A novel pH-sensitive antibacterial bilayer film for intelligent packaging

Abstract

Intelligent single-layer packaging is widely used in food monitoring and storage. However, most single-layer intelligent packaging has poor mechanical strength and water barrier properties. In this study, a bilayer intelligent detector film based on polyvinyl alcohol-chitosan (PVA-CS)/nano-ZnO/sodium alginate (SA) combined with anthocyanin extract (cyanidin chloride) was prepared using a layer-by-layer solution casting assembly technique. The effects of different levels of anthocyanin extracts on the physical and functional properties of the films, including microstructure, mechanical property, barrier property, pH sensitivity, and antibacterial property, were investigated. The results show that the bilayers exhibit excellent physical properties, lower water vapor permeability, better light transmission and UV-blocking properties, a broader pH sensitivity (ΔE > 10), and good antibacterial activity. In short, the bilayer films studied are superior to the single-layer films in terms of their packaging potential for products with low moisture content, offering new directions for active intelligent packaging and biodegradable materials for the food industry.

Graphical Abstract

Introduction

During storage, transportation, and distribution, food products will be damaged by environmental and other factors such as microorganisms and reducing freshness [1]. Moreover, food waste has been a further concern due to the spread of COVID-19 during the last 2 years. Intelligent packaging is aimed at protecting and controlling food products [2]. In particular, increased safety, quality, and information are provided by their ability to perform functions such as detection, tracking, and communication [3]. As a result, intelligent packaging films are widely used to detect food quality and extend food storage.

Currently, the development of colorimetric pH indicator films for application in intelligent packaging to monitor the freshness of food has attracted people’s attention. This indicator film changes color in response to the food’s pH change. Thus, consumers can quickly distinguish whether food is spoiled without opening the package, which may reduce the amount of food wasted [4]. Anthocyanins are a class of water-soluble natural pigments widely found in plants, producing blue, purple, red, and intermediate tones in plant tissues [5]. The colors of anthocyanins are sensitive to pH changes because of their structural transformations [6, 7]. Therefore, incorporating anthocyanins into food packaging films can effectively monitor varying pH resulting from food decay. Meanwhile, it is necessary to have good antibacterial properties for a well-behaved intelligent packaging film. So far, metal nanoparticles including zinc oxide nanoparticles, copper oxide nanoparticles, and silver nanoparticles have been applied to a wide range of packaging films, among which nano-ZnO has attracted more attention due to its good antibacterial properties and non-toxicity [7–9]. Therefore, nano-ZnO was added to the PVA-CS blended film as an enhanced antimicrobial agent to extend the food preservation time while detecting the food quality.

The traditional plastic-based food packaging films are derived from petroleum, which is not only non-degradable but has serious environmental concerns [10]. Recently, biomaterials have received increasing attention as intelligent packaging materials for food storage and quality monitoring. Among them, chitosan (CS) is derived from a non-toxic, renewable resource and has antimicrobial and antioxidant properties that prevent oxidative deterioration and prolong the shelf life of food products. In addition, it is widely used in many applications for its excellent biocompatibility and biodegradability [11]. Polyvinyl alcohol (PVA) is a non-toxic, biocompatible, biodegradable polymer with remarkable film-forming ability and barrier off-odors, which can be used in food packaging [12]. Pereira, Arruda, and Stefani reported that the mechanical properties of pure chitosan films are shallow, while the blending films from PVA and CS had enhanced physical properties and high antioxidant activities [13, 14]. Also, sodium alginate (SA) is a biocompatible, biodegradable, and non-toxic polysaccharide with sufficient fibrous chemistry to form gels in the presence of divalent cations and is a biopolymer that creates high-quality films [15].

Unlike single-layer films, bilayers could exhibit the uniqueness that single-layer films have even better. Ebrahimzadeh et al. prepared electrospun chitosan-polyvinyl alcohol bilayers containing essential oils by hybrid casting and electrostatic spinning methods, which exhibited better antimicrobial and physical properties than chitosan monolayers [16]. Moradi et al. developed plasma-improved chitosan/polyethylene bilayers containing summer savory essential oil, which also demonstrated reduced water vapor permeability (WVP) and increased mechanical strength of the bilayers compared to chitosan films [17]. However, sodium alginate is a hydrophilic group and cross-linking with multivalent cations is usually required to improve water resistance. Guo et al. reported that a high selectivity of Ca²⁺ was found due to its ability to form dense membrane networks by cross-linking only with G blocks [18]. Also, alginate’s high affinity and strong ionic bonding allow Ca²⁺ to obtain better film properties than Zn ²⁺, Mn ²⁺, and Mg ²⁺ when cross-linked. On the other hand, due to the electrostatic interaction between -NH₃⁺ on chitosan and -COO⁻ on sodium alginate forming a white insoluble polyelectrolyte polymer, a stable polyelectrolyte multilayer film is formed by a layer-by-layer casting method and this multilayer film can achieve even better performance [19]. To improve the performance of single-layer films, sodium alginate and chitosan solutions could be used to form multilayer composite films. Nevertheless, there are few reports on the ability to perform pH-responsive bilayer membranes for real-time monitoring of food products.

Therefore, the present study aims to prepare a bilayer film that could provide real-time monitoring of food quality and improve the shortcomings of single-layer films as intelligent packaging for food by extending the shelf life of the food and enhancing the physical properties of water resistance. In this work, we have developed a pH-sensitive antibacterial bilayer film. The inner SA layer and the outer PVA-CS layer are prepared by a solution casting method and cross-linked by calcium ions. The bilayer films are further enhanced for food storage by adding nano-ZnO as an antimicrobial agent. The performance optimization of the new bilayer films is comprehensively evaluated by comparing the nano-bilayer films with single-layer films. It is demonstrated to have broad applications in food preservation indication and intelligent bionic devices, especially for low moisture substances.

Experimental section

Materials

Polyvinyl alcohol (PVA, 1799, degree of alcoholics: 98–99%) and chitosan (degree of deacetylation 85%) were purchased from Tianjin Heowns Biochem, Ltd. Sodium alginate (AR grade), cyanidin chloride (purity: 25⁺%), nano-ZnO (purity: 99.9%, particle size: 30 ± 10 nm), glycerol (purity: 99.5%), and 1% acetic acid aqueous solution were purchased from Tianjin Kmart Chemical Technology Co., Ltd. and CaCl₂ (0.5%) solution was purchased from Nanjing SenbeiJia Biotechnology Co., Ltd.

Preparation of films

1.2 g of chitosan (CS) was dissolved in 110 mL of 1% aqueous acetic acid solution with glycerol (20% w/w based on chitosan) added as a plasticizer, and 0.3 g of nano-ZnO as an antibacterial agent. The solution was then stirred magnetically at room temperature until the chitosan powder was completely dissolved and the nano-ZnO was uniformly dispersed. In addition, 5.5 g of polyvinyl alcohol (PVA) with 1.1 g (20% w/w based on PVA) of glycerol added was dissolved in 110 ml before stirring at 95 ± 2 ℃, and cooled to room temperature. Subsequently, the anthocyanins were mixed at 0%, 5%, 10%, and 20% (w/w based on chitosan) content with the chitosan solution and PVA solution by magnetic stirring and vacuuming to remove air bubbles to obtain the outer film-forming solution. The inner film-forming solution was obtained by dissolving 2.2 g of sodium alginate (SA) in 220 mL of distilled water and adding 0.44 g of glycerol (20% w/w based on chitosan), followed by magnetic stirring at room temperature to obtain a clarified solution. Meanwhile, 20 g of the outer film-forming solution and 25 g of the inner film-forming solution were cast in separate plastic petri dishes (d = 35 mm) and dried at 55 °C. Then, the films were quickly wetted (t < 30 s) with 1 mL CaCl₂ (0.5%, w/v) solution and dried at 55℃ to obtain the corresponding films as a control group. The single-layer films obtained from the inner film-forming solution were named SA, and the single-layer films obtained from the outer film-forming solution were named CP-1, CP-2, CP-3, and CP-4, respectively.

The preparation of the bilayers was based on the multilayer solution casting method of Zhuang et al. [19]. Firstly, 22 g of the inner film-forming SA solution was cast on a medium plastic petri dish (d = 35 mm) and dried at 55℃ for 15 h. Subsequently, 10 g of the outer film-forming solution was decanted again into this petri dish and dried in an oven at 55℃ for 7 h. The resultant bilayer film was quickly wetted (t < 30 s) with 1 mL CaCl₂ (0.5%, w/v) solution and dried at 55℃ for 2 h. And they were named SA-CP-1, SA-CP-2, SA-CP-3, and SA-CP-4.

The dried films are stored at room temperature, and 50% relative humidity for at least 48 h before testing.

Structural characterization

Fourier transform infrared (FT-IR) spectroscopy

FTIR spectra were obtained by scanning films over a range of 400–4000 cm⁻¹ using a spectrometer (Nicolet IS 10, America).

X-ray diffraction

The phase structure of the films was measured by X-ray diffraction (SmartLab, Japan), with Cu produced at 40 kV and 200 mA radiation, and scanned at a 5°/min rate over a diffraction angle range of 5–60° (2θ) [20].

Scanning electron microscope

The surface and cross sections of the films were observed using SEM (Regulus 8100, S-4800, Hitachi, Japan) with an accelerating voltage of 3.00 kV.

Physical properties measurement

Thickness and mechanical properties

The thickness of the film was measured by selecting five random points using a digital micrometer. The tensile strength (TS) and elongation at break (EB) of films were measured by a universal mechanical testing machine (CTM6103, MTS, USA) with an initial grip separation of 40 mm and a strain rate of 60 mm/min. The films were cut into strips (60 mm × 6 mm), and the samples were taken five times in parallel [21].

Moisture content and swelling degree

The film samples (d = 35 mm) (M₀, g) were placed in an oven set at 70 °C for 24 h to evaporate the water contained in the film and weighed (M₁, g). The dried films were then immersed in 50 ml of distilled water at room temperature for 24 h. The excess water was wiped off the film’s surface with filter paper and weighed (M₂, g). Afterwards, they are dried again in an oven at 70 °C for 24 h and weighed (M₃, g) [20]. The moisture content (MC (%)) and swelling degree (Sw (%)) were calculated by the following equations:

$$MC\%=\left(M_0,-,M_1\right)/M_0\times 100$$

$$S_W\%=\left(M_2,-,M_3\right)/M_1\times 100$$

Water vapor permeability (WVP)

The film water vapor permeability was determined according to the method of Yong et al. with modifications [22]. Six grams g of anhydrous silica gels was poured into a test tube (d = 20 mm), and the mouth of the tube was sealed with a film. Then, the tubes were placed in a desiccator at a temperature maintained at 20 °C, containing distilled water (100% relative humidity), and weighed only once a day for 6 days. WVP was calculated as follows:

$$WVP=\frac{W{\times }_x}{t\times A\times \mathrm{\Delta }P}$$

where W is the change in weight of the tube (g), x is the film thickness(mm), t is the time interval (s), ΔP is the water vapor pressure difference across the film at 20 °C(KPa), and A is the area of m².

Contact angle test

The water contact angle of the film surface is measured using a contact angle tester (Do nano, China). The film sample was cut into a rectangle of 4 cm × 1 cm. A drop of ultrapure water (10 μL) was dropped onto the surface of the film samples fixed to the platform. The contact angle of the drop was recorded after 8 s of water drop contact. All films were selected at 5 different locations for measurement and the average value was reported.

Light transmittance and opacity [9]

The light transmittance of the films was determined by scanning the film samples in the 200–800 nm range using a UV-1601 spectrophotometer (Beifen Riley, China). The opacity of the film is calculated as follows:

$$Opacity=\frac{Abs600}{x}$$

where Abs600 and x are the film absorbance at 600 nm and the film thickness (mm), respectively.

Functional properties of food packing

pH sensitivity

According to Liu et al., the pH-sensitivity of this anthocyanin was assessed in the method with several modifications [23]. In simple terms, 5 mg of anthocyanins were dissolved separately in the 50 ml buffer of different pH for 20 min. The UV–Vis spectra of the extracted liquids could then be recorded on a UV–Vis spectrophotometer (TU-1900, Pu-Analysis, China) through a sweep of 450–700 nm.

To verify the pH-sensitivity of the films, the films (d = 35 mm) were dipped into the different buffer solutions (pH = 2–12) for 20 min. The chromatic parameters of the films were recorded with a colorimeter (CR-10, Konica Minolta, Japan) and photographed. The results were expressed in terms of the parameters L*, a*, b*, and ΔE [24]. ΔE was calculated as follows:

$$\mathrm{\Delta }E=\sqrt{{\left(L^{\ast },-,L_0\right)}^2+{\left(a^{\ast },-,a_0\right)}^2+{\left(b^{\ast },-,b_0\right)}^2}$$

where L₀, a₀, and b₀ are the original grey value of films. L* = lightness, a* = red to green, and b* = yellow to blue.

Ammonia sensitivity

Film samples (2 cm × 2 cm) were placed on top of the inner Petri dishes (d = 9 cm) containing 15 mL of 0.1 mg/mL ammonia solution at room temperature for 300 min. The color changes of the film samples were recorded at different time intervals (5, 10, 20, 30, 40, 50, 60, 180, 300 min).

Antibacterial activity

The antibacterial capacity of the film samples was determined by judging the inhibition circle (mm) against Staphylococcus aureus and Escherichia coli using the agar diffusion test described by Abral et al. with some modifications [25]. Bacterial colonies were counted based on the 0.5 McFarland standard. By comparing the turbidity between the 0.5 McFarland standard and the microbial culture, the microbial suspension cell density was approximately 1.5 × 10⁸ CFU/mL. Firstly, 100 μL of the microbial suspension was evenly spread on the Luria–Bertani agar. Then, using forceps, 10 mm film discs, sterilized under a UV lamp for 15 min, were placed in an orderly fashion on the microbial suspension to be tested. Finally, all the media were incubated in a biochemical incubator at 37 °C for 24 h before recording their inhibition circle diameter (mm). Three sets of parallel experiments were set.

Statistical analysis

All films were measured in parallel greater than three times, and results were expressed as average ± standard deviation (SD). All statistics were handled using SPSS software (25.0, SPSS Inc., Chicago, IL, USA), and Duncan’s multiple range test (p < 0.05) was used to compare differences among the data.

Results and discussion

Molecular bonds analysis

FTIR spectra of all films are shown in Fig. 1. The single-layer films exhibit similar peak areas, and both the SA and bilayer films also exhibit similar peak areas. Single-layer films which showed bands at 3302–3286 cm⁻¹, 2939 cm⁻¹, 1569 cm⁻¹, 1412 cm⁻¹, and 1045 cm⁻¹ were attributed to O–H stretching, C-H asymmetric stretching, amide II groups, CH-CH₂ bending in the basic carbon skeleton, and C-O stretching, respectively [23]. In addition, single-layer films presented at 1328 cm⁻¹ (amide III), 1142 cm⁻¹ (C–O–C stretching), 921 cm⁻¹ (C-O stretching), and 847 cm⁻¹ (C-H vibrational stretching) [26]. The bond at 3302 cm⁻¹ (O–H stretching) in the bilayer containing the SA layer shifted towards lower wave numbers, while the bands at 1594 cm⁻¹, 1406 cm⁻¹, and 1022 cm⁻¹ became sharper and broader, probably due to the presence of the SA layer and electrostatic interactions between the SA and CP layers [19]. Notably, with the addition of anthocyanin (cyanidin chloride), the waveband of both films at 3302 cm⁻¹ (O–H stretching) shifted to a lower wave number near 3290 cm⁻¹, as a result of the hydrogen bonds formed between the components of the film, altering the physical and chemical interactions between the aromatic rings of the anthocyanins and polysaccharides [9].

Fig. 1.

FT-IR spectra of SA and films with different contents

Phase structure analysis

The XRD patterns of CP-1, CP-2, CP-3, CP-4, SA-CP-1, SA-CP-2, SA-CP-3, and SA-CP-4 films are shown in Fig. 2. Both SA-CP and CP films showed broad peaks in the 2θ range of 19 to 20°. The intensity of the peaks was significantly lower in the SA-CP film than in the CP film, which was attributed to the lower content of the CP mixture in the SA-CP film. In terms of peak intensity, it is found that the peak intensity at 2θ = 19.7° decreases with the addition of anthocyanin, while it increases with the increase of anthocyanin content. This phenomenon may result from the formation of new hydrogen bonds between the anthocyanins and the two polymers (PVA, CS), thus disrupting the original interactions between the polymeric substrates and promoting the spatial reconfiguration of the polymer chains, which was supported by FTIR spectrograms, and secondly, the plasticization and electrostatic interactions between anthocyanins and all components [27, 28]. However, as the anthocyanin content increases, this could weaken the interaction between the two interpretations, thereby allowing an increase in the crystallinity of the polymer matrix.

Fig. 2.

XRD spectra of films with different contents

Cross-sectional micromorphologies observation

Figure 3 exhibited the surface and cross-sectional micromorphology of the films. For the surfaces (Fig. 3A), both the single-layers and bilayers are smooth, dense, and homogeneous, indicating a good compatibility between CS, nano-ZnO, PVA, and anthocyanin extracts. However, the smoothness of the bilayers is lower than the single-layer films, which may be caused by the uneven drying of the bilayers during the layer-by-layer casting process. It can be seen that all the films show relatively continuous, dense, and non-cracking cross sections (Fig. 3B). Moreover, all the single layers demonstrated a homogenous and smooth cross section without voids, indicating good compatibility between CS, nano-ZnO, PVA, cyanidin chloride, and glycerol. SA-CP-2 films are slightly rough in cross sections with small agglomerates, which could be caused by the cross-linking reaction of Ca²⁺ with sodium alginate to form insoluble calcium alginate. In the SA-CP-3 film, a stratification was observed between the top and bottom layers, which may result from the SA layer’s low moisture content prior to casting the CP layer, preventing the two layers from being well compatible [29]. Besides, the results show that the other cross sections do not clearly exhibit a bilayer structure; instead, the outer layer exhibits a continuous and dense microstructure tightly bound to the inner layer. The ability of CP and SA to form a stable bilayer is further demonstrated. It stems from the fact that CP and SA could coalesce to form polyelectrolyte complexes by electrostatic gravity, while Ca²⁺ chelated with the guluronic acid blocks of the SA chain, creating a physically cross-linked SA/Ca²⁺ network to enhance their electrostatic binding [19]. These results were also confirmed in the FTIR analysis.

Fig. 3.

SEM images of surface (A) and cross sections (B). SA (a), CP-1(b), CP-2 (c), CP-3 (d), and CP-4 (e) are single-layer films and SA-CP-1 (f), SA-CP-2 (g), SA-CP-3 (h), and SA-CP-4 (i) are bilayer films

Thickness and mechanical properties measurement

The thickness and mechanical properties of the films are presented in Table 1. There was no significant difference between the thickness of the SA-CP and CP films. Among them, the thickness of the SA layer (0.035 ± 0.003 mm) is the thinnest. The thicknesses of single-layer films are thicker than bilayers. Since the SA films obtained by casting and drying equal amounts of solution are thicker than the CP film, the bilayers with inner of a SA layer and the outer a CP layer are thus less thick than the CP films.

Table 1.

Summary of the physical properties of the films

Films	Thickness (mm)	Tensile strength (MPa)	Elongation at break (%)	Moisture content (%)	WVP (g mm/m2day Kpa)	Opacity (mm−1)
SA	0.035 ± 0.003d	60.55 ± 1.91a	8.33 ± 0.11f	17.30 ± 0.42a	3.64 ± 0.34e	0.05 ± 0.01e
SA-CP-1	0.055 ± 0.004c	22.94 ± 4.46de	20.25 ± 1.05e	11.31 ± 0.46bc	5.69 ± 0.92d	0.05 ± 0.04e
SA-CP-2	0.068 ± 0.009b	31.01 ± 2.81cde	27.90 ± 0.76de	12.05 ± 0.13b	7.41 ± 0.83c	0.35 ± 0.05d
SA-CP-3	0.063 ± 0.004bc	20.89 ± 0.68e	34.75 ± 0.12d	12.13 ± 0.26b	6.74 ± 0.70c	0.61 ± 0.02c
SA-CP-4	0.065 ± 0.012bc	26.07 ± 6.45cde	24.59 ± 0.15e	11.86 ± 0.42b	6.96 ± 0.43c	1.02 ± 0.05b
CP-1	0.124 ± 0.009a	32.96 ± 4.02bcd	116.45 ± 7.52a	11.35 ± 0.73bc	13.34 ± 0.73a	0.06 ± 0.02e
CP-2	0.116 ± 0.004a	33.08 ± 6.05bcd	109.51 ± 7.67ab	10.90 ± 0.44cd	12.64 ± 0.57ab	0.55 ± 0.09c
CP-3	0.114 ± 0.019a	35.56 ± 7.11bc	104.59 ± 10.72bc	10.14 ± 0.53de	12.31 ± 0.39b	1.07 ± 0.14b
CP-4	0.125 ± 0.011a	43.13 ± 8.24b	99.10 ± 8.84c	9.82 ± 0.51e	13.23 ± 0.65a	2.26 ± 0.06a

All data are shown as mean ± standard deviation (SD). Different letters indicate differences at the p < 0.05 level are significant.

Elongation at break (EB) and tensile strength (TS) reflect the flexibility and mechanical resistance of a food packaging material, respectively [30]. CP films have a higher elongation at break (EB) than SA-CP and SA films, while CP films have higher tensile strength (TS) than SA-CP films but are lower than SA films. Assis et al. observed that an increase in the polymer network’s randomization could diminish the generation of structural consistency, resulting in a lower value of TS [31]. With the anthocyanin incorporation, the EB of the CP films decreased, and the TS increased, reflecting the lower flexibility of the chitosan/PVA films and the enhanced mechanical resistance of the films by adding anthocyanin extract. One is that the interaction between the chains of film components is hindered by anthocyanins, thereby reducing flexibility [30]. Another is hydrogen bonds could be formed between anthocyanins and the film matrix and between OH⁻ in the SA layer and NH₃⁺ in the chitosan layer (Fig. 4), thus increasing the mechanical resistance [6]. The difference in mechanical properties is insignificant for SA-CP films containing less anthocyanin than CP films. Nevertheless, in terms of mechanical properties, the tensile strength of the bilayer films in this study was not as high as that of the single-layer films up to 32.34 ± 3.5 Mpa, but the tensile strength of the prepared films was 103% higher compared to the data reported by Liu et al. at 15.90 ± 2.86 MPa [23]. In addition, the elongation at the break of the bilayer films was similar to the reported value of 39.50 ± 5.51%, while the single-layer films were much higher than this value. Consequently, the mechanical properties were all improved and could meet the needs of food packaging.

Fig. 4.

Schematic diagram of ionic interactions between chitosan and alginate in a bilayer

Moisture content, swelling, and WVP

According to Table 1, SA film (7.30 ± 0.42%) has the highest moisture content of all films, and the water solubility is close to 100% due to its hydrophilicity. Other than this, SA-CP films exhibit similar water contents. In the case of CP films, for CP-1 without anthocyanin addition, the moisture content was higher than for CP films with anthocyanin addition. This phenomenon was related to forming molecular hydrogen bonds between CS/PVA bound to the anthocyanin and replaced a part of the CP interacting with moisture. In previous studies, it was also confirmed that the addition of anthocyanin extracts reduced the water content of the films [32, 33]. Swelling ability reflects the film’s hydrophilicity as an essential film property [34]. The swelling of all films is shown in Fig. 5a.The results indicate that the swelling of the SA-CP films is much more significant than that of the CP films, which could be related to the strong hydrophilic of the SA layer in the bilayer. The presence of an SA layer in the bilayer, which is strongly hydrophilic, and the interaction between the SA layer and the water molecules during swelling increase molecular spacing and volume.

Fig. 5.

Swelling index (a) and water contact angle (b) of films [Different letters indicate significant differences. (p < 0.5)]

The WVP of food packaging film is essential in reducing the ability to transfer moisture from food to the environment and is a vital reference for comparing moisture barrier performance [30]. The result of WVP for SA-CP, CP, and SA films is shown in Table 1. The WVP values of SA-CP films and SA films were significantly weaker than those of CP films among all the films tested. This study found no significant change in WVP values after adding anthocyanins to the CP/PVA solution. Generally, the WVP of laminated films depends mainly on the WVP value of each layer of the film, and SA has a lower WVP because of the thinness of its film [35]. The lower WVP of the bilayers may be related to the bilayers having fewer free hydroxyl groups compared to the single-layer films. The free hydroxyl group could facilitate the attachment of water molecules down the films and through the films by enhancing the interaction between the water molecules and the polysaccharide chains. For bilayers, hydrogen bonding between the bilayers and electrostatic interactions between them reduced the adsorption of hydrophilic groups to water, making them more water resistant. In terms of the addition of anthocyanins, no change in the corresponding groups was induced, so the water resistance was not significantly altered.

The hydrophobicity of all films was determined by the water contact angle and the results are shown in Fig. 5b. The water contact angle of the SA film was 41.77° (< 65°), indicating the hydrophilic nature of the sodium alginate film, indicated also by the swelling properties. As the anthocyanin content increased, the water contact angle increased from 77.85 to 84.39° for the singles and from 86.29 to 96.00° for the bilayers. This might be attributable to the reduction of surface-free energy through enhanced hydrogen bonding interactions between anthocyanin, chitosan, and polyvinyl alcohol molecules, resulting in the increased hydrophobicity of the films [36]. Compared to the singles, the water contact angle of the bilayers was smaller and the bottom area increased, which could be attributed to the fact that the inner layer of the bilayer was an SA film making it less hydrophobic.

Light transmittance

The UV–visible barrier capability is an essential factor in preventing UV–vis light-induced food oxidation to extend food storage [37, 38], and also an integral element in assessing whether the appearance could be used for practical packaging applications. Figure 6 presents the light transmission spectra of all films. It can be seen that SA, SA-CP-1, and CP-1 represented in black, pink, and navy, respectively, have a light transmission more excellent than 80% and thus are transparent films. The following curves represent the single-layer films with the addition of anthocyanins and the bilayer films. The light transmission of the films tends to decrease with the accumulation of anthocyanins, which indicates that the films with the addition of anthocyanins have better UV–visible light barrier properties [32]. It was also discovered that all films incorporating anthocyanins had a significantly lower light transmission than all films without anthocyanins, which indicated that films containing anthocyanins were expected to have better UV–visible light barrier properties.

Fig. 6.

Light transmittance of films

In addition, the films exhibited a gradual decrease in light transmission as the anthocyanin content increased. As a result, the light transmission of the SA-CP films was higher than that of the CP films for the added anthocyanins, which was attributed to the SA-CP containing fewer anthocyanins than the CP at the same level of incorporation. According to Table 1, the opacity of the bilayers is lower than that of the monolayers, and the CP-4 film has the highest opacity (2.26 ± 0.06^a), indicating that sufficient anthocyanins could effectively diminish the light transmission of the films [9]. According to Table 1, all films in this study have low opacity values and could be used for food packaging, but double-layer films have better transparency and UV–visible light barrier properties.

pH sensitivity evaluation

Color response analysis of anthocyanin extract

Anthocyanins can cause structural changes at a wide range of pH values, resulting in visible color changes [39]. According to Fig. 7, the color range of anthocyanin solutions was peach > pink > purple > celadon > yellow from acidic buffered solutions to basic buffered solutions. An increased pH from 2 to 6 decreased the intensity of the anthocyanin color until a minimum value of 6, after which an increase in pH increased the intensity of the color. This phenomenon is ascribed to the presence of anthocyanins as flavylium cation at pH values of approximately 3 or lower, producing structural shifts in carbinol pseudobase, quinone base, and quinone anion as the pH increases [26]. With the structural changes in anthocyanin, the maximum absorption peak shifted from 510 to 570 nm.

Fig. 7.

Color variations of cyanidin chloride extracts and UV–vis spectra in different buffer solutions (pH 2–12)

pH sensitivity of films

In this study, we used the changing color of the films to evaluate pH sensitivity, a crucial indicator for intelligent packaging, and the results are displayed in Fig. 8. The results indicate that both SA-CP and CP films were highly pH-sensitive. The film color appears pH = 2–3 in pink, pH = 4–6 in purple, pH = 7–11 in blue, and pH = 12 in green. In particular, the color of the film changes related to the anthocyanin content, and the color intensity increases with increasing anthocyanins. According to Table 2, ΔE values can be obtained for all films of CP and SA-CP. The color change (ΔE > 3) could be easily observed with the naked eye [34]. In this work, the ΔE values for both SA-CP and CP films are more significant than 3, which is to say, all films could be visually detected over a wide range of pH values. The ΔE values in this study are much greater than the reported, so the bilayer films in this study have better-naked eye detection performance [23]. In contrast, CP-4 exhibited a slightly smaller ΔE (p < 0.5) under basic conditions at the same pH, while the ΔE of the SA-CP films was slightly more significant than the CP films, indicating that the bilayer had a more pronounced visually observable color change. In addition, CP or SA-CP films (Fig. 8) and cyanidin chloride solution (Fig. 7) exhibited different color changes in the same pH buffer solution. The change is attributed to the strong electrostatic interaction between cyanidin chloride and chitosan, leading to a structural modification of the anthocyanin, and thus affecting the discoloring ability [40].

Fig. 8.

Visual color change of CP-2, CP-3, CP-4, SA-CP-2, SA-CP-3, and SA-CP-4 films at pH 2–12

Table 2.

The ΔE values of films after being soaked into pH 2 to 12 buffer solutions for 20 min

pH values	SA-CP-2	SA-CP-3	SA-CP-4	CP-2	CP-3	CP-4
pH = 2	32.68 ± 1.56a	35.16 ± 0.26a	37.73 ± 0.63a	35.89 ± 0.77a	44.91 ± 0.96a	39.79 ± 1.10a
pH = 3	31.73 ± 1.58a	30.36 ± 0.75b	31.36 ± 0.54b	31.18 ± 0.80b	42.69 ± 0.84a	35.08 ± 1.33b
pH = 4	24.64 ± 1.59b	29.57 ± 1.41b	26.60 ± 0.64c	27.73 ± 0.84c	31.57 ± 1.53b	36.14 ± 0.97b
pH = 5	18.76 ± 1.73c	26.69 ± 1.40c	22.76 ± 0.58d	23.43 ± 0.81d	26.61 ± 1.26c	24.14 ± 1.11c
pH = 6	19.23 ± 1.59c	15.61 ± 0.44d	16.05 ± 0.56fg	23.01 ± 1.22d	23.80 ± 1.91c	22.51 ± 0.84c
pH = 7	14.68 ± 1.71d	8.15 ± 1.01f	14.58 ± 0.75h	14.39 ± 0.49f	5.19 ± 0.97f	6.84 ± 0.74e
pH = 8	19.22 ± 2.36c	7.85 ± 0.98f	12.19 ± 0.71i	14.45 ± 0.90f	10.60 ± 1.72e	3.74 ± 0.57f
pH = 9	12.52 ± 2.25d	15.33 ± 2.25d	15.08 ± 0.67gh	8.55 ± 0.43h	7.36 ± 1.62f	3.98 ± 1.28f
pH = 10	12.88 ± 2.20d	8.28 ± 1.59f	15.44 ± 0.61gh	7.13 ± 0.43i	13.89 ± 2.17d	3.02 ± 1.46f
pH = 11	12.40 ± 2.14d	10.83 ± 1.68e	19.40 ± 0.63e	10.48 ± 0.68g	16.71 ± 2.90d	17.08 ± 1.03d
pH = 12	13.58 ± 1.49d	26.42 ± 0.36c	16.90 ± 0.08f	17.61 ± 0.99e	24.81 ± 2.10c	6.57 ± 1.40e

All data are shown as mean ± standard deviation (SD). Different letters indicate differences at p < 0.05 level are significant.

Ammonia sensitivity

In the action of bacteria and enzymes, the proteins in animal foods are gradually broken down into peptides and amino acids. Furthermore, the protein is degraded into low molecular compounds containing nitrogen, which is the source of the spoilage smell of animal food [41]. Thus, indicator films are essential for the detection of ammonia sensitivity. In this study, ammonia was used to test the pH sensitivity of the films. Ammonia could diffuse into the films and then be hydrolyzed to hydroxide ions, creating an alkaline environment in the films. As shown in Fig. 9, all films except CP-4 showed significant differences from the initial film after 10 min. CP-4 film did not show significant changes visible to the naked eye at 60 min, probably due to the high anthocyanin content of the film resulting in a darker color that was not easily observed. At 180 min and 300 min, the films showed significant changes. The color gradually changes from purple to grey-blue to yellow-green as the time increases. The bilayers had a large difference in ΔE, suggesting that the bilayers may have good ammonia sensitivity.

Fig. 9.

The colorimetric response of bilayer films SA-CP-2 (a), SA-CP-3 (b), SA-CP-4 (c), and single-layer films CP-2 (d), CP-3 (e), CP-4 (f) for 0.1 mg/mL ammonia for 60 min; the colorimetric response of all films for 0.1 mg/mL ammonia for 180 min and 300 min (g)

Antibacterial activity

To extend food products’ storage life, the films’ antibacterial properties play a vital role. E. coli and S. aureus are the most common Gram-negative and Gram-positive bacteria, respectively. They are common foodborne pathogenic microorganisms and a common source of disease infection in humans. The antibacterial activity of the samples was evaluated by the diameter of the inhibition circle for two bacteria, E. coli (Gram-negative) and S. aureus (Gram-positive). As shown in Fig. 10 and Table 3, the results revealed that the SA film did not inhibit two bacteria, while the other films all had varying degrees of bacteria inhibition. Meanwhile, the maximum inhibition circle diameters were 21.17 ± 1.04 mm and 13.05 ± 0.56 mm for S. aureus and E. coli, respectively, indicating that the remaining eight films had more effective antibacterial capacity against Gram-positive bacteria. The antimicrobial activity of nano-ZnO had been demonstrated in several previous studies, both by the release of Zn²⁺ from the film matrix and by the production of hydrogen peroxide on the surface of the ZnO nanoparticles [42, 43]. Furthermore, according to Fig. 10, the bilayers we prepared were as effective as the single layers in inhibiting bacteria. At the same time, the data for the single layers demonstrate a tendency for the diameter of inhibition to rise with increasing amounts of ZnO added. Therefore, in the subsequent work, we can adjust the ZnO content in the bilayers according to the requirements of specific food storage conditions for bacterial inhibition, thus regulating the performance of the composite films by design.

Fig. 10.

Inhibitory zone of the fabricated packaging films (SA is sodium alginate film, addition of different anthocyanins to the bilayers is SA-CP-1 to SA-CP-4, and the single-layer films are CP-1 to CP-4.)

Table 3.

Antibacterial activity of the films

Films	Diameter of inhibition zone (mm)
	Staphylococcus aureus ( +)	Escherichia coli ( −)
SA	0h	0e
SA-CP-1	13.98 ± 0.39g	10.59 ± 0.24d
SA-CP-2	15.75 ± 0.22f	11.81 ± 0.20c
SA-CP-3	16.38 ± 0.14ef	11.87 ± 0.47c
SA-CP-4	16.72 ± 0.28de	12.86 ± 0.16a
CP-1	17.23 ± 0.25d	12.63 ± 0.16ab
CP-2	19.47 ± 0.45b	12.56 ± 0.49ab
CP-3	18.23 ± 0.59c	12.08 ± 0.13c
CP-4	21.17 ± 1.04a	13.05 ± 0.56a

All data are shown as mean ± standard deviation (SD). Different letters indicate differences at the p < 0.05 level are significant.

Conclusions

In this study, a novel homogeneous and flexible bilayer film is developed by a calcium ion cross-linking method, which is designed for the food intelligent packing. The results show that the bilayers exhibit a dense and homogeneous monolayer structure and excellent physical properties. With the addition of anthocyanins, the films show a decrease in elongation at break, an increase in tensile strength, an enhancement in UV–visible light barrier properties, a decrease in water vapor permeability, and an increase in swelling properties. Under the same conditions of addition, the bilayer films offer better light transmission and excellent UV-blocking properties. Meanwhile, the bilayer films have antimicrobial properties, which prolong the storage life of the food. Last, the bilayers demonstrated a broader pH sensitivity (ΔE > 10) in different pH buffer solutions, which is critical for intelligent packaging that effectively monitors the freshness of food products in real-time. Ultimately, this study presented the overall performance of the bilayer pH-responsive films, providing a new direction for biodegradable active intelligent packaging.

Author contribution

Huiru Li: investigation, methodology, formal analysis, data curation, visualization, writing—original draft. Guozhao Liu: writing—review and editing. Kairu Ye: writing—review and editing. Wanping He: writing—review and editing. Hongyuan Wei: writing—review and editing. Leping Dang: writing—review and editing, supervision, funding acquisition, project administration.

Data availability

Not applicable.

Declarations

Ethical approval

Not applicable.

Competing interests

The authors declare no competing interests.