Storage-induced changes in plasticizer emigration and structural properties of soybean isolate protein-based composite films

Storage-induced changes in plasticizer emigration and structural properties of soybean isolate protein-based composite films | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Storage-induced changes in plasticizer emigration and structural properties of soybean isolate protein-based composite films Tingwei Zhu, Peipei Fan, Qianyu Li, Luyan Ma, Qian Wei, Xingfeng Guo, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1859866/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Previous study found soybean isolate protein (SPI)-based composite films added by corn starch (CS) (SPI:CS (4:1) composite film, SPI:CS (1:1) composite film) exhibited good mechanical property and better stability during the short-term storage pre-experiment. These properties were related to internal structure changes. In order to accordingly understanding the causes driving stability changes in performance and then controlling long-term stability of the SPI-based composite films, plasticizer emigration and structural properties of SPI-based composite films stored at different temperatures (5 ℃, 20 ℃, 35 ℃) and 54% relative humidity were systematically investigated over a period of time. High storage temperature (＞ 20 ℃) accelerated plasticizer emigration which reflected in increased migration rate of glycerol molecules and decreased moisture content. During the storage of SPI-based composite films, the decreased free SH partly formed new disulfide bonds and non-covalent inter-molecular interactions like hydrogen bonds changes significantly weakened the protein structure, which influenced mechanical properties changes during storage. PAGE and FTIR results explained the protein aggregation was bond with disulfide bonds and the reorganization of protein structure with prevalence of extended β-sheet conformation. Soybean isolate protein Corn starch Composite films Storage temperature Plasticizer emigration Structural property Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction With the serious depletion of petroleum-based polymer materials and the enhancement of people's environmental awareness, the growing demand of consumer to nature friendly packaging materials has caused more investigations focused on nature renewable, biodegradable polymers as raw materials for packaging [1]. In this sense, different kinds of biodegradable polymers like proteins, starches, polysaccharides and lipid biopolymers from renewable sources are considered as basic materials for packaging [2-3]. The ability to form a certain network structure is essential to maintain the mechanical properties and application of the films, as a result, with molecular structure of proteins covers a large number of active groups protein-based films has attracted attention in recent years [3]. Soy protein isolate (SPI) as a by-product of edible oil industry, has been valued in the field of packaging for its inexpensive, biocompatible and renewable advantages [4]. SPI-based films can be applied to degradable food packaging and coating with moderate mechanical properties and oil barrier properties [5]. However, it was worth noting that SPI-based films are generally using and stored under different environmental conditions, which influenced physical properties and application in packaging that are associated with storage-induced stability changes. Maryam et al found that the SPI film showed stable antioxidant activity for 90 days of storage at 25 ℃ [6]. While, Srikiatden et al studied that the mechanical properties of SPI films decreased after 2-3 months storage period, resulting in impossible to using widely in packaging [7]. In fact, the storage stability including mechanical properties, oil barrier property, water solubility and practical application of the films is related to the internal structure changes of films during storage. During the storage process of the films, variable storage environment greatly favored the components in films such as moisture and plasticizer migration, and interaction between the components in the films [8]. These eventually lead to changes in internal structure of films. In conclusion, it is essential to study the migration and interaction of the internal components in the films during storage so as to clarify the reasons of the performance changes of films during storage, which is crucial for predicting and regulating the stability of protein films and then reasonable application of the films. In the previous studies, we successfully prepared SPI-based composite films added by corn starch (CS) (SPI:CS (4:1) composite film, SPI:CS (1:1) composite film) with good mechanical properties [9]. Then further studied the effect of temperature on physical properties and application in packaging of the SPI-based composite films during storage (this manuscript is under submitting). In order to accordingly understanding the causes driving stability changes in performance of the SPI-based composite films and then controlling long-term stability of physical properties and practical application of the SPI-based composite films, in this ongoing work, storage-induced changes in plasticizer emigration and structural properties of SPI-based composite films were investigated in this study. Firstly, the three SPI-based composite films (SPI:CS (4:1) composite film, SPI:CS (1:1) composite film, SPI film) were prepared, and three storage temperatures (5 ℃, 20 ℃ and 35 ℃) were selected according to the actual seasoning package application. Then plasticizer emigration about changes of glycerol content and moisture content in the SPI-based composite films during storage was monitored. At the same time, changes in structural properties of the SPI-based composite films during storage were determined by free sulfhydryl group content test, intermolecular forces characterization, SDS-PAGE and FTIR, respectively. 2. Materials and methods 2.1 Materials Soy protein isolate (SPI, protein content: 92.1%) was supplied by Wandefu Co. (Shandong, China) and corn starch (CS) was obtained from a local grocery store. All other reagents and solvents which were purchased from Tianjin Ruijinte Co. Ltd. (Tianjin, China) were of analytically pure grade. 2.2 Preparation of SPI-based composite films The soy protein isolate-based composite films including SPI:CS composite films (SPI:CS 1:1, SPI:CS 4:1) and SPI film were prepared using a solvent-cast method described in our previous study [10] and the specific steps of making SPI-based composite films were shown in Figure 1 . 2.3 Storage protocol The SPI-based composite films including SPI:CS composite films (SPI:CS 1:1, SPI:CS 4:1) and the control film (SPI film) were cut to the same size and then stored at 5 ℃, 20 ℃ and 35 ℃ with relative humidity (RH) 54% for 24 weeks, 16 weeks, and 8 weeks, respectively. The stop of experiment after a period of storage at certain temperature environment was because too fragile or moldy phenomenon was occurred in the sample. The three selected temperature represented the common environments of the film during application or storage. At least three parallel experiments were performed. Then the glycerol and moisture emigration, structural properties changes of the SPI-based films were analyzed by different ways. 2.4 Glycerol emigration The glycerol content of the SPI-based composite films during storage was determined by the cold oxidation of sodium iodate method, according to the description of Guo and Huang [11]. The glycerol in the accurately weighed SPI-based composite film was extracted in a 250 mL conical flask with 150 mL distilled water for 4 h, and then the conical flask was adjusted to pH 7.9 ± 0.1 with NaOH solution (0.05 mol/L). Followed steps by the cold oxidation were: 5 mL sodium periodate solution (60 g/L) was added in the conical flask and then placed in a sealed dark condition at 35 ℃ for 30 min, then 5 mL ethylene glycol was add and placed for 20 min. Finally, 5 mL sodium formate solution (1 mol/L) was added and the solution was titrated with NaOH to 7.9 ± 0.1 by pH meter indication. The calculation formula of glycerol migration rate (K) in the SPI-based composite film was as follows: Where V 1 (mL) was the volume of NaOH standard solution consumed by the SPI-based composite film determination, V 2 (mL) was the volume of NaOH standard solution consumed by the blank without the SPI-based composite film, m (g) was the mass of the SPI-based composite film, C (mol/L) was the concentration of NaOH standard solution, 0.0921 (g/mmol) was the molar mass of glycerol. 2.5 Moisture content Consulted the description of Guo et al [12], the moisture content of the SPI-based composite films during storage was determined according to the ASTM D644-99 (ASTM standard test method for moisture content of paper and paperboard by oven drying) 2.6 Free sulfhydryl group content The free sulfhydryl group (-SH) of the SPI-based composite films during storage was measured by the Ellman’s method according to the determination process described by Chen and Wasseman (1993) [13]. 2.7 Solubility of proteins in different denaturing solvents The Solubility of protein in different denaturing solvents was utilized to evaluate the intermolecular forces of proteins in the films [14]. It was determined according to the procedure of Ciannamea et al [15] and Hager [16] with some modification. The denaturing solvents were: 0.086 moL/L Tris-HCl buffer containing 0.09 mol/L glycine and 4 mmol/L Na 2 EDTA (pH 8.0) (S1), 0.086 moL/L Tris-HCl buffer containing 0.09 mol/L glycine, 4 mmol/L Na 2 EDTA and 5 mg/mL SDS (S2), 0.086 moL/L Tris-HCl buffer containing 0.09 mol/L glycine, 4 mmol/L Na 2 EDTA and 8 mg/L urea (S3), 0.086 moL/L Tris-HCl buffer containing 0.09 mol/L glycine, 4 mmol/L Na 2 EDTA, 5 mg/mL SDS and 8 mg/L urea (S4), 0.086 moL/L Tris-HCl buffer containing 0.09 mol/L glycine, 4 mmol/L Na 2 EDTA, 5 mg/mL SDS, 8 mg/L urea and 25 mg/mL β -mercaptoethanol (S5). The specific determination steps were as follows. 100 mg of SPI-based composite film sample was uniformly dispersed in 5 mL above denaturing solvent (S1-S5) for 12 h, and then centrifuged at 5000 r/min for 20 min to collect the supernatant. Then 1 mL the supernatant was combined with 4 mL biuret reagent and the protein content was analyzed by biuret spectrophotometry method at 530 nm. 2.8 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE was conducted on a discontinuous buffered system using a separating gel (12%) and a stacking gel (5%) [17]. The protein sample (protein concentration 5 mg/mL) was prepared by adding a sample buffer (Reductive buffer: containing pH 6.8 Tris-HCl buffer 2.5 mL, glycerol 4 mL, SDS 4 mL, bromophenol blue 0.5 mL, 𝛽-mercaptoethanol 1 mL, Non-reductive buffer: containing pH 6.8 Tris-HCl buffer 2.5 mL, glycerol 4 mL, SDS 4 mL, bromophenol blue 0.5 mL) and heated for 5 min in boiling water before electrophoresis. Gels were run at a constant voltage of 80 V (lasting about 40 min) before changing to 120 V when the bromophenol blue indicator ran into the separating gel (lasting about 60 min). After electrophoresis, proteins in gels were stained with Coomassie Brilliant Blue R-250. 2.9 Fourier transform infrared spectrum (FTIR) A Nicolet fourier transform infrared spectrum equipped with a computer (Thermo Nicolet Corporation, USA) was used for FTIR analysis. The dried and broken film was mixed with potassium bromide (KBr) (1:100, w/w), ground with a mortar to form homogeneous powder, pressed into a sheet and scanned from 4,000 to 400 cm −1 within 16 scans [18]. 2.10. Statistical analysis The experiments were performed in triplicate (n = 3), and the data were expressed as mean ± standard deviation (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA). A value of P < 0.05 was considered statistically significant. 3. Results and Discussion 3.1 The select SPI-based films In our previous studies, we optimized the prepared conditions and fabricated the SPI-based composite films successfully by monitoring the evaluation indexes of the tensile strength and elongation at break [9]. The formed SPI:CS (1:1) composite film exhibited good mechanical property and the SPI:CS (4:1) composite film showed better stability during the short-term storage pre-experiment. Furthermore, during the study about effect of storage conditions on physical properties and application in packaging of the SPI-based composite films, we found that the glycerol and moisture as the plasticizers, which influenced the internal structure, played improvement role in the physical properties and application of the SPI-based composite films during storage. Therefore, the plasticizer emigration and structural properties of the SPI-based composite films during storage at different temperature and time were studies in order to expound the change mechanism of the SPI-based composite films during storage and provide theoretical guidance for the application. 3.2 Plasticizer migration in the SPI-based composite films during storage Polyols and water are often used as plasticizers in the hydrosol-based films [19]. Small molecules of glycerol and moisture in the films can migrate during storage, which may result in the property changes of the films [20]. Therefore, the effect of storage conditions on the plasticizer emigration was explored by measuring the changes of glycerol content and moisture content in the SPI-based composite film during storage at different temperature and time. 3.2.1 Glycerol migration changes in the SPI-based films during storage The glycerin content of the SPI-based composite films during storage was determined by the cold oxidation of sodium iodate method. The glycerol emigration changes in the SPI-based composite films during storage were shown in Figure 2 . As can be seen in Figure 2(A), the glycerol migration rate in the SPI film were 9.0%, 9.3%, 10.9% after 8 weeks of storage at 5 ℃, 20 ℃, 35 ℃, respectively. Furthermore, with the extension of storage time, the glycerol migration was faster at the higher temperature (35 ℃). It can be concluded that the migration rate of glycerol molecules in the film was accelerated when the film was stored at high temperature, which was due to the accelerated movement of the glycerol when the storage temperature increased. This was also consistent with the research result of Ket-On et al [21]. Similarly, the effect of storage conditions on the glycerol mobility in the other SPI-based composite films including the SPI:CS (1:1) composite film and SPI:CS (4:1) composite film was consistent with that in SPI film. While, under the same storage conditions, the glycerol mobility in the SPI:CS (1:1) composite film was higher than that in the other SPI-based composite films (SPI:CS (4:1) composite film, SPI film). As the glycerol migration rate in the SPI:CS (1:1) composite film were 10.6%, 10.6%, 12.3% after 8 weeks of storage at 5 ℃, 20 ℃, 35 ℃, respectively. It was speculated that when the SPI-based composite films with more corn starch addition, partial glycerol was combined with starch by weak hydrogen bonds and reduced the glass transition temperature, thus this part of glycerol was more likely to migrate during storage [22]. 3.2.2 Moisture content changes in the SPI-based films during storage The moisture content in the film also had influence on the performance of the film, and the moisture content changes were also affected by the storage conditions. Figure 3 depicted the changes of moisture content in the SPI-based composite films during storage. Generally, under certain humidity condition (RH 54%), low temperature storage (5 ℃) had little effect on the moisture content of the SPI-based composite films. While when SPI-based composite films stored at 20 ℃ or 35 ℃, the moisture content decreased more obvious with the increase of storage temperature, indicating that increased storage temperature promoted the evaporation of moisture content in SPI-based composite films. The moisture molecules moved slowly when the films stored at low temperature, which slowed down the moisture migration from the inner to the surface in the films. Chinma et al also found that less moisture was restricted when the film stayed at high temperature condition and the moisture was easier to migrate [23]. For the three SPI-based composite films were stored at 35 ℃ for 8 weeks, the moisture content in SPI:CS (1:1) composite film, SPI:CS (4:1) composite film and SPI film reduced to about 13.0%, 15.5%, 15.0%, respectively. This may be due to less binding sites between protein and water existed in the SPI:CS (1:1) composite film with more CS addition [24]. Combined with our previous study which was the physical properties changes of the SPI-based composite films during storage, it can be concluded that the mechanical properties and application of the SPI-based composite films were affected by glycerol and moisture changes. Therefore, the structural properties of the SPI-based composite films during storage were further studied to explain the mechanism of the plasticizer migration. 3.3 Structural properties of SPI-based composite films during storage The structural properties including the free sulfhydryl group content, intermolecular forces, secondary structure by SDS-PAGE and FTIR of the SPI-based composite films during storage were further investigated in this study. 3.3.1 Free sulfhydryl group content in the SPI-based composite films during storage The changes of sulfhydryl group content is a dynamic process during the storage of the SPI-based films, which reflected the network structure changes of the protein based films to a certain extent. Hence, changes of the free sulfhydryl (-SH) content in SPI-based composite films during storage were determined and the results were exhibited in Figure 4 . On the whole, the content of free SH in the three SPI-based composite films was decreased during storage, implying the formation of new disulfide bonds. This result was similar to those obtained by Ciannmea et al, which reported an increment of intermolecular disulfide bound in concentrate soy protein-based film during storage which developing of S-S covalent interaction [25]. What’s more, the free SH content in SPI:CS (1:1) composite film, SPI:CS (4:1) composite film and SPI film reduce about 0.93-1.14 μmol/g (Fig. 4C), 0.95-1.71 μmol/g (Fig. 4B), 0.89-1.62 μmol/g (Figure 4A), respectively, after the end of storage at high temperature (﹥ 20 ℃). The free SH content in the SPI-based films decreased along with the rising of storage temperature, indicating that higher storage temperature could facilitate S-S bonds formation during storage. Disulfide bond is an important form of covalent binding in the protein molecules. The protein network structure in protein-based films becomes denser and polymer formed in protein-based films fluidity becomes worse [26], which leads to the increase of tensile strength and decrease of elongation at break. This phenomenon was also consistent with the mechanical property changes of the SPI-based films during storage studied in our previous study. 3.3.2 Intermolecular forces in the SPI-based composite films during storage The maintenance of protein network structure in protein-based films was mainly depended on the intermolecular/intramolecular acting force such as hydrogen bonds, hydrophobic interactions and S-S bonds. The type of protein aggregation interactions in SPI-based composite films were described with the solubility of proteins in different reducing solvents. Among the reducing solvents, urea could disrupt hydrogen bonds, SDS could disrupt hydrogen bonds and hydrophobic interactions, and β -mercaptoethanol could cleave S-S [27]. As can be seen in Figure 5(A1-A3) , the protein solubility of the SPI film in S1, S2, S3 were about 18 mg/g, 21 mg/g, 46 mg/g, respectively, implying hydrogen bonds was the main force in maintaining network structure in the SPI film and disulfide bonds was also existed. Similarly, from Figure 5 (B1-B3) and Figure 5(C1-C3) , the decreased of protein solubility in the presence of SDS and urea (S4), urea (S3) and SDS (S2) after the end of storage at the same temperature, suggested that non-covalent inter-molecular interactions (hydrogen bonds and/or hydrophobic interactions) changes significantly weakened the protein structure in the two SPI-CS composite films. Compared with SPI film, the SPI-CS composite films showed low protein solubility in S3 under the same storage conditions. This may be attributed to diluted protein content with addition of starch to a certain extent inducing the protein molecular spacing in the film. 3.3.3 SDS-PAGE In order to further investigate the internal structure of the SPI-based composite films during storage, both non-reductive SDS-PAGE and reductive SDS-PAGE were used to characterize the protein structure in the SPI-based composite films during storage. SPI is mainly composed of glycinin (11S) and conglycinin (7S). 7S is aggregated into trimer that contains α (~67 kDa), α´ (~71 kDa), and β (~50 kDa) subunit and 11S is heterogeneous subunits associated with acidic subunits (A, 31-45 kDa) and basic subunits (B, 18-20 kDa) through disulfide bonds connection [28-29]. Figure 6(A) depicted the SDS-PAGE patterns of the SPI-based composite films before storage. The color of the lanes in reductive electrophoretic bands were significantly lighter than that in non-reductive electrophoretic bands, especially the lighter color of the bands located in the separation gal with a new band appeared near the A subunits, implying disulfide bonds existed in the protein network structure of the SPI-based composite films and protein polymer with high molecular weight were bond with disulfide bonds. Moreover, from the Figure 6 , the color of bands in the reductive electrophoretic condition gradually became light during the storage of the SPI-based composite films, indicating the development of crosslink with non-covalent bonds between the protein molecules in the SPI-based composite films during the storage. Also, this speculation was supported by the results in intermolecular forces results ( Figure 5 ). Compared to the SDS-PAGE patterns of the three SPI-based composite films (SPI:CS (1:1) composite film, SPI:CS (4:1) composite film, SPI film) ( Figure 6 ), it was found that the dark color of bands in the three lanes when SPI-based composite films were stored in the same condition showed the following order: lane 2 ﹥ lane 3 ﹥ lane 1. This can be explained by the polymerized of more protein molecules in the SPI film. With addition of starch in the SPI:CS composite films increased the spacing in protein molecules thus weakening the protein polymerization in the SPI:CS composite films during storage. What’s more, some study found that Maillard reaction could also occurred in glycerol- and sorbitol-plasticized film during storage and the higher storage temperature may accelerate the reaction [30-31]. It also can be seen from Figure 6 that smear band with high molecular weight appeared at the top of the stacking gel and the bands of protein subunits became shallow when the SPI-based composite films was stored at high temperature with the storage time. Therefore, it could be inferred the decrease in protein subunits of the films may be caused by the protein polymerization and aggregation via Maillard reaction. These results are correlated very well with the changes in light transmittance property of the SPI-based composite films during storage which was our simultaneous research result about the SPI-based composite films (this manuscript is under submitting). 3.3.4 FTIR The intermolecular/intramolecular interactions of the ingredients in SPI-based composite films were investigated by using FTIR. The FTIR spectra of SPI-based composite films during storage at diverse temperatures with different times were shown in Figure 7 . Previous study reported that the regions of 800-1200 cm −1 and 3000-3600 cm −1 were the absorption bands of glycerol and the main absorption peak of free and bound O-H and N-H groups, respectively, else it was a broad absorption band nearly 3200 cm −1 for all proteins [32]. Hence, during the storage of the three SPI-based composite films, the absorption of films in the regions of 1000-1200 cm -1 and 3200-3700cm -1 were weak ( Figure 7A-C ), indicating migration of glycerol and moisture molecules in the SPI-based composite films during storage weakened hydrogen bonding of intermolecular proteins. In addition, compared with the SPI film, the two SPI:CS composite films (SPI:CS 4:1, SPI:CS 1:1) showed strong peak in the region of 1000-1200 cm -1 and broadens in the range of 3200-3700cm -1 , indicating the cross-linking between SPI and CS increased the bound O-H. Furthermore, the higher the storage temperature was, the weaker the absorption in the regions of 1000-1200 cm -1 and 3200-3700cm -1 . This is also consistent with the above results in the study on plasticizer migration ( Figure 2, Figure 3 ) and intermolecular forces ( Figure 5 ) in the SPI-based films during storage. In the characteristic absorption band for protein amino I, the 1600-1700 cm −1 range was reflected protein secondary structure, and corresponding parameters used in the study were: 𝛼-helix structure, 1650-1660 cm −1 , 𝛽-sheet structure, 1600-1640 cm −1 , 𝛽-turn structure, 1660-1700 cm −1 and random coil structure, 1640-1650 cm −1 [33].The data about FTIR spectra ( Figure 7 ) were analyzed using Peak-Fit V4.12 software, which depicted the secondary structure as shown in Table 1 . The effects of storage conditions on secondary structure of proteins in the three SPI-based composite films were similar, which reflected in the β -sheet structure content increased during the storage of SPI-based composite films and high storage temperature increased the amount of β -sheet structure in the SPI-based composite films. Specifically, the β -sheet structure content in the SPI film, SPI:CS (1:1) composite film and SPI:CS (4:1) composite film increased to 39.2%, 39.3%, and 39.7%, respectively. The β -sheet structures were essential for network formation in the SPI-based composite films [34] and glycerol migration lead to an increase in β -sheet structures which glycerol as protein plasticizer was reported as helical agent [35]. Therefore, this result was interpreted as aging of the protein based films during the storage partly caused by plasticizer migration, promoted the reorganization of protein secondary structure in films into others with prevalence of extended β -sheet conformation. 4. Conclusions The continuous assessment of the storage-induced changes in plasticizer emigration and structural properties of SPI-based composite films provides valuable practical information about clarify the reasons of the performance changes of films during storage and regulate the stability of protein films. Results revealed that the migration rate of glycerol molecules in films was accelerated and the moisture content decreased more obvious when the SPI-based composite films were stored under 54% humidity conditions at high temperature (＞ 20 ℃). Free sulfhydryl group (SH) content and intermolecular forces results indicated that the decreased free SH partly formed new disulfide bonds and non-covalent inter-molecular interactions like hydrogen bonds changes significantly weakened the protein structure, which well explained the mechanical properties changes of the SPI-based films during storage. Finally, SDS-PAGE and FTIR results explained the protein polymerization and aggregation appeared in the protein network structure was bond with disulfide bonds and plasticizer migration caused by storage condition promoted the reorganization of protein structure in the SPI-based composite films into others with prevalence of extended β -sheet conformation. Declarations Funding This study was funded by the National Natural Science Foundation of China (No. U21A20270), the Science and Technology Project of Henan Province (212102110320) and the Science Foundation of Henan University of Technology (2020BS013). Conflict of Interest All authors have no competing interests of this manuscript. Author Contributions TW Zhu: Conceived and designed the experiment, analyzed the data and wrote the manuscript. PP Fan, LY Ma: Performed the format of the manuscript. YY Wang: Contributed the data of the manuscript. QY Li: Contributed the manuscript language. XF Guo: Supervised the study and helped to initiate the project. FS Chen: Supervised the study and helped to initiate the project. References R. Battisti, N. Fronza, Á.V. Júnior, S.M. da Silveira, M.S.P. Damas, M.G.N. Quadri, Gelatin-coated paper with antimicrobial and antioxidant effect for beef packaging. Food Packag. Shelf Life 11 , 115-124 (2017). E. Basiak, A. Lenart, F. Debeaufort, Effects of carbohydrate/protein ratio on the microstructure and the barrier and sorption properties of wheat starch-whey protein blend edible films. J. Sci. Food Agr. 97 , 858-867 (2017). W. Huo, D. Wei, W. Zhu, Z. Li, Y. 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Ma, Determination of glycinin in soybean and soybean products using a sandwich enzyme-linked immunosorbent assay. Food Chem. 162 , 27-33 (2014). A. Leerahawong, M. Tanaka, E. Okazaki, K. Osako, Stability of the physical properties of plasticized edible films from squid ( Todarodes pacificus ) mantle muscle during storage. J. Food Sci. 77 , E159-E165 (2012). E.M. Ciannamea, J.P. Espinosa, P.M. Stefani, R.A. Ruseckaite, Long-term stability of compression-molded soybean protein concentrate films stored under specific conditions. Food Chem. 4 3, 448-452 (2018). G.N. Piccirilli, M. Soazo, L.M. Pérez, N.J. Delorenzi, R.A. Verdini, Effect of storage conditions on the physicochemical characteristics of edible films based on whey protein concentrate and liquid smoke. Food Hydrocolls. 87 , 221-228 (2019). X.Y. Chen, Y. Ru, F.L. Chen, X.C. Wang, X.Y. Zhao, Q. Ao, FTIR spectroscopic characterization of soy proteins obtained through AOT reverse micelles. Food Hydrocoll. 3 1, 435-437 (2013). M. Subirade, I. Kelly, J. Guéguen, M. Pézolet, Molecular basis of film formation from a soybean protein: comparison between the conformation of glycinin in aqueous solution and in films. Int. J. Biol. Macromol. 23 , 241-249 (1998). E.M. Ciannamea, P.M. Stefani, R.A. Ruseckaite, Storage-induced changes in functional properties of glycerol plasticized-soybean protein concentrate films produced by casting. Food Hydrocoll. 45 , 247-255 (2015). Tables Table 1. Secondary structure of SPI-based composite films during storage by FTIR and analyzed using PeakFit V4.12 Samples Content of secondary structure (%) 𝛼-Helix 𝛽-Sheet 𝛽-Turns Random coil SPI 0w 12.58±0.20 38.13±1.20 36.55±0.98 12.75±0.01 5 ℃-12w 12.66±0.00 38.73±0.01 35.37±0.03 13.24±0.01 5 ℃-24w 12.57±0.11 38.79±0.35 35.68±0.06 12.96±0.18 20 ℃-12w 13.34±0.12 38.68±0.66 34.40±0.62 13.59±0.08 20 ℃-24w 13.15±0.02 38.91±0.16 34.45±0.16 13.50±0.01 35 ℃-12w 12.40±0.18 39.21±0.18 35.37±0.25 12.95±0.25 SPI:CS 1:1 0w 12.38±0.39 38.48±0.27 36.33±0.33 12.82±0.21 5 ℃-12w 12.81±0.09 37.97±0.21 36.19±0.30 13.04±0.01 5 ℃-24w 13.26±0.08 39.02±0.26 34.17±0.21 13.55±0.01 20 ℃-12w 12.64±0.02 38.83±0.18 35.32±0.19 13.22±0.03 20 ℃-24w 12.74±0.56 39.07±0.06 34.99±0.92 13.21±0.42 35 ℃-12w 12.62±0.08 39.29±0.20 34.76±0.03 13.37±0.08 SPI:CS 4:1 0w 12.59±0.16 38.19±1.05 36.96±0.18 12.76±0.01 5 ℃-12w 12.59±0.06 38.69±0.21 35.68±0.18 13.04±0.01 5 ℃-24w 12.40±0.18 39.29±0.18 35.37±0.25 13.30±0.02 20 ℃-12w 12.56±0.07 38.75±0.30 35.75±0.13 12.95±0.09 20 ℃-24w 12.34±0.06 39.51±0.16 35.19±0.49 12.96±0.28 35 ℃-12w 12.63±0.04 39.72±0.78 34.42±0.74 13.25 ±0.08 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 08 Nov, 2022 Reviews received at journal 27 Sep, 2022 Reviewers agreed at journal 14 Sep, 2022 Reviewers invited by journal 13 Sep, 2022 Submission checks completed at journal 18 Jul, 2022 Editor assigned by journal 18 Jul, 2022 First submitted to journal 14 Jul, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1859866","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":121850208,"identity":"d434c49c-d665-46be-9530-91e9279436e4","order_by":0,"name":"Tingwei Zhu","email":"","orcid":"","institution":"Henan University of Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tingwei","middleName":"","lastName":"Zhu","suffix":""},{"id":121850211,"identity":"794695a7-5da7-4e18-911f-59b2d7d4a095","order_by":1,"name":"Peipei Fan","email":"","orcid":"","institution":"Henan University of 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1","display":"","copyAsset":false,"role":"figure","size":196878,"visible":true,"origin":"","legend":"\u003cp\u003eScheme of preparing SPI-based composite films\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-1859866/v1/85025b4af0e39362adef32ef.png"},{"id":24288569,"identity":"1478e254-a779-4d9b-8fe8-933b633c7ca5","added_by":"auto","created_at":"2022-07-25 14:00:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":157350,"visible":true,"origin":"","legend":"\u003cp\u003eGlycerol emigration of SPI-based composite films during storage\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-1859866/v1/3030bc9260b8a4553e589c84.png"},{"id":24288567,"identity":"0fc0b66d-babd-4b13-981c-d96814bc1fb1","added_by":"auto","created_at":"2022-07-25 14:00:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":147963,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of moisture content in the SPI-based composite films during storage\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-1859866/v1/b38b0bd6cc7e36011b38329f.png"},{"id":24289715,"identity":"12b73f11-d007-4402-bc3d-a3ffb37d3cd3","added_by":"auto","created_at":"2022-07-25 14:10:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":141268,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of free sulfhydryl (SH) content in the SPI-based composite films during storage\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-1859866/v1/5ceca4c32ff790e75adcdbe5.png"},{"id":24288572,"identity":"a54f02a5-004d-4023-9311-2e9c6a286786","added_by":"auto","created_at":"2022-07-25 14:00:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":657582,"visible":true,"origin":"","legend":"\u003cp\u003eSolubility profiles of SPI-based composite films during storage in different reducing solvent (S1, Tris-HCl buffer, S2, Tris-HCl buffer + SDS, S3, Tris-HCl buffer + urea, S4, Tris-HCl buffer + SDS + urea, S5, Tris-HCl buffer + SDS + urea +\u003cem\u003eβ\u003c/em\u003e-mercaptoethanol)\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-1859866/v1/99fbfb0c3887a0e9da767ada.png"},{"id":24288571,"identity":"6f9020ba-1a6f-46fd-a3e9-723bb526ce9b","added_by":"auto","created_at":"2022-07-25 14:00:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":290426,"visible":true,"origin":"","legend":"\u003cp\u003eSDS-PAGE patterns of the SPI-based composite films s during storage (MW, marker, lane 0, SPI, lane 1-3, non-reductive condition: SPI film, SPI:CS (1:1) composite film, SPI:CS (4:1) composite film, respectively, lane 1’-3’, reduction condition: SPI film, SPI:CS (1:1) composite film, SPI:CS (4:1) composite film, respectively)\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-1859866/v1/eb810f64bdd112d3d83970a8.png"},{"id":24288573,"identity":"0b1103c1-aa12-48a4-bf4e-3b2a6b85c3d4","added_by":"auto","created_at":"2022-07-25 14:00:07","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":179846,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra of SPI-based composite films during storage\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-1859866/v1/e08d2e72a490562a58fd7da9.png"},{"id":24289716,"identity":"8af1f9cd-00b1-45c2-a641-4d850383cc9a","added_by":"auto","created_at":"2022-07-25 14:10:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1238613,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1859866/v1/c9fb2ecb-c49f-4a13-b5ec-b6902aec8c66.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Storage-induced changes in plasticizer emigration and structural properties of soybean isolate protein-based composite films","fulltext":[{"header":"1. Introduction ","content":"\u003cp\u003eWith the serious depletion of petroleum-based polymer materials and the enhancement of people\u0026apos;s environmental awareness, the growing demand of consumer to nature friendly packaging materials has caused more investigations focused on nature renewable, biodegradable polymers as raw materials for packaging [1]. In this sense, different kinds of biodegradable polymers like proteins, starches, polysaccharides and lipid biopolymers from renewable sources are considered as basic materials for packaging [2-3]. The ability to form a certain network structure is essential to maintain the mechanical properties and application of the films, as a result, with molecular structure of proteins covers a large number of active groups protein-based films has attracted attention in recent years [3].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSoy protein isolate (SPI) as a by-product of edible oil industry, has been valued in the field of packaging for its inexpensive, biocompatible and renewable advantages [4]. SPI-based films can be applied to degradable food packaging and coating with moderate mechanical properties and oil barrier properties [5]. However, it was worth noting that SPI-based films are generally using and stored under different environmental conditions, which influenced physical properties and application in packaging that are associated with storage-induced stability changes. Maryam et al found that the SPI film showed stable antioxidant activity for 90 days of storage at 25 ℃ [6]. While, Srikiatden et al studied that the mechanical properties of SPI films decreased after 2-3 months storage period, resulting in impossible to using widely in packaging [7].\u003c/p\u003e\n\u003cp\u003eIn fact, the storage stability including mechanical properties, oil barrier property, water solubility and practical application of the films is related to the internal structure changes of films during storage. During the storage process of the films, variable storage environment greatly favored the components in films such as moisture and plasticizer migration, and interaction between the components in the films [8]. These eventually lead to changes in internal structure of films. In conclusion, it is essential to study the migration and interaction of the internal components in the films during storage so as to clarify the reasons of the performance changes of films during storage, which is crucial for predicting and regulating the stability of protein films and then reasonable application of the films.\u003c/p\u003e\n\u003cp\u003eIn the previous studies, we successfully prepared SPI-based composite films added by corn starch (CS) (SPI:CS (4:1) composite film, SPI:CS (1:1) composite film) with good mechanical properties [9]. Then further studied the effect of temperature on physical properties and application in packaging of the SPI-based composite films during storage (this manuscript is under submitting). In order to accordingly understanding the causes driving stability changes in performance of the SPI-based composite films and then controlling long-term stability of physical properties and practical application of the SPI-based composite films, in this ongoing work, storage-induced changes in plasticizer emigration and structural properties of SPI-based composite films were investigated in this study. Firstly, the three SPI-based composite films (SPI:CS (4:1) composite film, SPI:CS (1:1) composite film, SPI film) were prepared, and three storage temperatures (5 ℃, 20 ℃ and 35 ℃) were selected according to the actual seasoning package application. Then plasticizer emigration about changes of glycerol content and moisture content in the SPI-based composite films during storage was monitored. At the same time, changes in structural properties of the SPI-based composite films during storage were determined by free sulfhydryl group content test, intermolecular forces characterization, SDS-PAGE and FTIR, respectively.\u0026nbsp;\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003ch2\u003e2.1 Materials\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eSoy protein isolate (SPI, protein content: 92.1%) was supplied by Wandefu Co. (Shandong, China) and corn starch (CS) was obtained from a local grocery store. All other reagents and solvents which were purchased from Tianjin Ruijinte Co. Ltd. (Tianjin, China) were of analytically pure grade.\u003c/p\u003e\n\u003ch2\u003e2.2 Preparation of SPI-based composite films\u003c/h2\u003e\n\u003cp\u003eThe soy protein isolate-based composite films including\u0026nbsp;SPI:CS\u0026nbsp;composite films (SPI:CS 1:1, SPI:CS 4:1)\u0026nbsp;and\u0026nbsp;SPI film\u0026nbsp;were prepared using a solvent-cast method described in our previous study [10] and the specific steps of making\u0026nbsp;SPI-based composite films were shown in \u003cstrong\u003eFigure 1\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e2.3 Storage protocol\u003c/h2\u003e\n\u003cp\u003eThe SPI-based\u0026nbsp;composite films\u0026nbsp;including SPI:CS\u0026nbsp;composite films\u0026nbsp;(SPI:CS 1:1, SPI:CS 4:1) and the control film (SPI film) were cut to the same size and then stored at\u0026nbsp;5 ℃, 20 ℃ and 35 ℃ with\u0026nbsp;relative humidity (RH) 54% for 24 weeks, 16 weeks, and 8 weeks, respectively. The stop of experiment after a period of storage at certain temperature environment was because too fragile or moldy phenomenon was occurred in the sample. The three selected temperature represented the common environments of the film during application or storage. At least three parallel experiments were performed. Then the\u0026nbsp;glycerol\u0026nbsp;and moisture emigration, structural properties changes of the SPI-based films were analyzed by different ways.\u003c/p\u003e\n\u003ch2\u003e2.4 Glycerol emigration\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThe glycerol content of the SPI-based composite films during storage was determined by the cold oxidation of sodium iodate method, according to the description of Guo and Huang [11]. The glycerol in the accurately weighed SPI-based composite film was extracted in a 250 mL conical flask with 150 mL distilled water for 4 h, and then the conical flask was adjusted to pH 7.9 \u0026plusmn; 0.1 with NaOH solution (0.05 mol/L). Followed steps by the cold oxidation were: 5 mL sodium periodate solution (60 g/L) was added in the conical flask and then placed in a sealed dark condition at 35 ℃ for 30 min, then 5 mL ethylene glycol was add and placed for 20 min. Finally, 5 mL sodium formate solution (1 mol/L) was added and the solution was titrated with NaOH to 7.9 \u0026plusmn; 0.1 by pH meter indication. The calculation formula of glycerol migration rate (K) in the SPI-based composite film was as follows:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/p\u003e\n\u003cp\u003eWhere \u003cem\u003eV\u003csub\u003e1\u003c/sub\u003e\u003c/em\u003e (mL) was the volume of NaOH standard solution consumed by the SPI-based composite film determination, \u003cem\u003eV\u003csub\u003e2\u003c/sub\u003e\u003c/em\u003e (mL) was the volume of NaOH standard solution consumed by the blank without the SPI-based composite film, m (g) was the mass of the SPI-based composite film, C (mol/L) was the concentration of NaOH standard solution, 0.0921 (g/mmol) was the molar mass of glycerol.\u003c/p\u003e\n\u003ch2\u003e2.5 Moisture content\u003c/h2\u003e\n\u003cp\u003eConsulted the description of Guo et al [12], the moisture content of the SPI-based composite films during storage was determined according to the ASTM D644-99 (ASTM standard test method for moisture content of paper and paperboard by oven drying)\u003c/p\u003e\n\u003ch2\u003e2.6 Free sulfhydryl group content\u003c/h2\u003e\n\u003cp\u003eThe free sulfhydryl group (-SH) of the SPI-based composite films during storage was measured by the Ellman\u0026rsquo;s method according to the determination process described by Chen and Wasseman (1993) [13].\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e2.7 Solubility of proteins in different denaturing solvents\u003c/h2\u003e\n\u003cp\u003eThe Solubility of protein in different denaturing solvents was utilized to evaluate the intermolecular forces of proteins in the films [14]. It was determined according to the procedure of\u0026nbsp;Ciannamea\u0026nbsp;et al [15] and\u0026nbsp;Hager\u0026nbsp;[16] with some modification. The denaturing solvents were:\u0026nbsp;0.086 moL/L Tris-HCl\u0026nbsp;buffer\u0026nbsp;containing 0.09 mol/L glycine and 4 mmol/L Na\u003csub\u003e2\u003c/sub\u003eEDTA (pH 8.0) (S1), 0.086 moL/L Tris-HCl\u0026nbsp;buffer\u0026nbsp;containing 0.09 mol/L glycine, 4 mmol/L Na\u003csub\u003e2\u003c/sub\u003eEDTA and 5 mg/mL SDS (S2), 0.086 moL/L Tris-HCl\u0026nbsp;buffer\u0026nbsp;containing 0.09 mol/L glycine, 4 mmol/L Na\u003csub\u003e2\u003c/sub\u003eEDTA and 8 mg/L urea (S3), 0.086 moL/L Tris-HCl\u0026nbsp;buffer\u0026nbsp;containing 0.09 mol/L glycine, 4 mmol/L Na\u003csub\u003e2\u003c/sub\u003eEDTA, 5 mg/mL SDS and 8 mg/L urea (S4), 0.086 moL/L Tris-HCl\u0026nbsp;buffer\u0026nbsp;containing 0.09 mol/L glycine, 4 mmol/L Na\u003csub\u003e2\u003c/sub\u003eEDTA, 5 mg/mL SDS, 8 mg/L urea and 25 mg/mL\u0026nbsp;\u003cem\u003e\u0026beta;\u003c/em\u003e-mercaptoethanol\u0026nbsp;(S5).\u003c/p\u003e\n\u003cp\u003eThe specific determination steps were as follows. 100 mg of SPI-based\u0026nbsp;composite film\u0026nbsp;sample was uniformly dispersed in 5 mL above\u0026nbsp;denaturing solvent (S1-S5) for 12 h, and then centrifuged at 5000 r/min for 20 min to collect the supernatant. Then 1 mL the supernatant was combined with 4 mL biuret reagent and the protein content was analyzed by biuret spectrophotometry method at 530 nm.\u003c/p\u003e\n\u003ch2\u003e2.8 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)\u003c/h2\u003e\n\u003cp\u003eSDS-PAGE was conducted on a discontinuous buffered system using a separating gel (12%) and a stacking gel (5%) [17]. The protein sample (protein concentration 5 mg/mL) was prepared by adding a sample buffer (Reductive buffer: containing pH 6.8 Tris-HCl buffer 2.5 mL, glycerol 4 mL, SDS 4 mL, bromophenol blue 0.5 mL,\u0026nbsp;𝛽-mercaptoethanol 1 mL, Non-reductive buffer: containing pH 6.8 Tris-HCl buffer 2.5 mL, glycerol 4 mL, SDS 4 mL, bromophenol blue 0.5 mL) and heated for 5 min in boiling water before electrophoresis. Gels were run at a constant voltage of 80 V (lasting about 40 min) before changing to 120 V when the bromophenol blue indicator ran into the separating gel (lasting about 60 min). After electrophoresis, proteins in gels were stained with Coomassie Brilliant Blue R-250.\u003c/p\u003e\n\u003ch2\u003e2.9 Fourier transform infrared spectrum (FTIR)\u003c/h2\u003e\n\u003cp\u003eA Nicolet fourier transform infrared spectrum equipped with a computer (Thermo Nicolet Corporation, USA) was used for FTIR analysis. The dried and broken film was mixed with potassium bromide (KBr) (1:100, w/w), ground with a mortar to form homogeneous powder, pressed into a sheet and scanned from 4,000 to 400 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e within 16 scans [18].\u003c/p\u003e\n\u003ch2\u003e2.10. Statistical analysis\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThe experiments were performed in triplicate (n = 3), and the data were expressed as mean \u0026plusmn; standard deviation (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA). A value of P \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"3. Results and Discussion","content":"\u003ch2\u003e3.1 The select SPI-based films\u003c/h2\u003e\n\u003cp\u003eIn our previous studies, we optimized the prepared conditions and fabricated the SPI-based composite films successfully by monitoring the evaluation indexes of the tensile strength and elongation at break [9]. The formed SPI:CS (1:1) composite film exhibited good mechanical property and the SPI:CS (4:1) composite film showed better stability during the short-term storage pre-experiment. Furthermore, during the study about effect of storage conditions on physical properties and application in packaging of the SPI-based composite films, we found that the glycerol and moisture as the plasticizers, which influenced the internal structure, played improvement role in the physical properties and application of the SPI-based composite films during storage. Therefore, the plasticizer emigration and structural properties of the SPI-based composite films during storage at different temperature and time were studies in order to expound the change mechanism of the SPI-based composite films during storage and provide theoretical guidance for the application.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e3.2 Plasticizer migration in the SPI-based composite films during storage\u003c/h2\u003e\n\u003cp\u003ePolyols and water are often used as plasticizers in the hydrosol-based films [19].\u0026nbsp;Small molecules of glycerol and moisture in the films can migrate during storage, which may result in the property changes of the films [20]. Therefore,\u0026nbsp;the effect of storage conditions on the plasticizer emigration was explored by measuring the changes of glycerol content and moisture content in the SPI-based composite film during storage at different temperature and time.\u003c/p\u003e\n\u003ch2\u003e3.2.1 Glycerol migration changes in the SPI-based films during storage\u003c/h2\u003e\n\u003cp\u003eThe\u0026nbsp;glycerin\u0026nbsp;content of the SPI-based composite films during storage was determined by the cold oxidation of sodium iodate method. The glycerol emigration changes in the SPI-based composite films during storage were shown in \u003cstrong\u003eFigure 2\u003c/strong\u003e. As can be seen in Figure 2(A), the glycerol migration rate in the SPI film were 9.0%, 9.3%, 10.9% after 8 weeks of storage at 5 ℃, 20 ℃, 35 ℃, respectively. Furthermore, with the extension of storage time, the\u0026nbsp;glycerol migration was faster at the higher temperature (35 ℃). It can be concluded that the migration rate of glycerol molecules in the film was accelerated when the film was stored at high temperature, which was due to the accelerated movement of the glycerol when the storage temperature increased. This was also consistent with the research result of Ket-On et al [21].\u003c/p\u003e\n\u003cp\u003eSimilarly, the effect of storage conditions on the glycerol mobility in the other SPI-based composite films including the SPI:CS (1:1) composite film and SPI:CS (4:1) composite film was consistent with that in SPI film. While, under the same storage conditions, the glycerol mobility in the SPI:CS (1:1) composite film was higher than that in the other SPI-based composite films (SPI:CS (4:1) composite film, SPI film). As the glycerol migration rate in the SPI:CS (1:1) composite film were 10.6%, 10.6%, 12.3% after 8 weeks of storage at 5 ℃, 20 ℃, 35 ℃, respectively. It was speculated that when the SPI-based\u0026nbsp;composite films\u0026nbsp;with more corn starch addition, partial glycerol was combined with starch by weak hydrogen bonds and reduced the glass transition temperature, thus this part of glycerol was more likely to migrate during storage [22].\u003c/p\u003e\n\u003ch2\u003e3.2.2 Moisture content changes in the SPI-based films during storage\u003c/h2\u003e\n\u003cp\u003eThe moisture content in the film also had influence on the performance of the film, and the moisture content changes were also affected by the storage conditions. \u003cstrong\u003eFigure 3\u003c/strong\u003e depicted\u0026nbsp;the changes of moisture content in the SPI-based composite films during storage. Generally, under certain humidity condition (RH 54%), low temperature storage (5 ℃) had little effect on the moisture content of the SPI-based composite films. While when SPI-based composite films stored at 20 ℃ or 35 ℃, the moisture content decreased more obvious with the increase of storage temperature, indicating that increased storage temperature promoted the evaporation of moisture content in SPI-based composite films. The moisture molecules moved slowly when the films stored at low temperature, which slowed down the moisture migration from the inner to the surface in the films. Chinma et al also found that less moisture was restricted when the film stayed at high temperature condition and the moisture was easier to migrate [23]. For the three SPI-based composite films were stored at 35 ℃ for 8 weeks, the moisture content in SPI:CS (1:1) composite film, SPI:CS (4:1) composite\u0026nbsp;film and SPI film reduced to about 13.0%, 15.5%, 15.0%, respectively. This may be due to less binding sites between protein and water existed in the SPI:CS (1:1) composite film with more CS addition [24].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCombined with our previous study which was the physical properties changes of the SPI-based composite films during storage, it can be concluded that the mechanical properties and application of the SPI-based composite films were affected by glycerol and moisture changes. Therefore, the structural properties of the SPI-based composite films during storage were further studied to explain the mechanism of the plasticizer migration.\u003c/p\u003e\n\u003ch2\u003e3.3 Structural properties of SPI-based composite films during storage\u003c/h2\u003e\n\u003cp\u003eThe structural properties including the free sulfhydryl group content, intermolecular forces, secondary structure by SDS-PAGE and FTIR of the SPI-based composite films during storage were further investigated in this study.\u003c/p\u003e\n\u003ch2\u003e3.3.1 Free sulfhydryl group content in the SPI-based composite films during storage\u003c/h2\u003e\n\u003cp\u003eThe changes of\u0026nbsp;sulfhydryl group content is a dynamic process during the storage of the SPI-based films, which reflected the network structure changes of the protein based films to a certain extent. Hence,\u0026nbsp;changes of the free sulfhydryl (-SH) content in SPI-based composite films during storage\u0026nbsp;were determined and the results were exhibited in \u003cstrong\u003eFigure 4\u003c/strong\u003e. On the whole, the content of free SH in the three SPI-based\u0026nbsp;composite films\u0026nbsp;was decreased during storage, implying the formation of new disulfide bonds. This result was similar to those obtained by Ciannmea et al, which reported an increment of intermolecular disulfide bound in concentrate soy protein-based film during storage which developing of S-S covalent interaction [25]. What\u0026rsquo;s more,\u0026nbsp;the free SH content in SPI:CS (1:1) composite film, SPI:CS (4:1) composite film and SPI film reduce about 0.93-1.14 \u0026mu;mol/g (Fig. 4C), 0.95-1.71 \u0026mu;mol/g (Fig. 4B), 0.89-1.62 \u0026mu;mol/g (Figure 4A), respectively, after the end of storage at high temperature (﹥\u0026nbsp;20 ℃).\u0026nbsp;The free SH content in the SPI-based films decreased along with the rising of storage temperature, indicating that higher storage temperature could facilitate S-S bonds formation during storage. Disulfide bond is an important form of covalent binding in the protein molecules. The protein network structure in protein-based films becomes denser and polymer formed in protein-based films fluidity becomes worse [26], which leads to the increase of tensile strength and decrease of elongation at break. This phenomenon was also consistent with the mechanical property changes of the SPI-based films during storage studied in our previous study.\u003c/p\u003e\n\u003ch2\u003e3.3.2 Intermolecular forces in the SPI-based composite films during storage\u003c/h2\u003e\n\u003cp\u003eThe maintenance of\u0026nbsp;protein network structure in protein-based films was mainly depended on the intermolecular/intramolecular acting force such as hydrogen bonds, hydrophobic interactions and S-S bonds. The type of\u0026nbsp;protein aggregation interactions in SPI-based\u0026nbsp;composite films\u0026nbsp;were described with the solubility of proteins in different reducing solvents. Among the reducing solvents, urea could disrupt hydrogen bonds, SDS could disrupt hydrogen bonds and hydrophobic interactions, and \u003cem\u003e\u0026beta;\u003c/em\u003e-mercaptoethanol could cleave S-S [27].\u003c/p\u003e\n\u003cp\u003eAs can be seen in \u003cstrong\u003eFigure 5(A1-A3)\u003c/strong\u003e, the protein solubility of the SPI film in S1, S2, S3 were about 18 mg/g, 21 mg/g, 46 mg/g, respectively, implying hydrogen bonds was the main force in maintaining network structure in the SPI film and disulfide bonds was also existed. Similarly, from \u003cstrong\u003eFigure 5 (B1-B3)\u0026nbsp;\u003c/strong\u003eand \u003cstrong\u003eFigure 5(C1-C3)\u003c/strong\u003e, the decreased of protein solubility in the presence of SDS and urea (S4), urea (S3) and SDS (S2) after the end of storage at the same temperature, suggested that non-covalent inter-molecular interactions (hydrogen bonds and/or hydrophobic interactions) changes significantly weakened the protein structure in the two SPI-CS composite films. Compared with SPI film, the SPI-CS composite films showed low protein solubility in S3 under the same storage conditions. This may be attributed to diluted protein content with addition of starch to a certain extent inducing the protein molecular spacing in the film.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e3.3.3 SDS-PAGE\u003c/h2\u003e\n\u003cp\u003eIn order to further investigate the internal structure of the SPI-based composite films during storage, both non-reductive SDS-PAGE and reductive SDS-PAGE were used to characterize the protein structure in the SPI-based composite films during storage. SPI is mainly composed of glycinin (11S) and conglycinin (7S). 7S is aggregated into trimer that contains \u0026alpha; (~67 kDa), \u0026alpha;\u0026acute; (~71 kDa), and \u0026beta; (~50 kDa) subunit and 11S is heterogeneous subunits associated with acidic subunits (A, 31-45 kDa) and basic subunits (B, 18-20 kDa) through disulfide bonds connection [28-29]. \u003cstrong\u003eFigure 6(A)\u003c/strong\u003e depicted the SDS-PAGE patterns of the SPI-based composite films before storage. The color of the lanes in reductive electrophoretic bands were significantly lighter than that in non-reductive electrophoretic bands, especially the lighter color of the bands located in the separation gal with a new band appeared near the A subunits, implying disulfide bonds existed in the protein network structure of the SPI-based composite films and protein polymer with high molecular weight were bond with disulfide bonds. Moreover, from the \u003cstrong\u003eFigure 6\u003c/strong\u003e, the color of bands in the reductive electrophoretic condition gradually became light during the storage of the SPI-based composite films, indicating the development of crosslink with non-covalent bonds between the protein molecules in the SPI-based composite films during the storage. Also, this speculation was supported by the results in intermolecular forces results (\u003cstrong\u003eFigure 5\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eCompared to the SDS-PAGE patterns of the three SPI-based composite films (SPI:CS (1:1) composite film, SPI:CS (4:1) composite film, SPI film) (\u003cstrong\u003eFigure 6\u003c/strong\u003e), it was found that the dark color of bands in the three lanes when SPI-based composite films were stored in the same condition showed the following order: lane 2\u0026nbsp;﹥\u0026nbsp;lane 3\u0026nbsp;﹥\u0026nbsp;lane 1. This can be explained by the polymerized of more protein molecules in the SPI film. With addition of starch in the SPI:CS composite films increased the spacing in protein molecules thus weakening the protein polymerization in the SPI:CS composite films during storage. What\u0026rsquo;s more, some study found that Maillard reaction could also occurred in glycerol- and sorbitol-plasticized film during storage and the higher storage temperature may accelerate the reaction [30-31]. It also can be seen from \u003cstrong\u003eFigure 6\u0026nbsp;\u003c/strong\u003ethat smear band with high molecular weight appeared at the top of the stacking gel and the bands of protein subunits became shallow when the SPI-based composite films was stored at high temperature with the storage time. Therefore, it could be inferred the decrease in protein subunits of the films may be caused by the protein polymerization and aggregation via Maillard reaction. These results are correlated very well with the changes in light transmittance property of the SPI-based composite films during storage which was our simultaneous research result about the SPI-based composite films (this manuscript is\u0026nbsp;under submitting).\u003c/p\u003e\n\u003ch2\u003e3.3.4 FTIR\u003c/h2\u003e\n\u003cp\u003eThe intermolecular/intramolecular interactions of the ingredients in SPI-based\u0026nbsp;composite films\u0026nbsp;were investigated by using FTIR. The FTIR spectra of SPI-based\u0026nbsp;composite films\u0026nbsp;during storage at diverse temperatures with different times were shown in \u003cstrong\u003eFigure 7\u003c/strong\u003e.\u0026nbsp;Previous study reported that the regions of 800-1200 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e and 3000-3600 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e were the absorption bands of glycerol and the main absorption peak of free and bound O-H and N-H groups, respectively, else it was a broad absorption band nearly 3200 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e for all proteins [32]. Hence,\u0026nbsp;during the storage of the three SPI-based\u0026nbsp;composite films, the absorption of films in the regions of 1000-1200 cm\u003csup\u003e-1\u003c/sup\u003e and 3200-3700cm\u003csup\u003e-1\u003c/sup\u003e were weak (\u003cstrong\u003eFigure 7A-C\u003c/strong\u003e), indicating migration of glycerol and moisture molecules in the SPI-based\u0026nbsp;composite films\u0026nbsp;during storage weakened hydrogen bonding of intermolecular proteins. In addition, compared with the SPI film, the two\u0026nbsp;SPI:CS composite films (SPI:CS 4:1, SPI:CS 1:1) showed strong peak in the region of 1000-1200 cm\u003csup\u003e-1\u003c/sup\u003e and broadens in the range of 3200-3700cm\u003csup\u003e-1\u003c/sup\u003e, indicating the cross-linking between SPI and CS increased the\u0026nbsp;bound O-H.\u0026nbsp;Furthermore, the higher the storage temperature was, the weaker the absorption in the regions of 1000-1200 cm\u003csup\u003e-1\u003c/sup\u003e and 3200-3700cm\u003csup\u003e-1\u003c/sup\u003e. This is also consistent with the above results in the study on plasticizer migration (\u003cstrong\u003eFigure 2, Figure 3\u003c/strong\u003e) and intermolecular forces (\u003cstrong\u003eFigure 5\u003c/strong\u003e)\u0026nbsp;in the SPI-based films during storage.\u003c/p\u003e\n\u003cp\u003eIn the characteristic absorption band for protein amino I, the 1600-1700 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e range was reflected protein secondary structure, and corresponding parameters used in the study were:\u0026nbsp;𝛼-helix structure, 1650-1660 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e,\u0026nbsp;𝛽-sheet structure, 1600-1640 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e,\u0026nbsp;𝛽-turn structure, 1660-1700 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e and\u0026nbsp;random coil\u0026nbsp;structure, 1640-1650 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e [33].The data about\u0026nbsp;FTIR spectra\u0026nbsp;(\u003cstrong\u003eFigure 7\u003c/strong\u003e) were analyzed using Peak-Fit V4.12 software, which depicted the secondary structure as shown in \u003cstrong\u003eTable 1\u003c/strong\u003e.\u0026nbsp;The effects of storage conditions on secondary structure of proteins in the three SPI-based\u0026nbsp;composite films\u0026nbsp;were similar, which reflected in the \u003cem\u003e\u0026beta;\u003c/em\u003e-sheet structure content increased during the storage of SPI-based\u0026nbsp;composite films\u0026nbsp;and high storage temperature increased the amount of \u003cem\u003e\u0026beta;\u003c/em\u003e-sheet structure in the SPI-based\u0026nbsp;composite films. Specifically, the \u003cem\u003e\u0026beta;\u003c/em\u003e-sheet structure content in the SPI film,\u0026nbsp;SPI:CS (1:1) composite film and SPI:CS (4:1) composite film increased to 39.2%, 39.3%, and 39.7%, respectively.\u0026nbsp;The \u003cem\u003e\u0026beta;\u003c/em\u003e-sheet structures were essential for network formation in the SPI-based\u0026nbsp;composite films\u0026nbsp;[34] and glycerol migration lead to an increase in\u0026nbsp;\u003cem\u003e\u0026beta;\u003c/em\u003e-sheet structures which\u0026nbsp;glycerol as protein plasticizer was reported as helical agent [35]. Therefore, this result was interpreted as aging of the protein based films during the storage partly caused by plasticizer migration, promoted the reorganization of protein secondary structure in films into others with prevalence of extended \u003cem\u003e\u0026beta;\u003c/em\u003e-sheet conformation.\u0026nbsp;\u003c/p\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eThe continuous assessment of the storage-induced changes in plasticizer emigration and structural properties of SPI-based composite films provides valuable practical information about clarify the reasons of the performance changes of films during storage and regulate the stability of protein films. Results revealed that the migration rate of glycerol molecules in films was accelerated and the moisture content decreased more obvious when the SPI-based composite films were stored under 54% humidity conditions at high temperature (＞\u0026nbsp;20\u0026nbsp;℃). Free sulfhydryl group (SH) content and intermolecular forces results indicated that the decreased free SH partly formed new disulfide bonds and non-covalent inter-molecular interactions like hydrogen bonds changes significantly weakened the protein structure, which well explained the mechanical properties changes of the SPI-based films during storage. Finally,\u0026nbsp;SDS-PAGE and FTIR results explained the\u0026nbsp;protein polymerization and aggregation appeared in the protein network structure was bond with disulfide bonds and\u0026nbsp;plasticizer migration caused by storage condition promoted the reorganization of protein structure in the SPI-based\u0026nbsp;composite films\u0026nbsp;into others with prevalence of extended \u003cem\u003e\u0026beta;\u003c/em\u003e-sheet conformation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the National Natural Science Foundation of China (No. U21A20270), the Science and Technology Project of Henan Province (212102110320) and the Science Foundation of Henan University of Technology (2020BS013).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have no competing interests of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTW Zhu: Conceived and designed the experiment, analyzed the data and wrote the manuscript.\u003c/p\u003e\n\u003cp\u003ePP Fan, LY Ma: Performed the format of the manuscript.\u003c/p\u003e\n\u003cp\u003eYY Wang: Contributed the data of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eQY Li: Contributed the manuscript language.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eXF Guo: Supervised the study and helped to initiate the project.\u003c/p\u003e\n\u003cp\u003eFS Chen: Supervised the study and helped to initiate the project.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eR. 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P\u0026eacute;zolet, Molecular basis of film formation from a soybean protein: comparison between the conformation of glycinin in aqueous solution and in films. Int. J. Biol. Macromol. \u003cstrong\u003e23\u003c/strong\u003e, 241-249 (1998).\u003c/li\u003e\n\u003cli\u003eE.M. Ciannamea, P.M. Stefani, R.A. Ruseckaite, Storage-induced changes in functional properties of glycerol plasticized-soybean protein concentrate films produced by casting. Food Hydrocoll. \u003cstrong\u003e45\u003c/strong\u003e, 247-255 (2015).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Secondary structure of SPI-based\u0026nbsp;composite films\u0026nbsp;during storage by FTIR and analyzed using PeakFit V4.12\u003c/p\u003e\n\u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" width=\"32.926829268292686%\"\u003e\n \u003cp\u003eSamples\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"top\" width=\"67.07317073170732%\"\u003e\n \u003cp\u003eContent of secondary structure (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"24.61139896373057%\"\u003e\n \u003cp\u003e𝛼-Helix\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"24.61139896373057%\"\u003e\n \u003cp\u003e𝛽-Sheet\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"24.61139896373057%\"\u003e\n \u003cp\u003e𝛽-Turns\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.16580310880829%\"\u003e\n \u003cp\u003eRandom coil\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"6\" width=\"16.493055555555557%\"\u003e\n \u003cp\u003eSPI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.493055555555557%\"\u003e\n \u003cp\u003e0w\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.493055555555557%\"\u003e\n \u003cp\u003e12.58\u0026plusmn;0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.493055555555557%\"\u003e\n \u003cp\u003e38.13\u0026plusmn;1.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.493055555555557%\"\u003e\n \u003cp\u003e36.55\u0026plusmn;0.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.53472222222222%\"\u003e\n \u003cp\u003e12.75\u0026plusmn;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"19.75051975051975%\"\u003e\n \u003cp\u003e5 ℃-12w\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.75051975051975%\"\u003e\n \u003cp\u003e12.66\u0026plusmn;0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.75051975051975%\"\u003e\n \u003cp\u003e38.73\u0026plusmn;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.75051975051975%\"\u003e\n \u003cp\u003e35.37\u0026plusmn;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.997920997920996%\"\u003e\n \u003cp\u003e13.24\u0026plusmn;0.01\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"19.75051975051975%\"\u003e\n \u003cp\u003e5 ℃-24w\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.75051975051975%\"\u003e\n \u003cp\u003e12.57\u0026plusmn;0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.75051975051975%\"\u003e\n \u003cp\u003e38.79\u0026plusmn;0.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.75051975051975%\"\u003e\n \u003cp\u003e35.68\u0026plusmn;0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.997920997920996%\"\u003e\n \u003cp\u003e12.96\u0026plusmn;0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"19.75051975051975%\"\u003e\n \u003cp\u003e20 ℃-12w\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.75051975051975%\"\u003e\n \u003cp\u003e13.34\u0026plusmn;0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.75051975051975%\"\u003e\n \u003cp\u003e38.68\u0026plusmn;0.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.75051975051975%\"\u003e\n \u003cp\u003e34.40\u0026plusmn;0.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.997920997920996%\"\u003e\n \u003cp\u003e13.59\u0026plusmn;0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"19.75051975051975%\"\u003e\n \u003cp\u003e20 ℃-24w\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.75051975051975%\"\u003e\n \u003cp\u003e13.15\u0026plusmn;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.75051975051975%\"\u003e\n \u003cp\u003e38.91\u0026plusmn;0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.75051975051975%\"\u003e\n \u003cp\u003e34.45\u0026plusmn;0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.997920997920996%\"\u003e\n \u003cp\u003e13.50\u0026plusmn;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"19.75051975051975%\"\u003e\n \u003cp\u003e35 ℃-12w\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd 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\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"food-biophysics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food Biophysics](https://www.springer.com/journal/11483)","snPcode":"11483","submissionUrl":"https://submission.nature.com/new-submission/11483/3","title":"Food Biophysics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Soybean isolate protein, Corn starch, Composite films, Storage temperature, Plasticizer emigration, Structural property","lastPublishedDoi":"10.21203/rs.3.rs-1859866/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1859866/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Previous study found soybean isolate protein (SPI)-based composite films added by corn starch (CS) (SPI:CS (4:1) composite film, SPI:CS (1:1) composite film) exhibited good mechanical property and better stability during the short-term storage pre-experiment. These properties were related to internal structure changes. In order to accordingly understanding the causes driving stability changes in performance and then controlling long-term stability of the SPI-based composite films, plasticizer emigration and structural properties of SPI-based composite films stored at different temperatures (5 ℃, 20 ℃, 35 ℃) and 54% relative humidity were systematically investigated over a period of time. High storage temperature (＞ 20 ℃) accelerated plasticizer emigration which reflected in increased migration rate of glycerol molecules and decreased moisture content. During the storage of SPI-based composite films, the decreased free SH partly formed new disulfide bonds and non-covalent inter-molecular interactions like hydrogen bonds changes significantly weakened the protein structure, which influenced mechanical properties changes during storage. PAGE and FTIR results explained the protein aggregation was bond with disulfide bonds and the reorganization of protein structure with prevalence of extended β-sheet conformation.","manuscriptTitle":"Storage-induced changes in plasticizer emigration and structural properties of soybean isolate protein-based composite films","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-07-25 14:00:05","doi":"10.21203/rs.3.rs-1859866/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-11-09T01:08:21+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-09-27T19:30:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"45ec03a0-704a-4e38-93b8-ca578e4a45c7","date":"2022-09-14T13:16:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-09-14T03:08:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-07-18T07:12:31+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-07-18T07:12:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Food Biophysics","date":"2022-07-15T02:48:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"food-biophysics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food Biophysics](https://www.springer.com/journal/11483)","snPcode":"11483","submissionUrl":"https://submission.nature.com/new-submission/11483/3","title":"Food Biophysics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"82bbde81-f77b-4645-8d53-3a263c1ca90b","owner":[],"postedDate":"July 25th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2023-05-25T13:29:19+00:00","versionOfRecord":[],"versionCreatedAt":"2022-07-25 14:00:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1859866","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1859866","identity":"rs-1859866","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}