Research on Properties of Edible Films Prepared from Zein, Soy Protein Isolate, Wheat Gluten Protein by Adding Beeswax

With the increasing awareness of environmental protection, the interest in the development of biodegradable materials has become increasingly popular. At present, wheat gluten protein (WGP), soybean protein isolate (SPI), and Zein have made some progress in the application of packaging materials. At the same time, beeswax (BW) is widely applied in the preparation of food coatings. In this study, composite films were prepared by adding BW to three different types of proteins. Various properties of the composite film, such as thickness, color, mechanical properties, and thermal stability, were tested. The thickness of the film was significantly increased after adding BW. Regarding the mechanical properties, the BW didn’t improve the composite film’s tensile strength. The results of the SEM indicated that the surface morphology of the composite films changed due to the interaction between BW and protein molecules. Besides, the addition of BW resulted in a decrease of thermal stability. The Td of the Zein film (77 ℃) and WGP film (106 ℃) were decreased to 72℃ and 98℃, respectively. The study shows that the composite films will have the opportunity to be applied in the food package field in the future and further replace the traditional petroleum-based films.


Introduction
Currently, the packaging films on the market are mainly made from petroleum-based materials, such as polyethylene, polypropylene, and polystyrene.These materials are nondegradable, which causes severe "white pollution" in the process of long-term use (Pei et al., 2020).With the increasing awareness of the importance of environmental protection, it is very urgent to develop and apply biodegradable films to replace petroleum-based polymers.Among all kinds of biodegradable materials, protein has attracted much attention because of its comprehensive source, low price, and natural and renewable properties.Due to its significant advantages, several researches have been done on a variety of proteinbased packaging materials, such as soybean protein isolated (SPI), wheat gluten protein (WGP), and Zein.
SPI is a kind of full-price protein produced from lowtemperature insoluble soybean meal (Rani & Kumar, 2019); the protein content is more than 90%, mainly existing in the form of 7S and 11S.It is primarily composed of globulin and albumin, which accounted for 90% and 5%, respectively.SPI-based materials are abundant, biocompatible, and renewable.The film made from it has high viscosity, plasticity, and elasticity, as well as some other excellent mechanical properties, so it is widely used in film-forming materials.In addition, SPI has excellent foaming, gel properties, and fine barrier properties (Erdem et al., 2021).Based on its foaming property, the film has great stretch and loose structure.This series of wonderful properties make it excellent film-forming material (RakeshKumar et al., 2019).In recent years, researchers have further improved the properties of SPI film and explored its application via combination with other components.For example, Manassero et al. (2018) modified the SPI film by incorporating calcium combined with high hydrostatic pressure (HHP) treatment.The ability of protein molecules to bind to each other increased, which may help improve the properties (such as gelation and emulsion) of the SPI film.Li et al. (2021) extracted feather keratin (FK) from the waste chicken feathers and prepared composite film with SPI.After adding FK, the thermal stability and the water resistance of SPI film were improved by constructing disulfide bonds cross-linked network structure in the protein system.These works provided a good reference to enhance property and application of SPI film by combining with other natural materials.
WGP is a kind of natural plant protein obtained from wheat and other grains after processing.The crude protein content is over 75%, and it is composed of gliadin and glutenin in a ratio of about 1:1.Glutenin is formed by the polymerization of peptide bonds by intermolecular disulfide bonds.It is fibrous and has good elasticity.Gliadin is spherical with great extensibility but little elasticity.Under the combined effect of gliadin and glutenin, the WGP has unique flexibility and extensibility (Zhang et al., 2021).Moreover, WGP is biodegradable and has good biological compatibility, which can be widely used in the food industry.However, there are some limitations to the application of WGP film.Due to the hydrophilic property and the high hydrogen bond content of wheat gluten protein, the WGP film has high water vapor permeability and low oxygen permeability (Khashayary et al., 2021).Considering the shortcomings of WGP, current researches focused on modifying WGP molecules in order to improve the application range of WGP film.Rovera et al. (2020) modified the WGP film by adding silica in order to overcome the inherent moisture sensitivity of wheat gluten protein.The results showed that the composite film exhibited a lower water contact angle value than that of the WGP film.Cheng et al. (2022) modified protein films by adding natural wax in order to change the physicochemical properties of films.The results showed that water vapor barrier property of films was depended on wax type.Besides, the high molecular polyphenol could cross-link protein, which improved the tensile strength of the WGP film.These researches were beneficial to improve the performance of WGP film.
Zein is a mixture of proteins with an average molecular weight of 25,000 to 45,000 (Nogueira & Martins, 2019).It is a by-product of corn yellow powder.It is insoluble in water and anhydrous alcohol but can dissolve in 60-90% alcoholwater solution, strong alkali, propylene glycol, and a variety of organic solvents.Zein is rich in sulfur-containing amino acids and has strong disulfide bonds and hydrophobic bonds between protein molecules, which are the basis of the film formation.It has advantages of low water vapor permeability and excellent gas barrier property.Therefore, it has great development potential in food packaging, nutrition delivery, and other fields (Alehosseini et al., 2019).The film forming technology of Zein is relatively mature; however, due to the strong intermolecular interaction between the Zein peptide chains, protein-based films are fragile and brittle.The fragility and brittleness shown in the hot forming process will lead to poor mechanical properties, affecting its processing performance and usage (De Vargas et al., 2022;Jiang et al., 2021).Accordingly, previous literatures focused on modification of Zein molecules to expand its application field to meet the needs of production and development (Zhan et al., 2020).Suwannasang et al. (2021) studied the effects of different amounts of surfactants (Tween 80 and lecithin) on the properties of Zein films.The results showed that the higher the concentration of surfactant, the smaller the Zein particles, and the higher the stability of the Zein film.
Beeswax (BW), a product derived from bee waxes, is a complex chemical substance.The structure of BW includes about 15 chemically independent components (Zhang et al., 2020).According to previous studies, the addition of BW greatly increased the hydrophobicity of the gelatin/starch film and promoted the compatibility of starch and gelatin to some extent (Cheng et al., 2021).As lipid-soluble agents, adding BW to the film can substantially modify the ultimate properties of the film such as water resistance properties and oxygen permeability (Bermúdez-Oria et al., 2018;Ochoa et al., 2017).The researches on BW and films are not comprehensive.In the past, the application of BW in protein film production was mainly cross-linked with whey protein and starch film.However, there are few experimental studies on the cross-linked of BW with other protein films.Moreover, the effects of BW on the properties of protein films are less studied (Jiménez et al., 2012).
We innovatively modified the protein film by adding BW into three inexpensive and common proteins (SPI, WGP, and Zein).In addition, we comprehensively evaluated the characteristics of the film from the perspectives of optical properties (color, opacity), mechanical properties (tensile strength, hardness), hydrophilicity (moisture content, water vapor permeability, water contact angle), and so on.On this basis, the composite protein films with excellently comprehensive performance were successfully prepared, which provided technical reference for the further study of protein films.

Materials
WGP was supplied by Midaner Trading Co., Ltd (Henan, China).SPI and Zein were purchased from Shanghai Yuanye Bio-Technology Co., Ltd (Shanghai, China).Tween 80 was provided by Tianli Chemical Reagent Co., Ltd (Tianjin, China).Glycerol was provided by Guanghua Sci-Tech Co., Ltd (Guangdong, China).Sodium hydroxide and hydrochloric acid were supplied by Tianjin Damao Chemical Reagent Factory.Unless mentioned otherwise, all other chemical reagents were analytical grade.

Film Preparation
According to different properties of three kinds of proteins (He et al., 2020), the process of film preparation was illustrated in Fig. 1.
First, 6.0 g of SPI was dispersed in 100 mL of deionized water, and the pH of solution was adjusted to 10.0 with 1 M NaOH and then magnetically stirred evenly.
Second, 6.0 g of WGP was dispersed in 100 mL of 60% ethanol solution, and the pH of solution was adjusted to 11.0 with 1 M NaOH, followed by magnetically stirring for 20 min at 60 °C.
Third, 10.0 g of Zein was dispersed in 100 mL of 75% ethanol solution, stirring evenly without adjusting pH at room temperature.
The BW was added to the Tween 80 emulsifier, the ratio of wax and emulsifier was 1:1, and it was magnetically stirred at 90 °C in order to form stable oil phase.Then, deionized water at 80 °C was added to make mixed solution; the ratio of emulsifier and water was 1:1.After stirring, 50% glycerol (w/w) was added.The BW emulsion was prepared.Then 3% BW emulsion (w/w) was dissolved and magnetically stirred for 30 min at 70 °C.Then, 10 mL of film-forming solution of SPI and WGP was poured and spread in a petri dish which has a diameter of 90 mm.The petri dish was dried in an oven at 50 °C for 5 h.All the films were peeled off after drying.The solution of Zein was harvested at 70 °C water bath and peeled of the film after drying.All the films were placed with 25 ℃ temperature for later testing.

Moisture Content
The film samples were placed in a clean petri dish.Then, they were dried at 30 °C to a constant weight and weighed three times for each film.The moisture content was calculated according to the following equation (Liu et al., 2017): In the equation, m 1 and m 2 refer to the initial weight (g) and final dry weight (g) of films, respectively.

Total Soluble Matter (TSM)
The film samples were placed in a sealed tube containing an appropriate amount of deionized water and evenly stirred with 25 °C for 24 h.After that, the films were kept at 50 °C to a constant weight and weighed for each film.The percentage of weight loss is defined as the total soluble matter (TSM) value (He et al., 2020).It was calculated as follows: where m 1 is the initial dry weight (g) and m 3 is final dry weight (g) of film samples. (1)

Water Vapor Permeability (WVP)
The bottom of the glass jars (10 mL) was filled with 40 g of anhydrous calcium chloride (0% relative humidity) and sealed with the films.After that, the jars covering the film samples were put in a desiccator and were weighed after a week.WVP was calculated as follows (Kai et al., 2021): where WVP (g mm) / (m 2 d kPa) is water vapor permeability; ∆m (g) refers to the quality difference of the wide-mouth bottle; T (mm) is the thickness of film; A (m 2 ) is the surface area of composite film; ∆P (kPa) refers to the water vapor pressure difference on both sides of the composite film; t (h) is the measurement time.

Water Contact Angle (WCA)
The surface contact angle of the films was measured by a contact angle measuring instrument (JY-PHb, Jinghe, China).The film sample (20 to 80 mm 2 ) was placed on a movable sample table and leveled horizontally; a drop of approximately 10 μL of deionized water was placed onto the surface of the film using a microsyringe.By drawing with the software, the value of the water contact angle could be directly measured (Andreuccetti et al., 2011).

Thickness and Morphology
The thickness of the film was measured by micrometer.
According to the previous method (Mandal & Chakrabarty, 2015), the surface morphology and cross-sectional images of all the films were observed using scanning electron microscope (SEM, HITACHI, Japan).The surface was magnified 1500 times, and the cross section was magnified 550 times.

Opacity
The transmittance of the film at 600 nm was measured by an ultraviolet spectrophotometer (UV-2550, SHIMADZU, Japan).Opacity was defined as the absorbance per unit thickness of the film at 600 nm: (3) WVP = △m × T × 24 where Abs refers to the absorbance of the film at 600 nm (dimensionless) and T is the thickness of film (mm).

Color
The color of all the film samples was measured by light color measurement instrument (CS-820, Caipu, China).The instrument was calibrated using air.The color parameters (ΔE*, L*, a* and b* value) were directly read from the instrument.

Tensile Test
The tensile strength (MPa) of the film was measured with a texture analyzer (TA.XT Plus, Stable Micro Systems Ltd, UK).The film was cut into the width of 1-cm rectangle shape.The initial distance of clamp was 10 mm, stretching speed was 1 mm s −1 , and tensile force was 10 g.The tensile distance of WGP was set to 80 mm, SPI was 60 mm, and Zein was 40 mm.The tensile strength was calculated with the following equation: In the equation, F stands for the force (kg) when the film was breaking, g is the local acceleration of gravity (m s −2 ), w is the width of film (m), and d (m) is the thickness of the film.

Puncture Test
Puncture strength tests were performed using a texture analyzer (TA.XT Plus, Stable Micro Systems Ltd, UK).The films with a diameter of 15 mm were fixed to a compression device with a diameter of 10-mm round hole.Then P/2 cylindrical probe was selected and moved perpendicularly to the film surface at a constant speed of 0.1 mm s −1 until the probe passed through the film.Force-deformation curves were obtained and force (N) at the puncture point was then recorded which was the hardness (N) of the films (Chen & Lai, 2008).

Thermal Property
The thermal property of films was detected by differential scanning calorimetry (DSC, TA Q2000, TA Instruments, USA).A certain quality of film was placed in an aluminum pan.The empty aluminum pan was used as a blank control during measurement.The sample was heated from 30 to 180 °C at 10 °C min −1 under a nitrogen atmosphere. (5)

Data Analysis
All the tests were carried out three times, the parallel mean value and standard deviation were reported.The OriginLab software was used for statistical analysis.Significant differences were carried out by using the Duncan test analysis in ANOVA.Unless otherwise specified, the significant differences are mentioned in the article based on 95% confidence level.

Water Resistance Performance
Moisture Content, WVP, and TSM Table 1 showed the data on moisture content, total soluble matter (TSM), and water vapor permeability (WVP) of the films.As shown in the Table 1, the moisture content of Zein film (2.20%) and SPI film (1.76%) was significantly higher than that of the WGP film (1.10%).After adding BW, the moisture content of the Zein-BW film (1.40%) was lower than that of the Zein film.This may be because that the combination of Zein and BW increased the hydrophobicity of the films due to their lipophilic substances (Khanzadi et al., 2015).For raw protein films, there was no significant difference of WVP value among the Zein film, SPI film, and WGP film.After adding BW, the WVP value of the three kinds of films didn't significantly change, indicating that BW did not improve the WVP of the protein films (Fabra et al., 2008).Cruces et al. (2021) prepared pectin-BW/rosinpectin (P-BC-P) multilayer films with different proportions of BW/rosin mixture.The results indicated that the addition of BW reduced the WVP value of the pectin film, which is different from our results.This may be because the mixture of BW and rosin formed an ordered crystal structure through hydrophobic interaction, while the single component of BW failed to produce this effect.Therefore, the WVP value of the three kinds of the films didn't significantly decrease after adding BW in our results.The TSM value of SPI film (100.00%) was significantly higher than that of the Zein film (51.74%) and WGP film (48.07%).

Water Contact Angle (WCA)
Figure 2 was the images of the WCA on the surface of the WGP film and SPI film.All surfaces of the films analyzed showed a hydrophilic nature, as no WCA value exceeded 65° whose WCA lower than 65° can be considered as hydrophilic surfaces (Vogler, 1998).Before the BW was added, the WCA of SPI film was significantly higher than that of the WGP film.This was probably because WGP had a tighter network structure compared with SPI, which bound fewer groups to lipids, leaving more lipids to improve the hydrophobicity of the film.After adding BW, the WCA of WGP film increased from 25.47 to 27.17°, but the SPI film decreased from 44.70 to 23.50°.In WGP, gliadin was embedded in the network structure formed by gluten, interacting with each other to form a three-dimensional network structure (Liao et al., 2013).After BW was added, the lipidbinding sites of the network system decreased, resulting in the increase of hydrophobicity of WGP film (Omar-Aziz et al., 2020).The WCA of SPI film decreased, it may be the result of glycerol migration (such as evaporation rate, uniaxial mass flux, contacting with hydrophobic carrier) during film drying, and solvent evaporation (Rocca-Smith et al., 2016).Besides, in the Zein film, loose structures with a lot of cavities and voids were observed (Ghanbarzadeh et al., 2007).Therefore, during the measurement water droplets rapidly penetrated the film, accurate data could not be obtained, and the WCA values were unavailable for the Zein films.

Thickness
The thickness of the film plays an essential role in the barrier performance of the film.As shown in Table 2, for raw protein films, there was significant difference in the thickness of the three films.Among the three raw protein films, the Zein film (126.33 μm) was thicker than the SPI film (105.67 μm) and WGP film (110.67 μm).After adding BW, the thickness of Zein-BW, SPI-BW, and WGP-BW films increased to 148.00 μm, 148.67 μm, and 160.33 μm, respectively.This may be due to new forms of cross-linking between BW and proteins during the process of the film formation, thus leading to an increased thickness of the films (Adilah et al., 2018).

Opacity
The results of the opacity were shown in Table 2.For raw protein films, Zein film presented the highest opacity (9.35 Abs/mm), which was due to the large amount of zeaxanthin that caused the yellow appearance.After adding BW, the opacity of the Zein-BW film decreased from 9.35 to 6.51 Abs/mm, whereas the opacity of the other two composite films significantly increased to 4.48 and 3.06 Abs/mm for SPI-BW and WGP-BW films, respectively.This may be because the chain mobility and intermolecular migration had influences on light permeability of the film (Zhang et al., 2012).Previous studies had reported that the addition of hydrophobic components could increase the opacity of the film (Wang et al., 2014a, b).Erdem et al. (2019) also discovered that whey protein isolate/sunflower seed oil film had an increased opacity due to the inclusion of sunflower seed oil.These were similar to the results of SPI-BW film and WGP-BW film in this work.Generally, films with lower opacity and higher luminosity can be used to observe the appearance of food more conveniently, while films with higher opacity are more suitable for the preservation of light-proof foods (Tang et al., 2019).Films with different properties are applied to different food packaging, so as to expand the application range of films.

Color
The color parameters of the films were also presented in Table 2.For raw protein films, there were significant differences in color among of the films made from the three proteins.The whiteness (L*) of the WGP film and SPI film was significantly higher than that of the Zein film.This was consistent with the result of opacity.The ΔE* value represents the change in the chromatic aberration, the ΔE* value and b* value (+ represents yellow) of the Zein film were significantly higher than those of the other two types of films, and this was the disadvantage of Zein film which may not be accepted by consumers.After adding BW, the L* value of the Zein film and WGP film was significantly reduced.BW acts as a surfactant which can increase the particles of Zein and WGP.The L* value reduced after adding BW which can be attributed by larger particles that decrease the light scattering (Suwannasang et al., 2021).The b* value and ΔE* value of the Zein-BW film and SPI-BW film were higher than those of the Zein film and SPI film, respectively.After BW was added, the a* value (-represents green) of SPI film and WGP film was significantly reduced, but there was no difference between Zein film and Zein-BW film.In addition, previous study also reported the effects of BW on the color parameters of protein film.Monedero et al. (2009) studied that the incorporation of BW softened the color of SPI film.And the more BW there are, the greater the softening effect.Generally, we should improve the transparency of the film as much as possible so as to make it more acceptable on the food packaging.

Morphology
The morphology of each film could be clearly observed by scanning electron microscopy (Fig. 3).The results of SEM showed that the morphology and structure of the three kinds of raw protein films were different.The surface of SPI film was generally flat.The surface of Zein film had small granules and appeared irregular folds.The WGP film exhibited relatively flat and smooth surface, and the structure was homogeneous.Compared with the raw films, many small granules appeared on the surface of the SPI-BW film and Zein-BW film.This was due to the interaction between BW and protein molecules, which was the aggregate of the protein and BW (Wang et al., 2014a, b).In the previous literature, Kang et al. (2016) studied the morphology of SPI film by SEM and AFM, and our results was similar to this study.However, the surface of the WGP-BW film appeared smoother.This may be due to a large number of hydrogen bonds in the molecular chains of WGP and BW, resulting in good compatibility of the composite films (Tanada-Palmu & Grosso, 2005).The cross-sectional image of all the films was also observed (Fig. 3).Before adding BW, the SPI film was denser than the other two films.Many stomata appeared on the WGP film and WGP-BW film.Among the WGP, the elasticity of glutenin affected the WGP film formation.In the process of film formation, enough pressure was applied to overcome the partial elasticity.However, it caused the air or water vapor to be surrounded by the continuous protein phase and fixed in the gluten network structure.So the WGP film and WGP-BW film formed a porous and fibrous structure with many pores (Li et al., 2019).The cross section produced more bubbles, and the structure became looser.This also explained the difference in the WGP film and WGP-BW film thickness from a microscopic point of view.

Tensile Test
Tensile strength results of raw films and composite films were shown in Fig. 4. The tensile strength of all three kinds of raw protein films was significantly different.The Zein film showed a higher tensile strength (9.00 MPa) than that of the SPI film (1.77MPa) and the WGP film (0.60 MPa).Generally, the Zein film is brittle and likely to break (Woods & Selling, 2010).The rigidity of protein molecules between Zein was strong, which made it easy to crack after film formation (Gontard et al., 2010).The intermolecular hydrogen bond between WGP was an essential factor in maintaining the tensile strength (Ma et al., 2012).But the intermolecular hydrogen bond network of WGP is weak, so the WGP film had a low tensile strength.
The tensile strength of the Zein-BW film (6.98 MPa) was lower than that of the Zein film (9.00 MPa).The tensile strength of the SPI-BW film (1.12 MPa) was also lower than that of the SPI film (1.77MPa).But there was no significant difference in the tensile strength between the WGP film (0.62 MPa) and WGP-BW film (0.64 MPa).BW can interact with the side chains of various amino acids in the polypeptide chain.Studies had shown that BW reduced the brittleness of Zein film, giving its softness and adhesion (Guo et al., 2015).Zein film was quite brittle.After adding BW, Zein-BW film became soft, so we found that BW can increase the flexibility of the film.The hydrogen bond interaction between SPI and BW is the main factor affecting the tensile properties of SPI-BW films (Zhang et al., 2012).Besides, the enhancement of disulfide bond in SPI film with BW will also reduce the tensile strength of SPI film (Erdem et al., 2021).When the BW was added, the threedimensional network structure of raw protein was changed and the interaction between the protein chains was deceased; hence, the fluidity of the chain increased (Meng et al., 2021).Thus, the strength of the film decreased, and the ductility increased.For WGP-BW film, the interaction between the BW and protein was weak (Cheng et al., 2021), so there was no significant change of the tensile strength.Our results are consistent with the other literatures (Kowalczyk al., 2015;Basch et al., 2013).The decrease in tensile strength of the protein film is attributed to the modification of film networks.The BW disrupted the associations of biopolymer chains and decreased the amounts of interchain hydrogen bonds.This resulted in a plastic strengthening of the film and thus reduced tensile strength.

Puncture Test
Figure 4 also showed the puncture test result of the films.There were significant difference among the Zein (5.48 N), SPI (3.36 N), and WGP films (2.08 N).For Zein film, the Zein molecules were cross-linked with each other under the action of hot water bath, finally resulting in strong bonding force between the molecules.We speculated that due to the intermolecular binding force, the hardness of Zein film was significantly higher than that of other films.Meanwhile, the WGP film exhibits the lowest hardness.This could be due to the presence of glutenin in WGP which were responsible for the elastic behavior (Jie et al., 2021).Thus, the hardness of WGP film is low.After adding BW, the WGP-BW and SPI-BW films exhibited lower hardness (2.76 N and 1.43 N, respectively).For SPI-BW film, it may have resulted from the interaction between BW and SPI molecules which weakened the bond between protein molecules (Fabra et al., 2009).This effect increased the fluidity of the chain and ultimately resulted in a lower hardness of the film.For WGP-BW film, the disulfide bonds, hydrogen bonds, and covalent bonds of the gluten protein in the WGP molecules formed a three-dimensional network structure.The added BW remained in the network structure, leading to the discontinuities of the protein matrix, which reduced the hardness of the films.Previous studies also found analogous results with whey protein puncture tests (Talens & Krochta, 2010).With the addition of the BW, it had great effect on the continuous matrix of the SPI and WGP, which disrupted the continuous matrix and induced the heterogeneous film structure (Chao et al., 2010).All of this led to a decrease in the hardness of the film.

Thermal Property
DSC is an analytical method to measure the energy changes in the denaturation process to reflect the structural characteristics of proteins.The DSC curves were carried out to observe the thermal properties of protein films (Fig. 5); all the films showed endothermic peaks.The endothermic process was caused by hydrogen bond breaking and molecular rearrangement of the helical structure of protein molecules (Oh et al., 2019).The endothermic process was associated with the vitrification transition of the protein phase (Thomas et al., 2016).The temperature at which the film denatured (T d ) was the important criterion for assessing the thermal stability of the film.It is known that T d is the temperature at which a film reversibly changes from a brittle state to a soft rubbery state.When temperatures were below T d , the material has a glassy structure, and at temperatures above T d , it is in a rubbery state (Erdem et al., 2021).For raw protein films, the WGP film exhibited the highest T d (106 °C), and the T d of Zein film was 77 °C, and the T d of SPI film was 86 °C.Therefore, the structure of the film became looser (Tavares et al., 2020).For WGP film, the T d was greatly reduced from 106 to 98 °C after BW was added.According to previous literature, the addition of BW would disturb the interaction between the molecules of the composite film and the fluidity of the molecular chain also increased.The T d of the Zein-BW and WGP-BW films was lower than that of the raw films because the hydrogen bonds of protein had been broken after adding BW.All the above results indicated that the molecular binding inside the protein film was looser and the thermal stability was decreased after adding the BW.

Conclusions
In this study, to expand the application of edible protein film and realize resource recycling, BW was added into Zein, SPI, and WGP for the first time, and the protein-lipid composite film was successfully prepared.The addition of BW improved the performance of the original protein films.After the BW was added, the thickness of all composite films was significantly improved.The opacity of the three composite films also changed significantly, and the Zein film had higher opacity than that of the other two films.Besides, the tensile strength all composite films showed a downward trend.The tensile strength and the hardness of the Zein-based films were still higher than those of the other two protein-based films.After BW was added, the thermal stability of the WGP and Zein films were decreased.Moreover, the thermal stability of the WGP-based films were higher than those of the other two protein-based films.This can enable different properties of protein film used in different food packaging, which expand the range of application of protein films.After adding BW, the water resistance performance of protein-based films didn't change significantly, which needs to be further discussed in the future.In summary, it is feasible to use protein as raw material and add BW to modify the film.This study lays a foundation for the production of biodegradable edible film and has good practical significance.

Fig. 1
Fig. 1 Schematic diagram of preparation and structure of three kinds of protein films

Fig. 2
Fig. 2 Water contact angle image of different films.A The SPI film, B the WGP film, C the SPI-BW film, D the WGP-BW film.Different letters in the same column indicate significantly different (p < 0.05)

Fig. 3
Fig. 3 Scanning electron microscopy (SEM) images of surface and cross section of different films.A The SPI film, B the WGP film, C the Zein film, D the SPI-BW film, E the WGP-BW film, F the Zein-BW film

Fig. 4
Fig. 4 Tensile strength and hardness of different protein films.Different superscript letters indicate significance at p < 0.05 For the SPI film,Shaikh et al. (2019) reported a lower T d , which is similar to our results.The hydrophilic property of SPI structure might cause lower T d .The hydrophilic nature might attract more water molecules into the film matrix, and water enhances interchain mobility and lowers T d of the film.After adding BW, the T d of the SPI film didn't change.The T d of Zein film was reduced (from 77 to 72 °C), which meant the mobility of molecular chain has increased.