Conformational Changes of Soy Proteins under High-intensity

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Conformational Changes of Soy Proteins under Highintensity Ultrasound and High-speed Shearing Treatments Jiahan Zou, Ngoc Thi-Hong Nguyen, Mae Devorah Biers, and Gang Sun ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b05713 • Publication Date (Web): 29 Mar 2019 Downloaded from http://pubs.acs.org on March 29, 2019

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ACS Sustainable Chemistry & Engineering

Conformational Changes of Soy Proteins under High-intensity Ultrasound and High-speed Shearing Treatments

Jiahan Zou†, Ngoc Nguyen‡, Mae Biers‡, Gang Sun*, †

† Division of Textiles and Clothing, ‡Department of Chemistry, University of California, One Shields Avenue, Davis, California, 95616, United States

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: (530) 752-0840

1 ACS Paragon Plus Environment

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ABSTRACT: Inspired by denatured globular protein conformational structures, soy protein isolate (SPI) aqueous solutions under neutral pH without the addition of any chemicals were studied under different mechanical treatments. Compared with the traditional magnetic stirring treatment (MS), high-intensity ultrasonication (US), highspeed mechanical shearing (SH) and their combined treatments (USSH, SHUS) were able to better dissolve SPI and further swell and deform the globular conformation. Higher solubility, lower turbidity, enlarged particle size, much uniform particle size distribution, higher free sulfhydryl groups content, increased fluorescence intensity, higher surface hydrophobicity and higher viscosity SPI solution properties were detected from all these US and SH treated protein samples. Among them, the USSH treated SPI solution was characterized to have the proteins in the best modified conformational structure. The polymer chain relaxation and degradation were also observed from the denatured protein solutions after up to 12 days of treatments. A schematic structure model focusing on the effect of dissolution and denaturation of SPI was established to better explain the results.


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KEYWORDS: Soy protein isolate, Aqueous solution, Denaturation, Ultrasound,

Mechanical shearing, Neutral pH

INTRODUCTION

Synthetic polymers are widely used in many products, but naturally abundant and biodegradable materials are potential sustainable alternatives to replace the traditional petroleum-based polymers. Bioplastics have gained considerable interest as a type of potential alternatives for the synthetic polymers,

1-6

especially in the field of food

packaging, bio-adhesive, skin care products, drug delivery, tissue engineering and biomedical products. 7-13

Vegetable proteins could be substantial sources of biopolymers, as an example, if they are processible and result in optimal mechanical and physical properties as intended products.

14

However, soybean proteins, as a representative of the most abundant and

inexpensive naturally renewable biomacromolecules, possesses very poor processability. According to a USDA report, in 2017, 4.43 billion bushels (around 156.11 billion liters) of soybeans were produced in the U.S.15 The vast number of soybeans was not entirely 3 ACS Paragon Plus Environment


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utilized, especially the soy protein byproducts from production of soybean oil (soy flour, SF, 54% protein; soy protein concentrate) as wastes.16 As a type of globulins, the subunits of soy proteins are associated via hydrophobic interactions and hydrogen bonding. βconglycinin (7S, 150-200 kDa), distributing 30% of the globulin components in soybeans, is a trimeric glycoprotein of three subunits, α (68 kDa), α’ (72 kDa) and β (52 kDa) associated by hydrogen bonding between the amine groups and carbonyl groups.

17-18

Glycinin (11S, 360kDa), the other major globulin components (40%) in soy seeds, is a hexamer composed of acidic (~37 kDa) and basic (~22 kDa) polypeptides linked by disulfide bonds. 18-19

To date, there have been many attempts in applying soy protein as a new source of raw material of bioplastics. However, there are still many obstacles in the manufacturing of soy protein-based bioplastics, especially caused by the low water solubility of soy proteins under neutral pH, low viscosity of protein solutions, which also comes along with a set of material defects in practical applications including the low mechanical properties, high water sensitivity, and visible material yellowness. While among the studies, there were


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limited studies focusing on the relationship between the properties of the protein solutions and the resulting products. Also, it is necessary to seek solutions of enhancing the soy protein processability from modifying the conformational structure of the globulins. 12-13,2026

In a previous work, Aghanouri et al. improved the solubility of β-conglycinin and glycinin

and modified their conformational structures by managing chemical forces in different solvent systems with Hansen solubility parameters as a prediction tool. It was confirmed that, neutral aqueous system could not break intermolecular and intramolecular interactions within soy protein domains or form more stable interactions with the protein molecules without extra physical forces. 27-29

In this study, conformational changes of soy proteins in an environmentally friendly aqueous system were investigated. Mechanical forces were applied to facilitate the denaturation of the globular structures of soy proteins according to a model of the proteins established by Aghanouri et al.

27

Thus, the structural changes of SPI aqueous systems

after different mechanical treatments were predicted and characterized by using different analytical methods. The research activities were planned in two sections, i.e. investigating



properties of SPI dispersions (5 wt. % SPI) and study conformational changes of soy proteins in diluted solutions (0.5 wt.%). The solubility of 5 wt.% SPI in neutral aqueous solution was measured by a Bradford test. Particle size and the size distribution of 0.5 wt.% SPI solutions were measured by dynamic light scattering (DLS) analyzer. The relative viscosity of SPI solutions and its change up to 12 days were investigated using a CannonFenske Viscometer. The free sulfhydryl group content was analyzed by Ellman’s reagent and a spectrophotometric analyzer. The turbidity of both 5 wt.% SPI dispersions and 0.5 wt.% SPI solutions was evaluated by their absorbance at 600nm under a spectrophotometer. To confirm the better processability of treated SPI dispersions, SPIcellophane films were cast out of selected SPI dispersions on commercial porous cellophane films and characterized with the apparent and mechanical properties.

MATERIALS AND METHODS

Materials. Soy protein isolate (SPI, MP Biomedicals, 92.0% protein content based on dry basis) in powder form was provided by MP Biomedicals, LLC (Solon, Ohio). Coomassie (Bradford) protein assay kit was purchased from Thermo Scientific (Rockford, IL). Mini-


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protean precast protein gels (12%, 10-well, 30 µL), protein dual color standards and 4x Laemmli sample buffer were purchased from Bio-Rad (Hercules, CA). 8-Anilino-1naphthalenesulfonic acid ammonium salt was purchased from the Millipore Sigma (St. Louis, MO). Ellman’s reagent (5,5’-dithiobis (2-nitrobenzoic acid), or DTNB) was purchased from Thermo Fisher Scientiﬁc Co. (Waltham, MA). Deionized (DI) water was used as the solvent for all the samples preparations and the blank samples in the tests. Porous cellophane film was purchased from GE Healthcare Bio-science Corp (Piscataway, NJ). All the other chemicals were purchased from Thermo Scientific (Rockford, IL).

SPI Dispersion Preparation. SPI powders at 5 wt.% were weighted through a stainlesssteel screen (40 mesh) and mixed with DI water at pH 7.5 during rapid stirring with a glass rod.

Uniform 5 wt.% SPI aqueous dispersions were achieved under room temperature.

Five Different Mechanical Treatments. Different treatments were applied to groups of 150 mL 5 wt.% SPI aqueous dispersions in 400mL beakers. Magnetic stirring (MS) was conducted by an isotemp stirring plate (Fisher Scientific, Pittsburgh, PA) at 1200 rpm for



60 minutes at room temperature. High-intensity ultrasound treatment (US) was applied using a Sonicator-4000 ultrasonic liquid processor (Misonix Sonicators, Newtown, CT) with a Microtip at 20 kHz with 50% amplitude for 10 min (see Fig-1S in supporting materials). High-speed mechanical shearing (SH) was applied by a Silverson L5M-A mixer (Silverson Machines, MA) at a maximum speed (10230 rpm) for 15 min. Two combinations of treatments were also incorporated with an emphasis on the sequence of the application of the different forces. The groups of the USSH treatments were conducted with 10 minutes of high-intensity ultrasound at 50% amplitude followed with 15 minutes of high-speed shearing at 10230 rpm; the groups of SHUS represent the treatments of the 15 minute high-speed shearing followed with the 10 minute highintensity ultrasound. Both high-intensity ultrasound and high-speed shearing caused a considerable amount of heat release which elevated the temperature of the SPI dispersions. According to multiple reports, the elevated temperature can cause protein denaturation and aggregation.

30

Thus, ice baths were applied to all treatments that

involve high-intensity ultrasound and high-speed shearing to control the SPI solution under the temperature of 35 C. 8 ACS Paragon Plus Environment

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Soy Protein Solubility. The soy protein solubility was measured according to a method reported by Aghanouri et al. 27. After one of the five treatments, the 5 wt.% SPI dispersions were further centrifuged for 15 min at 12000g under room temperature to remove insoluble large protein aggregates and particles to achieve uniform SPI solutions. To conduct the Bradford test, the achieved solutions were diluted 10-fold for MS groups and 40-fold for other treatment groups by DI water to appropriate concentrations. In the part of constructing a standard calibration curve, standard solutions made of bovine serum albumin (BSA) and DI water under a range of 0 – 2000 µg/mL were prepared. In a vial, 0.05 mL of either standard solution or diluted SPI solution and 1.5 mL of Coomassie Plus Reagent were well mixed and incubated under room temperature for 10 min before being tested under 595nm with a UV spectrophotometer (Evolution 600, Thermo). According to the established calibration curve (Fig-2S) of BSA standards, the concentration of each SPI solution samples was determined by measuring the absorbance at 595 nm. The relative solubility of SPI after different treatments were further calculated according to eq 1.



Relative Solubility (%) =

The concentration of SPI Supernatant The concentration of SPI Dispersion

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(1)

× 100%

Particle Sizes and Size Distribution. The particle sizes of soy proteins and their aggregates in the solutions were measured by a dynamic light scattering particle size analyzer (Zetasizer Nano-ZS, Malvern, MA). SPI solutions were diluted to 0.5 wt.% and lower concentrations with DI water for the measurements.

Viscosity Measurements. The viscosity test of SPI solutions after different treatments were performed following the American Society for Testing and Materials (ASTM) using a size 100 Kimax Cannon-Fenske Viscometer (Kimble, Rockwood, TN).

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Each test

sample was balanced in 25 C water bath for at least 15 min and then tested in the constant temperature water bath. The time of samples or DI water passing the capillary part of the Cannon-Fenske Viscometer was recorded by a stopwatch (Fisher brand, Waltham, MA). The relative viscosity of the samples was calculated based on eq.2. The SPI solution concentration was used at 0.5 wt.% in these tests.

Relative Viscosity =

Time of the solution passing the capillary tube Time of DI water passing the capillary tube


(2)

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Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). SDS-PAGE was performed according to the method of Xiong et.al

40.

A 12% Mini-Protein precast

protein gels were employed. SPI solutions after different treatment were diluted to 15 g/L. Each 9 µL protein solution was well mixed with 3 µL diluted Laemmli buffer and heated for 5 min in a boiling water bath. After centrifuge, 10 µL of the prepared protein sample was loaded to a well. The gel was stained with Coomassie brilliant blue R-250 and destained overnight before scanning. Protein standards of 10 – 250 kD were used as references.

Circular Dichroism (CD) Spectrum Measurement. A Chirascan CD (Applied Photophysics, United Kingdom) Spectrometer was used for characterization of the protein secondary structures after different treatments. Protein aqueous solutions at the concentration of 0.005 – 0.02 mg/mL were tested in a 10mm quartz cuvette. The scan wavelength ranges from 200 – 240 nm with the band width of 1.0 nm.

Fluorescence Measurement. The fluorescent properties of proteins were tested by a Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies, CA). Soy proteins



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solutions with 0.001 g/mL were tested under an excitation wavelength of 290 nm with the slit of 2.5 nm. The fluorescent spectrum was scanned under the range of 300 nm – 460 nm with the scan rate of 120 nm/min.

Protein Surface Hydrophobicity. The protein surface hydrophobicity after different treatments were tested with the Cary Eclipse Fluorescence Spectrophotometer. 8Anilino-1-naphthalene sulfonic acid (ANSA) was used as a fluorescent probe. To each 10 mL of SPI solution (0.001 g/mL, in PBS buffer), 100 µL of ANSA solution (2.4 mM, in PBS buffer) was added. The prepared samples were tested with an excitation wavelength of 390 nm with the slit of 5 nm. The scan range was 400 nm – 650 nm with a scan speed of 120 nm/min. Relative surface hydrophobicity of proteins in the solution was calculated according to eq. (3).

Ho = Area of sample solution ― Area of solvent

(3)

Turbidity. The turbidity of 5 wt.% SPI dispersion samples and 0.5 wt.% SPI solution samples was measured using an Evolution 600 UV–vis spectrophotometer (Thermo


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Scientific, WI). DI water was used and as a blank. The absorbance of the samples at 600 nm was selected to represent the turbidity.

Free Sulfhydryl Group Content of SPI Solutions. Free sulfhydryl group contents of 0.5 wt.% SPI solutions were analyzed by the Ellman’s reagent (4 mg/mL) in 0.1 M phosphate buffer (pH=8.0). Within each trial, 2.7 mL of the diluted SPI solution was mixed well with 50 µL of the prepared Ellman’s reagent and incubated for 15 min under room temperature before the test for a consistent chromogenic result. The color of the resulted samples was evaluated and converted to the amount of sulfhydryl groups using an Evolution 600 UV– vis spectrophotometer Thermo Scientific, WI) at 412 nm according to a calibration curve (Fig-3S). All solution properties were measured for at least 3 repetitions by 3 trials within each repetition.

Soy Protein Film Casting and Mechanical Properties. SPI-cellophane composite films were tailored based on commercial cellophane porous films (GE Healthcare Bio-science Corp, Piscataway, NJ). To avoid the film wrinkle caused by the swelling effect, the commercial cellophane films were wetted by DI water in advance. On a piece of the glass



plate, an aliquot of treated 5 wt.% SPI dispersion was poured onto wet cellophane films and repeatedly scraped along the lengthwise direction of the cellophane films to facilitate orientation of the protein molecules. Different mechanical treatments were applied to the 5 wt.% SPI dispersions to analyze the influence of protein dissolution degree on the properties of corresponding films. Different casting repetitions were selected to compare the film property difference brought by the degree of protein biopolymer orientations. The mechanical properties of the SPI-cellophane films were tested using an Instron 5566 tester (Norwood, MA) according to the ASTM-D882 method. Five repetitions of the test were applied.

RESULTS AND DISCUSSION

Figure 1. Relative solubility of SPI in DI water at pH 7.5 after different treatments.


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Solubility. Relative solubilities of SPI after mechanical treatments in neutral aqueous solutions were tested by the Bradford method, and the results are shown in Figure 1. MS, a typical method of preparing SPI solutions for film casting 21 did not show a satisfactory protein dissolution effect. However, US, SH, and the combination of the two treatments all increased the solubility of SPI persuasively, revealing that strong mechanical forces may have reduced SPI aggregation and assisted the dissolution of SPI. Among them, US and SH boosted the dissolution of SPI from 20.85% (control, no treatment) to 67.83% and 83.99%, respectively, due to their strong physical forces. Thus, both US and SH have substantial impacts on the relative solubility of SPI under the condition. Hu et al. proved that an increased solubility was observed on ultrasound treated soy proteins and suggested a partial disruption of the non-covalent bond among proteins including hydrogen bonds and hydrophobic interactions by the force.

32-33

The partial disruption of

H-bonds and hydrophobic bonds among the protein macromolecules and different subunits increased the swelling of protein macromolecular chains and allowed further deformation of the protein conformation under shearing forces. When combining US and SH, the extended total treatment duration further increased the dissolution of SPI. 15 ACS Paragon Plus Environment


However, the sequence of combinations was not an essential factor of the protein solubility since both of USSH and SHUS showed high solubility at 95.80% and 94.9%, respectively.

Figure 2. (a) Volume-weighted mean diameters of particles. (b) The PDI of particle size, of SPI after different treatments

Protein Particle Sizes and Size Distribution in Solutions. The sizes of soluble soy protein aggregates were examined by dynamic light scattering (DLS) and exprssed as hydrodynamic diameters, an indicator of the diameter of an equivalent sphere with the same translational diffusion coefficient with the particle being measured. The volumeweighted mean hydrodynamic diameters (nm) of the SPI solution samples after different treatments are shown in Figure 2 (a). Among the five treatments, the protein particles


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after MS treatment showed the smallest size of 227.5 nm, which can be regarded as the original mean hydrodynamic diameter of SPI particles. 34 For the rest of the treatments, it was found that the SH treatment resulted in a more significant effect on enlarging the size of soy protein units compared to the US. After US and SH treatment, the mean hydrodynamic diameters of SPI particles increased to 252.5 nm and 476.2 nm, respectively. As of USSH, the mean hydrodynamic diameter of the SPI should further increase on top of the US but ended up with a smaller size than SH at 334.5 nm, whereas the size of the SHUS treatment decreased compared to SH, and even smaller than USSH with a value of 297.2 nm.

Different treatments also showed an impact on the distribution of SPI particle sizes, which was represented by the polydispersity indexes (PDI). PDI ranges from 0 to 1.00. Larger PDI indicates a broader particle size distribution with a higher possibility of the presence of large particle aggregates. 27 According to Figure 2 (b), a wide particle size distribution (PDI = 0.634) was detected from MS samples, confirmed the deficient dissolution and dissociation of SPI, as well as the existence of large SPI aggregates in the solution. In



comparison, after all US and SH treatments, the particle size distribution tended to be narrow and uniform. The narrowest size distribution appeared after US treatment (PDI=0.193), followed by SHUS (PDI = 0.219), USSH (PDI = 0.247), and SH (PDI = 0.327).

Thus, according to the results of both hydrodynamic diameters and PDI, after the MS treatment, there were still large protein aggregates existing in the SPI solutions. On the contrary, the US treatment has a considerable mechanical force to break intermolecular interactions and swell the protein, as well as to reduce aggregation of the proteins. Similarly, the SH treatment can affect the intermolecular interactions and reduce SPI aggregates as well. In addition, the shearing force of SH treatment could deform the original globular structure of soy proteins into a flatter and stretched conformation, at the same time orient the macromolecular chains. Such a shape change of the SPI may have contributed to the increased hydrodynamic diameter and PDI. In other words, SH treatment could have a stronger unfolding effect than other treatments (MS and US) shown as the increase of the particle sizes. Thus, in the case of USSH treatments, the globular SPI were swollen during the US and further stretched and deformed by the SH


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treatment, resulted in a slightly higher hydrodynamic diameter and PDI. Similarly, in the case of SHUS treatments, after SH treatment, the oriented and deformed macromolecular chains were additionally ultrasonically relaxed and swollen, resulting in the reduced hydrodynamic diameter and PDI.

Figure 3. Relative viscosity changes of 0.5 wt.% SPI solution after specific treatments and relaxation time – rest 0 days (D0), rest 6 days (D6), rest 12 days (D12), treated again after 12 days (D12*).

Relative Viscosity (RV) and its Changes Over Time. Viscosity or flow properties of macromolecular solutions are profoundly influenced by the shape, size, concentration of the SPI and the solute-solvent interactions.35 As demonstrated in the study of hydrodynamic diameters and PDI values of SPI solutions under different treatments, it seems that the macromolecular chains were affected by different forces. The relative



viscosity measurements could further prove the changes. The SH treatments should make the protein chains deformed and oriented, causing the increased viscosity of the SPI solutions. Both the SH and USSH treated SPI revealed higher viscosities right after the treatments (D0 in Figure 3).

As shown in Figure 3, compared to the SPI viscosity after MS treatment (RV=1.248), the relative viscosity after USSH (RV=1.581), SH (RV=1.547), and US (RV=1.417) all significantly increased, indicating different degrees of unfolding and deformation effect of SPI molecules in the solutions. The viscosity results, agreed well with hydrodynamic diameter, suggest that the SPI molecules become swollen after the US treatment, increasing the possibility of intermolecular entanglements and intermolecular interactions of soy protein chains. After the SH treatment, the shearing force could break, deform and orientate the globular structure of soy proteins. Thus the shape of the soy protein units became more open and was able to form slight intermolecular entanglements in the solutions. Whereas to the USSH treatment, the swelling effect from the US treatment and the shearing effect of SH established well-denatured and unfolded soy protein chains. On


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the other hand, as of the SHUS treatment, the distorted proteins structures after the SH treatment was given extra energy and time to be relaxed during the US treatment. Compared to the MS and US treated samples, the hydrophobic intramolecular interactions of soy proteins after the SHUS treatment was significantly reduced. However, the intermolecular entanglement and interactions of SHUS-treated samples were much less than the USSH-treated samples.

The physically forced denaturation, unfolding, and deformation of the SPI molecules are recoverable if the forces are removed. The viscosity changes of the SPI solutions after 6 and 12 days were tracked and recorded, with the relative viscosities of US, SH and USSH decreased to 1.125, 1.197, and 1.229 in 6 days and further dropped to 1.030, 1.041, and 1.044 in 12 days, respectively. Such decreases are partially due to the relaxation of the polymer chains. However, when the solutions relaxed for 12 days were further treated again with the method that they were initially applied (D12*), the viscosity of USSH, US and SH slightly increased to 1.089, 1.068 and 1.067, respectively. The increases in the viscosities could be explained as the relaxed polymer chains were stretched back into the



more oriented chains in the solution system. However, the viscosities of the solutions at D12* were all much lower than that of at D0, due to the degradation of protein chains by bacteria and fungi during the storage.


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Figure 4. Effect of different treatments on the structures of SPI. (a) SDS-PAGE profile of treated SPI; (c) Emission fluorescent spectra of SPI solutions; (d) Surface hydrophobicity of proteins in the solutions after different treatments.



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Table 1. The secondary structural contents simulated by SELCON3 from Far- UV CD spectra

α-Helix

β-Sheet

Turns

Unorder

MS

9.064

46.11

10.84

33.99

US

9.118

45.89

11.10

33.89

SH

10.92

43.98

11.64

33.47

USSH

9.335

45.88

10.92

33.86

SHUS

9.335

45.88

10.92

33.86

Protein Structural Characterizations. To better illustrate the physical properties of soy protein solutions and confirm the proposed protein structural changes, SDS-PAGE, farUV CD spectra, and fluorescent emission spectra were employed to characterize the primary, secondary and tertiary structures of the treated proteins, respectively. As shown in Figure 4 (a), the SDS-PAGE profiles among the five treatments appears similar pattern, which indicates that the physical forces including US and SH are not aggressive enough to disrupt the primary structure of proteins in the dispersion. Similar results were reported by Hu et al. 36 and Xiong et al. 37.

As of the secondary structures of the soy proteins, the far-UV CD spectra were analyzed with SELCON3.

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Mean residue weight (MRW) of 115 g/mol used in the regression.39 24 ACS Paragon Plus Environment

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According to the calculated secondary content shown in Table 1, there was little change in the secondary structures of soy proteins after different treatments. Xiong et al. reported the limited secondary structural changes of pea proteins after ultrasound treatments37.

The tertiary structures of the soy proteins, as shown in Figure 4 (b), were dramatically changed after US, SH, USSH and SHUS treatments. It can be observed that the characterizing fluorescence intensity of the treated soy proteins prominently increased, and the characterizing peak shifted to lower wavelengths (λ MS=354.5 nm, λ US =350.4 nm, λ

SH

=348.9 nm, λ

USSH

=345.9 nm, λ

SHUS

=344.8nm. The results illustrated that the

tryptophan residues of proteins from US and SH treatments were more exposed to the hydrophilic solvent environment than that of proteins from MS treatment. Meanwhile, the protein surface hydrophobicity of soy proteins after treatments increased as shown in Figure 4 (c). The value of Ho corresponds to the exposed hydrophobic region in the protein particles. 36-37 The dramatically increased Ho values (Ho MS=4,480, Ho US =15,340, Ho SH =17,240, Ho USSH =18,086, Ho SHUS =18,200), further confirm the observed swelling effect of the US treatments and deformation effects of SH treatments in the viscosity tests.



Comparing the degree of the fluorescence intensity changes and surface hydrophobicity increases, it was also observed that SH has a stronger effect on destroying the tertiary structure of soy proteins compared to the US treatment.

Schematic Structures. Based on the above discussion, a schematic structural change of SPI in neutral aqueous solution is proposed, shown in Scheme 1 (a). To further improve the reliability of the proposed structure, turbidity and free sulfhydryl group content in the SPI solutions were evaluated. Besides, to better express the protein conformational changes of up to 12 days of viscosity tests, Scheme 1 (b) was designed.


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Scheme 1. (a) Schematic presentations of SPI conformation in neutral aqueous solution after different treatments. (

represents α-helix,

represents β-sheet,

represents hydrophobic interactions.) (b)

The polymer chain relaxation and degradation process of USSH treated SPI after specific resting time (Day 0 – Day 12) and further USSH treatment (Day 12*).



Figure 5. Turbidity and appearances of 5 wt.% SPI dispersion (a) and 0.5 wt.% SPI solution (b).

Turbidity. The turbidities of the SPI 5 wt.% dispersions and 0.5 wt.% solutions were characterized by the light absorbance at 600 nm and are shown in Figure 5. The visual appearances of different samples are shown respectively underneath the columns in Figure 5. Higher turbidity (or a higher absorbance at 600 nm) indicates a larger particle diameter in the solution. Like the particle size measurements by DLS, the larger particle diameter can be explained as either the larger size protein aggregates for 5 wt.% SPI dispersions or, the larger volume of the spatial structure of the denatured soy protein chains by swelling the original globular structures for 0.5 wt.% SPI solutions.


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The turbidity of 5 wt.% SPI dispersion in Figure 5 (a) agreed well with the results of the solubility tests. The turbidity of the SPI dispersion after the MS treatment was quite high (Abs=3.17), while the SPI solutions after both US and SH treatments showed decreased absorbance values of 3.00 and 2.79, respectively. Again, the SH treatment had a more substantial effect, agreed with the high solubility result. It was also noted that the combination of US and SH treatments provided stronger forces in deforming SPI molecules and reducing the aggregates of the biomolecules. At the same time, the turbidity of the SHUS treated SPI solution (Abs=2.25) was lower than that of the USSH treated one (Abs=2.71) due to less deformation of the molecules.

The turbidities of 0.5 wt.% SPI solutions are shown in Figure 5 (b), quite consistent with the hydrodynamic diameter results (Figure 2(a). Turbidities of both US (Abs=0.27) and SH (Abs=0.30) treated SPI solutions increased in large scales compared to that of the MS (Abs=0.14) treated one due to the swelling and deforming effect caused by the ultrasound and shearing forces. Consistent with the result of particle size measurement, the turbidity of the solution after the SHUS (Abs=0.14) treatment was also lower than that



of the USSH (Abs=0.24) treated one. This was because after the SH treatment, the soy protein aggregates were mostly separated, and the globular chains have been deformed to more-stretched structures, which highly increases the volume size of protein particles. With further high-intensity ultrasound, the high energy promoted the deformed protein polymer chain back into smaller size. However, if the treatments were conducted in the order of USSH, the high-intensity ultrasound could initially separate the soy protein aggregates and swell the globular macromolecular biopolymer chain. Later, the highspeed shearing could provide a strong shearing force to break intermolecular and intramolecular hydrophobic bonds, thus orientating and deforming large protein aggregates and outstretching the swelled macropolymer chain into deformed conformations.


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Figure 6. Free sulfhydryl group content of 0.5 wt.% SPI solution after treatments.

Free Sulfhydryl Group Content. The thiol groups or free sulfhydryl groups in SPI (µmol/g protein) represent the number of disulfide bonds being broken and exposed to chemicals. Due to the intrinsic reducing property of sulfhydryl groups and the existence of oxygen and oxygen free radical in the samples introduced by the US and SH treatments, the experimental data in all US and SH treated protein samples could be smaller than the real amounts of the thiol groups produced after the treatments. The test values of the treated SPI samples are shown in Figure 6. Agreed with the prior tests, the MS treated samples showed the lowest thiol group content at 12.7 µmol/g protein, indicating the subtle breakage of protein disulfide bonds by the MS treatment. While both US and SH treatments increased the thiol group contents in the protein solutions to 21.73 µmol/g protein and 23.25 µmol/g protein, respectively. Moreover, the USSH treatment brought the highest thiol group content of 27.58 µmol/g protein, indicating occurrence of considerable disulfide bond disruptions and more protein denaturation. However, with reverse order of the two treatments, SHUS, the thiol group contents decreased compared



to either US or SH individually to 20.51 µmol/g protein, which could be a result of a reduction of SH groups due to an oxidation with oxygen in air.

Figure 7. (a) Transparency of SPI-Cellophane composite films, cast out of 5 wt.% SPI-MS dispersion (iii.), and 5 wt.% SPI-USSH15 dispersion (iv.), with commercial Cellophane films (ii.). (b) The mechanical properties of SPI (USSH)-cellophane composite films

The Properties of SPI-Cellophane Films. To confirm improved industrial processability of the SPI aqueous dispersion after the proposed treatments, films were cast out of 5 wt.% SPI dispersion after the MS and USSH treatments. As shown in Figure 7 (a), compared with the background (Figure 7-a-i) and the commercial cellophane film (Figure 7-a-ii) the USSH SPI-Cellophane composite film (Figure 7-a-iv) was clear and uniform, whereas the film made from the MS dispersion (Figure 7-a-iii) had a grainy surface with large protein


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aggregates embedded in the composite films. The mechanical properties of SPICellophane composite films are shown in Figure 7 (b). To testify that after the USSH treatment, the SPI proteins were well dissolved and denatured, different repetitions of the film casting process were used to prepare the composite films. Since the commercial cellophane films have a production direction, the SPI dispersion was oriented during the casting process in order to achieve a same direction of polymer chain orientation with the cellophane film, thus to establish the highest possible film mechanical performance. Theoretically, the linear chain polymers would orient better under forces during the blade casting, and to produce a film with a higher tensile strength under the same tensile strain. From Figure 7 (b), the expected phenomenon was detected. The films after 10 times of the blade casting showed doubled tensile strength compared to the one cast only one time. This further confirmed that after USSH, the SPI protein structure was denatured and can be stabilized in a certain degree during industrial process.



CONCLUSIONS

Through different mechanical treatments, the soy proteins in neutral aqueous dispersions were modified in conformational structures. Comparing with the SPI film prepared by magnetic stirring, both high-intensity ultrasonication and high-speed mechanical shearing processes showed much more significant improvements in dissolution, denaturation and orientation process of SPI in the dispersion by overcoming the intermolecular and intramolecular interactions. Among all tested treatments, high-intensity ultrasonication followed by high-speed mechanical shearing showed the best protein solution properties with a relative solubility of 95.80%, uniform unfolded particle diameter at 334.5 nm, increased fluorescence intensity and protein surface hydrophobicity, low turbidity, high exposure of free sulfhydryl group at 27.58 µmol/g protein, and highly increased relative viscosity. Biopolymer molecular chain relaxation and decomposition was observed over up to 12 days of protein solution resting time. In this research, high-intensity ultrasonication was proved in the outstanding ability of dissolving large protein aggregates and swelling globular protein structures; high-speed mechanical shearing was confirmed


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in its competence of breaking intramolecular hydrophobic bonding and deforming and orienting the globular protein structure into more processable structures.

ACKNOWLEDGEMENTS

J.Z. acknowledges the Jastro Shields graduate student research fellowship for partial financial support.



SUPPORTING INFORMATION

Effects of high-intensity ultrasound to properties of SPI solutions;

Calibration curves of protein solubility and sulfhydryl groups in protein solutions.


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TOC A schematic structural change model of soy protein isolate was established based

on the effect of the sustainable physical treatments on its dispersion


Conformational Changes of Soy Proteins under High-intensity

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