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Physical and structural properties of peanut protein isolate-gum Arabic films prepared by various glycation time
Food Hydrocolloids 43 (2015) 322e328
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Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd
Physical and structural properties of peanut protein isolate-gum Arabic films prepared by various glycation time Chen Li, Weizhe Zhu, Haoran Xue, Zhiyan Chen, Yajing Chen, Xingguo Wang* State Key Laboratory of Food Science and Technology, Synergetic Innovation Center of Food Safety and Nutrition, School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, Jiangsu, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 November 2013 Accepted 3 June 2014 Available online 12 June 2014
Peanut protein isolate (PPI) was glycated with gum Arabic for 3, 6 and 9 days respectively to study the effect of different degree of graft on the physical and structural properties of glycated films. As the glycation continued, the degree of graft of PPI-gum Arabic conjugates increased gradually, the degree of crosslinking between PPI and gum Arabic in films also increased. Compared to PPI films, glycated films had increased tensile strength, decreased water vapor permeability (WVP), but decreased elongation. As the glycation proceeded from 3 to 9 days, the tensile strength of glycated films decreased, the WVP and the elongation increased gradually. This phenomenon may be caused by the decreased crystalline and increased amorphous structure of glycated films with the glycation time, which was confirmed by X-ray diffractometry and scanning electron microscopy. Disulfide bonds were the predominant interactive forces involved in all film networks. The result suggested that as the glycation continued, the level of disulfide bonds in PPI-gum Arabic films reached equilibrium after 6 days glycation. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Peanut protein isolate Gum Arabic Glycation Films
1. Introduction There is an increasing interest in developing biopolymers such as proteins, polysaccharides, lipids and their composites for packaging materials. They are considered to be a promising solution to environmental pollution and hold promise for innovative uses in food protection and preservation. Numerous proteins have been used to develop films, such as whey protein, soy protein, corn zein, sunflower protein, gelatin and others (Ferreira, Nunes, Delgadillo, & Lopes-da-Silva, 2009; Ma et al., 2012; Orliac, Rouilly, Silvestre, & Rigal, 2002; Parris & Coffin, 1997; Stuchell & Krochta, 1994; Teerakarn, Hirt, Acton, Rieck, & Dawson, 2002). Peanut is one of the most important oil crops in China. Peanut meal is a byproduct generated during oil extraction and is mainly used as animal feed and fertilizer due to the poor protein solubility, dark color, as well as unpleasant flavor (Su, Ren, Yang, Cui, & Zhao, 2011). Peanut protein isolate (PPI), extracted from peanut meal, has higher protein content and good functional properties, which could be a good source to develop films for various applications. Protein films have inferior physical and mechanical properties compared to synthetic films, thus numerous efforts have been
* Corresponding author. Tel./fax: þ86 510 85876799. E-mail address: [email protected] (X. Wang). http://dx.doi.org/10.1016/j.foodhyd.2014.06.003 0268-005X/© 2014 Elsevier Ltd. All rights reserved.
made to enhance the properties. Some researches focused on the optimization of film formulation and preparation conditions (Liu, Tellez-Garay, & Castell-Perez, 2004; Soazo, Rubiolo, & Verdini, 2011). Other researchers successfully developed stronger films via crosslinking (Patzsch, Riedel, & Pietzsch, 2010; Perez-gago & Krochta, 2001; Ustunol & Mert, 2004). The blend of proteins and polysaccharides appears to be a new approach for environmentally friendly and edible films. Jia, Fang, and Yao (2009) suggested that konjac glucomannan, chitosan and soy protein isolate can be applied to make a composite edible film. Galus, Mathieu, Lenart, and Debeaufort (2012) reported that incorporation of starch and maltodextrin could increase the water uptake of soy protein isolate films, but decrease the tensile strength and elongation at break. Gum Arabic can be used as stabilization of emulsion, encapsulation, and film formation (Krishnan, Kshirsagar, & Singhal, 2005). Therefore, gum Arabic could be a good choice for the formation of protein-polysaccharides films. Overall, there is still limited information available on the film properties formed by protein and polysaccharides components, and there are no reports about the protein films composited with gum Arabic. In recent years, conjugating proteins with polysaccharides through Maillard-type reactions has attracted much attention. Maillard-type reaction can also be called glycation, which occurs between ε-amino groups in protein and the reducing end carbonyl group in polysaccharides (Kato, 2002). Glycation, in contrast to
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acylation or phosphorylation, is safer because it does not produce health-hazardous secondary compounds during food applications, and the glycated proteins showed excellent emulsifying properties, thermal stability, and gelling properties (Kato, Mifuru, Matsudomi, & Kobayashi, 1992; Liu, Zhao, Zhao, Ren, & Yang, 2012; Sun, Hayakawa, & Izumori, 2004). The three-dimensional networks of the films were greatly dependent on the proteineprotein interactions as well as the interactions between protein and other constituents. These interactions, including low-energy bonds and covalent forces, may account for the macroscopic properties and ~o n, 2006). Therefore, it network formation of the films (Mauri & An is reasonable to expect that the physical and structural properties of films may relate to the structural modifications of proteins. In this research, PPI was glycated with gum Arabic to form PPIgum Arabic conjugates with different degree of graft. The barrier and mechanical properties, as well as the structural properties of films prepared from glycated PPI were compared. The relationship between structural modifications and properties of films was also examined. 2. Experimental 2.1. Materials Low-temperature defatted peanut meal was provided by Shandong Guangda Lvyuan Food Technology Co., Ltd (Shandong, China). Gum Arabic (MW 240,000e580,000) and glycerol were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). 5, 50 -dithiobis-(2-nitrobenzoic acid) (DTNB) were purchased from Aladdin Reagent Company (Shanghai, China). Bovine serum albumin (BSA), Folin & Ciocalteu's phenol reagent and other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).
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forming solutions were poured onto polystyrene Petri dishes (100 cm2 area). To control film thickness, the solutions with the same solid content were casted on each plate. Films were dried in an oven at 50 C for 12 h and peeled intactly. The peeled films were conditioned at 43% relative humidity for at least 48 h prior to analysis. 2.5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) SDSePAGE was carried out on a Bio-rad Mini-Protein Tetra Electrophoresis System (Bio-Rad Laboratories, Inc., Hercules, USA) with a 4% stacking gel and 12% separating gel. The protein content of all samples was 2 mg/mL. After electrophoresis, gels were stained for protein with Coomassie R250 dye (Yadav, Parris, Johnston, Onwulata, & Hicks, 2010). The SDS-PAGE pattern was analyzed by Image Lab Software 3.0 (Bio-Rad Laboratories, Inc., Hercules, USA). 2.6. Differential scanning calorimetry (DSC) DSC was applied using a Q2000 instrument (TA instruments, New Csatle, DE, USA). The samples were hermetically sealed in aluminum pans and were heated from 25 C up to 150 C under nitrogen atmosphere at the rate of 10 C min1. Denaturation temperature (Td) and enthalpy change of denaturation (DH) were computed. 2.7. Fourier transformed infrared (FT-IR) FT-IR spectra of the films were carried out on a Nicolet Nexus 470 FTIR (Thermo Fisher Scientific, Waltham, U.K.). A total of 64 scans were performed at 4 cm1 resolution. Scanning was carried out in the range 4000e400 cm1.
2.2. Preparation of peanut protein isolate (PPI) PPI was prepared according to the method of Liu et al. (2012) with slight modifications. Low-temperature defatted peanut meal was dispersed in deionised water (1:20 w/v), and the pH was adjusted to 8.0. The dispersions were stirred for 2 h at room temperature then centrifuged. The supernatants were adjusted to pH 4.5 and centrifuged again. The precipitate was washed and redispersed in deionised water, and the pH was adjusted to 7.0. The dispersions were frozen at 70 C for 24 h and then lyophilized at 20 Pa for 48 h (PPI). 2.3. Preparation of PPI-gum Arabic conjugates PPI was conjugated with gum arabic by adapting the method of Kato et al. (1992) with slight modifications. PPI and gum Arabic were mixed in water in a weight ratio of 1:1, the pH was adjusted to 7.0 and then the solutions were also lyophilized after 24 h prefrozen at 70 C. The mixture were incubated in a desiccator at 60 C and 79% relative humidity (over saturated potassium bromide) for 3, 6, 9 days, respectively. The lyophilized mixtures, without heating, were used as control.
2.8. X-ray diffractometry (XRD) XRD patterns were obtained using a D8 Advance powder diffractometer (Bruker AXS, Germany), CuKa1 (l ¼ 0.1541 nm) radiation was used and patterns were recorded at 2 min1 scan rate in the 2q range from 5 to 50 . 2.9. Water vapor permeability (WVP) The WVP of films was tested by using the ASTM E96-95 gravimetric method with modifications (McHugh, Avena-Bustillos, & Krochta, 1993). The films were located on the tops of glass cups containing P2O5 powder (0% RH). Tested cups were placed in a desiccator containing NaCl (75% RH) solution at the bottom. The weight gains of tested cups were recorded until the equilibrium was achieved. The WVP (g m1 s1 Pa1) of the films was calculated as WVP ¼ Dm x/(Dt A Dp), where Dm is the weight gain (g) of the cups during time Dt (s), x is the film thickness (m), A is the area of exposed film (1.49 104 m2), and Dp is the water vapour pressure differential across the film (Pa). At least three samples were taken from each film to obtain the WVP value.
2.4. Film preparation 2.10. Scanning electron microscopy (SEM) The film-forming solutions were prepared by dispersing PPI, PPI-gum Arabic mixtures and PPI-gum Arabic conjugates respectively in deionized water (10%, w/w). Glycerol was added at 25% of the total components as a plasticizer. The pH of the dispersions was adjusted to 8.0 and then the dispersions were magnetically stirred for 30 min at 90 C. After degassing under vacuum, the film-
Film samples were coated with gold/palladium using a Balzers evaporator (model SCD 050, Baltec Lichtenstein, Austria), and the morphology of samples was observed with a scanning electron microscope (Quanta-200, FEI, USA) at an accelerating voltage of 5 kV.
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2.11. Tensile strength (TS) and elongation (E) TS and E were determined using a TA-XT2i texture analyzer (Stable MicroSystems, London, U.K.). The initial distance of separation and cross-head speed were fixed at 50 mm and 1 mm/s, respectively. The TS was calculated by dividing the maximum force at break by initial transverse section, and E was calculated the ratio of increase in length to original length and multiplying by 100 (De Carvalho & Grosso, 2004). Each trail was replicated at least four times. 2.12. Protein solubility of the film Protein solubility of the films was determined according to the method of Yin, Tang, Wen, and Yang (2007) with slight modifications. Films (5 mg/mL) were dispersed in various solvent systems as follows: DW, deionized water at pH 8.0; B: Tris-glycine buffer (0.086 M Tris, 0.09 M glycine, and 4 mM Na2EDTA, pH 8.0); BSU: B with 0.5% sodium dodecyl sulphate (SDS) and 6 M urea; BSUM: BSU with 1% (v/v) b-mercaptoethanol (2-ME). The mixtures were shaken for 24 h at 25 C then centrifuged. The protein content in the supernatant was determined by the Lowry method using BSA as the standard (Lowry, Rosebrough, Farr, & Randall, 1951). Protein solubility was expressed as grams of supernatant protein per 100 g of protein. All determinations were conducted in triplicate. 2.13. Determination of free sulfhydryl content (SHf) The SHf content of proteins and films was determined according ~ on, 1995) with some modifications. to the method (Petruccelli & An Protein samples (5 mg/mL) and films (10 mg/mL) were dispersed in buffer (0.086 M Tris, 0.09 M glycine, 4 mM Na2EDTA, 0.5% SDS and 8 M urea, pH 8.0). The mixtures were shaken for 24 h at 25 C and the suspensions were then centrifuged. Ellman's reagent (4 mg DTNB in 1 mL methanol) was added to the supernatant. After reaction, the absorbance was determined at 412 nm. The extinction coefficient was 13,600 M1 cm1, and the content of SHf was expressed as mmol/g of protein. 2.14. Statistical analysis All the data obtained were analyzed by variance (One-Way ANOVA) using SPSS for Windows version 17.0 (SPSS Inc.,Chicago, IL). Values are means ± SD. Duncan's multiple range test was used to identify significant differences (p < 0.05) among the means of various incubation time. 3. Results and discussion 3.1. Characterization of the conjugates The SDSePAGE pattern of PPI, PPI-gum Arabic mixtures and PPIgum Arabic conjugates heated for 3, 6 and 9 days were shown in Fig. 1. The pattern suggested that PPI was composed of five components: conarachin protein (>50 kDa), acidic arachins (38e49.9 kDa), intermediate molecular weight proteins (23e37.9 kDa), basic arachins (18e22.9 kDa), and low molecular weight proteins (14e17.9 kDa) (Bianchi-Hall, Keys, Stalker, & Murphy, 1993). As the glycation reaction proceeded (from 3 to 9 days), a gradual disappearance of conarachin band (about 70 kDa marked by arrow) could be observed. The characteristic bands of arachin (about 40 kDa and 22 kDa marked by arrows) in 3 days incubated conjugates did not change significantly compared to arachin bands in PPI and PPI-gum Arabic mixtures. However, as the conjugation reaction proceeded for more than 6 days, a gradual
Fig. 1. SDSePAGE patterns of PPI and PPI-gum Arabic reaction products dry-heated for different times. (M) marker proteins; (1) PPI; (2)PPI-gum Arabic mixtures; (3) PPI-gum Arabic conjugates heated for 3 days; (4) PPI-gum Arabic conjugates heated for 6 days; (5) PPI-gum Arabic conjugates heated for 9 days.
decrease of arachin bands with increasing incubation time (6 days and 9 days, Lane 4e5) was seen. It was clear that more than 9 days dry-heating was required for the completion of glycation reaction. Furthermore, as the dry-heating started, a broad band appeared near the top of the running gel. Previous studies verified the formation of covalent linkage on dry-heating of proteinpolysaccharide mixtures by using SDS-PAGE analysis (Diftis & Kiosseoglou, 2006; Jimenez-Castano, Villamiel, & Lopez-Fandino, 2007). Therefore, our results demonstrated that PPI complexed with gum Arabic and formed higher molecular weight conjugates after dry-heating. Two typical endotherms of PPI were observed by DSC (Table 1), the first at 89.00 C, corresponding to conarachin denaturation, and the second at 101.77 C, corresponding to arachin. The results suggested that conarachin had a much lower denaturation temperature (Td) than arachin. Therefore, it was easier for conarachin to expose more available lysine residues for the occurrence of Maillard reaction during dry-heating (Liu et al., 2012). The higher Td is generally associated with higher thermal stability for a globular
Table 1 DSC characteristics of PPI and PPI-gum Arabic reaction products dry-heated for different time. Sample
Conarachin
PPI PPI-gum arabic mixtures PPI-gum arabic conjugates 3 days PPI-gum arabic conjugates 6 days PPI-gum arabic conjugates 9 days
Arachin
Td ( C)
DH (J/g)
89.00 ± 0.80c 92.55 ± 0.45b
0.89 ± 0.01a 101.77 ± 0.73c 4.63 ± 0.37d 0.67 ± 0.01b 108.65 ± 0.85b 8.36 ± 0.20a
Td ( C)
DH (J/g)
92.95 ± 0.45 ab 0.34 ± 0.03c
110.67 ± 0.63a 7.02 ± 0.69b
93.65 ± 0.45a
0.37 ± 0.03c
110.74 ± 0.46a 5.95 ± 0.54c
93.91 ± 0.59a
0.16 ± 0.02d 110.81 ± 0.69a 5.94 ± 0.39c
Mean values in the same column with different letters are significantly (p < 0.05) different, as determined by Duncan's multiple range test.
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protein. Therefore, the increase of Td indicated an improvement in thermal stability of PPI-gum Arabic mixtures and conjugates as compared to PPI. Disruption of hydrophobic interactions can reduce the observed enthalpy (Boye, Alli, & Ismail, 1996). The initial increase (p < 0.05) of the DH of arachin in PPI-gum Arabic mixtures may be the result of gum Arabic protecting PPI against aggregation by decreasing the hydrophobic interactions on protein surface (Ibanoglu, 2005). A previous study reported that the decrease of enthalpy was closely related to the development of glycation reaction (Manzocco, Nicoli, & Maltini, 1999). Compared to the mixtures, prolonged heating could cause the decrease of the DH of both conarachin and arachin, indicating the denaturation of proteins. Their unfolded structures allowed them to interact intermolecularly easily, leading them to crosslink with gum Arabic and form conjugates of high molecular weight. Therefore, the gel pattern and DSC results suggested that PPI could be glycated with gum Arabic under dry-heating, and with the increase of incubation time, the degree of graft increased. 3.2. FT-IR spectra of the films The FT-IR spectra of the films prepared from PPI, PPI-gum Arabic mixtures and conjugates was shown in Fig. 2. The broad absorption band observed in the wave number range 3600e3000 cm1 was mainly attributed to eOH groups from gum Arabic and the moisture. The characteristic stretching of eCH2 and eCH3 groups of saturated structures was observed in the range 2980e2850 cm1. The vibration bands at 1632 cm1 (amide I) and NeH bending at 1537 cm1 (amide II) were related to amino acid residues forming the protein structure. The absorption band at 1241e1472 cm1 was attributable to C(O)eO and CeN stretching and NeH bending (amide III) vibrations. The band at 1038 cm1 was attributed to vibrations such as out-of-plane CeH bending (from an aromatic structure) (Su, Huang, Yuan, Wang, & Li, 2010). It can be seen from the spectra that as the dry-heating proceeded, the absorbance associated with eOH groups (3600e3000 cm1) and NeH bending (amide I at 1632 cm1, amide II at 1537 cm1 and amide III at 1398 cm1) vibrations gradually decreased, which showed that eOH groups in gum Arabic and amino groups in PPI were gradually consumed. Because that glycation occurs between amino groups in protein and the carbonyl group in polysaccharides (Kato, 2002), the spectra verified the
Fig. 2. FTIR spectra of the films. From bottom to top: PPI films, films prepared from PPI-gum Arabic mixtures, films prepared from PPI-gum Arabic conjugates heated for 3 days, films prepared from PPI-gum Arabic conjugates heated for 6 days and films prepared from PPI-gum Arabic conjugates heated for 9 days.
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crosslinking between PPI and gum Arabic. With the increase of dryheating time, the degree of crosslinking increased. After 9 days dryheating, typical bands of the protein structure (amide I, II and III) were still observed, indicating that the protein was not fully degraded. However, the protein structure probably underwent structural rearrangement, such as dissociation into subunits (Schmidt & Soldi, 2006). This dissociation may facilitate the interactions among reactive side groups, such as eNH2, eOH, and eSH in PPI, to form crosslinks such as disulphide, lysinoalanine, and lanthionine. 3.3. XRD patterns of the films Fig. 3 showed the XRD patterns of films prepared from PPI, PPIgum Arabic mixtures and conjugates. XRD patterns were characterized by sharp peaks associated with crystalline diffraction and an amorphous zone (Garcia, Martino, & Zaritzky, 2000b). PPI films gave a strong characteristic reflection at 2q about 14 . After PPI was mixed or conjugated with gum Arabic, the films exhibited a large peak at about 20 2q. This result indicated that after blend, the crystalline structures were different. Moreover, the peak intensities of PPI-gum Arabic films decreased with incubation time, and showed a tendency towards a decrease in crystallinity, indicating more amorphous structure as the glycation proceeded (Su et al., 2010). Mixing/conjugating with polysaccharides could facilitate polymeric chain mobility of the protein and allow a rapid development of the film structures, thus reducing the crystalline structures by interfering with the protein chain arrangement (Garcia, Martino, & Zaritzky, 2000a; Miralles, Martínez-Rodríguez, Santiago, van de Lagemaat, & Heras, 2007). 3.4. Barrier properties of the films Physicochemical interactions between water and macromolecular networks could affect water diffusion. The WVP of the films was shown in Table 2. Compared to PPI-gum Arabic films, PPI films showed higher (p < 0.05) permeability. The low resistance of the films to water vapor transmission may be the result of protein hydrophilicity and the presence of glycerol (hydrophilic plasticizer), which favoured water molecule adsorption (Yoshida, Antunes, Antunes, & Antunes, 2003). After mixing with gum Arabic, the WVP of films significantly (p < 0.05) decreased. As the
Fig. 3. X-ray patterns of the films. (a) PPI films; (b) films prepared from PPI-gum Arabic mixtures; (c) films prepared from PPI-gum Arabic conjugates heated for 3 days; (d) films prepared from PPI-gum Arabic conjugates heated for 6 days and (e) films prepared from PPI-gum Arabic conjugates heated for 9 days.
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Table 2 Mechanical and barrier properties for the films: PPI films (PF), films prepared from PPI-gum Arabic mixtures (PGF), films prepared from PPI-gum Arabic conjugates heated for 3 days (PGF 3), films prepared from PPI-gum Arabic conjugates heated for 6 days (PGF 6) and films prepared from PPI-gum Arabic conjugates heated for 9 days (PGF 9). Film
WVP ( 1011 g m1 s1 Pa1)
PF PGF PGF 3 PGF 6 PGF 9
167.9 139.8 71.2 89.0 112.0
± ± ± ± ±
10.3a 8.2b 6.2e 7.5d 10.0c
TS (MPa) 0.77 1.18 1.76 1.29 1.15
± ± ± ± ±
0.06d 0.08bc 0.05a 0.08b 0.06c
E (%) 135.7 34.5 37.4 50.2 56.2
± ± ± ± ±
1.2a 0.9e 1.8d 0.9c 1.7b
WVP: water vapor permeability, TS: tensile strength, E: elongation. Mean values in the same column with different letters are significantly (p < 0.05) different, as determined by Duncan's multiple range test.
glycation continued to 3 days, the glycated films showed 2e2.5-fold lower permeability than PPI films. However, the permeability gradually increased in the glycated films as the glycation proceeded from 3 to 9 days. Permeability may be a complex phenomenon, depending on the composition and the microscopic structure of the filmogenic matrix. SEM was used to visualize the surface of each film, and could be an attempt to elucidate the film structure characteristics important for resistance to water vapor transmission (Kester & Fennema, 1989). The micrographs of the PPI and PPI-gum Arabic films were in Fig. 4. PPI-gum Arabic films had markedly different surface appearance than that of PPI films. It was apparent that PPIgum Arabic films had more dense structure than PPI films, which may be less easy for the moisture transmission. When glycation started, PPI gradually cross-linked with gum Arabic, the surface of
the films became more compact and smooth with less cracks and pores compared to PPI-gum Arabic films without glycation. Therefore, the WVP values significantly (p < 0.05) decreased. However, as the glycation continued, the surface of PPI-gum Arabic films became more even and smooth, indicating more homogeneous matrix. Conjugation with polysaccharides could facilitate protein chain mobility. Therefore, it can be predicted that with the increase of degree of graft, more polysaccharides could be incorporated into protein network, allowing larger degree of chain mobility. This rapid development of film structures would allow glycerol dispersing homogenously into film networks, which maximized the loosening effect, causing the films to trap relatively higher amount of moisture (Xia, Wang, & Chen, 2011). The SEM results also indicated more and more amorphous structure of PPIgum Arabic films as the glycation proceeded. 3.5. Mechanical properties of the films The mechanical properties of PPI and PPI-gum Arabic films were shown in Table 2. It is clear from this table that PPI-gum Arabic films have been observed to be at least 50% increase in tensile strength as compared to PPI films (p < 0.05). The 9 days incubation could decrease the tensile strength from 1.76 MPa to 1.15 MPa, but the values were still higher (p < 0.05) than PPI films. The elongation of films prepared from mixtures and conjugates was significantly (p < 0.05) lower than that of PPI films. The result suggested that by adding gum Arabic, the crosslinking between PPI and the polysaccharides would reinforce the film network in spite of the reduction of the crystallinity as mentioned before, thus the tensile strength increased while the elongation decreased. It was notable that as glycation of proteins proceeded to 9 days, the elongation of films prepared from conjugates significantly (p < 0.05) increased by 63% compared to that of films prepared from mixtures. The results were in accordance with previous study about the decreases in tensile strength and increases in elasticity relating to the degree of crosslinking for films modified with glutaraldehyde (Bigi, Cojazzi, Panzavolta, Rubini, & Roveri, 2001). As more gum Arabic incorporated into the protein network, homogenously dispersed glycerol in the film networks (in Section 3.4) would weaken the intermolecular force between the chains of adjacent macromolecules. Thus, the plasticizer could cause a reduction in tensile strength due to the decrease in intermolecular interactions between protein molecules and an increase in elongation due to the increase in the molecules mobility (Di Gioia & Guilbert, 1999). 3.6. Interactive forces of the films
Fig. 4. SEM surface morphologies of the films. (a) PPI films; (b) films prepared from PPI-gum Arabic mixtures; (c) films prepared from PPI-gum Arabic conjugates heated for 3 days; (d) films prepared from PPI-gum Arabic conjugates heated for 6 days and (e) films prepared from PPI-gum Arabic conjugates heated for 9 days.
Protein solubility in various buffer systems was analyzed to reveal the interactive forces present in PPI and PPI-gum Arabic films (Table 3). Buffer B affected protein electrostatic interactions; Buffer BSU disrupted hydrophobic interactions and hydrogen bonds; and ~o n, BSUM disrupted disulfide bonds (Lupano, 2000; Mauri & An 2006; Rangavajhyala, Ghorpade, & Hanna, 1997). As shown from the table, in the case of all films, the solubility in Buffer B was lower than that in water. The lower solubility of Buffer B has been attributed to an effect of salting-out, which also suggested that the electrostatic interactions were not significant for the film network formation. The solubility in Buffer BSU of films prepared from PPI and PPI-gum Arabic mixtures was significantly (p < 0.05) higher than that of other films, suggesting that hydrophobic interactions and hydrogen bonds still existed in the film network prepared from PPI and PPI-gum Arabic mixtures. However, with the development of glycation, hydrophobic interactions and hydrogen bonds decreased in the film network. Solubility of all films was increased to above 70% by the presence of 2-ME in Buffer BSUM, suggesting
C. Li et al. / Food Hydrocolloids 43 (2015) 322e328 Table 3 Solubility of the films in different buffers: PPI films (PF), films prepared from PPIgum Arabic mixtures (PGF), films prepared from PPI-gum Arabic conjugates heated for 3 days (PGF 3), films prepared from PPI-gum Arabic conjugates heated for 6 days (PGF 6) and films prepared from PPI-gum Arabic conjugates heated for 9 days (PGF 9). Film
Solubility (g/100 g) DW
PF PGF PGF 3 PGF 6 PGF 9
5.96 7.03 7.48 4.10 5.37
B ± ± ± ± ±
0.38b 0.65a 0.36a 0.23c 0.36b
2.94 4.03 3.49 1.82 0.42
BSU ± ± ± ± ±
0.18c 0.25a 0.22b 0.11d 0.01e
7.42 8.22 7.73 5.46 5.30
BSUM ± ± ± ± ±
0.31b 0.07a 0.43 ab 0.44c 0.40c
98.56 99.30 91.45 93.41 79.28
± ± ± ± ±
1.14a 0.50a 1.05b 1.41b 2.12c
DW, deionized water; B: 0.086 M Tris, 0.09 M glycine, and 4 mM Na2EDTA; BSU: B with 0.5% SDS and 6 M urea; BSUM: BSU with 1% 2-ME. All of them at pH 8.0. Mean values in the same column with different letters are significantly (p < 0.05) different, as determined by Duncan's multiple range test.
that disulfide bonds were the predominant interactive forces involved in the formation and maintenance of three-dimensional film network. Moreover, the addition of 2-ME in Buffer BSUM could increase the solubility of PPI films and films prepared from mixtures to almost 100%. However, as the glycation continued, the solubility of films prepared from conjugates in Buffer BSUM significantly decreased; and when the glycation continued to 9 days, there was about 20% protein in films which could not be dissolved. The decrease of BSUM solubility may be due to the presence of protein aggregates or formation of other covalent bonds in the films prepared from glycated proteins (Yin et al., 2007). The results discussed above indicated that covalent bonds (disulfide bonds) dominated in film network formation. As the glycation continued, the free sulfydryl groups (SHf) contents (Table 4) of all proteins significantly (p < 0.05) decreased. Dry-heating may help expose SH groups which were buried in the interior of protein molecules, and thus promoting the formation of disulfide bonds (Van der Plancken, Van Loey, & Hendrickx, 2005). The SHf contents of all films decreased compared to those of corresponding conjugates, suggesting the formation of new disulfide bonds in the film network. After 6 days glycation, SHf contents of the films were not significantly different from that of 9 days glycated films. The result suggested that the formation of disulfide bonds in the films gradually reached equilibrium. The formation of disulfide bonds requires two thiol groups to be brought into the correct orientation. Slower rate constant for disulfide bond formation were observed in unfolded and less compact structures. Therefore, the results of disulfide bonds in the films were consistent with the more Table 4 Free sulfydryl group content in PPI, PPI-gum Arabic mixtures, conjugates and films. Sample Proteins PPI PPI-gum PPI-gum PPI-gum PPI-gum Films PPI PPI-gum PPI-gum PPI-gum PPI-gum
Total free sulfydryl (mmol/g)
Arabic mixtures arabic conjugates 3 days arabic conjugates 6 days arabic conjugates 9 days
52.94 46.32 44.49 34.19 27.57
± ± ± ± ±
2.2a 1.47b 2.58b 1.1c 1.83d
Arabic mixtures arabic conjugates 3 days arabic conjugates 6 days arabic conjugates 9 days
26.03 23.75 17.72 15.29 14.04
± ± ± ± ±
0.25A 1.39B 0.38C 0.81D 0.62D
Letters (a-d) indicated significant (p < 0.05) difference within proteins; Letters (AeD) indicated significant (p < 0.05) difference within films, as determined by Duncan's multiple range test.
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homogeneous and amorphous structure with the increase of dryheating, and different level of disulfide bonds could reflect film properties and microstructure. 4. Conclusions The degree of graft of PPI-gum Arabic conjugates as well as the crosslinking between PPI and gum Arabic in glycated films both increased as the glycation continued. More than 9 days were needed for the completion of glycation reaction, and conarachin in PPI could more easily participate in glycation than arachin. Compared to PPI films, films prepared from conjugates showed better tensile strength and WVP but poorer elongation. As the glycation proceeded from 3 to 9 days, the glycated films showed increased WVP, decreased tensile strength but increased elongation. Structural analysis indicated that PPI-gum Arabic films had less crystalline and more amorphous structures than PPI films. Disulfide bonds played a predominant role in the film network and could not be further increased with the increase of glycation time. The result indicated that different degree of glycation had significant effect on the physical and structural properties of the PPI-gum Arabic films. Acknowledgements The authors are thankful to Dr. Feng Xue, College of Food Science and Engineering, Northwest A&F University, for his support in the work. This research was supported by the Key Projects in the National Science & Technology Pillar Program during the Twelfth Fiveyear Plan Period (2011BAD02B03). References Bianchi-Hall, C. M., Keys, R. D., Stalker, H. T., & Murphy, J. P. (1993). Diversity of seed storage protein patterns in wild peanut (Arachis, Fabaceae) species. Plant Systematics and Evolution, 186(1e2), 1e15. Bigi, A., Cojazzi, G., Panzavolta, S., Rubini, K., & Roveri, N. (2001). Mechanical and thermal properties of gelatin films at different degrees of glutaraldehyde crosslinking. Biomaterials, 22(8), 763e768. Boye, J. I., Alli, I., & Ismail, A. A. (1996). Interactions involved in the gelation of bovine serum albumin. Journal of Agricultural and Food Chemistry, 44(4), 996e1004. De Carvalho, R. A., & Grosso, C. R. F. (2004). Characterization of gelatin based films modified with transglutaminase, glyoxal and formaldehyde. Food Hydrocolloids, 18(5), 717e726. Diftis, N., & Kiosseoglou, V. (2006). Physicochemical properties of dry-heated soy protein isolate-dextran mixtures. Food Chemistry, 96, 228e233. Di Gioia, L., & Guilbert, S. (1999). Corn protein-based thermoplastic resins: effect of some polar and amphiphilic plasticizers. Journal of Agricultural and Food Chemistry, 47(3), 1254e1261. Ferreira, C. O., Nunes, C. A., Delgadillo, I., & Lopes-da-Silva, J. (2009). Characterization of chitosan-whey protein films at acid pH. Food Research International, 42(7), 807e813. Galus, S., Mathieu, H., Lenart, A., & Debeaufort, F. (2012). Effect of modified starch or maltodextrin incorporation on the barrier and mechanical properties, moisture sensitivity and appearance of soy protein isolate-based edible films. Innovative Food Science & Emerging Technologies, 16, 148e154. Garcia, M. A., Martino, M. N., & Zaritzky, N. E. (2000a). Lipid addition to improve barrier properties of edible starch-based films and coatings. Journal of Food Science, 65(6), 941e944. Garcia, M. A., Martino, M. N., & Zaritzky, N. E. (2000b). Microstructural character€rke, 52(4), 118e124. ization of plasticized starch-based films. Starch-Sta Ibanoglu, E. (2005). Effect of hydrocolloids on the thermal denaturation of proteins. Food Chemistry, 90(4), 621e626. Jia, D., Fang, Y., & Yao, K. (2009). Water vapor barrier and mechanical properties of konjac glucomannan-chitosan-soy protein isolate edible films. Food and Bioproducts Processing, 87(1), 7e10. Jimenez-Castano, L., Villamiel, M., & Lopez-Fandino, R. (2007). Glycosylation of individual whey proteins by Maillard reaction using dextran of different molecular mass. Food Hydrocolloids, 21, 433e443. Kato, A. (2002). Industrial applications of Maillard-type protein-polysaccharide conjugates. Food Science and Technology Research, 8(3), 193e199. Kato, A., Mifuru, R., Matsudomi, N., & Kobayashi, K. (1992). Functional caseinpolysaccharide conjugates prepared by controlled dry heating. Bioscience Biotechnology and Biochemistry, 56, 567e571.
328
C. Li et al. / Food Hydrocolloids 43 (2015) 322e328
Kester, J. J., & Fennema, O. (1989). An edible film of lipids and cellulose ethers: barrier properties to moisture vapor transmission and structural evaluation. Journal of Food Science, 54(6), 1383e1389. Krishnan, S., Kshirsagar, A. C., & Singhal, R. S. (2005). The use of gum arabic and modified starch in the microencapsulation of a food flavoring agent. Carbohydrate Polymers, 62(4), 309e315. Liu, C. C., Tellez-Garay, A. M., & Castell-Perez, M. E. (2004). Physical and mechanical properties of peanut protein films. LWT-Food Science and Technology, 37(7), 731e738. Liu, Y., Zhao, G., Zhao, M., Ren, J., & Yang, B. (2012). Improvement of functional properties of peanut protein isolate by conjugation with dextran through Maillard reaction. Food Chemistry, 131(3), 901e906. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193(1), 265e275. Lupano, C. E. (2000). Gelation of mixed systems whey protein concentrateegluten in acidic conditions. Food Research International, 33(8), 691e696. Ma, W., Tang, C. H., Yin, S. W., Yang, X. Q., Wang, Q., Liu, F., et al. (2012). Characterization of gelatin-based edible films incorporated with olive oil. Food Research International, 49(1), 572e579. Manzocco, L., Nicoli, M. C., & Maltini, E. (1999). DSC analysis of Maillard browning and procedural effects. Journal of Food Processing and Preservation, 23(4), 317e328. ~o n, M. C. (2006). Effect of solution pH on solubility and some Mauri, A. N., & An structural properties of soybean protein isolate films. Journal of the Science of Food and Agriculture, 86(7), 1064e1072. McHugh, T. H., Avena-Bustillos, R., & Krochta, J. (1993). Hydrophilic edible films: modified procedure for water vapor permeability and explanation of thickness effects. Journal of Food Science, 58(4), 899e903. Miralles, B., Martínez-Rodríguez, A., Santiago, A., van de Lagemaat, J., & Heras, A. (2007). The occurrence of a Maillard-type protein-polysaccharide reaction be^-lactoglobulin and chitosan. Food Chemistry, 100(3), 1071e1075. tween a Orliac, O., Rouilly, A., Silvestre, F., & Rigal, L. (2002). Effects of additives on the mechanical properties, hydrophobicity and water uptake of thermo-moulded films produced from sunflower protein isolate. Polymer, 43(20), 5417e5425. Parris, N., & Coffin, D. R. (1997). Composition factors affecting the water vapor permeability and tensile properties of hydrophilic zein films. Journal of Agricultural and Food Chemistry, 45(5), 1596e1599. Patzsch, K., Riedel, K., & Pietzsch, M. (2010). Parameter optimization of protein film production using microbial transglutaminase. Biomacromolecules, 11(4), 896e903. Perez-gago, M. B., & Krochta, J. M. (2001). Denaturation time and temperature effects on solubility, tensile properties, and oxygen permeability of whey protein edible films. Journal of Food Science, 66(5), 705e710.
~ on, M. C. (1995). Partial reduction of soy protein isolate disulfide Petruccelli, S., & An bonds. Journal of Agricultural and Food Chemistry, 43(8), 2001e2006. Rangavajhyala, N., Ghorpade, V., & Hanna, M. (1997). Solubility and molecular properties of heat-cured soy protein films. Journal of Agricultural and Food Chemistry, 45(11), 4204e4208. Schmidt, V., & Soldi, V. (2006). Influence of polycaprolactone-triol addition on thermal stability of soy protein isolate based films. Polymer Degradation and Stability, 91(12), 3124e3130. Soazo, M., Rubiolo, A. C., & Verdini, R. A. (2011). Effect of drying temperature and beeswax content on physical properties of whey protein emulsion films. Food Hydrocolloids, 25(5), 1251e1255. Stuchell, Y. M., & Krochta, J. M. (1994). Enzymatic treatments and thermal effects on edible soy protein films. Journal of Food Science, 59(6), 1332e1337. Su, G., Ren, J., Yang, B., Cui, C., & Zhao, M. (2011). Comparison of hydrolysis characteristics on defatted peanut meal proteins between a protease extract from Aspergillus oryzae and commercial proteases. Food Chemistry, 126(3), 1306e1311. Su, J. F., Huang, Z., Yuan, X. Y., Wang, X. Y., & Li, M. (2010). Structure and properties of carboxymethyl cellulose/soy protein isolate blend edible films crosslinked by Maillard reactions. Carbohydrate Polymers, 79(1), 145e153. Sun, Y., Hayakawa, S., & Izumori, K. (2004). Modification of ovalbumin with a rare ketohexose through the Maillard reaction: effect on protein structure and gel properties. Journal of Agricultural and Food Chemistry, 52(5), 1293e1299. Teerakarn, A., Hirt, D. E., Acton, J. C., Rieck, J. R., & Dawson, P. L. (2002). Nisin diffusion in protein films: effects of film type and temperature. Journal of Food Science, 67(8), 3019e3025. Ustunol, Z., & Mert, B. (2004). Water solubility, mechanical, barrier, and thermal properties of cross-linked whey protein isolate-based films. Journal of Food Science, 69(3), 129e133. Van der Plancken, I., Van Loey, A., & Hendrickx, M. E. (2005). Changes in sulfhydryl content of egg white proteins due to heat and pressure treatment. Journal of Agricultural and Food Chemistry, 53(14), 5726e5733. Xia, Y., Wang, Y., & Chen, L. (2011). Molecular structure, physicochemical characterization, and in vitro degradation of barley protein films. Journal of Agricultural and Food Chemistry, 59(24), 13221e13229. Yadav, M. P., Parris, N., Johnston, D. B., Onwulata, C. I., & Hicks, K. B. (2010). Corn fiber gum and milk protein conjugates with improved emulsion stability. Carbohydrate Polymers, 81(2), 476e483. Yin, S. W., Tang, C. H., Wen, Q. B., & Yang, X. Q. (2007). Properties of cast films from hemp (Cannabis sativa L.) and soy protein isolates. A comparative study. Journal of Agricultural and Food Chemistry, 55(18), 7399e7404. Yoshida, C. M., Antunes, A. C., Antunes, L. J., & Antunes, A. J. (2003). An analysis of water vapour diffusion in whey protein films. International Journal of Food Science & Technology, 38(5), 595e601.
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Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd
Physical and structural properties of peanut protein isolate-gum Arabic films prepared by various glycation time Chen Li, Weizhe Zhu, Haoran Xue, Zhiyan Chen, Yajing Chen, Xingguo Wang* State Key Laboratory of Food Science and Technology, Synergetic Innovation Center of Food Safety and Nutrition, School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, Jiangsu, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 November 2013 Accepted 3 June 2014 Available online 12 June 2014
Peanut protein isolate (PPI) was glycated with gum Arabic for 3, 6 and 9 days respectively to study the effect of different degree of graft on the physical and structural properties of glycated films. As the glycation continued, the degree of graft of PPI-gum Arabic conjugates increased gradually, the degree of crosslinking between PPI and gum Arabic in films also increased. Compared to PPI films, glycated films had increased tensile strength, decreased water vapor permeability (WVP), but decreased elongation. As the glycation proceeded from 3 to 9 days, the tensile strength of glycated films decreased, the WVP and the elongation increased gradually. This phenomenon may be caused by the decreased crystalline and increased amorphous structure of glycated films with the glycation time, which was confirmed by X-ray diffractometry and scanning electron microscopy. Disulfide bonds were the predominant interactive forces involved in all film networks. The result suggested that as the glycation continued, the level of disulfide bonds in PPI-gum Arabic films reached equilibrium after 6 days glycation. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Peanut protein isolate Gum Arabic Glycation Films
1. Introduction There is an increasing interest in developing biopolymers such as proteins, polysaccharides, lipids and their composites for packaging materials. They are considered to be a promising solution to environmental pollution and hold promise for innovative uses in food protection and preservation. Numerous proteins have been used to develop films, such as whey protein, soy protein, corn zein, sunflower protein, gelatin and others (Ferreira, Nunes, Delgadillo, & Lopes-da-Silva, 2009; Ma et al., 2012; Orliac, Rouilly, Silvestre, & Rigal, 2002; Parris & Coffin, 1997; Stuchell & Krochta, 1994; Teerakarn, Hirt, Acton, Rieck, & Dawson, 2002). Peanut is one of the most important oil crops in China. Peanut meal is a byproduct generated during oil extraction and is mainly used as animal feed and fertilizer due to the poor protein solubility, dark color, as well as unpleasant flavor (Su, Ren, Yang, Cui, & Zhao, 2011). Peanut protein isolate (PPI), extracted from peanut meal, has higher protein content and good functional properties, which could be a good source to develop films for various applications. Protein films have inferior physical and mechanical properties compared to synthetic films, thus numerous efforts have been
* Corresponding author. Tel./fax: þ86 510 85876799. E-mail address: [email protected] (X. Wang). http://dx.doi.org/10.1016/j.foodhyd.2014.06.003 0268-005X/© 2014 Elsevier Ltd. All rights reserved.
made to enhance the properties. Some researches focused on the optimization of film formulation and preparation conditions (Liu, Tellez-Garay, & Castell-Perez, 2004; Soazo, Rubiolo, & Verdini, 2011). Other researchers successfully developed stronger films via crosslinking (Patzsch, Riedel, & Pietzsch, 2010; Perez-gago & Krochta, 2001; Ustunol & Mert, 2004). The blend of proteins and polysaccharides appears to be a new approach for environmentally friendly and edible films. Jia, Fang, and Yao (2009) suggested that konjac glucomannan, chitosan and soy protein isolate can be applied to make a composite edible film. Galus, Mathieu, Lenart, and Debeaufort (2012) reported that incorporation of starch and maltodextrin could increase the water uptake of soy protein isolate films, but decrease the tensile strength and elongation at break. Gum Arabic can be used as stabilization of emulsion, encapsulation, and film formation (Krishnan, Kshirsagar, & Singhal, 2005). Therefore, gum Arabic could be a good choice for the formation of protein-polysaccharides films. Overall, there is still limited information available on the film properties formed by protein and polysaccharides components, and there are no reports about the protein films composited with gum Arabic. In recent years, conjugating proteins with polysaccharides through Maillard-type reactions has attracted much attention. Maillard-type reaction can also be called glycation, which occurs between ε-amino groups in protein and the reducing end carbonyl group in polysaccharides (Kato, 2002). Glycation, in contrast to
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acylation or phosphorylation, is safer because it does not produce health-hazardous secondary compounds during food applications, and the glycated proteins showed excellent emulsifying properties, thermal stability, and gelling properties (Kato, Mifuru, Matsudomi, & Kobayashi, 1992; Liu, Zhao, Zhao, Ren, & Yang, 2012; Sun, Hayakawa, & Izumori, 2004). The three-dimensional networks of the films were greatly dependent on the proteineprotein interactions as well as the interactions between protein and other constituents. These interactions, including low-energy bonds and covalent forces, may account for the macroscopic properties and ~o n, 2006). Therefore, it network formation of the films (Mauri & An is reasonable to expect that the physical and structural properties of films may relate to the structural modifications of proteins. In this research, PPI was glycated with gum Arabic to form PPIgum Arabic conjugates with different degree of graft. The barrier and mechanical properties, as well as the structural properties of films prepared from glycated PPI were compared. The relationship between structural modifications and properties of films was also examined. 2. Experimental 2.1. Materials Low-temperature defatted peanut meal was provided by Shandong Guangda Lvyuan Food Technology Co., Ltd (Shandong, China). Gum Arabic (MW 240,000e580,000) and glycerol were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). 5, 50 -dithiobis-(2-nitrobenzoic acid) (DTNB) were purchased from Aladdin Reagent Company (Shanghai, China). Bovine serum albumin (BSA), Folin & Ciocalteu's phenol reagent and other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).
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forming solutions were poured onto polystyrene Petri dishes (100 cm2 area). To control film thickness, the solutions with the same solid content were casted on each plate. Films were dried in an oven at 50 C for 12 h and peeled intactly. The peeled films were conditioned at 43% relative humidity for at least 48 h prior to analysis. 2.5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) SDSePAGE was carried out on a Bio-rad Mini-Protein Tetra Electrophoresis System (Bio-Rad Laboratories, Inc., Hercules, USA) with a 4% stacking gel and 12% separating gel. The protein content of all samples was 2 mg/mL. After electrophoresis, gels were stained for protein with Coomassie R250 dye (Yadav, Parris, Johnston, Onwulata, & Hicks, 2010). The SDS-PAGE pattern was analyzed by Image Lab Software 3.0 (Bio-Rad Laboratories, Inc., Hercules, USA). 2.6. Differential scanning calorimetry (DSC) DSC was applied using a Q2000 instrument (TA instruments, New Csatle, DE, USA). The samples were hermetically sealed in aluminum pans and were heated from 25 C up to 150 C under nitrogen atmosphere at the rate of 10 C min1. Denaturation temperature (Td) and enthalpy change of denaturation (DH) were computed. 2.7. Fourier transformed infrared (FT-IR) FT-IR spectra of the films were carried out on a Nicolet Nexus 470 FTIR (Thermo Fisher Scientific, Waltham, U.K.). A total of 64 scans were performed at 4 cm1 resolution. Scanning was carried out in the range 4000e400 cm1.
2.2. Preparation of peanut protein isolate (PPI) PPI was prepared according to the method of Liu et al. (2012) with slight modifications. Low-temperature defatted peanut meal was dispersed in deionised water (1:20 w/v), and the pH was adjusted to 8.0. The dispersions were stirred for 2 h at room temperature then centrifuged. The supernatants were adjusted to pH 4.5 and centrifuged again. The precipitate was washed and redispersed in deionised water, and the pH was adjusted to 7.0. The dispersions were frozen at 70 C for 24 h and then lyophilized at 20 Pa for 48 h (PPI). 2.3. Preparation of PPI-gum Arabic conjugates PPI was conjugated with gum arabic by adapting the method of Kato et al. (1992) with slight modifications. PPI and gum Arabic were mixed in water in a weight ratio of 1:1, the pH was adjusted to 7.0 and then the solutions were also lyophilized after 24 h prefrozen at 70 C. The mixture were incubated in a desiccator at 60 C and 79% relative humidity (over saturated potassium bromide) for 3, 6, 9 days, respectively. The lyophilized mixtures, without heating, were used as control.
2.8. X-ray diffractometry (XRD) XRD patterns were obtained using a D8 Advance powder diffractometer (Bruker AXS, Germany), CuKa1 (l ¼ 0.1541 nm) radiation was used and patterns were recorded at 2 min1 scan rate in the 2q range from 5 to 50 . 2.9. Water vapor permeability (WVP) The WVP of films was tested by using the ASTM E96-95 gravimetric method with modifications (McHugh, Avena-Bustillos, & Krochta, 1993). The films were located on the tops of glass cups containing P2O5 powder (0% RH). Tested cups were placed in a desiccator containing NaCl (75% RH) solution at the bottom. The weight gains of tested cups were recorded until the equilibrium was achieved. The WVP (g m1 s1 Pa1) of the films was calculated as WVP ¼ Dm x/(Dt A Dp), where Dm is the weight gain (g) of the cups during time Dt (s), x is the film thickness (m), A is the area of exposed film (1.49 104 m2), and Dp is the water vapour pressure differential across the film (Pa). At least three samples were taken from each film to obtain the WVP value.
2.4. Film preparation 2.10. Scanning electron microscopy (SEM) The film-forming solutions were prepared by dispersing PPI, PPI-gum Arabic mixtures and PPI-gum Arabic conjugates respectively in deionized water (10%, w/w). Glycerol was added at 25% of the total components as a plasticizer. The pH of the dispersions was adjusted to 8.0 and then the dispersions were magnetically stirred for 30 min at 90 C. After degassing under vacuum, the film-
Film samples were coated with gold/palladium using a Balzers evaporator (model SCD 050, Baltec Lichtenstein, Austria), and the morphology of samples was observed with a scanning electron microscope (Quanta-200, FEI, USA) at an accelerating voltage of 5 kV.
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2.11. Tensile strength (TS) and elongation (E) TS and E were determined using a TA-XT2i texture analyzer (Stable MicroSystems, London, U.K.). The initial distance of separation and cross-head speed were fixed at 50 mm and 1 mm/s, respectively. The TS was calculated by dividing the maximum force at break by initial transverse section, and E was calculated the ratio of increase in length to original length and multiplying by 100 (De Carvalho & Grosso, 2004). Each trail was replicated at least four times. 2.12. Protein solubility of the film Protein solubility of the films was determined according to the method of Yin, Tang, Wen, and Yang (2007) with slight modifications. Films (5 mg/mL) were dispersed in various solvent systems as follows: DW, deionized water at pH 8.0; B: Tris-glycine buffer (0.086 M Tris, 0.09 M glycine, and 4 mM Na2EDTA, pH 8.0); BSU: B with 0.5% sodium dodecyl sulphate (SDS) and 6 M urea; BSUM: BSU with 1% (v/v) b-mercaptoethanol (2-ME). The mixtures were shaken for 24 h at 25 C then centrifuged. The protein content in the supernatant was determined by the Lowry method using BSA as the standard (Lowry, Rosebrough, Farr, & Randall, 1951). Protein solubility was expressed as grams of supernatant protein per 100 g of protein. All determinations were conducted in triplicate. 2.13. Determination of free sulfhydryl content (SHf) The SHf content of proteins and films was determined according ~ on, 1995) with some modifications. to the method (Petruccelli & An Protein samples (5 mg/mL) and films (10 mg/mL) were dispersed in buffer (0.086 M Tris, 0.09 M glycine, 4 mM Na2EDTA, 0.5% SDS and 8 M urea, pH 8.0). The mixtures were shaken for 24 h at 25 C and the suspensions were then centrifuged. Ellman's reagent (4 mg DTNB in 1 mL methanol) was added to the supernatant. After reaction, the absorbance was determined at 412 nm. The extinction coefficient was 13,600 M1 cm1, and the content of SHf was expressed as mmol/g of protein. 2.14. Statistical analysis All the data obtained were analyzed by variance (One-Way ANOVA) using SPSS for Windows version 17.0 (SPSS Inc.,Chicago, IL). Values are means ± SD. Duncan's multiple range test was used to identify significant differences (p < 0.05) among the means of various incubation time. 3. Results and discussion 3.1. Characterization of the conjugates The SDSePAGE pattern of PPI, PPI-gum Arabic mixtures and PPIgum Arabic conjugates heated for 3, 6 and 9 days were shown in Fig. 1. The pattern suggested that PPI was composed of five components: conarachin protein (>50 kDa), acidic arachins (38e49.9 kDa), intermediate molecular weight proteins (23e37.9 kDa), basic arachins (18e22.9 kDa), and low molecular weight proteins (14e17.9 kDa) (Bianchi-Hall, Keys, Stalker, & Murphy, 1993). As the glycation reaction proceeded (from 3 to 9 days), a gradual disappearance of conarachin band (about 70 kDa marked by arrow) could be observed. The characteristic bands of arachin (about 40 kDa and 22 kDa marked by arrows) in 3 days incubated conjugates did not change significantly compared to arachin bands in PPI and PPI-gum Arabic mixtures. However, as the conjugation reaction proceeded for more than 6 days, a gradual
Fig. 1. SDSePAGE patterns of PPI and PPI-gum Arabic reaction products dry-heated for different times. (M) marker proteins; (1) PPI; (2)PPI-gum Arabic mixtures; (3) PPI-gum Arabic conjugates heated for 3 days; (4) PPI-gum Arabic conjugates heated for 6 days; (5) PPI-gum Arabic conjugates heated for 9 days.
decrease of arachin bands with increasing incubation time (6 days and 9 days, Lane 4e5) was seen. It was clear that more than 9 days dry-heating was required for the completion of glycation reaction. Furthermore, as the dry-heating started, a broad band appeared near the top of the running gel. Previous studies verified the formation of covalent linkage on dry-heating of proteinpolysaccharide mixtures by using SDS-PAGE analysis (Diftis & Kiosseoglou, 2006; Jimenez-Castano, Villamiel, & Lopez-Fandino, 2007). Therefore, our results demonstrated that PPI complexed with gum Arabic and formed higher molecular weight conjugates after dry-heating. Two typical endotherms of PPI were observed by DSC (Table 1), the first at 89.00 C, corresponding to conarachin denaturation, and the second at 101.77 C, corresponding to arachin. The results suggested that conarachin had a much lower denaturation temperature (Td) than arachin. Therefore, it was easier for conarachin to expose more available lysine residues for the occurrence of Maillard reaction during dry-heating (Liu et al., 2012). The higher Td is generally associated with higher thermal stability for a globular
Table 1 DSC characteristics of PPI and PPI-gum Arabic reaction products dry-heated for different time. Sample
Conarachin
PPI PPI-gum arabic mixtures PPI-gum arabic conjugates 3 days PPI-gum arabic conjugates 6 days PPI-gum arabic conjugates 9 days
Arachin
Td ( C)
DH (J/g)
89.00 ± 0.80c 92.55 ± 0.45b
0.89 ± 0.01a 101.77 ± 0.73c 4.63 ± 0.37d 0.67 ± 0.01b 108.65 ± 0.85b 8.36 ± 0.20a
Td ( C)
DH (J/g)
92.95 ± 0.45 ab 0.34 ± 0.03c
110.67 ± 0.63a 7.02 ± 0.69b
93.65 ± 0.45a
0.37 ± 0.03c
110.74 ± 0.46a 5.95 ± 0.54c
93.91 ± 0.59a
0.16 ± 0.02d 110.81 ± 0.69a 5.94 ± 0.39c
Mean values in the same column with different letters are significantly (p < 0.05) different, as determined by Duncan's multiple range test.
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protein. Therefore, the increase of Td indicated an improvement in thermal stability of PPI-gum Arabic mixtures and conjugates as compared to PPI. Disruption of hydrophobic interactions can reduce the observed enthalpy (Boye, Alli, & Ismail, 1996). The initial increase (p < 0.05) of the DH of arachin in PPI-gum Arabic mixtures may be the result of gum Arabic protecting PPI against aggregation by decreasing the hydrophobic interactions on protein surface (Ibanoglu, 2005). A previous study reported that the decrease of enthalpy was closely related to the development of glycation reaction (Manzocco, Nicoli, & Maltini, 1999). Compared to the mixtures, prolonged heating could cause the decrease of the DH of both conarachin and arachin, indicating the denaturation of proteins. Their unfolded structures allowed them to interact intermolecularly easily, leading them to crosslink with gum Arabic and form conjugates of high molecular weight. Therefore, the gel pattern and DSC results suggested that PPI could be glycated with gum Arabic under dry-heating, and with the increase of incubation time, the degree of graft increased. 3.2. FT-IR spectra of the films The FT-IR spectra of the films prepared from PPI, PPI-gum Arabic mixtures and conjugates was shown in Fig. 2. The broad absorption band observed in the wave number range 3600e3000 cm1 was mainly attributed to eOH groups from gum Arabic and the moisture. The characteristic stretching of eCH2 and eCH3 groups of saturated structures was observed in the range 2980e2850 cm1. The vibration bands at 1632 cm1 (amide I) and NeH bending at 1537 cm1 (amide II) were related to amino acid residues forming the protein structure. The absorption band at 1241e1472 cm1 was attributable to C(O)eO and CeN stretching and NeH bending (amide III) vibrations. The band at 1038 cm1 was attributed to vibrations such as out-of-plane CeH bending (from an aromatic structure) (Su, Huang, Yuan, Wang, & Li, 2010). It can be seen from the spectra that as the dry-heating proceeded, the absorbance associated with eOH groups (3600e3000 cm1) and NeH bending (amide I at 1632 cm1, amide II at 1537 cm1 and amide III at 1398 cm1) vibrations gradually decreased, which showed that eOH groups in gum Arabic and amino groups in PPI were gradually consumed. Because that glycation occurs between amino groups in protein and the carbonyl group in polysaccharides (Kato, 2002), the spectra verified the
Fig. 2. FTIR spectra of the films. From bottom to top: PPI films, films prepared from PPI-gum Arabic mixtures, films prepared from PPI-gum Arabic conjugates heated for 3 days, films prepared from PPI-gum Arabic conjugates heated for 6 days and films prepared from PPI-gum Arabic conjugates heated for 9 days.
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crosslinking between PPI and gum Arabic. With the increase of dryheating time, the degree of crosslinking increased. After 9 days dryheating, typical bands of the protein structure (amide I, II and III) were still observed, indicating that the protein was not fully degraded. However, the protein structure probably underwent structural rearrangement, such as dissociation into subunits (Schmidt & Soldi, 2006). This dissociation may facilitate the interactions among reactive side groups, such as eNH2, eOH, and eSH in PPI, to form crosslinks such as disulphide, lysinoalanine, and lanthionine. 3.3. XRD patterns of the films Fig. 3 showed the XRD patterns of films prepared from PPI, PPIgum Arabic mixtures and conjugates. XRD patterns were characterized by sharp peaks associated with crystalline diffraction and an amorphous zone (Garcia, Martino, & Zaritzky, 2000b). PPI films gave a strong characteristic reflection at 2q about 14 . After PPI was mixed or conjugated with gum Arabic, the films exhibited a large peak at about 20 2q. This result indicated that after blend, the crystalline structures were different. Moreover, the peak intensities of PPI-gum Arabic films decreased with incubation time, and showed a tendency towards a decrease in crystallinity, indicating more amorphous structure as the glycation proceeded (Su et al., 2010). Mixing/conjugating with polysaccharides could facilitate polymeric chain mobility of the protein and allow a rapid development of the film structures, thus reducing the crystalline structures by interfering with the protein chain arrangement (Garcia, Martino, & Zaritzky, 2000a; Miralles, Martínez-Rodríguez, Santiago, van de Lagemaat, & Heras, 2007). 3.4. Barrier properties of the films Physicochemical interactions between water and macromolecular networks could affect water diffusion. The WVP of the films was shown in Table 2. Compared to PPI-gum Arabic films, PPI films showed higher (p < 0.05) permeability. The low resistance of the films to water vapor transmission may be the result of protein hydrophilicity and the presence of glycerol (hydrophilic plasticizer), which favoured water molecule adsorption (Yoshida, Antunes, Antunes, & Antunes, 2003). After mixing with gum Arabic, the WVP of films significantly (p < 0.05) decreased. As the
Fig. 3. X-ray patterns of the films. (a) PPI films; (b) films prepared from PPI-gum Arabic mixtures; (c) films prepared from PPI-gum Arabic conjugates heated for 3 days; (d) films prepared from PPI-gum Arabic conjugates heated for 6 days and (e) films prepared from PPI-gum Arabic conjugates heated for 9 days.
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Table 2 Mechanical and barrier properties for the films: PPI films (PF), films prepared from PPI-gum Arabic mixtures (PGF), films prepared from PPI-gum Arabic conjugates heated for 3 days (PGF 3), films prepared from PPI-gum Arabic conjugates heated for 6 days (PGF 6) and films prepared from PPI-gum Arabic conjugates heated for 9 days (PGF 9). Film
WVP ( 1011 g m1 s1 Pa1)
PF PGF PGF 3 PGF 6 PGF 9
167.9 139.8 71.2 89.0 112.0
± ± ± ± ±
10.3a 8.2b 6.2e 7.5d 10.0c
TS (MPa) 0.77 1.18 1.76 1.29 1.15
± ± ± ± ±
0.06d 0.08bc 0.05a 0.08b 0.06c
E (%) 135.7 34.5 37.4 50.2 56.2
± ± ± ± ±
1.2a 0.9e 1.8d 0.9c 1.7b
WVP: water vapor permeability, TS: tensile strength, E: elongation. Mean values in the same column with different letters are significantly (p < 0.05) different, as determined by Duncan's multiple range test.
glycation continued to 3 days, the glycated films showed 2e2.5-fold lower permeability than PPI films. However, the permeability gradually increased in the glycated films as the glycation proceeded from 3 to 9 days. Permeability may be a complex phenomenon, depending on the composition and the microscopic structure of the filmogenic matrix. SEM was used to visualize the surface of each film, and could be an attempt to elucidate the film structure characteristics important for resistance to water vapor transmission (Kester & Fennema, 1989). The micrographs of the PPI and PPI-gum Arabic films were in Fig. 4. PPI-gum Arabic films had markedly different surface appearance than that of PPI films. It was apparent that PPIgum Arabic films had more dense structure than PPI films, which may be less easy for the moisture transmission. When glycation started, PPI gradually cross-linked with gum Arabic, the surface of
the films became more compact and smooth with less cracks and pores compared to PPI-gum Arabic films without glycation. Therefore, the WVP values significantly (p < 0.05) decreased. However, as the glycation continued, the surface of PPI-gum Arabic films became more even and smooth, indicating more homogeneous matrix. Conjugation with polysaccharides could facilitate protein chain mobility. Therefore, it can be predicted that with the increase of degree of graft, more polysaccharides could be incorporated into protein network, allowing larger degree of chain mobility. This rapid development of film structures would allow glycerol dispersing homogenously into film networks, which maximized the loosening effect, causing the films to trap relatively higher amount of moisture (Xia, Wang, & Chen, 2011). The SEM results also indicated more and more amorphous structure of PPIgum Arabic films as the glycation proceeded. 3.5. Mechanical properties of the films The mechanical properties of PPI and PPI-gum Arabic films were shown in Table 2. It is clear from this table that PPI-gum Arabic films have been observed to be at least 50% increase in tensile strength as compared to PPI films (p < 0.05). The 9 days incubation could decrease the tensile strength from 1.76 MPa to 1.15 MPa, but the values were still higher (p < 0.05) than PPI films. The elongation of films prepared from mixtures and conjugates was significantly (p < 0.05) lower than that of PPI films. The result suggested that by adding gum Arabic, the crosslinking between PPI and the polysaccharides would reinforce the film network in spite of the reduction of the crystallinity as mentioned before, thus the tensile strength increased while the elongation decreased. It was notable that as glycation of proteins proceeded to 9 days, the elongation of films prepared from conjugates significantly (p < 0.05) increased by 63% compared to that of films prepared from mixtures. The results were in accordance with previous study about the decreases in tensile strength and increases in elasticity relating to the degree of crosslinking for films modified with glutaraldehyde (Bigi, Cojazzi, Panzavolta, Rubini, & Roveri, 2001). As more gum Arabic incorporated into the protein network, homogenously dispersed glycerol in the film networks (in Section 3.4) would weaken the intermolecular force between the chains of adjacent macromolecules. Thus, the plasticizer could cause a reduction in tensile strength due to the decrease in intermolecular interactions between protein molecules and an increase in elongation due to the increase in the molecules mobility (Di Gioia & Guilbert, 1999). 3.6. Interactive forces of the films
Fig. 4. SEM surface morphologies of the films. (a) PPI films; (b) films prepared from PPI-gum Arabic mixtures; (c) films prepared from PPI-gum Arabic conjugates heated for 3 days; (d) films prepared from PPI-gum Arabic conjugates heated for 6 days and (e) films prepared from PPI-gum Arabic conjugates heated for 9 days.
Protein solubility in various buffer systems was analyzed to reveal the interactive forces present in PPI and PPI-gum Arabic films (Table 3). Buffer B affected protein electrostatic interactions; Buffer BSU disrupted hydrophobic interactions and hydrogen bonds; and ~o n, BSUM disrupted disulfide bonds (Lupano, 2000; Mauri & An 2006; Rangavajhyala, Ghorpade, & Hanna, 1997). As shown from the table, in the case of all films, the solubility in Buffer B was lower than that in water. The lower solubility of Buffer B has been attributed to an effect of salting-out, which also suggested that the electrostatic interactions were not significant for the film network formation. The solubility in Buffer BSU of films prepared from PPI and PPI-gum Arabic mixtures was significantly (p < 0.05) higher than that of other films, suggesting that hydrophobic interactions and hydrogen bonds still existed in the film network prepared from PPI and PPI-gum Arabic mixtures. However, with the development of glycation, hydrophobic interactions and hydrogen bonds decreased in the film network. Solubility of all films was increased to above 70% by the presence of 2-ME in Buffer BSUM, suggesting
C. Li et al. / Food Hydrocolloids 43 (2015) 322e328 Table 3 Solubility of the films in different buffers: PPI films (PF), films prepared from PPIgum Arabic mixtures (PGF), films prepared from PPI-gum Arabic conjugates heated for 3 days (PGF 3), films prepared from PPI-gum Arabic conjugates heated for 6 days (PGF 6) and films prepared from PPI-gum Arabic conjugates heated for 9 days (PGF 9). Film
Solubility (g/100 g) DW
PF PGF PGF 3 PGF 6 PGF 9
5.96 7.03 7.48 4.10 5.37
B ± ± ± ± ±
0.38b 0.65a 0.36a 0.23c 0.36b
2.94 4.03 3.49 1.82 0.42
BSU ± ± ± ± ±
0.18c 0.25a 0.22b 0.11d 0.01e
7.42 8.22 7.73 5.46 5.30
BSUM ± ± ± ± ±
0.31b 0.07a 0.43 ab 0.44c 0.40c
98.56 99.30 91.45 93.41 79.28
± ± ± ± ±
1.14a 0.50a 1.05b 1.41b 2.12c
DW, deionized water; B: 0.086 M Tris, 0.09 M glycine, and 4 mM Na2EDTA; BSU: B with 0.5% SDS and 6 M urea; BSUM: BSU with 1% 2-ME. All of them at pH 8.0. Mean values in the same column with different letters are significantly (p < 0.05) different, as determined by Duncan's multiple range test.
that disulfide bonds were the predominant interactive forces involved in the formation and maintenance of three-dimensional film network. Moreover, the addition of 2-ME in Buffer BSUM could increase the solubility of PPI films and films prepared from mixtures to almost 100%. However, as the glycation continued, the solubility of films prepared from conjugates in Buffer BSUM significantly decreased; and when the glycation continued to 9 days, there was about 20% protein in films which could not be dissolved. The decrease of BSUM solubility may be due to the presence of protein aggregates or formation of other covalent bonds in the films prepared from glycated proteins (Yin et al., 2007). The results discussed above indicated that covalent bonds (disulfide bonds) dominated in film network formation. As the glycation continued, the free sulfydryl groups (SHf) contents (Table 4) of all proteins significantly (p < 0.05) decreased. Dry-heating may help expose SH groups which were buried in the interior of protein molecules, and thus promoting the formation of disulfide bonds (Van der Plancken, Van Loey, & Hendrickx, 2005). The SHf contents of all films decreased compared to those of corresponding conjugates, suggesting the formation of new disulfide bonds in the film network. After 6 days glycation, SHf contents of the films were not significantly different from that of 9 days glycated films. The result suggested that the formation of disulfide bonds in the films gradually reached equilibrium. The formation of disulfide bonds requires two thiol groups to be brought into the correct orientation. Slower rate constant for disulfide bond formation were observed in unfolded and less compact structures. Therefore, the results of disulfide bonds in the films were consistent with the more Table 4 Free sulfydryl group content in PPI, PPI-gum Arabic mixtures, conjugates and films. Sample Proteins PPI PPI-gum PPI-gum PPI-gum PPI-gum Films PPI PPI-gum PPI-gum PPI-gum PPI-gum
Total free sulfydryl (mmol/g)
Arabic mixtures arabic conjugates 3 days arabic conjugates 6 days arabic conjugates 9 days
52.94 46.32 44.49 34.19 27.57
± ± ± ± ±
2.2a 1.47b 2.58b 1.1c 1.83d
Arabic mixtures arabic conjugates 3 days arabic conjugates 6 days arabic conjugates 9 days
26.03 23.75 17.72 15.29 14.04
± ± ± ± ±
0.25A 1.39B 0.38C 0.81D 0.62D
Letters (a-d) indicated significant (p < 0.05) difference within proteins; Letters (AeD) indicated significant (p < 0.05) difference within films, as determined by Duncan's multiple range test.
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homogeneous and amorphous structure with the increase of dryheating, and different level of disulfide bonds could reflect film properties and microstructure. 4. Conclusions The degree of graft of PPI-gum Arabic conjugates as well as the crosslinking between PPI and gum Arabic in glycated films both increased as the glycation continued. More than 9 days were needed for the completion of glycation reaction, and conarachin in PPI could more easily participate in glycation than arachin. Compared to PPI films, films prepared from conjugates showed better tensile strength and WVP but poorer elongation. As the glycation proceeded from 3 to 9 days, the glycated films showed increased WVP, decreased tensile strength but increased elongation. Structural analysis indicated that PPI-gum Arabic films had less crystalline and more amorphous structures than PPI films. Disulfide bonds played a predominant role in the film network and could not be further increased with the increase of glycation time. The result indicated that different degree of glycation had significant effect on the physical and structural properties of the PPI-gum Arabic films. Acknowledgements The authors are thankful to Dr. Feng Xue, College of Food Science and Engineering, Northwest A&F University, for his support in the work. This research was supported by the Key Projects in the National Science & Technology Pillar Program during the Twelfth Fiveyear Plan Period (2011BAD02B03). References Bianchi-Hall, C. M., Keys, R. D., Stalker, H. T., & Murphy, J. P. (1993). Diversity of seed storage protein patterns in wild peanut (Arachis, Fabaceae) species. Plant Systematics and Evolution, 186(1e2), 1e15. Bigi, A., Cojazzi, G., Panzavolta, S., Rubini, K., & Roveri, N. (2001). Mechanical and thermal properties of gelatin films at different degrees of glutaraldehyde crosslinking. Biomaterials, 22(8), 763e768. Boye, J. I., Alli, I., & Ismail, A. A. (1996). Interactions involved in the gelation of bovine serum albumin. Journal of Agricultural and Food Chemistry, 44(4), 996e1004. De Carvalho, R. A., & Grosso, C. R. F. (2004). Characterization of gelatin based films modified with transglutaminase, glyoxal and formaldehyde. Food Hydrocolloids, 18(5), 717e726. Diftis, N., & Kiosseoglou, V. (2006). Physicochemical properties of dry-heated soy protein isolate-dextran mixtures. Food Chemistry, 96, 228e233. Di Gioia, L., & Guilbert, S. (1999). Corn protein-based thermoplastic resins: effect of some polar and amphiphilic plasticizers. Journal of Agricultural and Food Chemistry, 47(3), 1254e1261. Ferreira, C. O., Nunes, C. A., Delgadillo, I., & Lopes-da-Silva, J. (2009). Characterization of chitosan-whey protein films at acid pH. Food Research International, 42(7), 807e813. Galus, S., Mathieu, H., Lenart, A., & Debeaufort, F. (2012). Effect of modified starch or maltodextrin incorporation on the barrier and mechanical properties, moisture sensitivity and appearance of soy protein isolate-based edible films. Innovative Food Science & Emerging Technologies, 16, 148e154. Garcia, M. A., Martino, M. N., & Zaritzky, N. E. (2000a). Lipid addition to improve barrier properties of edible starch-based films and coatings. Journal of Food Science, 65(6), 941e944. Garcia, M. A., Martino, M. N., & Zaritzky, N. E. (2000b). Microstructural character€rke, 52(4), 118e124. ization of plasticized starch-based films. Starch-Sta Ibanoglu, E. (2005). Effect of hydrocolloids on the thermal denaturation of proteins. Food Chemistry, 90(4), 621e626. Jia, D., Fang, Y., & Yao, K. (2009). Water vapor barrier and mechanical properties of konjac glucomannan-chitosan-soy protein isolate edible films. Food and Bioproducts Processing, 87(1), 7e10. Jimenez-Castano, L., Villamiel, M., & Lopez-Fandino, R. (2007). Glycosylation of individual whey proteins by Maillard reaction using dextran of different molecular mass. Food Hydrocolloids, 21, 433e443. Kato, A. (2002). Industrial applications of Maillard-type protein-polysaccharide conjugates. Food Science and Technology Research, 8(3), 193e199. Kato, A., Mifuru, R., Matsudomi, N., & Kobayashi, K. (1992). Functional caseinpolysaccharide conjugates prepared by controlled dry heating. Bioscience Biotechnology and Biochemistry, 56, 567e571.
328
C. Li et al. / Food Hydrocolloids 43 (2015) 322e328
Kester, J. J., & Fennema, O. (1989). An edible film of lipids and cellulose ethers: barrier properties to moisture vapor transmission and structural evaluation. Journal of Food Science, 54(6), 1383e1389. Krishnan, S., Kshirsagar, A. C., & Singhal, R. S. (2005). The use of gum arabic and modified starch in the microencapsulation of a food flavoring agent. Carbohydrate Polymers, 62(4), 309e315. Liu, C. C., Tellez-Garay, A. M., & Castell-Perez, M. E. (2004). Physical and mechanical properties of peanut protein films. LWT-Food Science and Technology, 37(7), 731e738. Liu, Y., Zhao, G., Zhao, M., Ren, J., & Yang, B. (2012). Improvement of functional properties of peanut protein isolate by conjugation with dextran through Maillard reaction. Food Chemistry, 131(3), 901e906. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193(1), 265e275. Lupano, C. E. (2000). Gelation of mixed systems whey protein concentrateegluten in acidic conditions. Food Research International, 33(8), 691e696. Ma, W., Tang, C. H., Yin, S. W., Yang, X. Q., Wang, Q., Liu, F., et al. (2012). Characterization of gelatin-based edible films incorporated with olive oil. Food Research International, 49(1), 572e579. Manzocco, L., Nicoli, M. C., & Maltini, E. (1999). DSC analysis of Maillard browning and procedural effects. Journal of Food Processing and Preservation, 23(4), 317e328. ~o n, M. C. (2006). Effect of solution pH on solubility and some Mauri, A. N., & An structural properties of soybean protein isolate films. Journal of the Science of Food and Agriculture, 86(7), 1064e1072. McHugh, T. H., Avena-Bustillos, R., & Krochta, J. (1993). Hydrophilic edible films: modified procedure for water vapor permeability and explanation of thickness effects. Journal of Food Science, 58(4), 899e903. Miralles, B., Martínez-Rodríguez, A., Santiago, A., van de Lagemaat, J., & Heras, A. (2007). The occurrence of a Maillard-type protein-polysaccharide reaction be^-lactoglobulin and chitosan. Food Chemistry, 100(3), 1071e1075. tween a Orliac, O., Rouilly, A., Silvestre, F., & Rigal, L. (2002). Effects of additives on the mechanical properties, hydrophobicity and water uptake of thermo-moulded films produced from sunflower protein isolate. Polymer, 43(20), 5417e5425. Parris, N., & Coffin, D. R. (1997). Composition factors affecting the water vapor permeability and tensile properties of hydrophilic zein films. Journal of Agricultural and Food Chemistry, 45(5), 1596e1599. Patzsch, K., Riedel, K., & Pietzsch, M. (2010). Parameter optimization of protein film production using microbial transglutaminase. Biomacromolecules, 11(4), 896e903. Perez-gago, M. B., & Krochta, J. M. (2001). Denaturation time and temperature effects on solubility, tensile properties, and oxygen permeability of whey protein edible films. Journal of Food Science, 66(5), 705e710.
~ on, M. C. (1995). Partial reduction of soy protein isolate disulfide Petruccelli, S., & An bonds. Journal of Agricultural and Food Chemistry, 43(8), 2001e2006. Rangavajhyala, N., Ghorpade, V., & Hanna, M. (1997). Solubility and molecular properties of heat-cured soy protein films. Journal of Agricultural and Food Chemistry, 45(11), 4204e4208. Schmidt, V., & Soldi, V. (2006). Influence of polycaprolactone-triol addition on thermal stability of soy protein isolate based films. Polymer Degradation and Stability, 91(12), 3124e3130. Soazo, M., Rubiolo, A. C., & Verdini, R. A. (2011). Effect of drying temperature and beeswax content on physical properties of whey protein emulsion films. Food Hydrocolloids, 25(5), 1251e1255. Stuchell, Y. M., & Krochta, J. M. (1994). Enzymatic treatments and thermal effects on edible soy protein films. Journal of Food Science, 59(6), 1332e1337. Su, G., Ren, J., Yang, B., Cui, C., & Zhao, M. (2011). Comparison of hydrolysis characteristics on defatted peanut meal proteins between a protease extract from Aspergillus oryzae and commercial proteases. Food Chemistry, 126(3), 1306e1311. Su, J. F., Huang, Z., Yuan, X. Y., Wang, X. Y., & Li, M. (2010). Structure and properties of carboxymethyl cellulose/soy protein isolate blend edible films crosslinked by Maillard reactions. Carbohydrate Polymers, 79(1), 145e153. Sun, Y., Hayakawa, S., & Izumori, K. (2004). Modification of ovalbumin with a rare ketohexose through the Maillard reaction: effect on protein structure and gel properties. Journal of Agricultural and Food Chemistry, 52(5), 1293e1299. Teerakarn, A., Hirt, D. E., Acton, J. C., Rieck, J. R., & Dawson, P. L. (2002). Nisin diffusion in protein films: effects of film type and temperature. Journal of Food Science, 67(8), 3019e3025. Ustunol, Z., & Mert, B. (2004). Water solubility, mechanical, barrier, and thermal properties of cross-linked whey protein isolate-based films. Journal of Food Science, 69(3), 129e133. Van der Plancken, I., Van Loey, A., & Hendrickx, M. E. (2005). Changes in sulfhydryl content of egg white proteins due to heat and pressure treatment. Journal of Agricultural and Food Chemistry, 53(14), 5726e5733. Xia, Y., Wang, Y., & Chen, L. (2011). Molecular structure, physicochemical characterization, and in vitro degradation of barley protein films. Journal of Agricultural and Food Chemistry, 59(24), 13221e13229. Yadav, M. P., Parris, N., Johnston, D. B., Onwulata, C. I., & Hicks, K. B. (2010). Corn fiber gum and milk protein conjugates with improved emulsion stability. Carbohydrate Polymers, 81(2), 476e483. Yin, S. W., Tang, C. H., Wen, Q. B., & Yang, X. Q. (2007). Properties of cast films from hemp (Cannabis sativa L.) and soy protein isolates. A comparative study. Journal of Agricultural and Food Chemistry, 55(18), 7399e7404. Yoshida, C. M., Antunes, A. C., Antunes, L. J., & Antunes, A. J. (2003). An analysis of water vapour diffusion in whey protein films. International Journal of Food Science & Technology, 38(5), 595e601.