Effect of addition of pullulan on the properties of native/waxy corn starch‐based films

Abstract

This article reports on the preparation of native/waxy corn starch (NCS/WCS)‐based films with the addition of pullulan (PUL). Our study investigated the effects of varying amounts of PUL and amylose contents on the structure, mechanical and physicochemical properties of corn starch films. Notably, it was observed that WCS films with low amylose content exhibited superior transparency, while NCS films with high amylose content demonstrated enhanced tensile strength (up to 7.35 ± 0.18 MPa). Fourier transform infrared spectroscopy (FTIR) analysis indicated that the addition of PUL did not change the molecular interactions within the corn starch films. The X‐ray diffraction (XRD) results supported that the WCS films were amorphous, while the NCS film exhibited B‐type and V‐type crystals. Moreover, an increase in PUL content led to a gradual reduction in the crystallinity of both WCS films and NCS films. The addition of PUL improved the mechanical properties and oxygen barrier characteristics of these films but had an adverse impact on their water vapor barrier properties. These findings offer valuable insights for the selection of additives for corn starch film, which can further enhance the practical application potential of corn starch films in food and other industries.

INTRODUCTION

The extensive use of nondegradable polyethylene and polypropylene films not only causes serious environmental pollution but also exacerbates the fossil energy consumption (Cheng et al., 2021). Given the recent global attention to pollution issues, the development of natural biodegradable film made from polysaccharides, protein, lipids, and other materials has become a focal point of research. Starch, a naturally occurring polysaccharide, has gained prominence in the preparation of biodegradable films due to its excellent film-forming properties and biocompatibility (Gu et al., 2021; Lu et al., 2019). However, the poor mechanical and barrier properties of starch-based biodegradable films have restricted their practical applications (Xu, Chen, Xu, et al., 2023). Various methods have been adopted to enhance the properties of starch-based biodegradable films, including starch modification (Hu, et al., 2019), the addition of plasticizers and polymers (Wu et al., 2023), and so on. Among these methods, the preparation of starch-based biodegradable films through the blending of starch with other natural polymers such as pullulan (PUL), chitosan, gelatin, and so on, is a promising method with simple operation and low cost.

PUL is an extracellular water-soluble microbial polysaccharide produced by the fungus-like yeast Aureobasidium pullulans in starch and sugar cultures (Kristo & Biliaderis, 2007; Liu et al., 2020). It is mainly composed of maltotriose units interconnected by α-1,6 glycosidic bonds, which gives it structural flexibility, good water solubility, and other unique properties (Kanmani & Lim, 2013; Leathers, 2003). PUL has been widely used in food, biological, pharmaceutical, chemical, and other fields (Ganie et al., 2021). Among these applications, PUL is particularly suitable for the preparation of biodegradable films due to its nontoxic, harmless, odorless, edible nature and its remarkable film-forming properties (Leathers, 2003). Moreover, PUL films have been reported to have excellent flexibility, smooth surface, high transparency, and effective barrier properties against oil and oxygen (Singh et al., 2008). However, the relatively high cost of PUL has limited its wide application in the field of biodegradable films (Tong et al., 2008). Recently, blending PUL with other low-cost and biocompatible polymers such as starch (Kanmani & Lim, 2013), sodium alginate (Xiao et al., 2012), and carboxymethyl cellulose (Tong et al., 2008) has emerged as an ideal strategy to prepare cost-effectively biodegradable films. Among these biopolymers, starch has garnered significant research interest as an affordable and compatible raw material for film preparation. Studies have investigated the preparation of biodegradable composite films by mixing potato starch (Liu et al., 2020) or tapioca starch (Kim et al., 2014) with PUL, and the results have shown that PUL can enhance the physical properties of composite films.

Corn starch, as one of the most common starch sources in daily life, is also considered an ideal raw material for preparing starch-based biodegradable films due to its affordability and large-scale production (Luchese et al., 2017). However, there has been limited research on blending corn starch with PUL to prepare biodegradable composite films. Additionally, it is important to investigate the differences in the properties of biodegradable films prepared by blending corn starch with different amylose content (native corn starch [NCS] and waxy corn starch [WCS]) with PUL. Therefore, in this study, we blended PUL with NCS and WCS separately to prepare biodegradable films, and subsequently characterized and analyzed the properties of these composite films.

MATERIALS AND METHODS

Materials

NCS (containing 28% amylose) was purchased from Zhucheng Xingmao Corn Development Co., Ltd. WCS (containing 2.48% amylose) was purchased from Suzhou Gaofeng Starch Technology Co., Ltd. PUL (molecular weight 200,000 Da) was purchased from Hayashibara Biochemical Inc. Glycerin was purchased from Sinopharm Chemical Reagent Co., Ltd. Unless otherwise stated, all chemicals used in this study were of analytical level from local suppliers.

Film preparation

The mixture of 3 g NCS or WCS, 1 g glycerol, and different ratios of PUL (0, 0.2, 0.4, 0.6 g/DL, w/v) was dispersed in 100 mL deionized water. The mixture was heated by magnetic stirring at 90°C for 30 min until the mixture was completely gelatinized. After degassing, 30 mL of the prepared film-forming solution was poured into 10 × 10 cm of acrylic petri dish, and then the petri dish containing the film-forming solution was placed in an oven and dried at 50°C for 8 h. Finally, all dried films were balanced at room temperature (25 ± 2°C) and a relative humidity of 52 ± 2% for 48 h. The composite films were designated as WCSPx and NCSPx, where x (1, 2, 3) corresponded to the amount of PUL added (0.2, 0.4, 0.6 g). The control samples WCS and NCS correspond to the pure WCS film and pure native corn starch film without PUL, respectively.

Scanning electronic microscopy (SEM)

The dried film sample was sputtered with a thin layer of gold in the vacuum evaporator, and then the surface morphology of the composite film was analyzed using an SEM (SU8220, SEM, Hitachi).

X-ray diffraction (XRD) analysis

The diffraction patterns of the film samples were analyzed by using an X-ray diffractometer (D2 PHASER, Bruker). The tension and current of the X-ray generator were 40 kV and 40 mA respectively. The diffraction patterns were obtained at room temperature over the 2θ range of 5–40° by step of 0.03°.

Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR)

The chemical structure of the film samples was analyzed by FTIR using a Nicolet Nexus 470 Fourier transform infrared spectrometer (Thermo Electron Corporation) with an ATR attachment. A spectrometer with an ATR accessory was used to carry out FT-IR analysis. Measurements were obtained in the range of 600–4000 cm⁻¹ as the average of 32 scans with a resolution of 4 cm⁻¹.

Thermo-gravimetric analysis (TGA)

The thermal stability of the film samples was determined by a thermo-gravimetric analyzer (TGA2, Mettler–Toledo). The film samples (3–5 mg) were heated from 50°C to 500°C at a heating rate of 10°C/min. A flow rate of 20 mL/min of nitrogen was used.

Mechanical properties

The tensile strength (TS) and elongation at break (EB) of the film samples were measured using a texture analyzer (TA, TA-XT Plus, Stable Microsystems) according to the method of ASTM D882-18. Before analysis, the film samples were cut into strips (80 mm × 15 mm). The test speed was 60 mm/min and the initial distance is 20 mm.

Thickness and transparency

The thickness (μm) of the film was measured using a digital micrometer at least five random points on the film sample at a time, and the average value was calculated.

The transparency of the film samples was measured with a UV spectrophotometer (Mapada Instruments) at a wavelength of 600 nm (Wu, et al., 2019). The film samples were cut into 10 mm × 40 mm strips, directly into the cuvette. Transparency is calculated by the following formula: $$\text{Transparency}({\text{mm}}^{-1})=\frac{{A}_{600}}{X},$$where A₆₀₀ is the absorbance at 600 (nm) and X is the thickness of film (mm).

Water vapor transmission rate (WVTR)

The film WVTR was determined according to the standard method of water vapor transmission (Xu, Chen, McClements, et al., 2023). Before the test, 3 g of anhydrous calcium chloride was put at the bottom of an open bottle with a diameter of 2 cm to achieve 0% relative humidity. Then the bottle was covered with the film sample (5.3093 × 10⁻⁴ m² of the film area exposed to permeation) and placed in a desiccator filled with deionized water to maintain 100% relative humidity at 25°C. After 24 h, the weight change of the bottle was measured. The measurements were conducted in triplicate. $$\mathrm{WVTR}=\frac{\bigtriangleup W}{t\times A},$$where the ΔW (g) is the increase in weight of the test bottles, t (s) is the time, and ΔW/t (g/h) is the water transmission rate through the film, which is calculated from the slope of the linear function ΔG = f(t), and A (cm²) is the permeation area of the film. Additionally, the weight of the tested films was measured before and after the WVTR test to exclude any absorption phenomena due to changes in the humidity of the film.

Oxygen permeability (OP)

OP test refers to the method of Xiao et al. (Zhou, et al., 2022), which is based on the oxidation mechanism of iron. Before the test, the bottom of a bottle with a diameter of 2 cm was filled with sodium chloride (1.5 g), activated carbon (1 g), and reduced iron powder (0.5 g). Then the bottle was covered with the film sample (5.3093 × 10⁻⁴ m² of the film area exposed to permeation) and placed in a desiccator filled with a saturated calcium chloride solution to achieve 90% relative humidity at 25°C. The test is repeated three times and the average value is determined. OP is calculated as follows: $$\mathrm{OP}=\frac{m-{m}_{0}}{t\times s},$$where the m₀ represents the initial weight of the whole bottle, m represents the final weight of the whole bottle, t represents the interval time, and s represents the bottle area covered with the film sample.

Statistical analysis

Statistical analysis was performed using the IBM SPSS Statistical Package Program (SPSS 25.0 for windows). The data were analyzed by analysis of variance (ANOVA), and the significant differences among samples were assessed by Duncan's multiple range test (p < 0.05). The Origin 2019 software was used to draw charts.

RESULTS AND DISCUSSION

SEM analysis

After the addition of PUL, the surface morphology of NCS and WCS was observed using SEM. As shown in Figure 1, both the NCS and WCS films, when supplemented with PUL, exhibit smooth and flat surfaces without noticeable holes or cracks. This result suggested that PUL has excellent compatibility with both types of corn starch. In addition, the surface of NCS films appeared more uniform and smoother than WCS films at low PUL content. However, NCSP3 in the figure shows that the smoothness of the surface of NCS films diminishes with increasing PUL content. This could be attributed to an excess of PUL molecules in the film-forming solution, causing rapid amylose in the NCS rapidly aggregate and coagulate during the cooling process after gelatinization, resulting in stripes on the film surface (Wang et al., 2017). This phenomenon does not appear in WCS films, which may be related to the lower amylose content in WCS.

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XRD analysis

XRD was used to examine both the crystal structure and amorphous structure of PUL/corn starch film. The XRD patterns of the film samples and their corresponding relative crystallinity are shown in Figure 2. It is evident from Figure 2 that the both pure WCS film and the WCS film added with PUL are amorphous, and all the film samples show a wide amorphous peak at 2θ = 19.5°. This observation may be attributed to the inclusion of glycerol, as well as the effect of heating and mechanical stirring during the gelatinization process destroyed the crystalline area of corn starch granules (Kong, et al., 2020). On the contrary, both pure NCS film and NCS film with the addition of PUL showed a partially recrystallized state, and the film samples had significant crystallization peaks near 2θ = 17.3° and 20°. This was consistent with the characteristic diffraction peak of the crystalline phase of corn starch film in the studies of (Wang, Xu, et al., 2022) and (Upadhyay et al., 2022). Corn starch belongs to A-type crystalline group, and its diffraction peaks are at 15°, 17°, 18°, and 23° (Bertoft, 2004). However, the NCS film shows a mixed crystallization peak of B-type and V-type, which indicates that the gelatinization of corn starch results in the replacement of A-type crystal of corn starch by B-type and V-type crystal (Wang, Xu, et al., 2022). On the one hand, the formation of V-type crystals in NCS films is related to the formation of lipid complexes from lipids in starch and amylose (Wang, Xu, et al., 2022). On the other hand, it is related to the addition of glycerol. Research by Zou et al. showed that glycerol can promote the formation of V-type crystallization of corn starch-based films (Zou, Yuan, Cui, Liu, et al., 2021). We can also find from Figure 2 that the relative crystallinity of both the WCS film with PUL addition and the NCS film with PUL addition decreased as the amount of PUL addition increased. This may be due to the uniform distribution of PUL molecules in the starch film and the interaction between PUL and starch molecules, which hindered the aggregation and retrogradation of starch chains (Zou, Yuan, Cui, Wang, et al., 2021).

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ATR-FTIR analysis

FTIR spectroscopy was used to study the interaction between different components in PUL/corn starch film. ATR-FTIR results of PUL/corn starch film samples are shown in Figure 3. In Figure 3, it is indicated that the addition of PUL does not change the molecular interaction of corn starch film. This may be because PUL and corn starch have similar elements and group composition. The wide overlapping peaks at 850.21 and 925.98 cm⁻¹ of the film indicate the existence of corn starch. These peaks are attributed to the vibration of glucose pyranose units and C–O vibration stretching of glucose unit, respectively (Ibrahim et al., 2019). The C–O–H group in the corn starch film appeared at 1078.47 cm⁻¹. Additionally, the coupling of C–C and C–O bond stretching appeared at 1149.99 cm⁻¹, which is the characteristic peak of polysaccharide molecules (Susmitha et al., 2021). The blending mode of CH₂ corresponds to a peak at 1364.95 cm⁻¹ (Cael et al., 1975). The oscillations of water adsorbed in the amorphous region of starch resulted in a wide infrared band at the peak of 1644.83 cm⁻¹ (Kizil et al., 2002). Additionally, the presence of C–H vibrational stretching gave rise to a sharp peak at 2925.57 cm⁻¹ (Ibrahim et al., 2019). Finally, an intense band at 3288.48 cm⁻¹ was also observed, attributed to the O–H group vibrational stretching (Pi-xin et al., 2009).

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TGA analysis

TGA was employed to analyze the thermal properties of PUL polysaccharide/corn starch film, with the weight loss rate and maximum decomposition temperature of the film depicted in the respective TGA thermal curves (Figure 4). It is evident from the TGA curve that the thermal degradation process of all film samples can be mainly divided into three stages. The first weight loss stage mainly occurs between 70°C and 120°C, corresponding to the evaporation of water in PUL/corn starch film. The second weight-loss stage mainly occurs between 140°C and 250°C, which is associated with the degradation of glycerol in PUL/corn starch film (Hu et al., 2019). The third weight loss stage mainly occurs between 280°C and 350°C, which is attributed to the decomposition of starch and PUL matrix in PUL/corn starch film.

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Mechanical properties

As shown in Table 1, TS and EB were used to evaluate the effect of different PUL polysaccharide addition on the mechanical properties of corn starch film with different amylose content. The mechanical strength of both PUL/WCS film and PUL/NCS film increased with the increase of PUL addition. However, the EB of both PUL/WCS film and PUL/NCS film decreased with the increase of PUL addition. This may be due to the excessive interaction between PUL and starch molecules, along with glycerol plasticizer when the amount of added PUL increase, which result in the decline of film toughness. In addition, on the whole, the mechanical strength of pure NCS film and PUL/NCS film is higher than that of pure WCS film and PUL/WCS film. This is because NCS contains more amylose content. It is widely appreciated that the more amylose content in the starch, the higher the mechanical strength of the starch film prepared.

Table 1 The thickness, opacity, tensile strength, and elongation at break of WCS, WCSP1, WCSP2, WCSP3, NCS, NCSP1, NCSP2, and NCSP3 films.

Films	Thickness (mm)	Opacity (mm−1)	Tensile strength (MPa)	Elongation at break (%)
WCS	0.0903 ± 0.0051b	0.49 ± 0.01d	1.22 ± 0.10a	197.04 ± 10.06a
WCSP1	0.0913 ± 0.0061b	0.70 ± 0.01c	2.61 ± 0.08b	162.34 ± 8.57b
WCSP2	0.0961 ± 0.0070b	0.78 ± 0.01a	3.87 ± 0.18c	146.74 ± 11.77bc
WCSP3	0.1043 ± 0.0049a	0.74 ± 0.01b	4.45 ± 0.05d	136.78 ± 8.57c
NCS	0.0902 ± 0.0036c	5.53 ± 0.01d	3.07 ± 0.20a	131.66 ± 8.58a
NCSP1	0.0971 ± 0.0024b	5.65 ± 0.01c	4.09 ± 0.12b	110.10 ± 7.39b
NCSP2	0.0985 ± 0.0058b	5.83 ± 0.01a	5.17 ± 0.08c	96.48 ± 8.04bc
NCSP3	0.1065 ± 0.0023a	5.88 ± 0.01b	7.35 ± 0.18d	90.82 ± 6.13c

Thickness and opacity

The color and transparency of films are important indicators to determine the appearance of films and consumer acceptance (Yu et al., 2022). Higher absorbance values in films correspond to increased opacity (Fu et al., 2022). As shown in Figure 5 our study reveals that all WCS films have higher transparency than NCS films. Notably, pure WCS films demonstrate the highest level of transparency. The transparency of starch film is related to the arrangement of starch chains in the film. The lower transparency of the NCS films than the WCS films may be due to the higher content of amylose in NCS. Amylose and amylopectin in common corn starch film undergo retrogradation to form an ordered structure through a synergistic effect (Zhou et al., 2010). The increase of these ordered structures hinders the light diffusion through the film. On the contrary, WCS film experience slower retrogradation, which makes it more transparent. Furthermore, the opacity of WCS films and NCS films increased with the addition of PUL polysaccharide. This effect may be attributed to the fact that PUL molecules are embedded into the chain space of the starch film matrix, thereby diminishing light transmittance through the film.

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WVTR

Water vapor transmission rate is an index to evaluate the water vapor barrier performance of a film, and it can be influenced by factors such as polysaccharide chain structure, additives, film-forming process, and the interaction of film components (Hu, et al., 2022). As shown in Figure 6, the addition of PUL had no significant effect on the water vapor transmission rate of the WCS film. However, the addition of PUL could increase the water vapor transmission rate of NCS films, likely owing to hydrophilic properties of PUL (Omar-Aziz et al., 2021). In addition, the figure indicates that the water vapor transmission rate of the films decreases with the increase of PUL in the NCS films with added PUL. This may be because more PUL molecules are uniformly dispersed in the starch molecules to form a tighter network structure, which reduces the free space in the film matrix and reduces the chance of water diffusion through the film (Wang, Yuan, et al., 2022).

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OP

OP is an important index to evaluate the ability of the film to resist oxygen penetration, with higher OP value indicating a lower the oxygen barrier effectiveness of the film (Gounga et al., 2007). Given the oxygen is a primary catalyst for food oxidative deterioration, we can determine the oxygen transmission rate of the film to evaluate the potential of the film to be used for food packaging applications. As shown in Figure 7, the oxygen transmission rate of the WCS film and the NCS film gradually decreased as the amount of PUL increased. This may be related to the good oxygen barrier properties of PUL (Tong et al., 2008). In addition, more PUL molecules in the film matrix can form a tighter network structure with starch molecules, which reduces the free space in the film matrix and also has a certain effect on the reduction of the oxygen transmission rate of the film. Overall, the addition of PUL enhanced the oxygen barrier properties of WCS films and NCS films.

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CONCLUSIONS

In this study, we prepared WCS films and NCS films with varying levels of added PUL and also investigated the effects of different PUL addition amounts and different amylose contents on the properties of corn starch films. FTIR analysis showed that the addition of PUL did not change the molecular interactions of corn starch films. The X-ray patterns indicated that the WCS film exhibited an amorphous structure, and the NCS film displayed both B-type and V-type crystals. Furthermore, the crystallinity of both WCS films and NCS films decreased with increasing levels of PUL incorporation. In addition, our findings demonstrated that the addition of PUL could improve the mechanical properties and oxygen barrier properties of WCS films and NCS films, but had a certain negative effect on the water vapor barrier properties of the films. This study provides valuable insights for the selection of additives for corn starch film, which is beneficial for expanding the potential application range of starch-based biodegradable films.

AUTHOR CONTRIBUTIONS

Hao Cheng: Conceptualization; data curation; methodology; writing—original draft. Long Chen: Conceptualization; data curation; funding acquisition; writing—review & editing. Zipei Zhang: Conceptualization; data curation; writing—review & editing. Ruojie Zhang: Conceptualization; data curation; formal analysis. David Julian McClements: Conceptualization; methodology. Hao Xu: Conceptualization; data curation; formal analysis. Zhenlin Xu: Data curation; formal analysis; methodology. Man Meng: Investigation; methodology. Zhengyu Jin: Formal analysis; funding acquisition; methodology; supervision.

ACKNOWLEDGMENTS

This work was financially supported by the National Natural Science Foundation of China (No. 32130084, No. 32101990, No. 32372475), Natural Science Foundation of Jiangsu Province - China (BK20200617), National Key Research and Development Program of China - China (No. 2022YFD2100602), the Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province, Jiangnan University (2022-1-1) and Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX23_2516).

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT

Data will be made available on request.

ETHICS STATEMENT

None declared.

References

Bertoft, E. (2004). Lintnerization of two amylose‐free starches of A‐ and B‐crystalline types, respectively. Starch ‐ Stärke, 56(5), 167–180. [DOI: https://dx.doi.org/10.1002/star.200300255]