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Relationship between the emulsifying properties and formation time of rice bran protein fibrils
LWT - Food Science and Technology 122 (2020) 108985
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Relationship between the emulsifying properties and formation time of rice bran protein fibrils
T
Shuxian Panga, Ping Shaob, Qingjie Suna, Chuanfen Pua,∗∗, Wenting Tanga,∗ a b
School of Food Science and Engineering, Qingdao Agricultural University, Qingdao, 266109, China Department of Food Science and Technology, Zhejiang University of Technology, Zhejiang, Hangzhou, 310014, China
ARTICLE INFO
ABSTRACT
Keywords: Rice bran protein fibrils RBPFs emulsion Rheological properties Formation time
In this study, oil-in-water emulsions stabilized with rice bran protein fibrils (RBPFs) were prepared, and the effect of the fibrils’ formation time on the structural and emulsion characteristics, including droplet size, zetapotential, and surface morphology, were analyzed. As the heating time increased to 420 min, the β-sheet structure content and surface hydrophobicity of the RBPFs increased. As the heating time continued to heat up to 600 min, a downward trend occurred. The formation time also affected the mean contour length of the fibrils, and the scale began to decrease at 540 min. The emulsion prepared by heating for 480 min showed the best emulsifying activity, and the 420-min fibril emulsion showed the best emulsion stability and the highest interfacial protein content. The rheological results showed that as the heating time increased (0–480 min), the storage modulus increased, and the loss modulus and the apparent viscosity were reduced. The emulsions in the range of 0–100 s−1 shear rate did not have gel properties and shear thinning. The results of this study will help to provide a new perspective to develop a stable food-grade emulsion in the future.
1. Introduction Oil-in-water (O/W) emulsions are widely used in food, nutrition, and medicine as a means of delivering lipophilic flavorings, antioxidants, functional lipids, and various other biologically active compounds (Ge et al., 2017). However, since the emulsion consists of water and a water-immiscible non-polar liquid (oil phase), the colloidal dispersion is thermodynamically unstable colloidal dispersion, with the oil phase dispersed within an aqueous phase. Therefore, interfacial stabilizers, including protein, starch and other polysaccharides, small molecule polymers, amphiphilic lipids, etc., have been used to stabilize the emulsion system. Among these stabilizers, natural food proteins have favorable interfacial film-forming properties when adsorbed to the oilwater interface, which reduce the interfacial tension. To date, an emulsion system is considered an important component in food processing (Feng, Cai, Wang, Li, & Liu, 2017). Recently, fibrils derived from globulin have received much attention and are used in materials science such as food and biopharmaceuticals (Serfert et al., 2014). At the pH away from the isoelectric point (pI, commonly at pH ≈ 2.0), thermal denaturation of the protein can lead to an aggregation of proteins into micron-length (1–10 μm), nano-diameter (1–10 nm) fibrils and multi-stranded twisted structures rich in β∗
sheets perpendicular to the fibril axis (Mohammadian & Madadlou, 2018). Acid heat treatment usually plays a role in unlocking protein molecules and exposing internal hydrophobic areas. The formation of linear fibrils is mainly controlled by the balance of intermolecular forces, including the attraction (mainly hydrophobic bonds) between pyrolytic folding molecules and repulsive forces. With high-aspect ratio anisotropic properties, fibrils have specific interfacial behaviors due to capillary forces. Most food proteins have the ability to form such fibrils, such as beta-lactoglobulin, whey protein isolate, and soy protein isolate (Gao et al., 2017). Studies have pointed out (Serfert et al., 2014) that as anisotropic particles, protein fibrils have better emulsifying effects than rigid-bound spherical particles due to a larger aspect ratio. In the interfacial shear rheology analysis, fibrils exhibited a high degree of elasticity at the O/W interface. Recently, protein fibrils have been widely used to prepare emulsions. Serfert et al. (2014) prepared antioxidized fish oil emulsions with β-lactoglobulin fibrils, which have better barrier properties than the original protein, resulting in lower permeability, effectively improving the oxidative stability of fish oil. Gao et al. (2017) studied the preparation of stable O/W Pickering emulsions of β-lactoglobulin fibrils at different fibril concentrations and pH. When the concentration of fibrils in O/W emulsion is above 5 mg/ mL, the emulsion has long-term stability. Although stability is
Corresponding author. Corresponding author. E-mail addresses: [email protected] (C. Pu), [email protected] (W. Tang).
∗∗
https://doi.org/10.1016/j.lwt.2019.108985 Received 22 September 2019; Received in revised form 4 December 2019; Accepted 21 December 2019 Available online 07 January 2020 0023-6438/ © 2020 Elsevier Ltd. All rights reserved.
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acceptable, an excessive concentration of fibrils can lead to larger emulsion droplet sizes. Rice bran protein (RBP) is composed of albumin, globulin, gluten, and prolamin. RBP is recognized as a high-quality vegetable protein with low hypoallergenic activity. Zhang, Huang, Liu & Wei (2014) prepared fibrils by heating RBP under pH 2.0 at 90 °C, and found that adding fibrils could significantly promote solution-thickening and gelhardening behavior. Liu and Tang (2014) studied the effect of ionic strength on the formation and characteristics of RBPFs, and pointed out that the presence of cellulose can change the structural properties of the RBP gel system, which is suitable for natural nano-scale gel materials. However, the emulsification characteristics of RBPFs are not clear. The objective of this study was to evaluate the effect of formation times on the structural properties and molecular flexibility of RBPFs. Then the emulsion properties of the RBPFs obtained under different times were evaluated. The physicochemical properties of fish oil emulsion stabilized by RBPFs prepare5d under different times were also compared, such as rheological properties, morphology, particle size potential, interface protein content.
Netto, and Cunha (2018) with a few modifications. ANS was dispersed in PBS buffer (pH = 7, 0.1 mol/L) to prepare a working solution (0.01 mol/L) with the RBPF sample, mixed at a ratio of 15:1; then the sample mixture was shaken for 1 min and determined. The excitation wavelength was 370 nm, the emission wavelength scope was 400–600 nm, the emission and excitation slits were set to 2.5 nm, the operating voltage was 250 mV, and the scanning rate was 500 nm/min. 2.3.3. Mean contour length and zeta-potential analysis The mean contour length and zeta-potential of the sample were measured by dynamic light scattering (DLS) using Zetasizer Nano-ZS (Malvern Instruments, UK) instrument at 25 °C. The sample was diluted appropriately to meet the requirement of the equipment. 2.3.4. Transmission electron microscope (TEM) The RBPF sample was dripped onto copper grids coated with amorphous carbon film and dyed with phosphotungstic acid (0.02 g/ mL). After natural air drying, the sample was observed using a transmission electron microscope (TEM, JEM-2200 FS, JEOL Ltd., Japan) with 80 kV acceleration voltage, the levels of magnification were 1500 times.
2. Materials and methods 2.1. Materials
2.3.5. Attenuated total reflectance fourier transform infrared spectroscopy (ATR/FTIR) An ATR infrared spectrum of the samples was taken using a Nicolet iS10 (Thermo Scientific, USA) instrument. RBPFs were freeze-dried and measured. The scanning was performed 32 times in the wave number range of 4000 cm-1 to 400 cm −1. Images and data were analyzed using OMNIC 8.0 software (Thermo Fisher Scientific, USA) and Peakfit software.
Rice bran protein with 0.98 g/mL purity was obtained from Xi'an Connor Chemical Co., China. Fish oil obtained from Shanxi Guanchen Biotech Co., Ltd (Xi'an, China). 8-Anilino-1-naphthalenesulfonic acid (ANS), Thioflavin T (Th T), trypsin was purchased from Sigma-Aldrich (St. Louis, Mo, U.S.A). Trichloroacetic acid (TCA) was obtained from Sangon Biotech Co., Ltd (Shanghai, China). All other reagents used were analytical grades.
2.3.6. Molecular flexibility This measure was conducted according to the method of Li et al. (2019) with some modifications. The trypsin solution (1 mg/mL) was prepared using Tris-HCl buffer (0.05 mol/L, pH = 8.0). Then, 4 mL of RBPFs (1 mg/mL) was mixed with 250 μL of the enzyme solution at 37 °C for 5 min, and the reaction was terminated with 4 mL of 5 mg/mL trichloroacetic acid (TCA). The supernatant was collected by centrifugation at 4000 rpm for 20 min. Molecular flexibility was measured at 280 nm and expressed by its absorbance.
2.2. Methods 2.2.1. Preparation of RBP RBP was dissolved in distilled water (0.06 g/mL) and magnetically stirred for 24 h to obtain RBP dispersion. This RBP dispersion was adjusted to pH 2.0 with 1 mol/L HCl (ionic strength, 150 mmol/L) and centrifuged at 3000×g for 30 min in order to remove insoluble materials. The supernatant was taken at 90 °C for a specific time (60, 180, 360, 420, 480, 540 and 600 min). Immediately after the end of the sample heating, the samples were placed in an ice bath and stored under 4 °C for further study.
2.4. Preparation of RBPFs emulsion An aqueous phase was prepared by dispersing 0.04 g/mL RBPFs in aqueous buffer solution (10 mmol/L phosphate, pH 7.0). Coarse fish O/ W emulsions containing 1/10 organic phase and 9/10 aqueous phase were formed using a high-shear mixer (D50, WIGDENNS) for 5 min at 10,000 rpm. Then, ultrasonic treatment (5 min) was carried out using a 6-mm probe in the ice bath with a sequence of 1 s of sonication and 1 s of rest (power: 540 W, frequency: 25 kHz). The prepared RBPFs emulsion was kept under 4 °C.
2.3. RBAFs evaluation 2.3.1. Th T fluorescence analysis The fluorescence analysis was performed according to Zhang et al.'s method (2014) with some modifications. The Th T stock solution was prepared by dispersing 8 mg of Th T into 10 mL of phosphate buffer solution (PBS, pH 7, 0.05 mol/L). Before the test, the Th T stock solution was diluted 50 times as a working solution. Then, 20 μL of the RBPF sample was mixed with 3-mL Th T working solution, shaken evenly, and allowed to stand for 1 min before the fluorescence analysis using a fluorescence spectrophotometer (F2700, Hitachi, Japan). The excitation wavelength was set to 460 nm, the emission wavelength was set to 470–600 nm, the excitation and emission slits were both 5 nm, the voltage was 700 mV, and the scan rate was 500 nm/min. The value of F0/F as a function of heating time was determined, in which F0 was the maximum fluorescence intensity of unheated protein at 496 nm, and F was the fluorescence intensity of protein fibrils at 496 nm for a different heating time.
2.5. Emulsifying activity index (EAI) and emulsifying stability (ES) Emulsification activity (EAI) and emulsion stability (EAI) were determined according to Tang et al.’s method (2005). The emulsion sample was centrifuged at 3500 r/min for 10 min, and the supernatant was taken for 100 μL mixed with 10 mL sodium dodecyl sulfate buffer (SDS, 1 mg/mL). The absorbance of the emulsion sample was measured at 500 nm using an ultraviolet spectrophotometer (TU-1810, PERSEE, China). EAI and ES were calculated by the following equations (Tang & Shen, 2013):
2.3.2. Surface hydrophobicity(H0) H0 was measured by 1-anililo-naphthalene-8-sulfonate (ANS) fluorescent probe, according to the method of Mantovani, Guilherme,
EAI =
2
2.303 × 2 × A500nm ×dilution multiple c × × L
(1)
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ES =
(EAImax
EAImax EAImin ) × 100
2.8. Dynamic rheological measurement
(2)
Frequency scanning was carried out from 0.1 to 100 Hz at 25 °C. The relationship between storage modulus (G′) and loss modulus (G″) was measured.
Where c is the concentration of protein in the sample solution, g/mL; EAImin and EAImax are the EAI values of the emulsion after 10 min and 0 min; ϕ is the fraction of the oil phase, which is 1/10 in this experiment; EAI is the largest EAI after emulsion formation; L is the cuvette path, 0.01 m.
2.8.1. Raman spectra analysis The Raman spectrum was recorded by Raman spectrometer (Thermo Fisher Scientific, USA) and excited by a near-infrared laser at 785 nm according to Niu et al.'s method (2016) with a modification. The laser output power was 334 mV and the data acquisition time was set to 40 s to obtain the best peak intensity. The scan range for each test was 300–3050 cm−1. Images and data were analyzed using OMNIC 8.0 software (Thermo Fisher Scientific, USA).
2.6. Emulsion characterization 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.
2.8.2. Differential scanning calorimetry (DSC) DSC-Q1000 differential scanning calorimetry (TA Instrument, USA) was used to identify the thermal transitions in the samples during heating according to Zhang, Pu, Tang, Wang, and Sun (2019)'s method with some modifications. The lyophilized sample (4–7 mg) was placed in a crucible and sealed, the temperature range was 25–300 °C, and the heating rate was 10 °C/min. Sample was tested under nitrogen flow, reaction gas set to 50–60 mL/min, dry gas was set to 200 mL/min.
2.6.1. Emulsion droplet size and zeta-potential analysis The physical stability of emulsion was quantified by measuring the average droplet diameter and zeta-potential of a freshly prepared emulsion by a Zetasizer Nano-ZS (Malvern Instruments, UK) instrument. Emulsion samples were moderately diluted with ultrapure water to avoid multiple scattering. All measurements were carried out at 25 °C.
2.9. Data analysis
2.6.2. Adsorption rate of protein (AP) and interfacial protein concentration (Γ) of RBPFs emulsion According to the method of Ye (2008), some modifications have been made. The fresh emulsion was centrifuged at 13,000 r/min for 15 min, and the top layer of the centrifuge tube was removed. Determination of protein content (Cf) was performed by the Bradford (Redmilegordon, Armenise, White, Hirsch, & Goulding, 2013) method.
Adsorption rate of protein: AP = (C0
Cf ) × 100/ C0
Interfacial protein concentration: (mg/ m2) = (C0
Cf ) × D3,2 /6
Data analysis and mapping were performed using Origin 8 software. The data were expressed as mean ± standard deviation (Means ± SD), and the significant level was p < 0.05. All tests were performed in triplicate. The correlation analysis was analyzed by SPSS 17.0 software and expressed as Pearson correlation coefficient.
(3)
3. Results and discussion
(4)
3.1. Th T fluorescence spectral analysis of fibrils
Where, C0 was total protein content in the emulsion; φ was the oil phase ratio. In this paper, the volume concentration of oil phase was 1/ 10. D3,2 was the average radius of emulsion surface.
Th T is a cationic benzothiazole dye that can bind in parallel to the β-sheet structure. This reaction results in a significant increase in the maximum fluorescence intensity of Th T. Based on this principle, this fluorescent probe was used to study the formation and the number of amyloid fibrils in order to study the reaction. Fig. 1A shows the Th T fluorescence intensity of RBPFs at specific heating times. As the heating time increased from 60 min to 420 min, the fluorescence intensity reached its highest point, and then heated from 480 min to 600 min, the β-sheet content in RBP began to decrease and entered the decline period of fibrils growth. This may be due to the loss of the β-sheet structure due to excessive heating. Fig. 1B shows the fluorescence intensity changes of F0/F. When heated to 420 min, the fluorescence intensity increased sharply, possibly due to the increase of β-sheets and the enhancement of hydrophobic interactions. The subsequent sharp decline may be due to the fibrosis reaction, the protein has reached a certain degree of hydrolysis, no more hydrophobic groups as the building unit continue to self-assemble into fibrils.
2.6.3. Optical microscopy observation One mL of Th T buffer was added to the RBPFs solution, shaken, and mixed. After 1 min, the new RBPFs emulsion was prepared according to section 2.4. The whole process of production was kept from the light. The emulsion sample was dripped onto the slide with a volume of 1 mL, and the appearance of the emulsions were observed by an optical microscope (BX 35, Olympus, Japan) under a bright and fluorescence field with a 40 × objective lens. 2.6.4. Rheology test The rheological properties of the RBPFs emulsion were evaluated by a MCR320 rheometer (Anton Paar, Austria), using a plate configuration and PP50 with 1-mm gap at 25 °C. Freshly prepared emulsions were used for all tests.
3.2. Surface hydrophobicity (H0)
2.7. Static rheological measurement
H0 is one of the most important factors affecting the interface properties of proteins. 1-Anililo-naphthalene-8-sulfonate (ANS) was used as the fluorescent probe to determine the H0 values. Increased surface hydrophobicity improved fibrillation of rice bran protein (Moayedzadeh, Madadlou, & Khosrowshahi, 2015). According to Fig. 1C, the protein fibrils heated for 420 min showed higher fluorescence intensity than the other times, indicating that the exposed hydrophobic groups varied with heating time, that acid-heat treatment exposed a large number of hydrophobic groups, and that the RBPFs exhibited an increase in hydrophobicity before heating for 420 min.
Viscosity was evaluated in the linear viscoelastic region (LVER) at 25 °C with a shear rate from 0.1 to 100 s−1. Viscoelastic data were fitted by the Ostwald de Waale model (Zhang et al., 2015):
=K
n 1
(5)
Where, η was apparent viscosity, Pa·s; γ was shear rate, S−1; K was the consistency index, Pa sn; and n was the index that provides information about the flow behavior affected by shear rate. 3
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resulting in a dramatic increase in fibrils. The mean contour length of the fibrils gradually increased with the increase of heating time from 60 min to 420 min, until the fibrils heated for 480 min showed a downward trend. This indicates that heat treatment promotes the fibrillation of RBP to form RBPFs with larger size, and excessive heating time may cause the further fibrillation. Zeta-potential is widely used to characterize the electrostatic interaction between charged particles. Fig. 2C shows the zeta-potential distribution of RBPFs, Fig. 2C shows the zeta-potential distribution of RBPFs. When heating for a short time (60 min), the zeta-potential becomes less negative, and then becomes more negative (180–360 min) as the heating time increases, and finally the zeta-potential becomes less negative as heating continues (420–600 min). This shows that when heated for 360 min, the absolute value of zeta-potential was the largest, the electrostatic interaction between protein molecules was the strongest, and the protein solution tends to be stable. Protein flexibility is an important aspect that affects its emulsification properties. The flexibility of proteins is a controlling factor, which plays an important role in the surface activity of proteins (Li et al., 2019; Razumovsky & Damodaran, 1999). The molecular flexibility of proteins is not only affected by covalent interactions but also by noncovalent interactions such as van der Waals forces, electrostatic bonds, and hydrophobic interactions (Li et al., 2019). Heat treatment may destroy some of the above bonds and expand the flexible structure of RBPFs, thus increasing flexibility with the increase of reaction time. The molecular flexibility of the protein slowly increased before heating for 180 min, and quickly increased to the maximum value (0.054) at 420 min (Fig. 2D). Then it gradually decreased after 420 min. Compared with RBP, the flexibility of RBPF increased significantly with prolonged heating time (P < 0.05), which indicates that the structure of RBP molecule was unfolded (Li et al., 2019). Thermal denaturation can destroy non-covalent effects, so heat treatment of the protein can improve its emulsifying properties (Damodaran, 2005). 3.4. TEM image of RBPFs The TEM image (Fig. 3) shows the profile length of the RBP fibrils, showing that the prepared fibrils are semi-flexible under these conditions. It can also be seen that the unheated protein (Fig. 3A) is a spherical particle, which gradually changes to the shape of fibrils as the heating time is further increased. In addition, the DLS results are smaller than the TEM size, probably because the DLS was hydrated, while the TEM sample was measured after drying. 3.5. ATR/FTIR analysis ATR/FTIR was widely used to analyze the secondary structure of various samples. The characteristic bands in the protein ATR/FTIR spectra mainly include amide I (1600―1700 cm−1) and amide II (1500―1600 cm−1) (Jung, Gunes, & Mezzenga, 2010) combined with the original map (Fig. 4) peak position obtained by deconvolution and secondary structure identification: α-helix, 1648―1660 cm−1; βfolding, 1626―1640 cm−1; β-turns, 1662―1684 cm−1; irregular curl, 1640―1650 cm−1. Table 1 shows that as the heating time increases, the β-sheet structure of the protein also increases, reaching the highest at 360 min, and then remaining stable. Gosal, Clark, Pudney & Ross (2002) studied the fibrillation of bovine serum albumin and also found that the number of β-sheets increased after heat-induced protein formation of amyloid fibrosis. The main component of RBP was β-sheets structure, accounting for 26% of the total secondary structure. The main secondary structure of RBP after the heat treatment was still βsheets structure, and the content of β-sheets structure of RBPFs was higher than that of RBP, the RBPFs of 360 min was the highest reaching 30%. However, the content of β-sheets structure of RBPFs 360 min was no longer increasing, which indicates that the further heat treatment of RBP induced the loss of β-sheets structure.
Fig. 1. Characterization 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.
After heating for 480 min, H0 of the RBPFs began to decline. This phenomenon may be due to the fact that proteins are no longer hydrolyzed to form a large number of beta-folded structures, but aggregate to form nuclei, which are linked to the construction units and increase the length of protein fibrils. 3.3. Mean contour length, zeta-potential and flexibility of RBPFs The distribution of RBPFs mean contour length was affected by the heat-induced environment (Feng et al., 2017). Fig. 2A―B show the mean contour length and distribution of fibrils at different heating times. A typical bimodal shape appeared in RBPFs heated for 420 min, 4
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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. 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. Table 1 Analysis of protein secondary structure base on attenuated total Fourier Transform infrared spectroscopy (ATR/FTIR).
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.
3.6. EAI and ES analysis EAI is an indicator of the emulsification efficiency of an emulsifier. The emulsification activity of a protein is measured by the emulsification efficiency of a protein molecule during homogenization. EAI represents the ability of a protein to participate in emulsion formation
Heat time (min)
α- helix (%)
0 60 180 360 420 480 540 600
22 21 21 22 21 22 23 21
± ± ± ± ± ± ± ±
0.38b 0.66a 0.03a 0.16b 0.23a 0.34b 0.24c 0.06a
β-folding (%) 26 26 27 30 26 26 26 26
± ± ± ± ± ± ± ±
0.19a 0.50a 0.36b 0.22c 0.25a 0.31a 0.02a 0.05a
β-turns (%) 16 17 18 16 18 16 16 16
± ± ± ± ± ± ± ±
0.42a 0.54b 1.55b 0.14a 0.18b 0.25a 0.34a 1.75ab
Irregular curl (%) 26 23 25 26 25 26 26 26
± ± ± ± ± ± ± ±
0.19a 1.28b 1.48b 0.16a 0.39b 0.43a 0.19a 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. 5
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emulsification properties of proteins. For the above reason, the relationship between the hydrophobicity and molecular flexibility of RBPFs and the EAI and ES between RBPFs emulsions was investigated. According to Table 3, the correlation coefficient between H0 and flexibility was 0.881 (p < 0.05), the correlation coefficient of ES was 0.899, and the correlation coefficient of EAI was 0.887. The correlation coefficient between flexibility and EAI was 0.962 (p < 0.01), and the correlation coefficient of ES was 0.685. Hydrophobicity H0 is positively correlated with flexibility, EAI and ES. This indicates that the trends in hydrophobicity and molecular flexibility are consistent. Surface hydrophobicity and flexibility are important factors affecting emulsification performance. In the previous studies, researchers focused on the relationship between H0 and protein emulsification properties and found a strong correlation (Li et al., 2019). Prak, Nakatani, Katsube, Adachi, Maruyama & Utsumi (2005) found the emulsifying ability of protein is not exactly the same as the emulsifying ability of surface hydrophobicity. Flexible protein molecules tend to denature at the interface, while rigid protein molecules are less susceptible. Studies by Kato, Komatsu, Fujimoto, and Kobayashi (1985) have shown that surface hydrophobicity is ultimately an important factor in achieving good foaming and emulsifying properties due to the high surface hydrophobicity imparted by the mild effector.
Table 2 Emulsifying activity index (EAI) and emulsifying stability (ES) of emulsions stabilized by different RBPFs. Heat time (min)
EAI (m2/g)
0 60 180 360 420 480
1.13 0.44 0.43 0.29 0.49 1.20
± ± ± ± ± ±
ES × 10−2
0.02e 0.06c 0.01b 0.01a 0.04d 0.03f
1.18 2.71 2.47 2.37 2.87 1.92
± ± ± ± ± ±
0.06a 0.04e 0.01d 0.01c 0.06f 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. Table 3 Correlation analysis between flexibility with surface hydrophobicity (H0), ES and EAI.
Correlation coefficient
H0 and flexibility
Flexibility and ES
Flexibility and EAI
H0 and ES
H0 and EAI
0.881*
0.685
0.962**
0.899
0.887
* 0.01 < p < 0.05, **p < 0.01.
3.7. Emulsion characterization
under external force. ES is the ability of emulsion droplets to maintain stable (Li et al., 2019). The higher the EAI, the higher the emulsification activity. The emulsification activity of a protein is related to the structure of its own molecule. At the same time, according to Table 2, in these six emulsion samples, the RBPFs emulsion heated for 420 min had the best stability, so it was believed that increasing the heating time within a certain range could improve emulsion stability. Tang and Shen (2013) found that after protein adsorption to the oil-water interface, structural rearrangement generally occurs, which affects the
3.7.1. Average droplet size and zeta-potential The droplet size of the emulsion is one of the most important qualitative properties of emulsions (Ge et al., 2017). According to our preliminary experiments (Fig. S1), 0.01–0.05 g/mL of the fish oil concentration was selected, and the results showed that the 0.04 g/mL protein concentration emulsion was the most stable. Liu and Tang (2014) found that increasing the concentration (0.005–0.06 g/mL) of protein particles can reduce the droplet size and increase the stability of
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. 6
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protein fibrils was not enough to cover the oil-water interface. For example, the length of the protein fibril may not be enough to coat the fish oil droplets, the interfacial tension between the O/W interface is great, and the oil droplets combine to make the particle size larger. Zeta-potential is an important indicator for evaluating the stability of the system. Generally, the absolute value of the zeta-potential is high, the protein molecules are repelled, and the solution tends to be stable. Fig. 5B shows the zeta-potential distribution of the RBPFs emulsion droplets, from +31 mV to +35 mV, and the emulsion stabilized by RBPFs was positive. Zeta―potential did not show significant changes indicating that the heating time has little effect on the RBPFs emulsion zeta-potential. It might be due to the length of the heating time has little effect on the zeta-potential of the RBPFs stabilized-emulsion, the overall emulsion potential does not change much. Compared with protein fibrils, the zeta-potential of the emulsion was converted into a positive charge, probably because the fibrils are negatively charged, and the adsorption on the droplets is not enough to change the positive charge of the droplets. 3.8. AP and Γ of RBPFs emulsion between O/W interface Some studies (Kato et al., 1985) have pointed out that proteins with softer structures are more likely to undergo structural transformation and thus adsorb at the interface. According to the interface adsorption principle of polyelectrolytes, the protein structure will be expanded and denatured during the interface adsorption process. It can be seen from Fig. 5C―D that as the heating time increases to 420 min, the AP and Γ of the protein are continuously increasing, and only the emulsion sample heated for 480 min shows a downward trend. This may be due to increased hydrophobicity of the protein, which is consistent with the results of molecular flexibility in Fig. 2. In the protein adsorption process, because the molecular weight of the protein is larger, the adsorption rate at the interface is much lower than that of the small molecule surfactant. After adsorption to the interface, the hydrophobic amino acid of the protein tends to stretch toward the oilcontaining direction and rearrange. Therefore, compared with small molecule surfactants, protein emulsifiers have weaker emulsifying ability, but the prepared emulsions have higher physical and chemical stability. 3.9. Rheology properties of fish oil emulsion stabilized with RBPFs Fig. 6 shows the rheological characteristics of the fish oil emulsion stabilized with RBPFs. Within the angular frequency range (0―100 s−1), the fact that the storage modulus G′ was less than the loss modulus G″ indicates that in this frequency range, the emulsion does not exhibit gel properties, but exists in a liquid state in which fluidity is good. As the heating time increases, the G′ increases gradually, and the G″ decreases. The data in the figure were fitted by the Ostwald de Waale model (the relevant parameters are shown in the insert table), and the K-value is lower, indicating lower viscosity, and the n-value is higher, indicating that the emulsion is closer to a Newtonian fluid (Wang, Li, Wang, & Adhikari, 2011). The table shows that the K-value was the smallest for the emulsion heated for 420 min, indicating that the viscosity was the lowest. The value n > 1 represents that the emulsion exhibits shear thickening, n = 1 means an ideal viscosity flow and n < 1 suggests shear thinning occurs (Wei & Gao, 2015). As seen in Fig. 6 within the set shear rate range (0-100 s−1), the viscosity of all samples gradually decreases, and shear thinning occurs. The reason for this phenomenon is that oil droplets gather together, the weak interaction force that maintains stable flocculation under high-speed shear was destroyed, and shearing occurs thinning phenomenon. In protein emulsions, when the protein concentration reaches a certain value, RBPFs that are not adsorbed to the oil-water interface are present. Due to the presence of unabsorbed and amphiphilic protein fibrils in the continuous phase, the fibrils tend to approach each other due to
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.
the emulsion. As result, the 0.04 g/mL RBPFs solution was chosen to prepared the emulsion. Fig. 5A shows that the average droplet size of the RBPFs emulsion did not change significantly within 0–360 min. While the average droplet size of the emulsion stabilized by 420 min RBPFs rose sharply, which was probably due to the more formation of fibrils, causing the increase of the length of RBPFs those were located at the O/W interface of the emulsion. In contrast, the concentration of 7
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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.
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 I2935/I2880
1.00 ± 0.009b 1.01 ± 0.006d
0.97 ± 0.002b 1.00 ± 0.005d
0.96 ± 0.004b 0.97 ± 0.01b
0.95 ± 0.006a 1.02 ± 0.004e
0.94 ± 0.009a 0.94 ± 0.002a
0.97 ± 0.007b 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.
secondary forces such as hydrophobic interactions, apparently increasing the viscosity of the emulsion. And this thickening enhances the stability of the emulsion (Voutsinas, Cheung, & Nakai, 1983) (see Fig. 7).
3.9.1. Morphology Fig. 8 shows the morphology of six emulsion samples under an optical microscope and a fluorescence microscope. According to the results of the fluorescence microscope, it can be seen that the emulsion 8
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the polymer degradation (Paula, Sombra, Cavalcante, Abreu, & Paula, 2011). This has been confirmed that RBPFs heated for 180 min will cause emulsion instability in section 3.6. 4. Conclusion This study better understands the effect of different heating times on the physicochemical properties of an RBPF-forming emulsion. When RBP were heated for 420 min, the mean contour length and hydrophobicity of the fibrils were highest and the corresponding RBPFs were exhibited favorable emulsifying properties. And the fibril-stabilized O/ W emulsion heated for 420 min had better emulsifying properties, and the good hydrophobicity and flexibility also improved emulsifying activity of the RBPFs emulsion. This study provides a theoretical basis for constructing protein fibrils suitable for stabilizing emulsions and these properties increase the potential of RBP to prepare emulsions as an effective delivery system for nutraceuticals in functional foods.
Fig. 9. DSC profiles of RBPFs emulsions stabilized by unheated RBP, RBPFs heated for 60 min, 180 min, 360 min and 420 min, respectively.
Author statement
droplets gradually increase as the heating time increases. And the unheated protein emulsion is darker, which may be caused by the combination of the Th T dye and the β-sheet in the protein, which is the same as that of the fluorescence spectrophotometer.
Pang shuxian: Conceptualization, Writing - Original Draft, Data Curation. Shaoping: Supervision. Sun qingjie: Project administration. Pu chuanfen: Methodology. Tang wenting: Writing - Review & Editing, Funding acquisition.
3.9.2. Raman spectroscopic analysis In order to examine the interaction between RBPFs and lipids, the structural properties of RBPFs emulsion were determined using Raman spectroscopy. The ratio of relative intensity ratio I2850/I2880 and I2935/ I2880 reflects the interaction between lipid chains and the state of order or disorder of acyl chains (Ruiz-Capillas, Carmona, Jiménez-Colmenero & Herrero, 2013). It can be visualized from Table 4 that the emulsion of 420 min RBPFs shows the lowest intensity ratios. The disorder of the acyl chain indicates that more proteins were inserted between the acyl chains of the oil. (Herrero, Ruiz, Pintado, Carmona, & Jiménez, 2018). Lower I2850/I2880 and I2930/I2880 intensity ratios were also related to hydrophobic interactions including the CH groups of the lipid with protein (Herrero, Ruiz-Capillas, Pintado, Carmona, & JiménezColmenero, 2018). In addition, combined with the results of infrared spectroscopy analysis, I2850/I2880 was negatively correlated with the number of β-sheets. Ruiz-Capillas et al. (2013) also reported that the intensity ratio of the emulsion (I2850/I2900) was lower than that of the separated fatty phase and pointed out that most of the hydrocarbon chains of milk fat globules and/or triglycerides are closely packed. This hydrocarbon chain accumulation was very important for the stability of the emulsion.
Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The authors thank Key Research and Development Program of Shandong Province of China (No. 2019GNC106051), China Scholarship Council (No. 201908370054), National Natural Science Foundation of China (NO. 31501578), University Science and Technology Project of Shandong Province of China (No. J16LE22), Doctoral Science Foundation of Shandong Province of China (No. BS2015SW019), Advanced Talents Foundation of Qingdao Agricultural University, China (No. 6631115030), and Special Funds for Taishan Scholars Project of Shandong Province of China (ts201712058) for financial support. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.lwt.2019.108985.
3.9.3. DSC analysis DSC were determined the thermal behavior of RBPFs stabilized emulsions with different heating times. The emulsion stabilized by unheated RBP showed an initial endothermic peak at 185 °C and a maximum exothermic peak at 146 °C. The endothermic peak was caused by dehydration associated with the hydrophilic group of the polymer, while the exothermic peak was associated with degradation of the polyelectrolyte due to dehydration and depolymerization reactions (Li et al., 2018). The exothermic peak of the RBPFs stabilized emulsion after heating was smaller and broader compared with those of RBP emulsion (Fig. 9). This can be explained as the hydrophobic interaction between RBPFs and fish oil (Sarmento, Ferreira, Veiga, & Ribeiro, 2006). The unheated RBP emulsion began to denature at about 85 °C, the RBPFs emulsion denature temperature began to gradually increase, and the 420 min RBPFs emulsion denature temperature reached about 140 °C, indicating that the corresponding emulsion was the most stable among all the samples. In addition, the emulsion stabilized by RBPFs heated for 180 min has a sharp exothermic peak near 176 °C, suggesting
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Relationship between the emulsifying properties and formation time of rice bran protein fibrils
T
Shuxian Panga, Ping Shaob, Qingjie Suna, Chuanfen Pua,∗∗, Wenting Tanga,∗ a b
School of Food Science and Engineering, Qingdao Agricultural University, Qingdao, 266109, China Department of Food Science and Technology, Zhejiang University of Technology, Zhejiang, Hangzhou, 310014, China
ARTICLE INFO
ABSTRACT
Keywords: Rice bran protein fibrils RBPFs emulsion Rheological properties Formation time
In this study, oil-in-water emulsions stabilized with rice bran protein fibrils (RBPFs) were prepared, and the effect of the fibrils’ formation time on the structural and emulsion characteristics, including droplet size, zetapotential, and surface morphology, were analyzed. As the heating time increased to 420 min, the β-sheet structure content and surface hydrophobicity of the RBPFs increased. As the heating time continued to heat up to 600 min, a downward trend occurred. The formation time also affected the mean contour length of the fibrils, and the scale began to decrease at 540 min. The emulsion prepared by heating for 480 min showed the best emulsifying activity, and the 420-min fibril emulsion showed the best emulsion stability and the highest interfacial protein content. The rheological results showed that as the heating time increased (0–480 min), the storage modulus increased, and the loss modulus and the apparent viscosity were reduced. The emulsions in the range of 0–100 s−1 shear rate did not have gel properties and shear thinning. The results of this study will help to provide a new perspective to develop a stable food-grade emulsion in the future.
1. Introduction Oil-in-water (O/W) emulsions are widely used in food, nutrition, and medicine as a means of delivering lipophilic flavorings, antioxidants, functional lipids, and various other biologically active compounds (Ge et al., 2017). However, since the emulsion consists of water and a water-immiscible non-polar liquid (oil phase), the colloidal dispersion is thermodynamically unstable colloidal dispersion, with the oil phase dispersed within an aqueous phase. Therefore, interfacial stabilizers, including protein, starch and other polysaccharides, small molecule polymers, amphiphilic lipids, etc., have been used to stabilize the emulsion system. Among these stabilizers, natural food proteins have favorable interfacial film-forming properties when adsorbed to the oilwater interface, which reduce the interfacial tension. To date, an emulsion system is considered an important component in food processing (Feng, Cai, Wang, Li, & Liu, 2017). Recently, fibrils derived from globulin have received much attention and are used in materials science such as food and biopharmaceuticals (Serfert et al., 2014). At the pH away from the isoelectric point (pI, commonly at pH ≈ 2.0), thermal denaturation of the protein can lead to an aggregation of proteins into micron-length (1–10 μm), nano-diameter (1–10 nm) fibrils and multi-stranded twisted structures rich in β∗
sheets perpendicular to the fibril axis (Mohammadian & Madadlou, 2018). Acid heat treatment usually plays a role in unlocking protein molecules and exposing internal hydrophobic areas. The formation of linear fibrils is mainly controlled by the balance of intermolecular forces, including the attraction (mainly hydrophobic bonds) between pyrolytic folding molecules and repulsive forces. With high-aspect ratio anisotropic properties, fibrils have specific interfacial behaviors due to capillary forces. Most food proteins have the ability to form such fibrils, such as beta-lactoglobulin, whey protein isolate, and soy protein isolate (Gao et al., 2017). Studies have pointed out (Serfert et al., 2014) that as anisotropic particles, protein fibrils have better emulsifying effects than rigid-bound spherical particles due to a larger aspect ratio. In the interfacial shear rheology analysis, fibrils exhibited a high degree of elasticity at the O/W interface. Recently, protein fibrils have been widely used to prepare emulsions. Serfert et al. (2014) prepared antioxidized fish oil emulsions with β-lactoglobulin fibrils, which have better barrier properties than the original protein, resulting in lower permeability, effectively improving the oxidative stability of fish oil. Gao et al. (2017) studied the preparation of stable O/W Pickering emulsions of β-lactoglobulin fibrils at different fibril concentrations and pH. When the concentration of fibrils in O/W emulsion is above 5 mg/ mL, the emulsion has long-term stability. Although stability is
Corresponding author. Corresponding author. E-mail addresses: [email protected] (C. Pu), [email protected] (W. Tang).
∗∗
https://doi.org/10.1016/j.lwt.2019.108985 Received 22 September 2019; Received in revised form 4 December 2019; Accepted 21 December 2019 Available online 07 January 2020 0023-6438/ © 2020 Elsevier Ltd. All rights reserved.
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acceptable, an excessive concentration of fibrils can lead to larger emulsion droplet sizes. Rice bran protein (RBP) is composed of albumin, globulin, gluten, and prolamin. RBP is recognized as a high-quality vegetable protein with low hypoallergenic activity. Zhang, Huang, Liu & Wei (2014) prepared fibrils by heating RBP under pH 2.0 at 90 °C, and found that adding fibrils could significantly promote solution-thickening and gelhardening behavior. Liu and Tang (2014) studied the effect of ionic strength on the formation and characteristics of RBPFs, and pointed out that the presence of cellulose can change the structural properties of the RBP gel system, which is suitable for natural nano-scale gel materials. However, the emulsification characteristics of RBPFs are not clear. The objective of this study was to evaluate the effect of formation times on the structural properties and molecular flexibility of RBPFs. Then the emulsion properties of the RBPFs obtained under different times were evaluated. The physicochemical properties of fish oil emulsion stabilized by RBPFs prepare5d under different times were also compared, such as rheological properties, morphology, particle size potential, interface protein content.
Netto, and Cunha (2018) with a few modifications. ANS was dispersed in PBS buffer (pH = 7, 0.1 mol/L) to prepare a working solution (0.01 mol/L) with the RBPF sample, mixed at a ratio of 15:1; then the sample mixture was shaken for 1 min and determined. The excitation wavelength was 370 nm, the emission wavelength scope was 400–600 nm, the emission and excitation slits were set to 2.5 nm, the operating voltage was 250 mV, and the scanning rate was 500 nm/min. 2.3.3. Mean contour length and zeta-potential analysis The mean contour length and zeta-potential of the sample were measured by dynamic light scattering (DLS) using Zetasizer Nano-ZS (Malvern Instruments, UK) instrument at 25 °C. The sample was diluted appropriately to meet the requirement of the equipment. 2.3.4. Transmission electron microscope (TEM) The RBPF sample was dripped onto copper grids coated with amorphous carbon film and dyed with phosphotungstic acid (0.02 g/ mL). After natural air drying, the sample was observed using a transmission electron microscope (TEM, JEM-2200 FS, JEOL Ltd., Japan) with 80 kV acceleration voltage, the levels of magnification were 1500 times.
2. Materials and methods 2.1. Materials
2.3.5. Attenuated total reflectance fourier transform infrared spectroscopy (ATR/FTIR) An ATR infrared spectrum of the samples was taken using a Nicolet iS10 (Thermo Scientific, USA) instrument. RBPFs were freeze-dried and measured. The scanning was performed 32 times in the wave number range of 4000 cm-1 to 400 cm −1. Images and data were analyzed using OMNIC 8.0 software (Thermo Fisher Scientific, USA) and Peakfit software.
Rice bran protein with 0.98 g/mL purity was obtained from Xi'an Connor Chemical Co., China. Fish oil obtained from Shanxi Guanchen Biotech Co., Ltd (Xi'an, China). 8-Anilino-1-naphthalenesulfonic acid (ANS), Thioflavin T (Th T), trypsin was purchased from Sigma-Aldrich (St. Louis, Mo, U.S.A). Trichloroacetic acid (TCA) was obtained from Sangon Biotech Co., Ltd (Shanghai, China). All other reagents used were analytical grades.
2.3.6. Molecular flexibility This measure was conducted according to the method of Li et al. (2019) with some modifications. The trypsin solution (1 mg/mL) was prepared using Tris-HCl buffer (0.05 mol/L, pH = 8.0). Then, 4 mL of RBPFs (1 mg/mL) was mixed with 250 μL of the enzyme solution at 37 °C for 5 min, and the reaction was terminated with 4 mL of 5 mg/mL trichloroacetic acid (TCA). The supernatant was collected by centrifugation at 4000 rpm for 20 min. Molecular flexibility was measured at 280 nm and expressed by its absorbance.
2.2. Methods 2.2.1. Preparation of RBP RBP was dissolved in distilled water (0.06 g/mL) and magnetically stirred for 24 h to obtain RBP dispersion. This RBP dispersion was adjusted to pH 2.0 with 1 mol/L HCl (ionic strength, 150 mmol/L) and centrifuged at 3000×g for 30 min in order to remove insoluble materials. The supernatant was taken at 90 °C for a specific time (60, 180, 360, 420, 480, 540 and 600 min). Immediately after the end of the sample heating, the samples were placed in an ice bath and stored under 4 °C for further study.
2.4. Preparation of RBPFs emulsion An aqueous phase was prepared by dispersing 0.04 g/mL RBPFs in aqueous buffer solution (10 mmol/L phosphate, pH 7.0). Coarse fish O/ W emulsions containing 1/10 organic phase and 9/10 aqueous phase were formed using a high-shear mixer (D50, WIGDENNS) for 5 min at 10,000 rpm. Then, ultrasonic treatment (5 min) was carried out using a 6-mm probe in the ice bath with a sequence of 1 s of sonication and 1 s of rest (power: 540 W, frequency: 25 kHz). The prepared RBPFs emulsion was kept under 4 °C.
2.3. RBAFs evaluation 2.3.1. Th T fluorescence analysis The fluorescence analysis was performed according to Zhang et al.'s method (2014) with some modifications. The Th T stock solution was prepared by dispersing 8 mg of Th T into 10 mL of phosphate buffer solution (PBS, pH 7, 0.05 mol/L). Before the test, the Th T stock solution was diluted 50 times as a working solution. Then, 20 μL of the RBPF sample was mixed with 3-mL Th T working solution, shaken evenly, and allowed to stand for 1 min before the fluorescence analysis using a fluorescence spectrophotometer (F2700, Hitachi, Japan). The excitation wavelength was set to 460 nm, the emission wavelength was set to 470–600 nm, the excitation and emission slits were both 5 nm, the voltage was 700 mV, and the scan rate was 500 nm/min. The value of F0/F as a function of heating time was determined, in which F0 was the maximum fluorescence intensity of unheated protein at 496 nm, and F was the fluorescence intensity of protein fibrils at 496 nm for a different heating time.
2.5. Emulsifying activity index (EAI) and emulsifying stability (ES) Emulsification activity (EAI) and emulsion stability (EAI) were determined according to Tang et al.’s method (2005). The emulsion sample was centrifuged at 3500 r/min for 10 min, and the supernatant was taken for 100 μL mixed with 10 mL sodium dodecyl sulfate buffer (SDS, 1 mg/mL). The absorbance of the emulsion sample was measured at 500 nm using an ultraviolet spectrophotometer (TU-1810, PERSEE, China). EAI and ES were calculated by the following equations (Tang & Shen, 2013):
2.3.2. Surface hydrophobicity(H0) H0 was measured by 1-anililo-naphthalene-8-sulfonate (ANS) fluorescent probe, according to the method of Mantovani, Guilherme,
EAI =
2
2.303 × 2 × A500nm ×dilution multiple c × × L
(1)
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ES =
(EAImax
EAImax EAImin ) × 100
2.8. Dynamic rheological measurement
(2)
Frequency scanning was carried out from 0.1 to 100 Hz at 25 °C. The relationship between storage modulus (G′) and loss modulus (G″) was measured.
Where c is the concentration of protein in the sample solution, g/mL; EAImin and EAImax are the EAI values of the emulsion after 10 min and 0 min; ϕ is the fraction of the oil phase, which is 1/10 in this experiment; EAI is the largest EAI after emulsion formation; L is the cuvette path, 0.01 m.
2.8.1. Raman spectra analysis The Raman spectrum was recorded by Raman spectrometer (Thermo Fisher Scientific, USA) and excited by a near-infrared laser at 785 nm according to Niu et al.'s method (2016) with a modification. The laser output power was 334 mV and the data acquisition time was set to 40 s to obtain the best peak intensity. The scan range for each test was 300–3050 cm−1. Images and data were analyzed using OMNIC 8.0 software (Thermo Fisher Scientific, USA).
2.6. Emulsion characterization 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.
2.8.2. Differential scanning calorimetry (DSC) DSC-Q1000 differential scanning calorimetry (TA Instrument, USA) was used to identify the thermal transitions in the samples during heating according to Zhang, Pu, Tang, Wang, and Sun (2019)'s method with some modifications. The lyophilized sample (4–7 mg) was placed in a crucible and sealed, the temperature range was 25–300 °C, and the heating rate was 10 °C/min. Sample was tested under nitrogen flow, reaction gas set to 50–60 mL/min, dry gas was set to 200 mL/min.
2.6.1. Emulsion droplet size and zeta-potential analysis The physical stability of emulsion was quantified by measuring the average droplet diameter and zeta-potential of a freshly prepared emulsion by a Zetasizer Nano-ZS (Malvern Instruments, UK) instrument. Emulsion samples were moderately diluted with ultrapure water to avoid multiple scattering. All measurements were carried out at 25 °C.
2.9. Data analysis
2.6.2. Adsorption rate of protein (AP) and interfacial protein concentration (Γ) of RBPFs emulsion According to the method of Ye (2008), some modifications have been made. The fresh emulsion was centrifuged at 13,000 r/min for 15 min, and the top layer of the centrifuge tube was removed. Determination of protein content (Cf) was performed by the Bradford (Redmilegordon, Armenise, White, Hirsch, & Goulding, 2013) method.
Adsorption rate of protein: AP = (C0
Cf ) × 100/ C0
Interfacial protein concentration: (mg/ m2) = (C0
Cf ) × D3,2 /6
Data analysis and mapping were performed using Origin 8 software. The data were expressed as mean ± standard deviation (Means ± SD), and the significant level was p < 0.05. All tests were performed in triplicate. The correlation analysis was analyzed by SPSS 17.0 software and expressed as Pearson correlation coefficient.
(3)
3. Results and discussion
(4)
3.1. Th T fluorescence spectral analysis of fibrils
Where, C0 was total protein content in the emulsion; φ was the oil phase ratio. In this paper, the volume concentration of oil phase was 1/ 10. D3,2 was the average radius of emulsion surface.
Th T is a cationic benzothiazole dye that can bind in parallel to the β-sheet structure. This reaction results in a significant increase in the maximum fluorescence intensity of Th T. Based on this principle, this fluorescent probe was used to study the formation and the number of amyloid fibrils in order to study the reaction. Fig. 1A shows the Th T fluorescence intensity of RBPFs at specific heating times. As the heating time increased from 60 min to 420 min, the fluorescence intensity reached its highest point, and then heated from 480 min to 600 min, the β-sheet content in RBP began to decrease and entered the decline period of fibrils growth. This may be due to the loss of the β-sheet structure due to excessive heating. Fig. 1B shows the fluorescence intensity changes of F0/F. When heated to 420 min, the fluorescence intensity increased sharply, possibly due to the increase of β-sheets and the enhancement of hydrophobic interactions. The subsequent sharp decline may be due to the fibrosis reaction, the protein has reached a certain degree of hydrolysis, no more hydrophobic groups as the building unit continue to self-assemble into fibrils.
2.6.3. Optical microscopy observation One mL of Th T buffer was added to the RBPFs solution, shaken, and mixed. After 1 min, the new RBPFs emulsion was prepared according to section 2.4. The whole process of production was kept from the light. The emulsion sample was dripped onto the slide with a volume of 1 mL, and the appearance of the emulsions were observed by an optical microscope (BX 35, Olympus, Japan) under a bright and fluorescence field with a 40 × objective lens. 2.6.4. Rheology test The rheological properties of the RBPFs emulsion were evaluated by a MCR320 rheometer (Anton Paar, Austria), using a plate configuration and PP50 with 1-mm gap at 25 °C. Freshly prepared emulsions were used for all tests.
3.2. Surface hydrophobicity (H0)
2.7. Static rheological measurement
H0 is one of the most important factors affecting the interface properties of proteins. 1-Anililo-naphthalene-8-sulfonate (ANS) was used as the fluorescent probe to determine the H0 values. Increased surface hydrophobicity improved fibrillation of rice bran protein (Moayedzadeh, Madadlou, & Khosrowshahi, 2015). According to Fig. 1C, the protein fibrils heated for 420 min showed higher fluorescence intensity than the other times, indicating that the exposed hydrophobic groups varied with heating time, that acid-heat treatment exposed a large number of hydrophobic groups, and that the RBPFs exhibited an increase in hydrophobicity before heating for 420 min.
Viscosity was evaluated in the linear viscoelastic region (LVER) at 25 °C with a shear rate from 0.1 to 100 s−1. Viscoelastic data were fitted by the Ostwald de Waale model (Zhang et al., 2015):
=K
n 1
(5)
Where, η was apparent viscosity, Pa·s; γ was shear rate, S−1; K was the consistency index, Pa sn; and n was the index that provides information about the flow behavior affected by shear rate. 3
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resulting in a dramatic increase in fibrils. The mean contour length of the fibrils gradually increased with the increase of heating time from 60 min to 420 min, until the fibrils heated for 480 min showed a downward trend. This indicates that heat treatment promotes the fibrillation of RBP to form RBPFs with larger size, and excessive heating time may cause the further fibrillation. Zeta-potential is widely used to characterize the electrostatic interaction between charged particles. Fig. 2C shows the zeta-potential distribution of RBPFs, Fig. 2C shows the zeta-potential distribution of RBPFs. When heating for a short time (60 min), the zeta-potential becomes less negative, and then becomes more negative (180–360 min) as the heating time increases, and finally the zeta-potential becomes less negative as heating continues (420–600 min). This shows that when heated for 360 min, the absolute value of zeta-potential was the largest, the electrostatic interaction between protein molecules was the strongest, and the protein solution tends to be stable. Protein flexibility is an important aspect that affects its emulsification properties. The flexibility of proteins is a controlling factor, which plays an important role in the surface activity of proteins (Li et al., 2019; Razumovsky & Damodaran, 1999). The molecular flexibility of proteins is not only affected by covalent interactions but also by noncovalent interactions such as van der Waals forces, electrostatic bonds, and hydrophobic interactions (Li et al., 2019). Heat treatment may destroy some of the above bonds and expand the flexible structure of RBPFs, thus increasing flexibility with the increase of reaction time. The molecular flexibility of the protein slowly increased before heating for 180 min, and quickly increased to the maximum value (0.054) at 420 min (Fig. 2D). Then it gradually decreased after 420 min. Compared with RBP, the flexibility of RBPF increased significantly with prolonged heating time (P < 0.05), which indicates that the structure of RBP molecule was unfolded (Li et al., 2019). Thermal denaturation can destroy non-covalent effects, so heat treatment of the protein can improve its emulsifying properties (Damodaran, 2005). 3.4. TEM image of RBPFs The TEM image (Fig. 3) shows the profile length of the RBP fibrils, showing that the prepared fibrils are semi-flexible under these conditions. It can also be seen that the unheated protein (Fig. 3A) is a spherical particle, which gradually changes to the shape of fibrils as the heating time is further increased. In addition, the DLS results are smaller than the TEM size, probably because the DLS was hydrated, while the TEM sample was measured after drying. 3.5. ATR/FTIR analysis ATR/FTIR was widely used to analyze the secondary structure of various samples. The characteristic bands in the protein ATR/FTIR spectra mainly include amide I (1600―1700 cm−1) and amide II (1500―1600 cm−1) (Jung, Gunes, & Mezzenga, 2010) combined with the original map (Fig. 4) peak position obtained by deconvolution and secondary structure identification: α-helix, 1648―1660 cm−1; βfolding, 1626―1640 cm−1; β-turns, 1662―1684 cm−1; irregular curl, 1640―1650 cm−1. Table 1 shows that as the heating time increases, the β-sheet structure of the protein also increases, reaching the highest at 360 min, and then remaining stable. Gosal, Clark, Pudney & Ross (2002) studied the fibrillation of bovine serum albumin and also found that the number of β-sheets increased after heat-induced protein formation of amyloid fibrosis. The main component of RBP was β-sheets structure, accounting for 26% of the total secondary structure. The main secondary structure of RBP after the heat treatment was still βsheets structure, and the content of β-sheets structure of RBPFs was higher than that of RBP, the RBPFs of 360 min was the highest reaching 30%. However, the content of β-sheets structure of RBPFs 360 min was no longer increasing, which indicates that the further heat treatment of RBP induced the loss of β-sheets structure.
Fig. 1. Characterization 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.
After heating for 480 min, H0 of the RBPFs began to decline. This phenomenon may be due to the fact that proteins are no longer hydrolyzed to form a large number of beta-folded structures, but aggregate to form nuclei, which are linked to the construction units and increase the length of protein fibrils. 3.3. Mean contour length, zeta-potential and flexibility of RBPFs The distribution of RBPFs mean contour length was affected by the heat-induced environment (Feng et al., 2017). Fig. 2A―B show the mean contour length and distribution of fibrils at different heating times. A typical bimodal shape appeared in RBPFs heated for 420 min, 4
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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. 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. Table 1 Analysis of protein secondary structure base on attenuated total Fourier Transform infrared spectroscopy (ATR/FTIR).
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.
3.6. EAI and ES analysis EAI is an indicator of the emulsification efficiency of an emulsifier. The emulsification activity of a protein is measured by the emulsification efficiency of a protein molecule during homogenization. EAI represents the ability of a protein to participate in emulsion formation
Heat time (min)
α- helix (%)
0 60 180 360 420 480 540 600
22 21 21 22 21 22 23 21
± ± ± ± ± ± ± ±
0.38b 0.66a 0.03a 0.16b 0.23a 0.34b 0.24c 0.06a
β-folding (%) 26 26 27 30 26 26 26 26
± ± ± ± ± ± ± ±
0.19a 0.50a 0.36b 0.22c 0.25a 0.31a 0.02a 0.05a
β-turns (%) 16 17 18 16 18 16 16 16
± ± ± ± ± ± ± ±
0.42a 0.54b 1.55b 0.14a 0.18b 0.25a 0.34a 1.75ab
Irregular curl (%) 26 23 25 26 25 26 26 26
± ± ± ± ± ± ± ±
0.19a 1.28b 1.48b 0.16a 0.39b 0.43a 0.19a 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. 5
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emulsification properties of proteins. For the above reason, the relationship between the hydrophobicity and molecular flexibility of RBPFs and the EAI and ES between RBPFs emulsions was investigated. According to Table 3, the correlation coefficient between H0 and flexibility was 0.881 (p < 0.05), the correlation coefficient of ES was 0.899, and the correlation coefficient of EAI was 0.887. The correlation coefficient between flexibility and EAI was 0.962 (p < 0.01), and the correlation coefficient of ES was 0.685. Hydrophobicity H0 is positively correlated with flexibility, EAI and ES. This indicates that the trends in hydrophobicity and molecular flexibility are consistent. Surface hydrophobicity and flexibility are important factors affecting emulsification performance. In the previous studies, researchers focused on the relationship between H0 and protein emulsification properties and found a strong correlation (Li et al., 2019). Prak, Nakatani, Katsube, Adachi, Maruyama & Utsumi (2005) found the emulsifying ability of protein is not exactly the same as the emulsifying ability of surface hydrophobicity. Flexible protein molecules tend to denature at the interface, while rigid protein molecules are less susceptible. Studies by Kato, Komatsu, Fujimoto, and Kobayashi (1985) have shown that surface hydrophobicity is ultimately an important factor in achieving good foaming and emulsifying properties due to the high surface hydrophobicity imparted by the mild effector.
Table 2 Emulsifying activity index (EAI) and emulsifying stability (ES) of emulsions stabilized by different RBPFs. Heat time (min)
EAI (m2/g)
0 60 180 360 420 480
1.13 0.44 0.43 0.29 0.49 1.20
± ± ± ± ± ±
ES × 10−2
0.02e 0.06c 0.01b 0.01a 0.04d 0.03f
1.18 2.71 2.47 2.37 2.87 1.92
± ± ± ± ± ±
0.06a 0.04e 0.01d 0.01c 0.06f 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. Table 3 Correlation analysis between flexibility with surface hydrophobicity (H0), ES and EAI.
Correlation coefficient
H0 and flexibility
Flexibility and ES
Flexibility and EAI
H0 and ES
H0 and EAI
0.881*
0.685
0.962**
0.899
0.887
* 0.01 < p < 0.05, **p < 0.01.
3.7. Emulsion characterization
under external force. ES is the ability of emulsion droplets to maintain stable (Li et al., 2019). The higher the EAI, the higher the emulsification activity. The emulsification activity of a protein is related to the structure of its own molecule. At the same time, according to Table 2, in these six emulsion samples, the RBPFs emulsion heated for 420 min had the best stability, so it was believed that increasing the heating time within a certain range could improve emulsion stability. Tang and Shen (2013) found that after protein adsorption to the oil-water interface, structural rearrangement generally occurs, which affects the
3.7.1. Average droplet size and zeta-potential The droplet size of the emulsion is one of the most important qualitative properties of emulsions (Ge et al., 2017). According to our preliminary experiments (Fig. S1), 0.01–0.05 g/mL of the fish oil concentration was selected, and the results showed that the 0.04 g/mL protein concentration emulsion was the most stable. Liu and Tang (2014) found that increasing the concentration (0.005–0.06 g/mL) of protein particles can reduce the droplet size and increase the stability of
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. 6
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protein fibrils was not enough to cover the oil-water interface. For example, the length of the protein fibril may not be enough to coat the fish oil droplets, the interfacial tension between the O/W interface is great, and the oil droplets combine to make the particle size larger. Zeta-potential is an important indicator for evaluating the stability of the system. Generally, the absolute value of the zeta-potential is high, the protein molecules are repelled, and the solution tends to be stable. Fig. 5B shows the zeta-potential distribution of the RBPFs emulsion droplets, from +31 mV to +35 mV, and the emulsion stabilized by RBPFs was positive. Zeta―potential did not show significant changes indicating that the heating time has little effect on the RBPFs emulsion zeta-potential. It might be due to the length of the heating time has little effect on the zeta-potential of the RBPFs stabilized-emulsion, the overall emulsion potential does not change much. Compared with protein fibrils, the zeta-potential of the emulsion was converted into a positive charge, probably because the fibrils are negatively charged, and the adsorption on the droplets is not enough to change the positive charge of the droplets. 3.8. AP and Γ of RBPFs emulsion between O/W interface Some studies (Kato et al., 1985) have pointed out that proteins with softer structures are more likely to undergo structural transformation and thus adsorb at the interface. According to the interface adsorption principle of polyelectrolytes, the protein structure will be expanded and denatured during the interface adsorption process. It can be seen from Fig. 5C―D that as the heating time increases to 420 min, the AP and Γ of the protein are continuously increasing, and only the emulsion sample heated for 480 min shows a downward trend. This may be due to increased hydrophobicity of the protein, which is consistent with the results of molecular flexibility in Fig. 2. In the protein adsorption process, because the molecular weight of the protein is larger, the adsorption rate at the interface is much lower than that of the small molecule surfactant. After adsorption to the interface, the hydrophobic amino acid of the protein tends to stretch toward the oilcontaining direction and rearrange. Therefore, compared with small molecule surfactants, protein emulsifiers have weaker emulsifying ability, but the prepared emulsions have higher physical and chemical stability. 3.9. Rheology properties of fish oil emulsion stabilized with RBPFs Fig. 6 shows the rheological characteristics of the fish oil emulsion stabilized with RBPFs. Within the angular frequency range (0―100 s−1), the fact that the storage modulus G′ was less than the loss modulus G″ indicates that in this frequency range, the emulsion does not exhibit gel properties, but exists in a liquid state in which fluidity is good. As the heating time increases, the G′ increases gradually, and the G″ decreases. The data in the figure were fitted by the Ostwald de Waale model (the relevant parameters are shown in the insert table), and the K-value is lower, indicating lower viscosity, and the n-value is higher, indicating that the emulsion is closer to a Newtonian fluid (Wang, Li, Wang, & Adhikari, 2011). The table shows that the K-value was the smallest for the emulsion heated for 420 min, indicating that the viscosity was the lowest. The value n > 1 represents that the emulsion exhibits shear thickening, n = 1 means an ideal viscosity flow and n < 1 suggests shear thinning occurs (Wei & Gao, 2015). As seen in Fig. 6 within the set shear rate range (0-100 s−1), the viscosity of all samples gradually decreases, and shear thinning occurs. The reason for this phenomenon is that oil droplets gather together, the weak interaction force that maintains stable flocculation under high-speed shear was destroyed, and shearing occurs thinning phenomenon. In protein emulsions, when the protein concentration reaches a certain value, RBPFs that are not adsorbed to the oil-water interface are present. Due to the presence of unabsorbed and amphiphilic protein fibrils in the continuous phase, the fibrils tend to approach each other due to
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.
the emulsion. As result, the 0.04 g/mL RBPFs solution was chosen to prepared the emulsion. Fig. 5A shows that the average droplet size of the RBPFs emulsion did not change significantly within 0–360 min. While the average droplet size of the emulsion stabilized by 420 min RBPFs rose sharply, which was probably due to the more formation of fibrils, causing the increase of the length of RBPFs those were located at the O/W interface of the emulsion. In contrast, the concentration of 7
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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.
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 I2935/I2880
1.00 ± 0.009b 1.01 ± 0.006d
0.97 ± 0.002b 1.00 ± 0.005d
0.96 ± 0.004b 0.97 ± 0.01b
0.95 ± 0.006a 1.02 ± 0.004e
0.94 ± 0.009a 0.94 ± 0.002a
0.97 ± 0.007b 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.
secondary forces such as hydrophobic interactions, apparently increasing the viscosity of the emulsion. And this thickening enhances the stability of the emulsion (Voutsinas, Cheung, & Nakai, 1983) (see Fig. 7).
3.9.1. Morphology Fig. 8 shows the morphology of six emulsion samples under an optical microscope and a fluorescence microscope. According to the results of the fluorescence microscope, it can be seen that the emulsion 8
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the polymer degradation (Paula, Sombra, Cavalcante, Abreu, & Paula, 2011). This has been confirmed that RBPFs heated for 180 min will cause emulsion instability in section 3.6. 4. Conclusion This study better understands the effect of different heating times on the physicochemical properties of an RBPF-forming emulsion. When RBP were heated for 420 min, the mean contour length and hydrophobicity of the fibrils were highest and the corresponding RBPFs were exhibited favorable emulsifying properties. And the fibril-stabilized O/ W emulsion heated for 420 min had better emulsifying properties, and the good hydrophobicity and flexibility also improved emulsifying activity of the RBPFs emulsion. This study provides a theoretical basis for constructing protein fibrils suitable for stabilizing emulsions and these properties increase the potential of RBP to prepare emulsions as an effective delivery system for nutraceuticals in functional foods.
Fig. 9. DSC profiles of RBPFs emulsions stabilized by unheated RBP, RBPFs heated for 60 min, 180 min, 360 min and 420 min, respectively.
Author statement
droplets gradually increase as the heating time increases. And the unheated protein emulsion is darker, which may be caused by the combination of the Th T dye and the β-sheet in the protein, which is the same as that of the fluorescence spectrophotometer.
Pang shuxian: Conceptualization, Writing - Original Draft, Data Curation. Shaoping: Supervision. Sun qingjie: Project administration. Pu chuanfen: Methodology. Tang wenting: Writing - Review & Editing, Funding acquisition.
3.9.2. Raman spectroscopic analysis In order to examine the interaction between RBPFs and lipids, the structural properties of RBPFs emulsion were determined using Raman spectroscopy. The ratio of relative intensity ratio I2850/I2880 and I2935/ I2880 reflects the interaction between lipid chains and the state of order or disorder of acyl chains (Ruiz-Capillas, Carmona, Jiménez-Colmenero & Herrero, 2013). It can be visualized from Table 4 that the emulsion of 420 min RBPFs shows the lowest intensity ratios. The disorder of the acyl chain indicates that more proteins were inserted between the acyl chains of the oil. (Herrero, Ruiz, Pintado, Carmona, & Jiménez, 2018). Lower I2850/I2880 and I2930/I2880 intensity ratios were also related to hydrophobic interactions including the CH groups of the lipid with protein (Herrero, Ruiz-Capillas, Pintado, Carmona, & JiménezColmenero, 2018). In addition, combined with the results of infrared spectroscopy analysis, I2850/I2880 was negatively correlated with the number of β-sheets. Ruiz-Capillas et al. (2013) also reported that the intensity ratio of the emulsion (I2850/I2900) was lower than that of the separated fatty phase and pointed out that most of the hydrocarbon chains of milk fat globules and/or triglycerides are closely packed. This hydrocarbon chain accumulation was very important for the stability of the emulsion.
Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The authors thank Key Research and Development Program of Shandong Province of China (No. 2019GNC106051), China Scholarship Council (No. 201908370054), National Natural Science Foundation of China (NO. 31501578), University Science and Technology Project of Shandong Province of China (No. J16LE22), Doctoral Science Foundation of Shandong Province of China (No. BS2015SW019), Advanced Talents Foundation of Qingdao Agricultural University, China (No. 6631115030), and Special Funds for Taishan Scholars Project of Shandong Province of China (ts201712058) for financial support. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.lwt.2019.108985.
3.9.3. DSC analysis DSC were determined the thermal behavior of RBPFs stabilized emulsions with different heating times. The emulsion stabilized by unheated RBP showed an initial endothermic peak at 185 °C and a maximum exothermic peak at 146 °C. The endothermic peak was caused by dehydration associated with the hydrophilic group of the polymer, while the exothermic peak was associated with degradation of the polyelectrolyte due to dehydration and depolymerization reactions (Li et al., 2018). The exothermic peak of the RBPFs stabilized emulsion after heating was smaller and broader compared with those of RBP emulsion (Fig. 9). This can be explained as the hydrophobic interaction between RBPFs and fish oil (Sarmento, Ferreira, Veiga, & Ribeiro, 2006). The unheated RBP emulsion began to denature at about 85 °C, the RBPFs emulsion denature temperature began to gradually increase, and the 420 min RBPFs emulsion denature temperature reached about 140 °C, indicating that the corresponding emulsion was the most stable among all the samples. In addition, the emulsion stabilized by RBPFs heated for 180 min has a sharp exothermic peak near 176 °C, suggesting
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