Aggregation and emulsifying properties of soybean protein isolate pretreated by combination of dual-frequency ultrasound and ionic liquids

Journal Pre-proof Aggregation and emulsifying properties of soybean protein isolate pretreated by combination of dual-frequency ultrasound and ionic liquids

Liurong Huang, Shifang Jia, Wenxue Zhang, Lixin Ma, Xiaona Ding PII:

S0167-7322(19)35330-9

DOI:

https://doi.org/10.1016/j.molliq.2019.112394

Reference:

MOLLIQ 112394

To appear in:

Journal of Molecular Liquids

Received date:

24 September 2019

Revised date:

11 December 2019

Accepted date:

24 December 2019

Please cite this article as: L. Huang, S. Jia, W. Zhang, et al., Aggregation and emulsifying properties of soybean protein isolate pretreated by combination of dual-frequency ultrasound and ionic liquids, Journal of Molecular Liquids(2019), https://doi.org/10.1016/ j.molliq.2019.112394

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© 2019 Published by Elsevier.

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Aggregation and emulsifying properties of soybean protein isolate pretreated by combination of dual-frequency ultrasound and ionic liquids

Liurong Huang1, 2*, Shifang Jia1, Wenxue Zhang1, Lixin Ma1, Xiaona Ding1 School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road,

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1

Zhenjiang 212013, Jiangsu, China

Institute of Food Physical Processing, Jiangsu University, 301 Xuefu Road,

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2

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Zhenjiang 212013, Jiangsu, China

*Corresponding author: Liurong Huang E-mail address: [email protected]

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Abstract The objectives of this study were to investigate the changes in the aggregation and emulsifying properties of soy protein isolate (SPI) pretreated with dual-frequency ultrasound and three different ionic liquids (ILs), tributyl methyl ammonium chloride ([TBMA]Cl),

1-butyl-3-methyl

imidazolium

chloride

([BMIM]Cl)

and

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1-butyl-3-methyl imidazolium tetrafluoroborate ([BMIM]BF4). Results showed that dual-frequency ultrasound and ILs could not change the primary structure of SPI. The

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changes in UV–vis spectra indicated the unfolding of SPI as pretreated by [TBMA]Cl

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and [BMIM]Cl combined with dual-frequency ultrasound, followed by formation of

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soluble aggregates. The thermal stability analysis of the regenerated SPI suggested

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that [BMIM]BF4 presumably broke most of the hydrogen bonds within adjacent SPI

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molecules, leading to the decrease of thermal stability. Transmission electron microscopy showed major changes in protein morphology upon protein–protein

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interactions. SPI emulsion pretreated with dual-frequency ultrasound and [BMIM]BF4 had a higher emulsion stability index than the other samples (P < 0.05). Furthermore, as compared to untreated protein, all ultrasound and ILs pretreatments decreased the creaming index of SPI emulsion. These results indicated that combination of dual-frequency ultrasound and ionic liquids could be a potential method to change the aggregation and emulsifying properties of SPI. Keywords: aggregation; emulsifying property; dual-frequency ultrasound; ionic liquid;

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1. Introduction Soy protein isolate is a commercial soy protein product which has been widely applied in many protein-based food formulations due to its high nutritional value and good functional properties. However, certain characteristics of native SPI, such as

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compact globular structure, low molecular flexibility and lack of solubility, make its commercial applications limited. Thus, many various methods have been used to

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modify SPI and improve its functional properties [1, 2]. It was reported that the

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functional and physicochemical properties of protein solutions changed with the

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aggregation state of subunits and molecules [3]. Therefore, knowledge of aggregation

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behavior of protein molecules is of vital importance and provides useful guidance for

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the utilization of SPI in food industry.

The effect of ultrasound on protein modification has been the subject of many

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researches in recent years. They showed that ultrasound destroyed the noncovalent interactions such as hydrophobic interactions and hydrogen bonding, which induced the dissociation and aggregation of protein subunits [4, 5]. The aggregation state is believed to endow protein with special functional and physical properties, such as solubility, gelation, emulsification and foam ability [6, 7]. Compared to mono-frequency ultrasound, dual-frequency ultrasound could lead to more structural changes due to higher shear force and bubble temperature [8]. Dual-frequency ultrasound also has the ability to save pretreatment time. Generally, single modification treatment has relative low efficiency. Recently, most researches focus 3

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on the combination of different modification methods to improve the functional properties of protein [9, 10]. Ionic liquids (ILs) have received considerable attention over the past decade as desirable green solvents for many natural polymers. This is mainly because of their negligible vapor pressures, good thermal stabilities, enhanced reaction rates, and can recovered

after

the

process.

It

was

reported

that

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1-ethyl-3-methylimidazoliumacetate ([EMIM][Ac]) could alter the aggregation

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behavior of collagen molecules by disrupting the hydrogen bonds or ionic interactions

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between inter- and intra-molecules [3, 11]. As a result, changes in the interaction of

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protein molecules can lead to the variations in aggregation behavior. Xie et al. [12]

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used 1-butyl-3-methylimidazolium chloride ([BMIM]Cl) to dissolve and regenerate

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wool keratin, and reported that the thermal stability of the regenerated wool keratin was improved compared to the native wool keratin fibers. In the view of these results,

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it can be inferred that the combination of dual-frequency and ionic liquid solvent pretreatment is a good choice for modifying the aggregation state of protein subunits and molecules.

ILs used in this study are tributyl methyl ammonium chloride ([TBMA]Cl), 1-butyl-3-methyl

imidazolium

chloride

([BMIM]Cl)

and

1-butyl-3-methyl

imidazolium tetrafluoroborate ([BMIM]BF4) . Components of SPI were firstly verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) experiment. Subsequently, changes in the aggregation structure of SPI were further investigated by the means of ultraviolet-visible technique, fourier transform infrared 4

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spectroscopy, differential scanning calorimetry and transmission electron microscopy. The emulsifying properties were investigated in terms of emulsifying activity, emulsion stability index and creaming stability. To the best of our knowledge, this is the first study to use the combination of dual-frequency ultrasound and ILs to alter the aggregation state and emulsifying properties of SPI. These data will be helpful for

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producing SPI with excellent emulsifying properties that will broaden its applications. 2. Materials and methods

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2.1. Materials

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Soybean protein isolate was bought from Shansong Biological Products Co., Ltd.

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(Shandong, China). The protein content is 92% as determined by the Kjeldahl method.

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Tributyl methyl ammonium chloride ([TBMA]Cl) and 1-butyl-3-methyl imidazolium

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chloride ([BMIM]Cl) with high purity (≥98.0%) were obtained from Tokyo Chemical Industry Co. Ltd (Tokyo, Japan). 1-butyl-3-methyl imidazolium tetrafluoroborate

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([BMIM]BF4) was purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China) and were all of analytical grade. 2.2. Pretreatment of SPI by dual-frequency ultrasound and ionic liquids SPI was firstly stirred with deionized water (1:90, w/v) for 5 min at a temperature of 25°C. Next, ionic liquid was added into SPI suspension and mixed by magnetic stirring for 1 h, using ratio SPI/ ionic liquid of 1:1 (by weight). The pH of mixture was maintained at 7.0 with addition of 0.1 M NaOH or HCl during stirring process. And then final volume of the mixture was set to be 100 times of SPI weight 5

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(v/w). The mixture was sonicated in an ultrasound generator, which was constructed at Jiangsu University. The dual-frequency sonoreactor was equipped with two probes operating at two frequencies (20 and 28 kHz), which can work simultaneously. The frequencies were in a pulse mode, in which the on-time of pulsed ultrasound was 2 s

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and off-time was 2 s. Additionally, the temperature and circulation of the sample solution can be programmed by a temperature control system and liquid circulating

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pump, respectively. An aliquot of 1400 mL mixture was poured into the ultrasonic

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reaction vessel, and the ultrasonic probes were dipped to 2.0 cm. The following

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parameters were used: power density of 100 W/L, pretreatment time of 30 min,

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temperature of 25°C and pump circulation speed of 180 rpm. For control, the SPI

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suspension without ionic liquid and ultrasonic treatment was kept at 25°C while stirring for 90 min using magnetic string apparatus. The effect of dual-frequency

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ultrasound pretreatment (U0) was compared with the combination effect of dual-frequency ultrasound and ILs of [TBMA]Cl, [BMIM]BF4, [BMIM]Cl, repectively (hereinafter called UA, UB and UC treatment). 2.3. Regeneration of SPI After ultrasonic treatment, the mixture was condensed to 30-40 mL in vacuum rotary evaporation system. The concentrated sample was mixed with 95% (1:4, v/v) ethanol at 4°C for 1 h and subjected to centrifugation at 3500 g for 10 min. The resulting precipitation was washed with 95% ethanol until ionic liquid was completely washed away. The residue was collected and freeze-dried to obtain regenerated SPI. 6

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2.4. Analysis of protein components by SDS-PAGE Reducing and non-reducing conditions were carried out to examine the status of protein. 12% separating gel and 5% stacking gel were used according to the method of Laemmli [13] with some modifications. Regenerated SPI samples were mixed with deionized water (0.5%, w/v) and then stirred at 25°C for 1 h, followed by

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centrifugation at 4500 g for 10 min to remove undissolved debris. The supernatant was mixed with SDS-PAGE sample buffer (1:1, v/v) in the presence (reducing

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conditions) or absence (non-reducing conditions) of 2% β-mercaptoethanol. After

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heating for 5 min in a boiling water bath, each sample (10 μL) was loaded onto a

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polyacrylamide gel and subjected to electrophoresis at a constant current of 20 mA.

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After electrophoresis, the gel was stained with 0.25% Coomassie blue R-250 solution,

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and destained in a solution containing 45% methanol and 10% acetic acid. Protein markers were used to estimate the molecular weight of the protein bands.

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2.5. Ultraviolet-visible (UV-vis) spectroscopy The ultraviolet-visible spectra of the samples were recorded in the wavelength range of 240-500 nm using a Varian Cary 100 UV-vis spectrophotometer (Varian Inc., USA) at 25 °C with 100 nm/min scan rate in a 1.0 cm path length quartz cuvette. 2.6. Fourier transform infrared (FTIR) spectroscopy FTIR spectrum analyses were carried out using a FTIR spectrometer (Nicolet iS50, Nicolet instrument Co., USA) according to the method of Li et al. [14]. The SPI sample was mixed with KBr by the ratio 1:100 and pressed into a tablet. FTIR spectra were obtained in the wave number region ranging from 4000 to 400 cm-1 at 0.5 cm-1 7

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interval. A total of 32 scans was measured and averaged. The KBr spectrum was taken as background, and then spectra were obtained. 2.7. Calorimetric measurements The thermal stability was determined using a differential scanning calorimeter (TA instruments, USA) over a temperature range from 30 to 300 °C at a constant heating rate of 10 °C/min. Measurements were performed while samples were

used as the reference.

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2.8. Transmission electron microscopy (TEM)

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constantly purged with nitrogen at 30 cm3/min. An empty sealed aluminum pan was

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The microstructures of samples were examined with transmission electron

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microscope (JEM-1200 EX) at 80 kV acceleration voltage. One drop (5–10 μL) of the freshly-prepared suspension was deposited onto a TEM copper grid. The excess liquid

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was absorbed by a piece of filter paper, and a drop of 2% uranyl acetate negative stain

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was applied for 2 min before drying at room temperature. Then the grid was examined under the microscope.

2.9. Emulsifying activity and emulsion stability index Emulsifying activity (EA) and emulsion stability index (ESI) were determined by the method of Hou et al. [15] with some modifications. At a speed of 10,000 rpm for 1 min, soybean oil (40 g) and 0.5% (w/v) of proteins suspension (160 mL) were homogenized in a mechanical homogenizer (HG-15A, Daihan Scientific Co., Ltd, Korea) to produce the emulsion. 50 μL of emulsion was pipetted from the bottom of the container at 0 min and 60 min and mixed with 5 mL of 0.1% SDS, respectively. Absorbance of emulsion was measured at 500 nm with the spectrophotometer (Unic 8

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7200, Unocal Corporation, China) to obtain EA and ESI.

EA(m2 / g) 

ESI 

2* 2.303* A0 * D 2 C 2 * I *(1  φ)*10000

A0 * 60 A0  A60

where D2 is dilution factor, C2 is protein concentration (g/mL), I is optical path (I = 1

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cm), φ is the fraction of the oil phase (φ= 0.2). A0 and A60 are the absorbance of the sample at 0 min and 60 min, respectively.

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2.10. Creaming index (CI) of emulsion

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10 mL of fresh emulsion was added into a sample bottle (1.4 cm internal

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diameter ×15 cm height) and then stored at 4 °C. The heights of emulsion (Ht) and

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serum phase (Hs) were recorded at different times for 7 days. The creaming index

2.11. Data analysis

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(CI, %) was calculated as (Hs/Ht)×100.

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Data are presented as mean ± SD (three replicates). Difference between means was compared by analysis of variance (ANOVA) at a significance level of P <0.05. All graphs were plotted using OriginPro 8.0 (OriginLab Corporation, MA, USA). 3. Results and discussion 3.1. Electrophoresis analysis of protein components SDS-PAGE analysis was performed to estimate the components of untreated and treated SPI under reducing and non-reducing conditions. As shown in Fig. 1, control SPI shows a typical SDS-PAGE pattern under reducing condition with α’, α and β subunits as well as AS and BS polypeptides [1]. The bands became stronger 9

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after the treatment of UC and U0, indicating that more soluble aggregates might be formed. When SPI was treated by UB, the intensity of the bands was clearly lower than that of control. The result of solubility showed that UB treatment decreased the SPI solubility significantly (data not shown). Therefore, it can be inferred that more large and insoluble aggregates were formed with the treatment of UB. A possible

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reason might be due to that [BMIM]BF4 could strongly affect the electrostatic interactions, resulting in the enhancement of intermolecular interactions. However,

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the relative molecular mass of protein subunit did not change, suggesting that

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dual-frequency ultrasound and ILs could not change the primary structure of SPI. The

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result was in agreement with the report of Hu et al. [4] and Chen et al. [16], who also

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isolate electrophoretic patterns.

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reported that ultrasonic treatment did not induce major changes in the soy protein

Under non-reducing condition, the SPI pretreated by single ultrasound (U0)

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contains the highest content of high molecular weight subunits (more than 80 kDa). However, the content of subunits with low molecular weight (less than 40 kDa) increased when SPI was pretreated by UA. Furthermore, compared to control and U0, a band at about 30 kDa was disappeared when SPI was pretreated by UA. This result indicates that the aggregation state was changed by the induction of [TBMA]Cl. However, it is difficult to conclude whether the disappeared molecule was aggregated or dissociated, which needs to be further explored. 3.2. Effects of different pretreatments on the UV–visible spectroscopy of SPI Fig. 2 shows the effect of dual-frequency ultrasound and ionic liquids 10

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pretreatments on UV–visible spectroscopy of SPI. The characteristic absorption at about 265 nm was mainly due to the presence of tyrosine, tryptophan and phenylalanine [17]. Results in Fig. 2 illustrated that the absorbance intensity of SPI increased after pretreated by U0, UA and UC. The increase of intensity indicates that more chromophores of protein were exposed to solvent due to molecular unfolding [9].

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Furthermore, as far as UA and UC pretreatments were concerned, the increase of intensity was accompanied by red shift of maximum absorbance wavelength. It was

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reported that ILs could affect the electrostatic interactions and exhibit aggregate

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dissociation behavior of native proteins, which is similar to molecule heparin [18].

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Sonication could disrupt hydrogen bonds and lead to the exposure of more charged

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groups (NH4+, COO−), resulting in the enhancement of protein-water interactions [19,

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20]. The changes in absorbance intensity and maximum absorbance wavelength might be attributed to the synergistic effect of ultrasound and ILs. However, when SPI was

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pretreated by UB, the absorbance intensity decreased significantly, indicating the embedding of some hydrophobic groups and forming of large aggregates. This result was in agreement with the result of SDS-PAGE. It can be concluded that different ionic liquids have different influences on the aggregation state of SPI. Some can cause aggregation and others can hinder aggregation of SPI molecules. 3.3. FTIR spectral analysis FTIR spectroscopy is based on molecular vibrations, which can detect changes in the molecular groups and their surroundings [21, 22]. The infrared radiation spectra of SPI pretreated by dual-frequency ultrasound and different ILs are shown in Fig. 3. 11

Journal Pre-proof It is seen that a strong absorption in the wave numbers between 3300 and 3100 cm-1, representing the stretching vibration of the hydrogen-bonded NH groups, do not shift with kinds of ILs. Compared to control and U0, the intensity of SPI at about 3300 and 3100 cm-1 was decreased by the pretreatment of UA and UC, indicating that hydrogen-bonded

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NH groups were reduced. This kind of hydrogen bonds is presented between amide groups of the residue at X position and the ester oxygen of the Y residue [3]. The

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addition of ILs could influence the inter- and intra-molecular hydrogen bonds. Just

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like the dissolution mechanism of collagen proposed by Meng et al. [23], the Cl− ions

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associated with the amino hydroxyl of SPI and [BMIM]+ complexes with the ester

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oxygen. This interaction disrupted hydrogen bonding in SPI. The ionic bonds,

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between the two oppositely charged groups close to each other, could also be impaired by ILs [23]. As the [BMIM]Cl was washed away by ethanol, new hydrogen bonds and

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ionic bonds were rebuilt in the regenerated SPI. However, the quantity and position of the new bonds were no longer the same as the original ones. It can therefore be concluded that ILs induced the changes in the structure and properties of regenerated SPI. 3.4. Thermal analysis of SPI The changes of aggregation structure are always accompanied by the changes in thermal properties. The DSC thermograms of SPI pretreated by dual-frequency ultrasound and different ILs are presented in Fig. 4. As can be seen from the DSC curves, all samples displayed two typical thermal denaturation peaks. The first peak 12

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(Tm1) at the lower temperature was related to hydrogen bonds and electrostatic interactions among SPI aggregates, and the second main peak (Tm2) at the higher temperature is connected with the continued conformational changes and destruction of materials [24, 25]. Tm1 values of UA (122°C), UB (86°C), UC (105°C), U0 (101°C) and control

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(96°C), were obtained. The lowest Tm1 of UB suggests that [BMIM]BF4 solvent presumably broke most of the hydrogen bonds within adjacent SPI molecules, leading

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to the formation of large aggregates. The Tm1 values of UA and UC are both higher

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than those of control and U0, which means that more hydrogen bonds or increased

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electrostatic interactions were formed in SPI molecules. Result of FTIR indicates that

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hydrogen-bonded NH groups of SPI were reduced by UA and UC treatments.

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Therefore, although some hydrogen bonds in the native SPI were disrupted by combination of dual-frequency ultrasound and ionic liquids, strong electrostatic

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interactions would be formed during regeneration process, which is highly dependent on the kinds of ionic liquids.

The value of Tm2 is an indicator of the thermal stability of protein [26, 27], which is related to the structural characteristic. Higher Tm2 (Tm2=253°C) were observed in UC than in other samples, indicating that SPI treated by UC possessed higher thermal stability and greater structural stability. The increase of structural stability was due to the form of new inter- and intra-molecular hydrogen bonds and ionic bonds. Therefore, the thermal stability of the SPI tended to become stronger as pretreated by combination of dual-frequency ultrasound and [BMIM] Cl. These 13

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results are consistent with the study by Yang et al. [3], who observed that [EMIM][Ac] changed the structural and thermal properties of collagen. 3.5. Transmission electron microscopic analysis The morphological changes of SPI after the pretreatments of ultrasound and ILs can be observed visually by using transmission electron microscopy (TEM). The

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TEM images of SPI in deionized water are shown in Fig. 5. TEM photograph of control SPI shows major aggregates (Fig. 5A). The particle size was decreased after

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the pretreatment of ultrasound (Fig. 5B). When SPI was pretreated by UA and UC,

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the micrographs show that the particles were having homogenous and more ordered

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shape (Fig. 5C and E) as compared to control, revealing that ILs caused decrease in

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the protein aggregation. The TEM images also show that the UB induced the

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formation of a greater number of larger protein aggregates (Fig. 5D). 3.6. Emulsifying activity of SPI

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Protein can lower the tension at the oil-water interfaces and control the aggregation of oil droplets by forming adsorption layer [14]. This ability endows SPI with emulsifying effect. The emulsifying effect included emulsifying activity (EA) and emulsion stability index (ESI). As shown in Fig. 6, control, U0, UA and UC pretreated SPI have similar EA while the emulsifying activity of SPI pretreated with UB were the lowest. This difference might be due to their different solubility. Protein with high solubility can rapidly diffuse and adsorb at the interface [28]. However, SPI pretreated with UB has a significant superior ESI to the other samples. The regenerated SPI induced by UB can enhance the interaction between 14

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protein and lipid owing to the new aggregation structure [29]. 3.7. Creaming stability The creaming stability is reflected by the creaming index. Fig. 7 presents changes in creaming index for all emulsions upon storage for up to 1 week. The emulsions formed by control and pretreated SPI rapidly developed into two layers in

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one day. And then the creaming rate slowed down in the following days. The creaming index of protein emulsions pretreated by dual-frequency ultrasound and ILs

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was lower than that of control emulsions. Furthermore, the emulsion with SPI

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pretreated by UB had the lowest creaming index. Analysis of structure indicates that

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UB induced the aggregation and denaturation of SPI more significantly than UA and

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UC. It was also reported that the creaming index of emulsions prepared with

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denatured protein was smaller than that of control emulsions [30, 31]. This observation also indicates that re-aggregated SPI induced by UB improved the ESI

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and creaming stability of emulsions. 4. Conclusion

Combination of dual-frequency ultrasound and ILs affects non-covalent interactions such as hydrophobic interactions, electrostatic interactions and hydrogen bonds. Different ILs have different influences on the structure of SPI. Combination of ultrasound and [BMIM]BF4 induced the embedding of some hydrophobic groups, while [TBMA]Cl and [BMIM]Cl caused the exposure of more chromophores and unfolding of SPI, which further influenced the protein-protein and protein-water interactions. These changes are believed to be associated with aggregation and 15

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emulsifying properties of SPI. The results of electrophoretic patterns and solubility showed that insoluble and large aggregates were formed under the pretreatment of dual-frequency ultrasound and [BMIM]BF4. On the reverse side, [TBMA]Cl and [BMIM]Cl combined with ultrasound induced the formation of soluble aggregates. The thermal stability of the regenerated SPI was highly dependent on the kinds of

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ionic liquids. Dual-frequency ultrasound and [BMIM]Cl pretreatment caused the increase of thermal stability of the SPI. The SPI regenerated from [BMIM]BF4 had the

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lowest emulsifying activity due to the solubility, but it possessed the highest

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emulsifying stability and creaming stability. Therefore, compared to protein

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concentration, the protein structure significantly affected the flocculation stability of

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protein stabilized emulsions. Overall, application of dual-frequency ultrasound

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combined with appropriate ionic liquid can alter the aggregation state of SPI, leading to the improvement of emulsifying properties for industrial purposes.

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Acknowledgements

The authors wish to express their appreciation for the support by National Natural Science Foundation of China (31701540), Natural Science Foundation of Jiangsu Province (BK20170539) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References [1] Liu, B., Wang, H., Hu, T., Zhang, P., Zhang, Z., Pan, S., & Hu, H. (2017). Ball-milling changed the physicochemical properties of SPI and its cold-set gels. Journal of Food Engineering, 195, 158–165. 16

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[2] Yang, F., Liu, X., Ren, X., Huang, Y., Huang, C., & Zhang, K. (2018). Swirling cavitation improves the emulsifying properties of commercial soy protein isolate. Ultrasonics Sonochemistry, 42, 471–481. [3] Yang, H., Li, Q., Liu, S., & Li, G. (2018). Acetic acid/1-ethyl-3-methyl imidazolium acetate as a biphasic solvent system for altering the aggregation behavior

of

of collagen molecules. Journal of Molecular Liquids, 262, 78–85. [4] Hu, H., Wu, J., Li-Chan, E. C., Zhu, L., Zhang, F., Xu, X., Fan, G., Wang, L.,

ro

Huang, X., & Pan, S. (2013). Effects of ultrasound on structural and physical

-p

properties of soy protein isolate (SPI) dispersions. Food Hydrocolloids, 30(2), 647–

re

655.

lP

[5] Jin, J., Ma, H., Wang, W., Luo, M., Wang, B., Qu, W, He, R., John, Q., & Li, Y.

na

(2016). Effects and mechanism of ultrasound pretreatment on rapeseed protein enzymolysis. Journal of the Science of Food & Agriculture, 96(4), 1159–1166.

Jo ur

[6] Huang, L., Ding, X., Li, Y., & Ma, H. (2019). The aggregation, structures and emulsifying properties of soybean protein isolate induced by ultrasound and acid. Food Chemistry, 279, 114–119. [7] Xu, H. N., Liu, Y., & Zhang, L. (2015). Salting-out and salting-in: competitive effects of salt on the aggregation behavior of soy protein particles and their emulsifying properties. Soft Matter, 11(29), 5926–5932. [8] Cheng, Y., Donkor, P. O., Ren, X., Wu, J., Agyemang, K., Ayim, I., & Ma, H. (2019). Effect of ultrasound pretreatment with mono-frequency and simultaneous dual frequency on the mechanical properties and microstructure of whey protein emulsion 17

Journal Pre-proof

gels. Food Hydrocolloids, 89, 434–442. [9] Li, S., Yang, X., Zhang, Y., Ma, H., Liang, Q., Qu, W., He, R., Zhou, C., & Mahunu, G. K. (2016). Effects of ultrasound and ultrasound assisted alkaline pretreatments

on

the

enzymolysis

and

structural

characteristics

of

rice

protein. Ultrasonics Sonochemistry, 31, 20–28.

of

[10] Ahmadi, Z., Razavi, S. M. A., & Varidi, M. (2017). Sequential ultrasound and transglutaminase treatments improve functional, rheological, and textural properties

ro

of whey protein concentrate. Innovative Food Science & Emerging Technologies, 43,

-p

207–215.

re

[11] Hu, Y., Liu, L., Dan, W., Dan, N., & Gu, Z. (2013). Evaluation of

lP

1-ethyl-3-methylimidazolium acetate based ionic liquid systems as a suitable solvent

na

for collagen, Journal of Applied Polymer Science, 130 (4), 2245–2256. [12] Xie, H., Li, S., & Zhang, S. (2005). Ionic liquids as novel solvents for the

Jo ur

dissolution and blending of wool keratin Fibers. Green Chemistry, 7 (8), 606–608. [13] Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259), 680–685. [14] Li, K., Ma, H., Li, S., Zhang, C., & Dai, C. (2017). Effect of ultrasound on alkali extraction protein from rice dreg flour. Journal of Food Process Engineering, 40(2), 1–9. [15] Hou, F., Ding, W., Qu, W., Oladejo, A. O., Xiong, F., Zhang, W., He, R., & Ma, H. (2017). Alkali solution extraction of rice residue protein isolates: Influence of alkali concentration on protein functional, structural properties and lysinoalanine formation. 18

Journal Pre-proof

Food Chemistry, 218, 207–215. [16] Chen, L., Chen, J., Ren, J., & Zhao, M. (2011). Effects of ultrasound pretreatment on the enzymatic hydrolysis of soy protein isolates and on the emulsifying properties of hydrolysates. Journal of Agricultural and Food Chemistry, 59(6), 2600–2609.

of

[17] Ren, X., Zhang, X., Liang, Q., Hou, T., & Zhou, H. Effects of different working modes of ultrasound on structural characteristics of zein and ACE inhibitory activity

ro

of hydrolysates. Journal of Food Quality, 2017, 11, 1–8.

-p

[18] Rawat, K., & Bohidar, H. B. (2015). Heparin-like native protein aggregate

re

dissociation by 1-alkyl-3-methyl imidazolium chloride ionic liquids. International

lP

Journal of Biological Macromolecules, 2015, 73, 23–30.

na

[19] Liu, R., Liu, Q., Xiong, S., Fu, Y., & Chen, L. (2017). Effects of high intensity ultrasound on structural and physicochemical properties of myosin from silver carp.

Jo ur

Ultrasonics Sonochemistry, 37, 150–157. [20] Mirmoghtadaie, L., Aliabadi, S. S., & Hosseini, S. M. (2016). Recent approaches in physical modification of protein functionality. Food Chemistry, 199, 619–627. [21] Ellepola, S. W., Choi, S. M., & Ma, C. Y. (2005). Conformational study of globulin from rice (Oryza sativa) seeds by Fourier-transform infrared spectroscopy. International Journal of Biological Macromolecules, 37(1-2), 12-20. [22] Zhang, Y. , Ma, H. , Wang, B. , Qu, W. , Li, Y. , & He, R. , et al. (2015). Effects of ultrasound pretreatment on the enzymolysis and structural characterization of wheat gluten. Food Biophysics, 10(4), 385-395. 19

Journal Pre-proof

[23] Meng, Z., Zheng, X., Tang, K., Liu, J., Ma, Z., & Zhao, Q. (2012). Dissolution and regeneration of collagen fibers using ionic liquid. International Journal of Biological Macromolecules, 51(4), 440–448. [24] Zhang, M., Ding, C. C., Yang, J. H., Lin, S., Chen, L. H., & Huang, L. L. (2016). Study of interaction between water-soluble collagen and carboxymethyl cellulose in

of

neutral aqueous solution. Carbohydrate Polymers, 137, 410–417. [25] Safandowska, M., & Pietrucha, K. (2013). Effect of fish collagen modification on

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its thermal and rheological properties. International Journal of Biological

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Macromolecules, 53, 32–37.

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[26] Huang, C.Y., Kuo, J. M., Wu, S. J., & Tsai, H. T. (2016). Isolation and

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characterization of fish scale collagen from tilapia (Oreochromis sp.) by a novel

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extrusion–hydro-extraction process. Food Chemistry, 190, 997–1006. [27] Ren, X., Wei, X., Ma, H., Zhou, H., Guo, J., Mao, S., & Hu, A. (2015). Effects of

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a dual-frequency frequency-dweeping ultrasound treatment on the properties and structure of the zein protein. Cereal Chemistry Journal, 92(2), 193–197. [28] Foh, M. B. K., Wenshui , X., Amadou, I., & Jiang, Q. (2012). Influence of pH shift on functional properties of protein isolated of Tilapia (Oreochromis niloticus) muscles and of soy protein isolate. Food and Bioprocess Technology, 5(6), 2192– 2200. [29] Bandyopadhyay, K., Misra, G., & Ghosh, S. (2008). Preparation and characterisation of protein hydrolysates from Indian defatted rice bran meal. Journal of Oleo Science, 57(1), 47–52. 20

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[30] Peng, W., Kong, X., Chen, Y., Zhang, C., Yang, Y., & Hua, Y. (2016) . Effects of heat treatment on the emulsifying properties of pea proteins. Food Hydrocolloids, 2016, 52, 301–310. [31] Shao, Y., & Tang, C. H. (2014). Characteristics and oxidative stability of soy protein stabilized oil-in-water emulsions: influence of ionic strength and heat

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pretreatment. Food Hydrocolloids, 37, 149–158.

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Figures

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Fig. 1 SDS-PAGE of SPI under reducing and non-reducing conditions. Lane 1: U0

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(ultrasound); Lane 2: marker; Lane 3: UA (ultrasound-[TBMA]Cl); Lane 4: UB

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(ultrasound-[BMIM]BF4); Lane 5: UC (ultrasound-[BMIM] Cl); Lane 6: Control.

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Fig. 2 Effects of dual-frequency ultrasound and ionic liquids pretreatments on the UV

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-[BMIM]BF4; UC, ultrasound-[BMIM] Cl.

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spectra of SPI. U0, ultrasound; UA, ultrasound-[TBMA]Cl; UB, ultrasound

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Fig. 3 FTIR spectra of SPI pretreated by combination of dual-frequency ultrasound

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-[BMIM]BF4; UC, ultrasound-[BMIM] Cl.

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and ionic liquids. U0, ultrasound; UA, ultrasound-[TBMA]Cl; UB, ultrasound

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Fig. 4 DSC thermograms of SPI pretreated by combination of dual-frequency

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ultrasound and ionic liquids. U0, ultrasound; UA, ultrasound-[TBMA]Cl; UB,

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ultrasound -[BMIM]BF4; UC, ultrasound-[BMIM] Cl.

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Fig.5 TEM photographs of SPI with different pretreatments. A, Control; B, ultrasound

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(U0); C, ultrasound-[TBMA]Cl (UA); D, ultrasound -[BMIM]BF4 (UB); E,

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ultrasound-[BMIM] Cl (UC).

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EA (m2/g)

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EA ESI

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140

4

120

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100

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ESI

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80 Control U0

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Method Fig. 6 Emulsifying activity of SPI pretreated by combination of dual-frequency

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ultrasound and ionic liquids. U0, ultrasound; UA, ultrasound-[TBMA]Cl; UB,

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ultrasound -[BMIM]BF4; UC, ultrasound-[BMIM] Cl.

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Fig. 7. Creaming index of the SPI emulsion with ultrasound and ionic liquids

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-[BMIM]BF4; UC, ultrasound-[BMIM] Cl.

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pretreatments. U0, ultrasound; UA, ultrasound-[TBMA]Cl; UB, ultrasound

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Author statement

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The material presented in this manuscript has not been previously published, except in abstract form, nor is it simultaneously under consideration by any other journal.

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Journal Pre-proof Highlights

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►Dual-frequency ultrasound and ILs changed the aggregation of SPI. ►Large aggregates were formed by dual-frequency ultrasound and [BMIM]BF4 treatment. ►Stability of emulsions were improved by appropriate ILs and ultrasound.

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