0.1 mol/L) to prepare a
130
working solution (0.01 mol/L) with the RBPF sample, mixed at a ratio of 15:1; then
131
the sample mixture was shaken for 1 min and determined. The excitation wavelength
132
was 370 nm, the emission wavelength scope was 400-600 nm, the emission and
133
excitation slits were set to 2.5 nm, the operating voltage was 250 mV, and the
134
scanning rate was 500 nm/min.
135
2.3.3. Mean contour length and zeta- potential analysis
136
The mean contour length and zeta-potential of the sample were measured by
137
dynamic light scattering (DLS) using Zetasizer Nano-ZS (Malvern Instruments, UK)
138
instrument at 25℃. The sample was diluted appropriately to meet the requirement of
139
the equipment.
140
2.3.4. Transmission electron microscope (TEM)
141
The RBPF sample was dripped onto copper grids coated with amorphous carbon
142
film and dyed with phosphotungstic acid (0.02 g/mL). After natural air drying, the
143
sample was observed using a transmission electron microscope (TEM, JEM-2200 FS,
144
JEOL Ltd., Japan) with 80 kV acceleration voltage, the levels of magnification were
145
1500 times. 7
146
2.3.5. Attenuated total reflectance Fourier Transform infrared spectroscopy
147
(ATR/FTIR)
148
An ATR infrared spectrum of the samples was taken using a Nicolet iS10 (Thermo
149
Scientific, USA) instrument. RBPFs were freeze-dried and measured. The scanning
150
was performed 32 times in the wave number range of 4000 cm - 1 to 400 cm -1. Images
151
and data were analyzed using OMNIC 8.0 software (Thermo Fisher Scientific，USA)
152
and Peakfit software.
153
2.3.6. Molecular flexibility
154
This measure was conducted according to the method of Li et al. (2019) with some
155
modifications. The trypsin solution (1 mg/ml) was prepared using Tris-HCl buffer
156
(0.05 mol/L, pH = 8.0). Then, 4 ml of RBPFs (1 mg/ml) was mixed with 250 µL of
157
the enzyme solution at 37 °C for 5 min, and the reaction was terminated with 4 ml of
158
5 mg/mL trichloroacetic acid (TCA). The supernatant was collected by centrifugation
159
at 4000 rpm for 20 min. Molecular flexibility was measured at 280 nm and expressed
160
by its absorbance.
161
2.4. Preparation of RBPFs emulsion
162
An aqueous phase was prepared by dispersing 0.04 g/mL RBPFs in aqueous buffer
163
solution (10 mmol/L phosphate, pH 7.0). Coarse fish O/W emulsions containing 1/10
164
organic phase and 9/10 aqueous phase were formed using a high-shear mixer (D50,
165
WIGDENNS) for 5 min at 10,000 rpm. Then, ultrasonic treatment (5 min) was carried
166
out using a 6-mm probe in the ice bath with a sequence of 1 s of sonication and 1 s of 8
167
rest (power: 540 W, frequency: 25 kHz). The prepared RBPFs emulsion was kept
168
under 4 °C.
169
2.5. Emulsifying activity index (EAI) and emulsifying stability (ES)
170
Emulsification activity (EAI) and emulsion stability (EAI) were determined
171
according to Tang et al.’s method (2005). The emulsion sample was centrifuged at
172
3500 r/min for 10 min, and the supernatant was taken for 100 µL mixed with 10 ml
173
sodium dodecyl sulfate buffer (SDS, 1 mg/mL). The absorbance of the emulsion
174
sample was measured at 500 nm using an ultraviolet spectrophotometer (TU-1810,
175
PERSEE, China). EAI and ES were calculated by the following equations (Tang et al.,
176
2005):
177
(1)
178 179
(2)
180
Where c is the concentration of protein in the sample solution, g/mL;
181
EAImin and EAImax are the EAI values of the emulsion after 10 min and 0 min;
182
φ is the fraction of the oil phase, which is 1/10 in this experiment;
183
EAI is the largest EAI after emulsion formation;
184
L is the cuvette path, 0.01 m.
185
2.6. Emulsion characterization
186 187
Based on the results of the RBPFs, we selected the samples heated for 60 min, 180 min, 360 min, 420 min and 480 min to prepare the emulsion. 9
188
2.6.1. Emulsion droplet size and zeta-potential analysis
189
The physical stability of emulsion was quantified by measuring the average
190
droplet diameter and zeta-potential of a freshly prepared emulsion by a Zetasizer
191
Nano-ZS (Malvern Instruments, UK) instrument. Emulsion samples were
192
moderately diluted with ultrapure water to avoid multiple scattering. All
193
measurements were carried out at 25℃.
194
2.6.2. Adsorption rate of protein (AP) and interfacial protein concentration (Γ) of
195
RBPFs emulsion
196
According to the method of Ye (2008), some modifications have been made. The
197
fresh emulsion was centrifuged at 13000 r/min for 15 min, and the top layer of the
198
centrifuge tube was removed. Determination of protein content (Cf) was performed by
199
the Bradford (Redmilegordon, Armenise, White, Hirsch, & Goulding, 2013) method.
200
Adsorption rate of protein:
201
Interfacial protein concentration:
(3) (4)
202
Where, C0 was total protein content in the emulsion; ϕ was the oil phase ratio. In
203
this paper, the volume concentration of oil phase was 1/10. D3,2 was the average
204
radius of emulsion surface.
205
2.6.3. Optical microscopy observation
206
One mL of Th T buffer was added to the RBPFs solution, shaken, and mixed. After
207
1 min, the new RBPFs emulsion was prepared according to section 2.4. The whole
208
process of production was kept from the light. The emulsion sample was dripped onto 10
209
the slide with a volume of 1 mL, and the appearance of the emulsions were observed
210
by an optical microscope (BX 35, Olympus, Japan) under a bright and fluorescence
211
field with a 40×objective lens.
212
2.6.4. Rheology test
213
The rheological properties of the RBPFs emulsion were evaluated by a MCR320
214
rheometer (Anton Paar, Austria), using a plate configuration and PP50 with 1-mm gap
215
at 25 °C. Freshly prepared emulsions were used for all tests.
216
2.6.4.1. Static rheological measurement
217
Viscosity was evaluated in the linear viscoelastic region (LVER) at 25℃ with a
218
shear rate from 0.1―100 s-1. Viscoelastic data were fitted by the Ostwald de Waale
219
model (Zhang, Tan, Abbas, Eric, Xia, & Zhang, 2015):
220
(5)
221
Where, η was apparent viscosity, Pa·s; γ was shear rate, S-1; K was the consistency
222
index, Pa sn; and n was the index that provides information about the flow behavior
223
affected by shear rate.
224
2.6.4.2. Dynamic rheological measurement
225
Frequency scanning was carried out from 0.1 to 100 Hz at 25℃. The relationship
226
between storage modulus (G') and loss modulus (G'') was measured.
227
2.6.5. Raman spectra analysis
228
The Raman spectrum was recorded by Raman spectrometer (Thermo Fisher
229
Scientific, USA) and excited by a near-infrared laser at 785 nm according to Niu et 11
230
al’s method (2016) with a modification. The laser output power was 334 mV and the
231
data acquisition time was set to 40 s to obtain the best peak intensity. The scan range
232
for each test was 300 to 3050 cm-1. Images and data were analyzed using OMNIC 8.0
233
software (Thermo Fisher Scientific，USA).
234
2.6.6. Differential scanning calorimetry (DSC)
235
DSC-Q1000 differential scanning calorimetry (TA Instrument, USA) was used to
236
identify the thermal transitions in the samples during heating according to Zhang, Pu,
237
Tang, Wang & Sun (2019)’s method with some modifications. The lyophilized sample
238
(4-7 mg) was placed in a crucible and sealed, the temperature range was 25-300 ℃,
239
and the heating rate was 10 ℃/min. Sample was tested under nitrogen flow, reaction
240
gas set to 50-60 mL/min, dry gas was set to 200 mL/min.
241
2.7. Data analysis
242
Data analysis and mapping were performed using Origin 8 software. The data were
243
expressed as mean ± standard deviation (Means ± SD), and the significant level was p
244
< 0.05. All tests were performed in triplicate. The correlation analysis was analyzed
245
by SPSS 17.0 software and expressed as Pearson correlation coefficient.
246
3. Results and discussion
247
3.1. Th T fluorescence spectral analysis of fibrils
248
Th T is a cationic benzothiazole dye that can bind in parallel to the β-sheet
249
structure. This reaction results in a significant increase in the maximum fluorescence
250
intensity of Th T. Based on this principle, this fluorescent probe was used to study the 12
251
formation and the number of amyloid fibrils in order to study the reaction. Figure 1A
252
shows the Th T fluorescence intensity of RBPFs at specific heating times. As the
253
heating time increased from 60 min to 420 min, the fluorescence intensity reached its
254
highest point, and then heated from 480 min to 600 min, the β-sheet content in RBP
255
began to decrease and entered the decline period of fibrils growth. This may be due to
256
the loss of the β-sheet structure due to excessive heating. Figure 1B shows the
257
fluorescence intensity changes of F0/F. When heated to 420 min, the fluorescence
258
intensity increased sharply, possibly due to the increase of β-sheets and the
259
enhancement of hydrophobic interactions. The subsequent sharp decline may be due
260
to the fibrosis reaction, the protein has reached a certain degree of hydrolysis, no more
261
hydrophobic groups as the building unit continue to self-assemble into fibrils.
262
3.2. Surface hydrophobicity (H0)
263
H0 is one of the most important factors affecting the interface properties of
264
proteins. 1-anililo-naphthalene-8-sulfonate (ANS) was used as the fluorescent probe
265
to determine the H0 values. Increased surface hydrophobicity improved fibrillation of
266
rice bran protein (Moayedzadeh, Madadlou, & Khosrowshahi, 2015). According to
267
Fig. 1C, the protein fibrils heated for 420 min showed higher fluorescence intensity
268
than the other times, indicating that the exposed hydrophobic groups varied with
269
heating time, that acid-heat treatment exposed a large number of hydrophobic groups,
270
and that the RBPFs exhibited an increase in hydrophobicity before heating for 420
271
min. After heating for 480 min, H0 of the RBPFs began to decline. This phenomenon 13
272
may be due to the fact that proteins are no longer hydrolyzed to form a large number
273
of beta-folded structures, but aggregate to form nuclei, which are linked to the
274
construction units and increase the length of protein fibrils.
275
3.3. Mean contour length, zeta-potential and flexibility of RBPFs
276
The distribution of RBPFs mean contour length was affected by the heat-induced
277
environment (Feng et al., 2018). Figures 2A―B show the mean contour length and
278
distribution of fibrils at different heating times. A typical bimodal shape appeared in
279
RBPFs heated for 420 min, resulting in a dramatic increase in fibrils. The mean
280
contour length of the fibrils gradually increased with the increase of heating time from
281
60 min to 420 min, until the fibrils heated for 480 min showed a downward trend.
282
This indicates that heat treatment promotes the fibrillation of RBP to form RBPFs
283
with larger size, and excessive heating time may cause the further fibrillation.
284
Zeta-potential is widely used to characterize the electrostatic interaction between
285
charged particles. Figure 2C shows the zeta-potential distribution of RBPFs, Figure
286
2C shows the zeta-potential distribution of RBPFs. When heating for a short time (60
287
min), the zeta-potential becomes less negative, and then becomes more negative
288
(180-360 min) as the heating time increases, and finally the zeta-potential becomes
289
less negative as heating continues (420-600 min). This shows that when heated for
290
360 min, the absolute value of zeta-potential was the largest, the electrostatic
291
interaction between protein molecules was the strongest, and the protein solution
292
tends to be stable. 14
293
Protein flexibility is an important aspect that affects its emulsification properties.
294
The flexibility of proteins is a controlling factor, which plays an important role in the
295
surface activity of proteins (Li et al., 2019; Razumovsky, & Damodaran, 1999).The
296
molecular flexibility of proteins is not only affected by covalent interactions but also
297
by non-covalent interactions such as van der Waals forces, electrostatic bonds, and
298
hydrophobic interactions (Li et al., 2019). Heat treatment may destroy some of the
299
above bonds and expand the flexible structure of RBPFs, thus increasing flexibility
300
with the increase of reaction time. The molecular flexibility of the protein slowly
301
increased before heating for 180 min, and quickly increased to the maximum value
302
(0.054) at 420 min (Fig.2D).
303
with RBP, the flexibility of RBPF increased significantly with prolonged heating time
304
(P <0.05), which indicates that the structure of RBP molecule was unfolded (Li et al.,
305
2019). Thermal denaturation can destroy non-covalent effects, so heat treatment of the
306
protein can improve its emulsifying properties (Damodaran, 2005).
307
3.4. TEM image of RBPFs
Then it gradually decreased after 420 min. Compared
308
The TEM image (Fig. 3) shows the profile length of the RBP fibrils, showing that
309
the prepared fibrils are semi-flexible under these conditions. It can also be seen that
310
the unheated protein (Fig. 3A) is a spherical particle, which gradually changes to the
311
shape of fibrils as the heating time is further increased. In addition, the DLS results
312
are smaller than the TEM size, probably because the DLS was hydrated, while the
313
TEM sample was measured after drying. 15
314
3.5. ATR/FTIR analysis
315
ATR/FTIR was widely used to analyze the secondary structure of various samples.
316
The characteristic bands in the protein ATR/FTIR spectra mainly include amide I
317
(1600―1700 cm-1) and amide II (1500―1600 cm-1) (Jung, Gunes, & Mezzenga, 2010)
318
combined with the original map (Fig. 4) peak position obtained by deconvolution and
319
secondary structure identification: α- helix, 1648―1660 cm-1; β-folding, 1626―1640
320
cm-1; β-turns, 1662―1684 cm-1; irregular curl, 1640―1650 cm-1. Table 1 shows that
321
as the heating time increases, the β-sheet structure of the protein also increases,
322
reaching the highest at 360 min, and then remaining stable. Gosal, Clark, Pudney &
323
Ross (2002) studied the fibrillation of bovine serum albumin and also found that the
324
number of β-sheets increased after heat-induced protein formation of amyloid fibrosis.
325
The main component of RBP was β-sheets structure, accounting for 26% of the total
326
secondary structure. The main secondary structure of RBP after the heat treatment
327
was still β-sheets structure, and the content of β-sheets structure of RBPFs was higher
328
than that of RBP, the RBPFs of 360 min was the highest reaching 30%. However, the
329
content of β-sheets structure of RBPFs 360 min was no longer increasing, which
330
indicates that the further heat treatment of RBP induced the loss of β-sheets structure.
331
3.6. EAI and ES analysis
332
EAI is an indicator of the emulsification efficiency of an emulsifier. The
333
emulsification activity of a protein is measured by the emulsification efficiency of a
334
protein molecule during homogenization. EAI represents the ability of a protein to 16
335
participate in emulsion formation under external force. ES is the ability of emulsion
336
droplets to maintain stable (Li et al., 2019). The higher the EAI, the higher the
337
emulsification activity. The emulsification activity of a protein is related to the
338
structure of its own molecule. At the same time, according to Table 2, in these six
339
emulsion samples, the RBPFs emulsion heated for 420 min had the best stability, so it
340
was believed that increasing the heating time within a certain range could improve
341
emulsion stability. Tang et al. (2013) found that after protein adsorption to the
342
oil-water interface, structural rearrangement generally occurs, which affects the
343
emulsification properties of proteins.
344
For the above reason, the relationship between the hydrophobicity and molecular
345
flexibility of RBPFs and the EAI and ES between RBPFs emulsions was investigated.
346
According to Table 3, the correlation coefficient between H0 and flexibility was 0.881
347
(p<0.05), the correlation coefficient of ES was 0.899, and the correlation coefficient
348
of EAI was 0.887. The correlation coefficient between flexibility and EAI was 0.962
349
(p<0.01), and the correlation coefficient of ES was 0.685. Hydrophobicity H0 is
350
positively correlated with flexibility, EAI and ES. This indicates that the trends in
351
hydrophobicity and molecular flexibility are consistent. Surface hydrophobicity and
352
flexibility are important factors affecting emulsification performance. In the previous
353
studies, researchers focused on the relationship between H0 and protein emulsification
354
properties and found a strong correlation (Li et al., 2019). Prak, Nakatani, Katsube,
355
Adachi, Maruyama & Utsumi (2005) found the emulsifying ability of protein is not 17
356
exactly the same as the emulsifying ability of surface hydrophobicity. Flexible protein
357
molecules tend to denature at the interface, while rigid protein molecules are less
358
susceptible. Studies by Kato, Komatsu, Fujimoto & Kobayashi (1985) have shown
359
that surface hydrophobicity is ultimately an important factor in achieving good
360
foaming and emulsifying properties due to the high surface hydrophobicity imparted
361
by the mild effector.
362
3.7. Emulsion characterization
363
3.7.1. Average droplet size and zeta-potential
364
The droplet size of the emulsion is one of the most important qualitative
365
properties of emulsions (Ge et al., 2017). According to our preliminary experiments
366
(Fig. S1), 0.01―0.05 g/mL of the fish oil concentration was selected, and the results
367
showed that the 0.04 g/mL protein concentration emulsion was the most stable. Liu &
368
Tang (2014) found that increasing the concentration (0.005―0.06 g/mL) of protein
369
particles can reduce the droplet size and increase the stability of the emulsion. As
370
result, the 0.04 g/mL RBPFs solution was chosen to prepared the emulsion. Figure 5A
371
shows that the average droplet size of the RBPFs emulsion did not change
372
significantly within 0-360 min. While the average droplet size of the emulsion
373
stabilized by 420 min RBPFs rose sharply, which was probably due to the more
374
formation of fibrils, causing the increase of the length of RBPFs those were located at
375
the O/W interface of the emulsion. In contrast, the concentration of protein fibrils was
376
not enough to cover the oil-water interface. For example, the length of the protein 18
377
fibril may not be enough to coat the fish oil droplets, the interfacial tension between
378
the O/W interface is great, and the oil droplets combine to make the particle size
379
larger.
380
Zeta-potential is an important indicator for evaluating the stability of the system.
381
Generally, the absolute value of the zeta-potential is high, the protein molecules are
382
repelled, and the solution tends to be stable. Figure 5B shows the zeta-potential
383
distribution of the RBPFs emulsion droplets, from +31 mV to +35 mV, and the
384
emulsion stabilized by RBPFs was positive. Zeta―potential did not show significant
385
changes indicating that the heating time has little effect on the RBPFs emulsion
386
zeta-potential. It might be due to the length of the heating time has little effect on the
387
zeta-potential of the RBPFs stabilized-emulsion, the overall emulsion potential does
388
not change much. Compared with protein fibrils, the zeta-potential of the emulsion
389
was converted into a positive charge, probably because the fibrils are negatively
390
charged, and the adsorption on the droplets is not enough to change the positive
391
charge of the droplets.
392
3.7.2. AP and Γ of RBPFs emulsion between O/W interface
393
Some studies (Kato et al., 1985) have pointed out that proteins with softer
394
structures are more likely to undergo structural transformation and thus adsorb at the
395
interface. According to the interface adsorption principle of polyelectrolytes, the
396
protein structure will be expanded and denatured during the interface adsorption
397
process. 19
398
It can be seen from Fig. 5C―D that as the heating time increases to 420 min, the
399
AP and Γ of the protein are continuously increasing, and only the emulsion sample
400
heated for 480 min shows a downward trend. This may be due to increased
401
hydrophobicity of the protein, which is consistent with the results of molecular
402
flexibility in Fig. 2. In the protein adsorption process, because the molecular weight of
403
the protein is larger, the adsorption rate at the interface is much lower than that of the
404
small molecule surfactant. After adsorption to the interface, the hydrophobic amino
405
acid of the protein tends to stretch toward the oil-containing direction and rearrange.
406
Therefore, compared with small molecule surfactants, protein emulsifiers have
407
weaker emulsifying ability, but the prepared emulsions have higher physical and
408
chemical stability.
409
3.7.3. Rheology properties of fish oil emulsion stabilized with RBPFs
410
Figure 6 shows the rheological characteristics of the fish oil emulsion stabilized
411
with RBPFs. Within the angular frequency range (0―100 s-1), the fact that the storage
412
modulus G' was less than the loss modulus G'' indicates that in this frequency range,
413
the emulsion does not exhibit gel properties, but exists in a liquid state in which
414
fluidity is good. As the heating time increases, the G' increases gradually, and the G''
415
decreases. The data in the figure were fitted by the Ostwald de Waale model (the
416
relevant parameters are shown in the insert table), and the K-value is lower, indicating
417
lower viscosity, and the n-value is higher, indicating that the emulsion is closer to a
418
Newtonian fluid (Wang, Li, Wang, & Adhikari, 2011). The table shows that the 20
419
K-value was the smallest for the emulsion heated for 420 min, indicating that the
420
viscosity
421
the emulsion exhibits shear thickening, n=1 means an ideal viscosity flow and n<1
422
suggests shear thinning occurs (Wei, & Gao, 2015). As seen in Figure 6 within the set
423
shear rate range (0-100 s-1), the viscosity of all samples gradually decreases, and shear
424
thinning occurs. The reason for this phenomenon is that oil droplets gather together,
425
the weak interaction force that maintains stable flocculation under high-speed shear
426
was destroyed, and shearing occurs thinning phenomenon. In protein emulsions, when
427
the protein concentration reaches a certain value, RBPFs that are not adsorbed to the
428
oil-water interface are present. Due to the presence of unabsorbed and amphiphilic
429
protein fibrils in the continuous phase, the fibrils tend to approach each other due to
430
secondary forces such as hydrophobic interactions, apparently increasing the viscosity
431
of the emulsion. And this thickening enhances the stability of the emulsion (Voutsinas
432
et al., 1983).
433
3.7.4. Morphology
was
the
lowest.
The
value
n
＞
1
represents
that
434
Figure 8 shows the morphology of six emulsion samples under an optical
435
microscope and a fluorescence microscope. According to the results of the
436
fluorescence microscope, it can be seen that the emulsion droplets gradually increase
437
as the heating time increases. And the unheated protein emulsion is darker, which may
438
be caused by the combination of the Th T dye and the β-sheet in the protein, which is
439
the same as that of the fluorescence spectrophotometer. 21
440
3.7.5. Raman spectroscopic analysis.
441
In order to examine the interaction between RBPFs and lipids, the structural
442
properties of RBPFs emulsion were determined using Raman spectroscopy. The ratio
443
of relative intensity ratio I2850 / I2880 and I2935 / I2880 reflects the interaction between
444
lipid chains and the state of order or disorder of acyl chains (Ruiz, Carmona, Jiménez,
445
& Herrero, 2013). It can be visualized from Table 4 that the emulsion of 420 min
446
RBPFs shows the lowest intensity ratios. The disorder of the acyl chain indicates that
447
more proteins were inserted between the acyl chains of the oil. (Herrero, Ruiz,
448
Pintado, Carmona, & Jiménez, 2018). Lower I2850/ I2880 and I2930/ I2880 intensity ratios
449
were also related to hydrophobic interactions including the CH groups of the lipid
450
with protein (Herrero et al., 2018). In addition, combined with the results of infrared
451
spectroscopy analysis, I2850 / I2880 was negatively correlated with the number of
452
β-sheets. Ruiz et al. (2013) also reported that the intensity ratio of the emulsion (I2850 /
453
I2900) was lower than that of the separated fatty phase and pointed out that most of the
454
hydrocarbon chains of milk fat globules and / or triglycerides are closely packed. This
455
hydrocarbon chain accumulation was very important for the stability of the emulsion.
456
3.7.6. DSC analysis
457
DSC were determined the thermal behavior of RBPFs stabilized emulsions with
458
different heating times. The emulsion stabilized by unheated RBP showed an initial
459
endothermic peak at 185 °C and a maximum exothermic peak at 146 °C. The
460
endothermic peak was caused by dehydration associated with the hydrophilic group of 22
461
the polymer, while the exothermic peak was associated with degradation of the
462
polyelectrolyte due to dehydration and depolymerization reactions (Li et al.,
463
2018).The exothermic peak of the RBPFs stabilized emulsion after heating was
464
smaller and broader compared with those of RBP emulsion (Fig. 9). This can be
465
explained as the hydrophobic interaction between RBPFs and fish oil (Sarmento,
466
Ferreira, Veiga, & Ribeiro, 2006).
467
The unheated RBP emulsion began to denature at about 85 °C, the RBPFs emulsion
468
denature temperature began to gradually increase, and the 420 min RBPFs emulsion
469
denature temperature reached about 140 °C, indicating that the corresponding
470
emulsion was the most stable among all the samples. In addition, the emulsion
471
stabilized by RBPFs heated for 180 min has a sharp exothermic peak near 176 °C,
472
suggesting the polymer degradation (Paula, Sombra, Cavalcante, Abreu, & Paula,
473
2011). This has been confirmed that RBPFs heated for 180 min will cause emulsion
474
instability in section 3.6
475
4. Conclusion
476
This study better understands the effect of different heating times on the
477
physicochemical properties of an RBPF-forming emulsion. When RBP were heated
478
for 420 min, the mean contour length and hydrophobicity of the fibrils were highest
479
and the corresponding RBPFs were exhibited favorable emulsifying properties. And
480
the fibril-stabilized O/W emulsion heated for 420 min had better emulsifying
481
properties, and the good hydrophobicity and flexibility also improved emulsifying 23
482
activity of the RBPFs emulsion. This study provides a theoretical basis for
483
constructing protein fibrils suitable for stabilizing emulsions and these properties
484
increase the potential of RBP to prepare emulsions as an effective delivery system for
485
nutraceuticals in functional foods.
486
Acknowledgements
487
The authors thank Key Research and Development Program of Shandong Province of
488
China (No. 2019GNC106051), National Nature Science Foundation of China (No.
489
31501578), University Science and Technology Project of Shandong Province of
490
China (No. J16LE22), Doctoral Science Foundation of Shandong Province of China
491
(No. BS2015SW019), Advanced Talents Foundation of Qingdao Agricultural
492
University (No. 6631115030), and Special Funds for Taishan Scholars Project of
493
Shandong Province of China (ts201712058) for financial support.
494
24
495
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FIGURE CAPTIONS Fig.1. Characterisation of unheated RBP and RBPFs. A, Th T fluorescence intensity of unheated RBP and RBPFs; B, the emission fluorescence intensity ratios F0/F at 496 nm, F0 is the maximum fluorescence intensity of unheated protein at 496 nm, and F is the fluorescence intensity of protein fibril at 496 nm for different heating time; C, the surface hydrophobicity (H0) of RBPFs Fig. 2. Average droplet size distribution (A), mean hydrodynamic diameter (B), zeta- potential (C) and flexibility (D) of RBPFs. Different letters: a, b, c in different columns represent significant differences when P < 0.05. Fig. 3. TEM images of RBPFs, A, B, C D and E were unheated RBP, RBPFs heated for 60 min, 180 min, 360 min and 420 min, respectively. Fig. 4. ATR/FTIR of unheated RBP and RBPFs heated for 60 min, 180 min, 360 min, 420 min, 480 min, 540 min and 600 min, respectively. Fig. 5. Average droplet size (A), zeta-potential (B) and adsorption rate of protein (AP%, C), interfacial protein concentration (Г, D) of the emulsions stabilized by different RBPFs. Different letters: a, b in different columns represent significant differences when P < 0.05. Fig. 6. Storage modulus (A), loss modulus (B) and rheological properties (C) of RBPFs emulsion, the inserted table is the rheological parameters obtained from fitting using power law model for RBPFs emulsion.
32
Fig. 7. Visual appearance of the emulsions stabilized by different RBPFs, A, B, C, D and E were the emulsions stabilized by unheated RBP, RBPFs heated for 60 min, 180 min, 360 min and 420 min, respectively. Fig. 8. Optical microscope and fluorescence microscope images of the emulsions stabilized by different RBPFs, A, B, C, G, H, and I was the bright field, D, E, F, J, K, and L was the fluorescence field.
These were emulsions stabilized by unheated
RBP, RBPFs heated for 60 min, 180 min, 360 min and 420 min, respectively. Fig.9. DSC profiles of RBPFs emulsions stabilized by unheated RBP, RBPFs heated for 60 min, 180 min, 360 min and 420 min, respectively.
33
Table 1 Analysis of protein secondary structure base on attenuated total Fourier Transform infrared spectroscopy (ATR/FTIR). Heat time (min)
α- helix (%)
β-folding (%)
β-turns (%)
Irregular curl (%)
0
22±0.38b
26±0.19a
16±0.42a
26±0.19a
60
21±0.66a
26±0.50a
17±0.54b
23±1.28b
180
21±0.03a
27±0.36b
18±1.55b
25±1.48b
360
22±0.16b
30±0.22c
16±0.14a
26±0.16a
420
21±0.23a
26±0.25a
18±0.18b
25±0.39b
480
22±0.34b
26±0.31a
16±0.25a
26±0.43a
540
23±0.24c
26±0.02a
16±0.34a
26±0.19a
600
21±0.06a
26±0.05a
16±1.75ab
26±0.23a
The data are expressed as ‘mean + standard deviation’. Different letters: a, b, c, d, e in the same line represent significant differences when P < 0.05.
34
Table 2 Emulsifying activity index (EAI) and emulsifying stability (ES) of emulsions stabilized by different RBPFs. Heat time (min)
EAI (m²/g)
ES×10-2
0
1.13±0.02e
1.18±0.06a
60
0.44±0.06c
2.71±0.04e
180
0.43±0.01b
2.47±0.01d
360
0.29±0.01a
2.37±0.01c
420
0.49±0.04d
2.87±0.06f
480
1.20±0.03f
1.92±0.08b
The data are expressed as ‘mean + standard deviation’. Different letters: a, b, c, d, e in the same line represent significant differences when P < 0.05.
35
Table 3 Correlation analysis between flexibility with surface hydrophobicity (H0), ES and EAI H0 and flexibility Flexibility and ES Correlation
0.881*
0.685
Flexibility and
H0 and ES
H0 and EAI
0.899
0.887
EAI 0.962**
coefficient * 0.01
36
Table 4 The relative intensity ratios of I2850/I2880 and I2935/I2880 of RBPFs emulsions Parameters 0 min
60 min
180 min
360 min
420 min
480 min
I2850/I2880
1.00±0.009b
0.97±0.002b
0.96±0.004b
0.95±0.006a
0.94±0.009a
0.97±0.007b
I2935/I2880
1.01±0.006d
1.00±0.005d
0.97±0.01b
1.02±0.004e
0.94±0.002a
0.99±0.002c
The data are expressed as ‘mean + standard deviation’. Different letters: a, b, c, d, e in the same line represent significant differences when P < 0.05.
37
Fig.1
38
Fig. 2
39
Fig. 3
40
Fig. 4
41
Fig. 5
42
Fig. 6
43
Fig. 7
44
Fig. 8
45
Fig.9
46
Highlights
-
Suitable heating time exerted effect on structural properties of rice bran protein
fibrils (RBPFs). -
The molecular flexibility of RBPFs was influenced by formation time.
-
Appropriate formation time induced to RBPFs favorable physic chemical and
emulsifying properties. -
RBPFs structure was closely related to their emulsification properties.