# Preparation and Characterization of Biodegradable Composited Films Based on Potato Starch/Glycerol/Gelatin

Authors: Xiaoqing Niu; Qianyun Ma; Shuiling Li; Wenxiu Wang; Yajuan Ma; Hongqian Zhao; Jianfeng Sun; Jie Wang
Journal: Journal of Food Quality
 (2021)

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## Abstract

The use of plastics is resisted worldwide. Therefore, finding alternatives to plastic packaging products is an urgent issue. This work was dedicated to the preparation of biodegradable composited films with potato starch, glycerol, and gelatin. The formulation of the biodegradable film was first optimized via response surface methodology combined with the multi-index comprehensive evaluation method that considered physical properties (thickness, water solution (WS), tensile strength (TS) and elongation at break (E%)) and barrier property (light transmittance (T%)). Results indicated that the optimal conditions were 2.5% starch, 2.0% glycerol, and 1.5% gelatin (based on water). The optimized film presented excellent properties with TS of 4.47 MPa, E% of 109.91%, WS of 43.64%, and T% of 41.21% at 500 nm, and the comprehensive evaluation score of the composite film was 28.68. Moreover, a model verification experiment was further conducted, which proved that the predicted value highly matched experimental values, indicting the credibility and accuracy of the model. The resulting films were further characterized on the basis of rheological measurements, Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). The rheological measurements proved that the film-forming solution exhibited low shear viscosity and non-Newtonian fluid behavior. FTIR and SEM revealed excellent compatibility among starch, glycerol, and gelatin. Hence, the resulting optimized film may be expected to provide theoretical basis and technical support for the food packing industry.


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## Body

## 1. Introduction

In the food industry, food packaging is required to enhance food protection and guarantee shelf life on the basis of their appropriate physicochemical features [1, 2]. Petroleum-based plastic packaging is still widely used due to its cost-effectiveness. However, with the worsening of environmental issues caused by nonrenewable and nondegradable plastic packaging and the serious harm to human health due to the accumulation of plastic waste [3, 4], some prohibitions about petroleum-based plastic packaging have been enforced. Internationally, the European Chemicals Agency has limited the use of conventional synthetic plastic polymers for food packaging [5]. In the domestic setting, plastic waste materials have been prohibited from importation due to their toxicity [6, 7]. Therefore, to overcome these issues, replacing traditional plastic packaging with renewable, biodegradable materials has attracted considerable attention in recent decades. Generally, biodegradable packaging films were produced by natural biodegradable materials included polysaccharides, proteins, lipids, and their complexes, which make up for the drawbacks of plastic and solve those root environmental issues caused by plastic waste. Among these natural biodegradable films, starch-based films have incomparable advantages included abundant, low-cost, high film transparency, over other food packaging materials, which has been proved that starch-based film has exceed 60% of the total biodegradable film [8].Starch is a highly annually renewable and high-molecular-weight polymer material that is composed of two types ofα-glucan, namely amylose and amylopectin [9, 10]. Amylose is a linear polysaccharide that is connected by α-1,4-glycosidic bonds, and that presents excellent film-forming property similar to fibers [11]. Moreover, the α-1,4-glycosidic bonds and α-1,6-glycosidic bonds present in amylopectin are conducive to the thickening effect and freeze-thaw stability [12]. The proportion of amylose and amylopectin in various sources of starch is also different and directly affects film-forming properties [13, 14]. Therefore, increasing efforts have been devoted to the research of various resources of starch-based films [9, 15, 16]. The maize starch matrix film was produced and it was found that the films possessed low tensile strength (TS) and water vapor permeability (WVP), which could be enhanced by zein blended, and great compatibility with maize starch matrix [10]. Chollakup et al. [17] investigated cassava starch film, who proved the hydrophobic interactions and hydrogen bonding present in cassava starch and whey protein blended films to explain their barrier mechanisms at the molecular level. Lin et al. [18] utilized cellulose nanocrystals and PVA (polyvinyl alcohol)/glycerol to modify the formability of oxidized starch films with excellent WVP, surface free energy, and oxygen resistance. The characteristic properties of starch–PLA (polylactic acid) composite films [19] significantly improved with the use of cellulosic strengthening agents (cellulosic fibers and cellulose nanocrystals) and coffee husk extracts to enhance tensile properties, reduced barrier permeability, and endowed additional antioxidant capacity to food packaging systems. These results indicated that the research on starch-based film from various resources had been continued, and the contribution of physicochemical and mechanical properties by a single starch is limited and needed to blend with other components to acquire desirable performances for improving product texture and resistance to various physical changes.Furthermore, as potato staple food strategy initiated, potatos have been the largest noncereal food crop worldwide [20], and potato planting area expanded 6.7 million hectares (2020) [21]. The yield of potato starch also increased up to 96 million tonnes (2016) [22], which has been a promising natural carbohydrate for food packing material. The size of potato starch granules is relatively large (25–100 μm), and exists in typile B-type crystallinity [23]. Also, in the presence of plasticizers and under high temperature and shearing conditions, potato starch would acquire thermoplastic property which was suitable for preparing biodegradable film [24]. However, the performance of potato starch-based films still needs to be improved to extend the application in food package because of their poor water resistance. To address such issue, plasticizing agents and other active ingredients were incorporated to endow various advantages to films. Glycerol has been reported as the most starch-compatible plasticizing agent that can endow polymers with flexibility and resilience [25, 26]. Moreover, gelatin, a protein of animal origin, exhibits excellent thermoreversible capability and thermal stability, presenting possible barrier performance [27, 28].Although a range of various composite films has been investigated, few pieces of research were conducted about the optimized formulation of the composite film via response surface methodology (RSM) combined with the multi-index comprehensive evaluation method that considered physical properties (thickness, water solution (WS), tensile strength (TS), and elongation at break (E%)) and barrier property (light transmittance (T%)). Thus, this study aimed to prepare a durable biodegradable film with excellent comprehensive performance based on potato starch, glycerol, and gelatin. The optimized films were further characterized by scanning electron microscopy (SEM), rheological measurements, and Fourier transform infrared spectroscopy (FTIR).


## 2. Materials and Methods

### 2.1. Materials

Potato starch was purchased from a local market in Baoding, China. Glycerol was purchased from Tianjin Kemiou Chemical Reagent Co., Ltd (Tianjin, China). Edible gelatin, extracted from pig skin, was obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).


### 2.2. Preparation of Complex Films

Potato starch was dispersed in 100 mL of distilled water and stirred for 30 min at 80°C until completely gelatinized. Glycerol and gelatin were successively added to the film-forming solution. The mixtures were degassed for another 30 min by using an ultrasonic device (SK5200H, Ningbo Scientz Biotechnology Co., Ltd. Zhejiang, China), then casting onto a plate, and dried at 40°C for 36 h in an incubator [29]. After this period, the films were peeled for further analysis.


### 2.3. Comprehensive Score Calculation

Membership degree and principal component analysis (PCA) were used to evaluate the films comprehensively. In accordance with importance, the indexes of the films were normalized to optimize the production process. Each index weight was determined. Indexes with high membership, such as TS and elongation at break (E%), were calculated using equation (1). WS (water solution) and light transmittance (T%) were calculated with equation (2):(1)P=Ai−AminAmax−Amin,(2)P=Amax−AiAmax−Amin,where P is the index membership degree; Ai is the actual value; Amin is the minimum of the same index; Amax is the maximum of the same index.The comprehensive score (Y) was calculated with the following equation:(3)Y=a1×P1+a2×P2+a3×P3+a4×P4+a5×P5,where a1, a2, a3, a4, and a5 are the weights of thickness, TS, E%, WS, and T%, respectively, and P1, P2, P3, P4, P5 are membership degrees of thickness, TS, E%, WS, and T%, respectively.


### 2.4. Design of Experiments

Film concentration ratios were optimized by using RSM to investigate the synergistic effect of the variables. Three-level (starch concentrationX1 [1–6 g/100 mL], glycerol concentration X2 [0.5–3 g/100 mL], and gelatin concentration X3 [0.5–3 g/100 mL]) factorial design was applied to investigate the effects of the parameters on WVP, TS, T%, and E%. After preliminary tests, the appropriate levels of the independent variables needed to obtain the optimal characteristic properties of the biodegradable composite film were measured.A total of 17 formulas with five replicates at the center point were evaluated to ensure the repeatability of the RSM model. These formulas are listed in Table1, which clearly records the coded and uncoded levels of starch, glycerol, and gelatin concentrations. The response for each formulation was independently evaluated with three replicates. A random experimental design was used to eliminate the effects of possible uncontrolled factors.Table 1
Experiments design with coded and uncoded independent variables.
RunCode variablesUncoded variablesX1X2X3Starch concentrationGlycerol concentrationGelatin concentration11.0000.0001.00042220.0001.000−1.00032.5130.0000.0000.000321.540.0001.0001.00032.525−1.0000.0001.00022260.0000.0000.000321.57−1.0000.000−1.0002218−1.0001.0000.00022.51.59−1.000−1.0000.00021.51.5100.0000.0000.000321.5110.0000.0000.000321.5121.0000.000−1.000421130.000−1.000−1.00031.51141.000−1.0000.00041.51.5150.0000.0000.000321.5160.000−1.0001.00031.52171.0001.0000.00042.51.5


### 2.5. Verification Test

Verification procedures were applied to determine the optimal level of composite films with excellent properties. The experimental data and fitted value predicted by the model were compared to verify the validity and adequacy of the RSM model.


### 2.6. Acquisition of Light Transmission Curves

UV-vis spectrophotometer (α-1500, Shanghai, China) was used to acquire the light transmittance spectra of the complex films. Briefly, the film was cut into strips (1 × 4 cm) and placed closely to the inner surface of the cuvette to record the T% with three replicates ranging from 400 nm to 800 nm.


### 2.7. Rheology of Film-Forming Solutions

The static rheological properties of the film-forming solutions were determined by using a rotational rheometer (RheolabQC, Anton Paar Instruments Ltd., Japan) equipped with a concentric cylinder (CC 39, 40 mm diameter, Austria) and temperature control system (TEZ 150P-C). The shear rate was varied from 1 s−1 to 300 s−1 with the Ramp log procedure at 25°C and the flow behavior was recorded every 30 s.The rheological properties of films were further stimulated by the Cross model [30]:(4)η=η∞+η0−η∞1+KγΡ,where η, η0, and η∞ represented viscosity, the initial shear viscosity, and infinite shear viscosity, respectively. γ indicated shear rate (s−1), and K and Þ were time constant and dimensionless exponent, respectively.


### 2.8. Physical Properties

The thicknesses of the films was recorded by using an electronic thickness gauge (EC770, Shanghai, China) at 10 random positions. An autotensile tester (PARAM XLW-PC, Jinan, China) was used to measure the TS and elongation at break (E%) of the films at a cross-head speed of 300 mm/s [31]. T% was measured with a UV-vis spectrophotometer at 500 nm.WVP of the film was measured by the gravimetric method [32]. The film (8 cm × 8 cm) was sealed in a circular cup with 20 g anhydrous CaCl2 (0% RH (relative humidity)). The cups were placed inside of desiccator with distilled water (100% RH). The difference in RH promoted the transport of water vapor, which caused weight changes.WS was tested according to the modified weight loss method [33] which was performed with three replicates to determine the WS of films. Briefly, at room temperature, a film (2 cm × 2 cm) was weighed prior to dissolution in distilled water for 24 h and then removed and dried at 90°C to a constant weight. WS was calculated by the following equation:(5)WS=M1−M2M1×100%,where M1 and M2 are the initial and terminal weights of the films (dry basis), respectively.


### 2.9. Morphology of Films

The microstructures of cross-sections and surfaces were measured by using a scanning electron microscope (JSM-7900F, Japan) with an accelerating voltage of 12.5 kV. Before the observation, the films were coated with a thin layer of gold.


### 2.10. FTIR Spectroscopy

FTIR spectra were acquired over the range of 500–4000 cm−1 in attenuated total reflection mode with a resolution of 4 cm−1 by using an FTIR spectrometer (Shimadzu Instrument Co. Ltd., Japan).


## 2.1. Materials

Potato starch was purchased from a local market in Baoding, China. Glycerol was purchased from Tianjin Kemiou Chemical Reagent Co., Ltd (Tianjin, China). Edible gelatin, extracted from pig skin, was obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).


## 2.2. Preparation of Complex Films

Potato starch was dispersed in 100 mL of distilled water and stirred for 30 min at 80°C until completely gelatinized. Glycerol and gelatin were successively added to the film-forming solution. The mixtures were degassed for another 30 min by using an ultrasonic device (SK5200H, Ningbo Scientz Biotechnology Co., Ltd. Zhejiang, China), then casting onto a plate, and dried at 40°C for 36 h in an incubator [29]. After this period, the films were peeled for further analysis.


## 2.3. Comprehensive Score Calculation

Membership degree and principal component analysis (PCA) were used to evaluate the films comprehensively. In accordance with importance, the indexes of the films were normalized to optimize the production process. Each index weight was determined. Indexes with high membership, such as TS and elongation at break (E%), were calculated using equation (1). WS (water solution) and light transmittance (T%) were calculated with equation (2):(1)P=Ai−AminAmax−Amin,(2)P=Amax−AiAmax−Amin,where P is the index membership degree; Ai is the actual value; Amin is the minimum of the same index; Amax is the maximum of the same index.The comprehensive score (Y) was calculated with the following equation:(3)Y=a1×P1+a2×P2+a3×P3+a4×P4+a5×P5,where a1, a2, a3, a4, and a5 are the weights of thickness, TS, E%, WS, and T%, respectively, and P1, P2, P3, P4, P5 are membership degrees of thickness, TS, E%, WS, and T%, respectively.


## 2.4. Design of Experiments

Film concentration ratios were optimized by using RSM to investigate the synergistic effect of the variables. Three-level (starch concentrationX1 [1–6 g/100 mL], glycerol concentration X2 [0.5–3 g/100 mL], and gelatin concentration X3 [0.5–3 g/100 mL]) factorial design was applied to investigate the effects of the parameters on WVP, TS, T%, and E%. After preliminary tests, the appropriate levels of the independent variables needed to obtain the optimal characteristic properties of the biodegradable composite film were measured.A total of 17 formulas with five replicates at the center point were evaluated to ensure the repeatability of the RSM model. These formulas are listed in Table1, which clearly records the coded and uncoded levels of starch, glycerol, and gelatin concentrations. The response for each formulation was independently evaluated with three replicates. A random experimental design was used to eliminate the effects of possible uncontrolled factors.Table 1
Experiments design with coded and uncoded independent variables.
RunCode variablesUncoded variablesX1X2X3Starch concentrationGlycerol concentrationGelatin concentration11.0000.0001.00042220.0001.000−1.00032.5130.0000.0000.000321.540.0001.0001.00032.525−1.0000.0001.00022260.0000.0000.000321.57−1.0000.000−1.0002218−1.0001.0000.00022.51.59−1.000−1.0000.00021.51.5100.0000.0000.000321.5110.0000.0000.000321.5121.0000.000−1.000421130.000−1.000−1.00031.51141.000−1.0000.00041.51.5150.0000.0000.000321.5160.000−1.0001.00031.52171.0001.0000.00042.51.5


## 2.5. Verification Test

Verification procedures were applied to determine the optimal level of composite films with excellent properties. The experimental data and fitted value predicted by the model were compared to verify the validity and adequacy of the RSM model.


## 2.6. Acquisition of Light Transmission Curves

UV-vis spectrophotometer (α-1500, Shanghai, China) was used to acquire the light transmittance spectra of the complex films. Briefly, the film was cut into strips (1 × 4 cm) and placed closely to the inner surface of the cuvette to record the T% with three replicates ranging from 400 nm to 800 nm.


## 2.7. Rheology of Film-Forming Solutions

The static rheological properties of the film-forming solutions were determined by using a rotational rheometer (RheolabQC, Anton Paar Instruments Ltd., Japan) equipped with a concentric cylinder (CC 39, 40 mm diameter, Austria) and temperature control system (TEZ 150P-C). The shear rate was varied from 1 s−1 to 300 s−1 with the Ramp log procedure at 25°C and the flow behavior was recorded every 30 s.The rheological properties of films were further stimulated by the Cross model [30]:(4)η=η∞+η0−η∞1+KγΡ,where η, η0, and η∞ represented viscosity, the initial shear viscosity, and infinite shear viscosity, respectively. γ indicated shear rate (s−1), and K and Þ were time constant and dimensionless exponent, respectively.


## 2.8. Physical Properties

The thicknesses of the films was recorded by using an electronic thickness gauge (EC770, Shanghai, China) at 10 random positions. An autotensile tester (PARAM XLW-PC, Jinan, China) was used to measure the TS and elongation at break (E%) of the films at a cross-head speed of 300 mm/s [31]. T% was measured with a UV-vis spectrophotometer at 500 nm.WVP of the film was measured by the gravimetric method [32]. The film (8 cm × 8 cm) was sealed in a circular cup with 20 g anhydrous CaCl2 (0% RH (relative humidity)). The cups were placed inside of desiccator with distilled water (100% RH). The difference in RH promoted the transport of water vapor, which caused weight changes.WS was tested according to the modified weight loss method [33] which was performed with three replicates to determine the WS of films. Briefly, at room temperature, a film (2 cm × 2 cm) was weighed prior to dissolution in distilled water for 24 h and then removed and dried at 90°C to a constant weight. WS was calculated by the following equation:(5)WS=M1−M2M1×100%,where M1 and M2 are the initial and terminal weights of the films (dry basis), respectively.


## 2.9. Morphology of Films

The microstructures of cross-sections and surfaces were measured by using a scanning electron microscope (JSM-7900F, Japan) with an accelerating voltage of 12.5 kV. Before the observation, the films were coated with a thin layer of gold.


## 2.10. FTIR Spectroscopy

FTIR spectra were acquired over the range of 500–4000 cm−1 in attenuated total reflection mode with a resolution of 4 cm−1 by using an FTIR spectrometer (Shimadzu Instrument Co. Ltd., Japan).


## 3. Results and Discussion

### 3.1. Light Transmission Analysis

The light transmittance of the film is an important index to directly affect consumer acceptance. And the light transmission curve of a resulting optimized film (starch + glycerol + gelatin) is shown in Figure1(a). The remarkable UV barrier capability shown by the film over the UV range of 200–280 nm would protect food from oxidative deterioration [32]. This characteristic was due to the intermolecular hydrogen bond between starch and glycerol during the film-forming process, resulting in good miscibility [34]. The optical property of film is significantly reinforced with wavelength increasing. Moreover, over the visible range (at 500 nm), the T% of films reached the maximum value then remained constant at approximately 90%, indicating excellent transparency. Such high transmittance indicated the uniform of film. Perfectly clear food packaging would not adversely affect the consumer experience. The picture covered by these films could be clearly observed (Figure 1(b)), and further proved good transmittance.Figure 1
(a) The light transmission curve. (b) The pictures of the films.
(a)(b)


### 3.2. Comprehensive Evaluation of the Composite Film

PCA was performed by using SPSS19.0 software on six randomly selected sets of data (Table2) to evaluate film comprehensive performance. According to the criterion of eigenvalues exceeding 1, the results of PCA (Table 3) showed that the three principal components (PC1, PC2, and PC3) are extracted and the variance contributions of PC1, PC2, and PC3 were 40.425%, 29.065%, and 20.892%, respectively. In addition, the cumulative variance of the three components reached 90.382%, indicating that PC1, PC2, and PC3 could contain most of the information (more than 90%) of all indexes (TS, E%, WS, thickness, and T%) and reflect the comprehensive performance of the films. Therefore, PC1, PC2, and PC3 could replace the original complex indexes.Table 2
Test data randomly selected for principal component analysis.
IndexesThickness (mm)E%TS (MPa)T%WS %10.071403.3500.2840.267420.060198.954.1300.4140.434330.066102.1511.6400.2980.325640.07366.171.2700.4390.433150.070166.21.9870.1230.349660.066198.952.9670.3610.3197Table 3
Eigenvalues and variance contribution rates of related components.
Principal componentsEigenvalueVariance contribution rate (%)Cumulative variance contribution rate (%)PC12.02140.42540.425PC21.45329.06569.489PC31.04520.89290.382PC40.3887.76698.148PC50.0931.852100.000Table4 lists the factor loading matrix and the contribution rate of thickness, TS, E%, and WS to PC1, PC2, and PC3 (eigenvalue > 1). As shown in Table 4, thickness, E%, and WS mainly affected PC1; TS mainly affected PC2; T% and TS mainly affected PC3. After normalization, the percentages of thickness, TS, E%, WS, and T%, were 13%, 34%, 18%, 30%, and 5%, respectively. The comprehensive score (Y) was calculated by using the following equation in accordance with equations (1)–(3):(6)Y=13%×P1+34%×P2+18%×P3+30%×P4+5%×P5.Table 4
The factor loading matrix of three components.
IndexPrincipal componentsPercentage (%)123T%0.514−0.5380.57513Thickness(mm)0.8440.4750.06234E%0.7720.313−0.52018TS (MPa)0.0380.7050.66230WS (%)0.668−0.5860.0415− indicated negative correlative.


### 3.3. RSM Analysis

RSM was applied to investigate the linear, quadratic, and interaction effects of each independent variable (starch, glycerol, and gelatin concentrations) on response values. All the response experimental data are listed in Table5.Table 5
Experiments data of the responses.
RunsResponseThickness (mm)E%TS(MPa)T% (500 nm)Water solubility%Comprehensive scores (Y)10.07687.6339.87336.452.625.9020.057121.0334.63044.960.733.0430.071138.3002.21738.051.336.7740.073109.8009.22331.152.231.4650.079101.73310.80033.158.529.8560.096115.4335.51735.558.131.7370.08466.9009.57336.146.720.4180.09788.13311.20731.831.826.3990.056157.4002.32752.835.841.77100.096169.6006.80034.955.846.30110.079195.30018.23038.760.256.35120.095184.3007.91732.352.150.44130.08260.86722.40032.242.322.68140.101201.7006.04537.365.154.44150.084173.2006.90735.553.647.26160.096144.2505.98550.546.139.47170.056197.7333.75048.948.752.70Each value represents the mean value of three determinations.The different evaluation indexes, including the F value, the lack-of-fit value, and the correlation coefficientR2, are presented in Table 6 to verify the model’s adequacy and accuracy. The predictive model was extremely significant (p<0.01). A low p value indicates the high significance and applicability of the regression model. Moreover, the p values of the linear (X1), quadratic (X13), and interactive (X12, X13, and X23) models were all less than 0.05, which indicated that the model fits well with the measured data. R2 and adj-R2 were used to prove the adequacy and goodness of fit of the applied model. A high R2 value (R2 > 0.9) indicated the high reliability of the models and acceptable error between the calculated and experimental results. The insignificance between R2 and R2-adj indicated the adequate accuracy and applicability of the selected polynomial model. The response comprehensive score (Y) was fitted with the regression equation as shown in the following equation:(7)Y=50.96−7.92X1+4.12X2+2.71X3+0.012X1X2−5.48X1X3+0.95X2X3−8.20X12−10.53X22−8.70X32.Table 6
ANOVA study for the model fitting.
SourceSSDfF valuep valueSignificantModel2010.48911.310.0021∗∗X1501.49125.390.0015∗∗X2135.8016.880.0343∗X358.5412.970.1288X1X20.00062513.16E − 050.9957X1X3120.0116.080.0431∗X2X33.5910.1880.6826X12282.79114.320.0069∗∗X22466.45123.620.0018∗∗X32318.35116.120.0051∗∗Residual138.257Lack-of-fit61.4131.070.4571Pure error76.844Cor. total2148.7316R2 = 0.9357R2Adj = 0.8529∗represented significant (p<0.05), ∗∗represented extremely significant (p<0.01).3D response surfaces were used (Figure2) to further interpret the effects of interactions among independent variables (starch, glycerol, and gelatin concentrations) on response value. The closer the bottom curve, which is the projection of the 3D response surface, to the ellipse, the more obvious the interaction effect and vice versa. In accordance with Table 5, the figure shows significant interaction between X1 and X2 and no significant interaction between X1 and X2 and between X2 and X3. Such results further indicated that the RSM model was reliable and veritable.Figure 2
3D responses surfaces.
(a)(b)(c)


### 3.4. Verification Experiments

Verification experiments on the optimal variable concentrations (2.40% starch, 2.11% glycerol, and 1.68% gelatin) and experimental concentrations (2.5% starch, 2.0% glycerol, and 1.5% glycerin) were performed to validate the reliability and validity of the model equations. The results of the verification experiments are shown in Table7. The acceptable percentage error between the predicted value and experimental value further demonstrated the reasonable adequacy of the response surface equations for responses and the reliability of the experiments.Table 7
Predicted and experimental data for the responses at the optimum point.
IndexesPredicted valueExperimental valuePercentage error (%)T%44.3741.21 ± 0.137.12Thickness (mm)0.0730.071 ± 0.022.73E%102.15109.91 ± 2.317.59TS (MPa)4.524.47 ± 1.211.11WS %39.1343.64 ± 0.0311.53Comprehensive score27.4828.68 ± 0.794.35Rheology of film-forming solutions.Figure3(a) depicts the variation curve of viscosity with the shear rate of 3 film solutions. Film thickness and uniformity are affected by the fluidity (viscosity) of film-forming solutions. The figure shows that the viscosity of the film-forming solutions decreased with the increase in shear rate, exhibiting non-Newtonian fluid behavior that might be attributed to the rearrangement of entangled molecules under high-speed shear [35, 36]. Such a result was in accordance with the finding of Glusac et al. [37], who proved that potato starch and zein proteins exhibited shear-thinning behavior. And the rheological behavior of the three films was successfully fitted by the Cross model (Table 8). The p value of all films was lower than 1, which further proved the pseudoplastic fluids. K-value was related to the destruction of the molecular structure inside the fluid [30]. And the three-component film showed the smallest K-value, which indicated the least time needed to form a new network structure and hydrogen bonding [38].Figure 3
(a) Shear viscosity curves. (b) Pictures of film-forming solutions.Table 8
The correlation coefficient fitted by the Cross model.
Sampleη0 (Pa·s)Κ (s)pR2Starch + glycerol + gelatin0.28202 ± 0.001170.03936 ± 4.68937E − 40.98729 ± 0.01470.99975Starch + glycerol1.46702 ± 0.011550.18258 ± 0.004530.91593 ± 0.012070.99991Starch + gelatin0.68782 ± 0.118940.55409 ± 0.380860.47097 ± 0.069550.99778The different viscosities of the film-forming solutions indicated different entanglement networks [30]. In principle, film-forming solutions become viscous with the increase in solute concentration. However, Figure 3(a) indicates that the film solution of the three mixed matrixes (starch + glycerol + gelatin) showed low viscosity. This result was consistent with Hussain et al. [39], who believed that the viscosity decrease could be attributed to the alteration in the macromolecular organization of the blended gelatin at a high shear rate.


### 3.5. The Physical Properties of Films

The physical properties including thickness, TS,E%, T%, WS, and WVP of the three films were listed in Table 9. The optimized film (starch + glycerol + gelatin) showed high thickness due to the blend of three components. Meanwhile, lower WVP of the optimized three-component film was possibly attributed to the forming of intermolecular hydrogen bonds between glycerol and starch with the matrix, resulting in strong interfacial adhesion [16] and lower WS indicated water resistance enhanced. Meanwhile, the lower TS and E% of the three-component film may probably be due to the agglomerates formed by the incorporation of gelatin, which disrupted the compactness of the hydrogen bonds [40]. Moreover, the T% showed no significance, which may be explained by the great miscibility of three components.Table 9
The physical performance of the optimized film.
SampleThickness (mm)TS (MPa)E%T%WS (%)WVP (×10−10gs−1m−1Pa−1)Starch + glycerol + gelatin0.071 ± 0.02a4.47 ± 1.21b109.91 ± 2.31b41.21 ± 0.13a43.64 ± 0.03a1.50 ± 0.02cStarch + glycerol0.064 ± 0.01b4.83 ± 0.5a138.32 ± 11.94a41.12 ± 0.34a44.83 ± 0.31c1.56 ± 0.04bStarch + gelatin0.068 ± 0.03b4.15 ± 0.07c89.62 ± 3.64c41.09 ± 0.18a43.77 ± 0.53b1.67 ± 0.02aDifferent letters indicated significant (p<0.05).


### 3.6. SEM Analysis

As shown in Figure4, SEM was applied to observe the microstructures of the surface and cross section of the optimized film. The smoothness and homogeneity of the surfaces presented in Figure 4(a) indicated that the components dispersed well within each other. The appearance of surface wrinkles did not mean that the film was uneven and discontinuous. Such wrinkles might be caused by adhesion between the film and the plastic plate when the film was peeled off. Additionally, as shown in Figure 4(b), distinctive ridged structures were presented in the cross-sections of the composite biodegradable film. The high uniformity of the biodegradable film was attributed to the plasticizing capability of glycerol and the thickening capability of gelatin. Furthermore, the numerous intermolecular hydrogen bonds that formed between hydroxyl groups in glycerol with the hydroxyl groups present in starch promoted close bonding in polymers [41] and further enhanced miscibility among starch, gelatin, and glycerol.Figure 4
The micrographs of optimized complex film (a) Surface. (b) Cross section.
(a)(b)


### 3.7. FTIR Analysis

The spectra of the single starch film and optimized biocomposite films are shown in Figure5. Typically, the broadening of the O–H and the decreasing of wavenumber could be possible due to the characteristic of hydrogen bonding interaction in the film [42]. For the control film (single starch film), the characteristic band around 3267 cm−1 was originally from the O–H stretching vibration, and the band absorption intensity was broadened with incorporation with gelatin and glycerol. Also, the characteristic peak at 1644 cm−1 slightly shifts to 1636 cm−1. This phenomenon indicated the formation of hydrogen bonds.Figure 5
FTIR spectra of optimized films.The similar characteristic peaks at approximately 2940 and 2875 cm−1 in the three biocomposite film spectra were attributed to C–H vibration and C=C vibration, respectively [43]. The peaks at 1636 and 1023 cm−1 represented the C=O and C–O stretching vibrations of glycoside bonds in the starch branch. The intensity of the peak at 1023 cm−1 increased with the incorporation of glycerol, and the enhancement in the –OH stretching vibration at approximately 3267 cm−1 suggested the occurrence of association [44]. Cross-linking between polymers tightened in the presence of intermolecular hydrogen and hydrogen-oxygen bonds (Figure 6). Such results illustrated the good compatibility between matrixes and were consistent with the SEM and rheology measurements.Figure 6
The schematic diagram of composite film.


## 3.1. Light Transmission Analysis

The light transmittance of the film is an important index to directly affect consumer acceptance. And the light transmission curve of a resulting optimized film (starch + glycerol + gelatin) is shown in Figure1(a). The remarkable UV barrier capability shown by the film over the UV range of 200–280 nm would protect food from oxidative deterioration [32]. This characteristic was due to the intermolecular hydrogen bond between starch and glycerol during the film-forming process, resulting in good miscibility [34]. The optical property of film is significantly reinforced with wavelength increasing. Moreover, over the visible range (at 500 nm), the T% of films reached the maximum value then remained constant at approximately 90%, indicating excellent transparency. Such high transmittance indicated the uniform of film. Perfectly clear food packaging would not adversely affect the consumer experience. The picture covered by these films could be clearly observed (Figure 1(b)), and further proved good transmittance.Figure 1
(a) The light transmission curve. (b) The pictures of the films.
(a)(b)


## 3.2. Comprehensive Evaluation of the Composite Film

PCA was performed by using SPSS19.0 software on six randomly selected sets of data (Table2) to evaluate film comprehensive performance. According to the criterion of eigenvalues exceeding 1, the results of PCA (Table 3) showed that the three principal components (PC1, PC2, and PC3) are extracted and the variance contributions of PC1, PC2, and PC3 were 40.425%, 29.065%, and 20.892%, respectively. In addition, the cumulative variance of the three components reached 90.382%, indicating that PC1, PC2, and PC3 could contain most of the information (more than 90%) of all indexes (TS, E%, WS, thickness, and T%) and reflect the comprehensive performance of the films. Therefore, PC1, PC2, and PC3 could replace the original complex indexes.Table 2
Test data randomly selected for principal component analysis.
IndexesThickness (mm)E%TS (MPa)T%WS %10.071403.3500.2840.267420.060198.954.1300.4140.434330.066102.1511.6400.2980.325640.07366.171.2700.4390.433150.070166.21.9870.1230.349660.066198.952.9670.3610.3197Table 3
Eigenvalues and variance contribution rates of related components.
Principal componentsEigenvalueVariance contribution rate (%)Cumulative variance contribution rate (%)PC12.02140.42540.425PC21.45329.06569.489PC31.04520.89290.382PC40.3887.76698.148PC50.0931.852100.000Table4 lists the factor loading matrix and the contribution rate of thickness, TS, E%, and WS to PC1, PC2, and PC3 (eigenvalue > 1). As shown in Table 4, thickness, E%, and WS mainly affected PC1; TS mainly affected PC2; T% and TS mainly affected PC3. After normalization, the percentages of thickness, TS, E%, WS, and T%, were 13%, 34%, 18%, 30%, and 5%, respectively. The comprehensive score (Y) was calculated by using the following equation in accordance with equations (1)–(3):(6)Y=13%×P1+34%×P2+18%×P3+30%×P4+5%×P5.Table 4
The factor loading matrix of three components.
IndexPrincipal componentsPercentage (%)123T%0.514−0.5380.57513Thickness(mm)0.8440.4750.06234E%0.7720.313−0.52018TS (MPa)0.0380.7050.66230WS (%)0.668−0.5860.0415− indicated negative correlative.


## 3.3. RSM Analysis

RSM was applied to investigate the linear, quadratic, and interaction effects of each independent variable (starch, glycerol, and gelatin concentrations) on response values. All the response experimental data are listed in Table5.Table 5
Experiments data of the responses.
RunsResponseThickness (mm)E%TS(MPa)T% (500 nm)Water solubility%Comprehensive scores (Y)10.07687.6339.87336.452.625.9020.057121.0334.63044.960.733.0430.071138.3002.21738.051.336.7740.073109.8009.22331.152.231.4650.079101.73310.80033.158.529.8560.096115.4335.51735.558.131.7370.08466.9009.57336.146.720.4180.09788.13311.20731.831.826.3990.056157.4002.32752.835.841.77100.096169.6006.80034.955.846.30110.079195.30018.23038.760.256.35120.095184.3007.91732.352.150.44130.08260.86722.40032.242.322.68140.101201.7006.04537.365.154.44150.084173.2006.90735.553.647.26160.096144.2505.98550.546.139.47170.056197.7333.75048.948.752.70Each value represents the mean value of three determinations.The different evaluation indexes, including the F value, the lack-of-fit value, and the correlation coefficientR2, are presented in Table 6 to verify the model’s adequacy and accuracy. The predictive model was extremely significant (p<0.01). A low p value indicates the high significance and applicability of the regression model. Moreover, the p values of the linear (X1), quadratic (X13), and interactive (X12, X13, and X23) models were all less than 0.05, which indicated that the model fits well with the measured data. R2 and adj-R2 were used to prove the adequacy and goodness of fit of the applied model. A high R2 value (R2 > 0.9) indicated the high reliability of the models and acceptable error between the calculated and experimental results. The insignificance between R2 and R2-adj indicated the adequate accuracy and applicability of the selected polynomial model. The response comprehensive score (Y) was fitted with the regression equation as shown in the following equation:(7)Y=50.96−7.92X1+4.12X2+2.71X3+0.012X1X2−5.48X1X3+0.95X2X3−8.20X12−10.53X22−8.70X32.Table 6
ANOVA study for the model fitting.
SourceSSDfF valuep valueSignificantModel2010.48911.310.0021∗∗X1501.49125.390.0015∗∗X2135.8016.880.0343∗X358.5412.970.1288X1X20.00062513.16E − 050.9957X1X3120.0116.080.0431∗X2X33.5910.1880.6826X12282.79114.320.0069∗∗X22466.45123.620.0018∗∗X32318.35116.120.0051∗∗Residual138.257Lack-of-fit61.4131.070.4571Pure error76.844Cor. total2148.7316R2 = 0.9357R2Adj = 0.8529∗represented significant (p<0.05), ∗∗represented extremely significant (p<0.01).3D response surfaces were used (Figure2) to further interpret the effects of interactions among independent variables (starch, glycerol, and gelatin concentrations) on response value. The closer the bottom curve, which is the projection of the 3D response surface, to the ellipse, the more obvious the interaction effect and vice versa. In accordance with Table 5, the figure shows significant interaction between X1 and X2 and no significant interaction between X1 and X2 and between X2 and X3. Such results further indicated that the RSM model was reliable and veritable.Figure 2
3D responses surfaces.
(a)(b)(c)


## 3.4. Verification Experiments

Verification experiments on the optimal variable concentrations (2.40% starch, 2.11% glycerol, and 1.68% gelatin) and experimental concentrations (2.5% starch, 2.0% glycerol, and 1.5% glycerin) were performed to validate the reliability and validity of the model equations. The results of the verification experiments are shown in Table7. The acceptable percentage error between the predicted value and experimental value further demonstrated the reasonable adequacy of the response surface equations for responses and the reliability of the experiments.Table 7
Predicted and experimental data for the responses at the optimum point.
IndexesPredicted valueExperimental valuePercentage error (%)T%44.3741.21 ± 0.137.12Thickness (mm)0.0730.071 ± 0.022.73E%102.15109.91 ± 2.317.59TS (MPa)4.524.47 ± 1.211.11WS %39.1343.64 ± 0.0311.53Comprehensive score27.4828.68 ± 0.794.35Rheology of film-forming solutions.Figure3(a) depicts the variation curve of viscosity with the shear rate of 3 film solutions. Film thickness and uniformity are affected by the fluidity (viscosity) of film-forming solutions. The figure shows that the viscosity of the film-forming solutions decreased with the increase in shear rate, exhibiting non-Newtonian fluid behavior that might be attributed to the rearrangement of entangled molecules under high-speed shear [35, 36]. Such a result was in accordance with the finding of Glusac et al. [37], who proved that potato starch and zein proteins exhibited shear-thinning behavior. And the rheological behavior of the three films was successfully fitted by the Cross model (Table 8). The p value of all films was lower than 1, which further proved the pseudoplastic fluids. K-value was related to the destruction of the molecular structure inside the fluid [30]. And the three-component film showed the smallest K-value, which indicated the least time needed to form a new network structure and hydrogen bonding [38].Figure 3
(a) Shear viscosity curves. (b) Pictures of film-forming solutions.Table 8
The correlation coefficient fitted by the Cross model.
Sampleη0 (Pa·s)Κ (s)pR2Starch + glycerol + gelatin0.28202 ± 0.001170.03936 ± 4.68937E − 40.98729 ± 0.01470.99975Starch + glycerol1.46702 ± 0.011550.18258 ± 0.004530.91593 ± 0.012070.99991Starch + gelatin0.68782 ± 0.118940.55409 ± 0.380860.47097 ± 0.069550.99778The different viscosities of the film-forming solutions indicated different entanglement networks [30]. In principle, film-forming solutions become viscous with the increase in solute concentration. However, Figure 3(a) indicates that the film solution of the three mixed matrixes (starch + glycerol + gelatin) showed low viscosity. This result was consistent with Hussain et al. [39], who believed that the viscosity decrease could be attributed to the alteration in the macromolecular organization of the blended gelatin at a high shear rate.


## 3.5. The Physical Properties of Films

The physical properties including thickness, TS,E%, T%, WS, and WVP of the three films were listed in Table 9. The optimized film (starch + glycerol + gelatin) showed high thickness due to the blend of three components. Meanwhile, lower WVP of the optimized three-component film was possibly attributed to the forming of intermolecular hydrogen bonds between glycerol and starch with the matrix, resulting in strong interfacial adhesion [16] and lower WS indicated water resistance enhanced. Meanwhile, the lower TS and E% of the three-component film may probably be due to the agglomerates formed by the incorporation of gelatin, which disrupted the compactness of the hydrogen bonds [40]. Moreover, the T% showed no significance, which may be explained by the great miscibility of three components.Table 9
The physical performance of the optimized film.
SampleThickness (mm)TS (MPa)E%T%WS (%)WVP (×10−10gs−1m−1Pa−1)Starch + glycerol + gelatin0.071 ± 0.02a4.47 ± 1.21b109.91 ± 2.31b41.21 ± 0.13a43.64 ± 0.03a1.50 ± 0.02cStarch + glycerol0.064 ± 0.01b4.83 ± 0.5a138.32 ± 11.94a41.12 ± 0.34a44.83 ± 0.31c1.56 ± 0.04bStarch + gelatin0.068 ± 0.03b4.15 ± 0.07c89.62 ± 3.64c41.09 ± 0.18a43.77 ± 0.53b1.67 ± 0.02aDifferent letters indicated significant (p<0.05).


## 3.6. SEM Analysis

As shown in Figure4, SEM was applied to observe the microstructures of the surface and cross section of the optimized film. The smoothness and homogeneity of the surfaces presented in Figure 4(a) indicated that the components dispersed well within each other. The appearance of surface wrinkles did not mean that the film was uneven and discontinuous. Such wrinkles might be caused by adhesion between the film and the plastic plate when the film was peeled off. Additionally, as shown in Figure 4(b), distinctive ridged structures were presented in the cross-sections of the composite biodegradable film. The high uniformity of the biodegradable film was attributed to the plasticizing capability of glycerol and the thickening capability of gelatin. Furthermore, the numerous intermolecular hydrogen bonds that formed between hydroxyl groups in glycerol with the hydroxyl groups present in starch promoted close bonding in polymers [41] and further enhanced miscibility among starch, gelatin, and glycerol.Figure 4
The micrographs of optimized complex film (a) Surface. (b) Cross section.
(a)(b)


## 3.7. FTIR Analysis

The spectra of the single starch film and optimized biocomposite films are shown in Figure5. Typically, the broadening of the O–H and the decreasing of wavenumber could be possible due to the characteristic of hydrogen bonding interaction in the film [42]. For the control film (single starch film), the characteristic band around 3267 cm−1 was originally from the O–H stretching vibration, and the band absorption intensity was broadened with incorporation with gelatin and glycerol. Also, the characteristic peak at 1644 cm−1 slightly shifts to 1636 cm−1. This phenomenon indicated the formation of hydrogen bonds.Figure 5
FTIR spectra of optimized films.The similar characteristic peaks at approximately 2940 and 2875 cm−1 in the three biocomposite film spectra were attributed to C–H vibration and C=C vibration, respectively [43]. The peaks at 1636 and 1023 cm−1 represented the C=O and C–O stretching vibrations of glycoside bonds in the starch branch. The intensity of the peak at 1023 cm−1 increased with the incorporation of glycerol, and the enhancement in the –OH stretching vibration at approximately 3267 cm−1 suggested the occurrence of association [44]. Cross-linking between polymers tightened in the presence of intermolecular hydrogen and hydrogen-oxygen bonds (Figure 6). Such results illustrated the good compatibility between matrixes and were consistent with the SEM and rheology measurements.Figure 6
The schematic diagram of composite film.


## 4. Conclusions

A comprehensive performance film was successfully fabricated with biodegradable materials (starch, glycerol, and gelatin), and its formulation was optimized by using RSM. Responses, including mechanical, physical, and barrier properties, were subjected to a comprehensive multi-index evaluation. The RSM results verified the reliability of the model, and the optimized film exhibited excellent performance. In addition, the verification result indicated that the experimental values were highly consistent with the model-predicted values, and the optimal preparation conditions were 2.5% starch, 2% glycerol, and 1.5% gelatin. Rheological measurements proved that the film-forming solution exhibited non-Newtonian fluid behavior with low shear viscosity. Moreover, FTIR and SEM analysis demonstrated that the optimized films showed homogeneous with smooth microstructures, indicating good compatibility. In this work, the resulting biodegradable starch-based film with great comprehensive performance may be expected to become a green food packaging.



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*Source: 6633711-2021-01-28.xml*