Effect of ethanol extract of black soybean coat on physicochemical properties and biological activities of chitosan packaging film

Abstract

Chitosan (CS) with an ethanol extract of black soybean coat (EBSC) was prepared, and its physicochemical properties and antioxidant and antibacterial activities were tested. The results showed that EBSC significantly increased the thickness and UV–Vis light barrier ability of the CS-based films, while the swelling degree, water vapor permeability, and tensile strength decreased. The CS-EBSC films had smooth surfaces, compact cross-sections, and no cracks, and they had higher crystallinity than the CS film. Fourier transform-infrared spectroscopy indicated that there were noncovalent bonds (hydrogen bonds) between EBSC and CS. Furthermore, the CS-EBSC III film presented a stronger ABTS radical scavenging ability (66.58%) and could effectively inhibit Bacillus subtilis, Escherichia coli, and Staphylococcus aureus. The lipid oxidation test proved that CS-EBSC films significantly reduced the peroxide value of lard. The results above indicate that CS-EBSC films could be used as an active packaging material to improve the shelf life of food.

Introduction

Plastic is widely used in the food packaging industry because of its low cost (Cui et al., 2020). Nevertheless, plastic materials are not easy to degrade, bringing about a series of environmental problems, such as ocean and soil pollution (Guo et al., 2019; Riaz et al., 2020a). Therefore, natural renewable film substrates have gradually attracted the attention of researchers. At present, renewable materials such as polysaccharides (starch, cellulose, and chitosan), proteins (soy protein, corn zein, and whey protein), and lipids (paraffin, acetoglyceride, and shellac resins) have been used to prepare biodegradable films (Hassan et al., 2018). Chitosan (CS) is the product of chitin deacetylation and has been widely used in food packaging films due to its good film-forming characteristics, biocompatibility, biodegradability and nontoxic properties (Wang et al., 2020).

However, the application of CS film in the food packaging industry is limited by its low antioxidant ability and high permeability to water vapor (Riaz et al., 2020a). Many studies show that adding natural plant extracts or essential oils can significantly improve the antioxidant and antibacterial activities of CS film, which is related to the abundant polyphenols in these natural compounds, such as, thymol and carvacrol are the major components of thymus kotschyanus essential oil and oregano essential oil (Alves et al., 2018; Bi et al., 2019; Mehdizadeh et al., 2020; Riaz et al., 2020b). In addition, intermolecular hydrogen bonds or electrostatic interactions may be formed between polyphenols and hydroxyl or amino groups in CS molecular chains, thus changing the physical properties of the CS film (Bi et al., 2019; Liu et al., 2017a). For example, curcumin, protocatechuic acid, apple peel polyphenols, and chrysanthemum essential oil have better effects on improving the antioxidant and antibacterial activities and increasing the water blocking capacity of CS composite films (Liu et al., 2016;2017b; Lin et al., 2019; Riaz et al., 2018;). Moreover, there are few reports about the effect of black soybean coat extract on the physical and chemical properties of CS films.

As dark-colored foods, black soybean has more active substances than soybean and mung bean (Peng et al., 2017). These natural compounds mainly exist on the black soybean coat, and the content of anthocyanins reaches 696.5 mg/100 g (Wang and Xie, 2019). Studies show that anthocyanins have antioxidant and anti-inflammatory effects, which also exhibit good effects on the treatment of diabetes and obesity (Kumar et al., 2019; Yuan et al., 2019). In recent years, natural degradable bioactive films prepared by incorporating anthocyanins into CS substrates have also been favored by consumers (Yong et al., 2019a). This kind of active film presents good antioxidant capacity and has broad application prospects as a food packaging material (Yong et al., 2019b).

The aim of this work was to develop antioxidant and antibacterial active food packaging films based on CS and ethanol Extracts of black soybean coats (EBSCs). The UV–Vis light barrier properties, microstructure, and crystallinity of CS composite films were studied by UV spectrophotometry, scanning electron microscopy, Fourier transform infrared spectroscopy, and X-ray diffraction. Then, the physicochemical properties and antioxidant and antibacterial activities of the CS-EBSC films were characterized and compared with those of the CS film. In addition, the potential application of CS-EBSC films as active food packaging materials in the food industry was evaluated by simulated lipid oxidation experiments.

Materials and methods

Materials and reagents

CS with a degree of deacetylation of ≥ 80% and viscosity > 400 mPa.s was purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2’-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were supplied by Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Bacillus subtilis (ATCC 6633), Escherichia coli (ATCC 8739), and Staphylococcus aureus (CMCC 26003) were acquired from the College of Food and Biological Engineering, Qiqihar University (Qiqihar, China). Pork fat was obtained from a local market (Qiqihar, China). All reagents were used of analytical grade.

Preparation of EBSC

EBSCs were prepared using the method reported by Wang et al. (2018) with some modifications. Black soybean coats were procured from a local market (Qiqihar, China), ground with a multifunctional pulverizer (Sufeng Industry and Trade Co., Ltd, Zhejiang, China), and sifted through 60 mesh. The crushed black soybean coat powder was immersed in 70% ethanol solution at a ratio of 1:20 (w:v). After extraction at 25 °C for 2 h, the extract solution was centrifuged at 4000 g, and the precipitate was extracted again by the above method. Then, the two supernatants were combined, concentrated, and lyophilized in a vacuum freeze dryer (2.5 L freeze dry system, Labconco Co., Kansas, Missouri, USA) (− 40 °C for 48 h). EBSC powder was stored at − 20 °C.

Film preparation.

The CS-EBSC films were prepared by the casting method, according to Riaz et al. (2020a), with slight modifications. First, CS film-forming solution was prepared by dissolving 2 g CS in 80 mL 1% (v/v) acetic acid solution under continuous stirring for 4 h. Then, 1 g glycerol, as a plasticizer, was added into CS film-forming solution with stirring for 0.5 h. After that, different weights of EBSC (0, 1, 3, and 5 wt% on CS basis) were dissolved in 20 mL 1% (v/v) acetic acid solution and added into CS film-forming solutions with stirring for 0.5 h. The bubbles in the film-forming solutions were removed by vacuum degassing. The CS-EBSC film-forming solutions with the same quantity (30 mL) were poured onto 15 cm × 15 cm plexiglass plates. After 12 h at 25 °C, they were moved to a 60 °C drying oven for 2 h, and then the CS-EBSC films were carefully removed from the plexiglass plates. Finally, the prepared films containing 0, 1, 3, and 5 wt% EBSC were designated CS, CS-EBSC I, CS-EBSC II, and CS-EBSC III films, respectively, and were equilibrated in a desiccator with saturated sodium bromide solution (56% RH, 25 °C) for 48 h.

Characterization of CS-EBSC films

Thicknesses of films

The thicknesses of the films were measured using a digital thickness micrometer (SM-114, Teclock, Okaya, Japan). The average thicknesses of five randomly selected locations of each film were taken as the film thickness.

Moisture content

The film pieces (2 × 2 cm²) were weighed and dried to constant weight at 105 °C according to the method of Riaz et al. (2018). The moisture content of CS-based films was calculated according to the following equation:

$$\text{Moisture}\text{content}\left(\%\right)=\frac{M_1-M_2}{M_1}\times 100$$ 1

where M₁ is the initial mass of films, and M₂ is the mass when dried to constant weight.

Determinations of water solubility (DVP)

According to the method of Riaz et al. (2018), the films were cut into 2 × 2 cm pieces and dried to constant weight at 105 °C, and the mass (M₁) was measured. Then, they were immersed in 50 mL distilled water and held at room temperature for 24 h. Finally, they were removed and dried to constant weight at 105 °C to obtain mass (M₂). The water solubility is calculated by using the following equation:

$$\text{Water}\text{solubility}\left(\%\right)=\frac{M_1-M_2}{M_1}\times 100$$ 2

where M₁ is initial dry mass, and M₂ is final dry mass.

Determination of water vapor permeability (WVP)

The WVP of films was determined by the gravimetrical method of Crizel et al. (2018) with some modifications. The films (10 cm in diameter) were sealed on top of 6 cm diameter plastic cups filled with anhydrous calcium chloride particles (maintaining 0% RH). The cups were weighed and then put into a desiccator with saturated sodium chloride solution while maintaining constant humidity (75%) and temperature (25 °C). The weight change of overall cups was recorded every 24 h and continued for seven days. The WVP is calculated by the following equation:

$$WVP=\frac{W\times x}{A\times t\times \mathrm{\Delta }P}$$ 3

where W is the overall weight gain of the cup (g), x is the film thickness (mm), A is the permeation area (m²), t is the elapsed time (h), and ∆P is the difference in the partial vapor pressure of the film (3200 Pa at 25 °C). The results were expressed as g mm/m² h Pa.

Analysis of film color and transparency

A colorimeter (CR-10 Plus, Konica Minolta optics Co., Ltd, Shanghai, China) was used to determine the color parameters of the films. The lightness (L*), red (a*), and yellow (b*) values were used to characterize the film color based on Hunter scaling. White tile was used as a standard (L = 91.8, a = − 2.2, b = − 2.7). Each film was measured at three different sites. The total color difference (ΔE), Yellowness index (YI), and Whiteness index (WI) were calculated according to the method of Mehdizadeh et al. (2018):

$$\mathrm{\Delta }E=\sqrt{{\left(L^{\ast }-L\right)}^2+{\left(a^{\ast }-a\right)}^2+{\left(b^{\ast }-b\right)}^2}$$ 4

$$YI=\frac{142.86\times b^{\ast }}{L^{\ast }}$$ 5

$$WI=100-\sqrt{{\left(100-L^{\ast }\right)}^2+a^{\ast 2}+b^{\ast 2}}$$ 6

The transmittance was determined according to the method of Riaz et al. (2018). A UV spectrophotometer (UV-5100, Metash Instruments Co., Ltd, Shanghai, China) was used to measure the absorption spectra. In addition, the opacity was expressed by the following equation:

$$Opacity\left(A.m{m}^{-1}\right)=\frac{Ab{s}_{600}}{x}$$ 7

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

Analysis of mechanical properties

The mechanical properties of the films were evaluated by using a texture analyzer (TA. XT plusC, Stable Micro System Co., UK) according to Liu et al. (2017a) with modification. The samples were pretreated into 2 cm × 8 cm rectangles and mounted between the grip with a 50 mm initial distance. The falling speed of the crosshead was set at 60 mm/min, and three parallel experiments were conducted for each film sample. The Tensile strength (TS) and Elongation at break (EB) of films were evaluated by the following equations:

$$TS\left(\mathit{MPa}\right)=\frac{F}{x\times W}$$ 8

$$EB\left(\%\right)=\frac{\mathrm{\Delta }L}{L_0}\times 100$$ 9

where F is the maximum stretching strength (N), x is the film thickness (mm), W is the film width (mm), ΔL is the elongation when the film breaks (mm) and L₀ is the initial length of the film (mm).

Scanning electron microscopy (SEM)

The microstructure of the film surface and cross-section were observed by scanning electron microscopy (S-4300, Hitachi Co., Japan). SEM micrographs of the samples were obtained at 5 kV accelerating voltage and 2000 × or 1000 × magnification. Before that, the films were broken in liquid nitrogen and sputter-coated with gold.

Fourier transform-infrared (FT-IR) spectroscopy

To investigate the chemical structure of the films, FT-IR spectra were obtained using FT-IR/NIR spectroscopy (Spotlight 400, Perkin Elmer Co., Waltham, Mass, USA) according to the method of Wang et al. (2018). Each spectrum result was obtained for a frequency range of 4000–500 cm⁻¹ with sixteen scans.

X-ray diffraction (XRD)

The crystalline characteristics of films were analyzed via XRD (SmartLab, Rigaku Co., Japan) according to the method described by Guo et al. (2019) with some modifications. The instrument's parameters were set to 45 kV, 200 mA, diffraction angle 2θ from 5–60°, and scan speed 4°/min.

Assay of the antioxidant and antibacterial activity of films

DPPH free radical scavenging activity of films

The DPPH free radical scavenging activity of the films was measured by using the method of Riaz et al. (2020b) with slight modifications. The film (10 mg) was mixed into 3 mL of methanolic DPPH solution (0.2 mM) and stored in a dark environment for 1 h. Afterward, the absorbance of the reaction mixture at 517 nm was measured (UV–5100, Metash Instruments Co., Ltd, Shanghai, Chain). The DPPH radical scavenging activity of the films was calculated based on the following equation:

$$\text{DPPH}\text{free}\text{radical}\text{scavening}\text{activity}\left(\%\right)=\frac{A_0-A_1}{A_0}\times 100$$ 10

where A₀ is the absorbance of the reaction mixture of the films and A₁ is the absorbance of the methanolic DPPH solution.

ABTS free radical scavenging activity of films

The antioxidant capacity of films was evaluated by the method of scavenging ABTS free radicals (Riaz et al., 2020b). ABTS was dissolved in 20 mM pH 4.5 acetic acid buffer solution to obtain a 7 mm ABTS solution, which was mixed with 2.45 mM potassium persulfate solution (1:1, v:v) and then kept for 12–16 h in the dark at room temperature. The absorbance of the stable ABTS⁺ radical solution at 734 nm was adjusted to 0.70 ± 0.02 with acetic acid buffer solution when it was used. The film (10 g) and 3 mL ABTS working solution were mixed and incubated for 6 min before the absorbance at 734 nm was measured (UV-5100, Metash Instruments Co., Ltd, Shanghai, Chain). The ABTS radical scavenging activity of the films was calculated by the following equation:

$$\text{ABTS}\text{free}\text{radical}\text{scavenging}\text{activity}\left(\%\right)=\frac{A_0-A_1}{A_0}\times 100$$ 11

where A₀ is the absorbance of the reaction mixture of the films and A₁is the absorbance of the ABTS working solution.

Application of films in lard packaging

The Schaal oven method was used to evaluate the potential application value of CS-EBSC films (Shanooba et al., 2020). The lard refined from pork fat was packed in film bags. Each bag was 6 g and sealed by a heat sealer. Then, they were stored in an electric blast drying oven (GZX-9146 MBE, Boxun Industrial Co., Ltd, Shanghai, China) at 60 ± 1 °C for 7 days. The peroxide value of the lard was determined every 24 h and compared with that of lard exposed to air.

Determination of peroxide value (POV)

The POV was determined by Meng et al. (2019) with some modifications. An aliquot of lard (3 g) was added to iodine flasks containing a 30 ml mixing solution of chloroform and acetic acid (2:3, v:v). To completely dissolve the lard, the iodine flasks were gently shaken. Afterward, the iodine flasks were transferred into 1 mL saturated potassium iodide solution and placed in a dark room. After 3 min, 100 mL distilled water was added and shaken well and then immediately titrated with 0.01 M sodium thiosulfate solution to light yellow. Then, 1 mL starch indicator was added to this solution, titrated, and vigorously shaken until the blue solution disappeared. At the same time, the blank reagent experiment was performed. The POV was calculated as follows:

$$POV\left(\mathit{mmol}/\mathit{kg}\right)=\frac{\left(V-V_0\right)\times c}{2\times m}\times 1000$$ 12

where V is the volume of sodium thiosulfate consumed solution by the sample (mL), V₀ is the volume of sodium thiosulfate consumed solution with the reagent blank experiment (mL), c is the concentration of sodium thiosulfate consumed solution (mol/L), and m is the lard weight (g).

Microbial analysis

The disc diffusion method was used to determine the antimicrobial activity of films by Liu et al., (2020) with slight modifications. Bacillus subtilis and Escherichia coli were expanded with sterilized 0.5% sodium chloride broth, and Staphylococcus aureus was expanded with sterilized 7.5% sodium chloride broth. Prior to use, the cell density of the obtained bacterial suspension was adjusted to 10⁴ CFU/ml with sterile normal saline. The films were cut into discs with a diameter of 10 mm and sterilized for 10 min under ultraviolet irradiation. After that, the discs were placed on an agar medium coated with 0.2 ml bacterial diluent and incubated at 37 °C for 48 h. The diameter of the inhibition zones was measured by a sliding caliper, and the size of the inhibition zone reflected the bacteriostatic effect of the films.

Statistical Analysis

All experiments were done in triplicate. Statistical analysis was performed using the IBM Statistical Program (SPSS 23, IMB, USA). At the 95% confidence level, a two-way Analysis of variance (ANOVA) was used to determine the significant difference among the groups. The significant difference between data means was analyzed by Duncan's test. p < 0.05 means significant difference.

Results and Discussion

Physical properties of CS-EBSC films

Thicknesses of CS-EBSC films

The film thickness significantly influences the mechanical properties, water vapor permeability, UV and light barrier properties of the film (Yong et al., 2019b), which is an important parameter to determine them. The film thickness is affected not only by the properties of the film materials, drying conditions, and preparation methods but also by the types of additives (Adilah et al., 2018; Liu et al., 2016). The addition of active substances may affect the molecular arrangement of CS and change the thickness of the CS film (Liu e et al., 2016).

Table 1 shows that the thickness of the CS film was significantly changed by adding EBSCs (p < 0.05). The thickness of the CS film increased as the EBSC concentration increased. A similar conclusion was obtained in both studies of CS composite films with Chinese chive root extract or apple peel polyphenols (Riaz et al., 2018;2020a). Nevertheless, Ciannamea et al. (2016) and Liu et al. (2016) found that the addition of red grape extract and curcumin had no significant effect on the thickness of soybean protein film and CS film. The increase in film thickness might be due to the increase in solid content introduced into the film matrix (Yuan et al., 2019). On the other hand, the polyhydroxy structure of EBSC promoted the binding of polyphenols to more than one CS molecule through intermolecular interactions (hydrogen) (Riaz et al., 2018; Yuan et al., 2019). The result was that the CS film was more compact and thicker. Furthermore, the change trend of film thickness was related to the different film materials, additives, and concentrations of additives (Yuan et al., 2019).

Table 1.

Physical properties of CS-based films with different concentration of EBSC

Index	Film sample
	CS film	CS-EBSC I film	CS-EBSC II film	CS-EBSC III film
Thickness (mm)	0.047 ± 0.0006c	0.055 ± 0.0040b	0.063 ± 0.0020a	0.065 ± 0.0023a
Moisture content (%)	47.91 ± 0.13a	38.88 ± 0.82a	38.49 ± 0.27a	38.30 ± 0.14a
Water solubility (%)	16.81 ± 2.57a	17.90 ± 0.73a	18.58 ± 0.14a	18.83 ± 0.19a
WVP(× 10–5 g mm/m2 h Pa)	2.21 ± 0.18a	1.57 ± 0.12bc	1.26 ± 0.15c	1.66 ± 0.30b
L*	90.4 ± 0.12a	83.0 ± 0.15b	74.6 ± 1.29c	67.2 ± 0.21d
a*	− 2.0 ± 0.06b	− 2.5 ± 0.06c	− 2.0 ± 0.21b	0.5 ± 0.31a
b*	− 0.8 ± 0.06b	− 1.7 ± 0.06c	− 1.0 ± 0.15b	0.3 ± 0.01a
ΔE	2.3 ± 0.11d	8.8 ± 0.15c	17.2 ± 1.35b	24.9 ± 0.58a
WI	90.2 ± 0.11a	82.8 ± 0.16b	74.5 ± 1.26c	67.2 ± 0.62d
Transmittance (%)	88.58 ± 0.77a	78.77 ± 0.38b	66.35 ± 2.21c	58.02 ± 2.34d
Opacity (A. mm−1)	1.11 ± 0.07d	1.89 ± 0.15c	2.84 ± 0.29b	3.63 ± 0.30a
Tensile strength (MPa)	35.68 ± 0.93a	27.76 ± 0.81ab	23.31 ± 1.67b	23.26 ± 0.77b
Elongation at break (%)	40.34 ± 0.46a	34.46 ± 0.16a	34.42 ± 0.54a	33.18 ± 0.63a

Values are given as mean ± standard deviation. a-d: Different lower case letters in the same line indicate significantly different (p < 0.05)

Moisture contents and water solubility of CS-EBSC films.

As a polysaccharide film, the sensitivity of CS film to water should be considered by researchers (Liu et al., 2017b). The moisture content and water solubility reflect the film's water absorption capacity and water resistance, respectively (Zhang et al., 2020). As observed in Table 1, the incorporation of EBSCs had no significant effect on the moisture content and water solubility of the CS film (p > 0.05). The reason why the CS film presented the highest moisture content (47.91%) was due to the formation of intermolecular hydrogen bonds between water molecules and the hydrophilic groups (e.g., hydroxyl and amino groups) of CS (Liu et al., 2017a). With increasing EBSC content, the moisture content of CS-EBSC films decreased, but the water solubility showed the opposite trend. The increase in the water solubility of CS-EBSC films was related to the hydrophilicity of EBSC (Liu et al., 2017a). The anthocyanins in EBSC could improve the binding ability of CS to water because the hydrophilic groups of anthocyanins easily interact with water, elevating the binding ability of the matrix with water and thus improving the hydrophilicity of the CS membrane (Riaz et al., 2018). A similar conclusion was obtained by Riaz et al. (2018), who added apple peel polyphenols to CS film.

WVP of CS-EBSC films

The barrier property of biodegradable films to water vapor is evaluated by measuring WVP (Bi et al., 2019). As food packaging materials, it should be able to effectively prevent water transfer between food and the surrounding environment (Riaz et al., 2018). The reason is that the loss of food moisture or the change in water balance causes a decline in food quality, thus affecting the shelf life of food (Riaz et al., 2020b). Usually, the WVP of films is affected by the thickness and the type and concentration of additives (Alves et al., 2018; Mehdizadeh et al., 2020).

Table 1 shows that there was a significant difference in WVP between the CS and CS-EBSC films (p < 0.05). The addition of EBSC reduced the WVP of the CS film, which was consistent with the study of Wang et al. (2018). The WVP of the CS-EBSC II film was the minimum (1.26 × 10^–5 g mm/m² h Pa) when the addition of EBSC reached 3% wt. On the one hand, it was related to the thicknesses of films, which increased the time for water vapor to pass through the films (Riaz et al., 2020a); on the other hand, it was due to the formation of dense networks between polyphenols and CS molecular chains (Wang et al., 2018). Nevertheless, when the EBSC amount increased to 5 wt%, the WVP of the CS-EBSC III film was higher than that of the CS-EBSC II film but still lower than that of the CS film. This might be attributed to the uneven distribution of EBSCs in the CS film, which resulted in the destruction of some dense networks (Yong et al., 2019a). In general, the addition of EBSC could reduce the WVP of the CS film. This was in agreement with the conclusions of Riaz et al. (2018) and Yong et al. (2019a;b) on CS films with apple peel polyphenols or purple-fleshed sweet potato extracts. In contrast, the incorporation of mangosteen rind powder increased the WVP of the CS film, which was related to the types of additives.

Color, opacity, and transmittance of CS-EBSC films

As an active packaging material, the color of the film also extremely affects the acceptability of consumers (Liu et al., 2017a). It is generally believed that the color difference of films is related to the pigments contained in additives and film base materials (Bi et al., 2019). As shown in Table 1, the CS film was colorless and transparent, and the addition of EBSC made the CS film dark. This change could also be obtained from the data in Table 1, in which the L* and WI values decreased with increasing EBSC content. This showed that EBSC had a great influence on the color of the CS film. At the same time, a*, b*, and ΔE of the CS-EBSC films increased gradually. The positive values of a* and b* of the CS-EBSC III film indicated that it turned red and yellow. The color change of CS-EBSC films might be related to the pigments of the black soybean coat itself. In previous studies, the incorporation of mango peel extracts could improve the b* value of gelatin film, and CS film turned red and yellow with the addition of grape seed extract-carvacrol microcapsules (Adilah et al., 2018; Alves et al., 2018).

As one of the factors inducing oxygen free radicals, visible and UV light accelerates the oxidation deterioration of food and affects food quality (Crizel et al., 2018; Meng et al., 2020). Therefore, as a food packaging film, it should have the function of blocking UV light. The light barrier properties of the films were measured, and the results are shown in Table 2. The CS film exhibited poor UV barrier properties, which could be significantly changed by the addition of EBSC (p < 0.05). The UV transmittance of CS-EBSC films decreased with increasing EBSC content. The enhanced UV blocking ability of the CS film might be due to the anthocyanin rich in EBSC, which could absorb light with wavelengths between 270–280 nm and 465–550 nm (Ciannamea et al., 2016). Moreover, compared with the CS film, the opacity of the CS-EBSC III film increased by two times. This result was related to the darkening of the CS film with the addition of EBSC. Bi et al. (2019) and Crizel et al. (2018) demonstrated that proanthocyanidins and olive pomace also improved the light barrier properties of CS films.

Table 2.

Antimicrobial activity of CS-based films against three foodborne pathogens

Film sample	Diameter of inhibition zone (mm)
	Bacillus subtilis	Escherichia coli	Staphylococcus aureus
CS film	12.27 ± 0.21b	11.60 ± 0.61ab	11.27 ± 0.21b
CS-EBSC I film	12.53 ± 0.15b	11.20 ± 0.36b	11.87 ± 0.55b
CS-EBSC II film	13.07 ± 0.29a	11.57 ± 0.12ab	12.40 ± 0.82b
CS-EBSC III film	13.37 ± 0.25a	12.33 ± 0.38a	16.83 ± 0.37a

Values are expressed as mean ± standard deviation (n = 3). a-d: Different lower case letters in the same column indicate significantly different (p < 0.05)

Mechanical property of CS-EBSC films

The mechanical properties reflect the mechanical resistance and flexibility of films (Liu et al., 2017b). The structural integrity and barrier properties of films are evaluated by tensile strength and elongation at break (Liu et al., 2017a). In practical applications, the integrity and deformability of films greatly influence food packaging, transportation, and storage (Riaz et al., 2020b; Souza et al., 2015). In addition, several factors affect the mechanical properties of composite films: the type and content of natural active compounds, the variety of biological substrates, and the interaction between them (Liu et al., 2017a).

The tensile strength and elongation at break of the CS and CS-EBSC films are shown in Table 1. The CS film displayed the highest tensile strength (35.68 MPa) and elongation at break (40.34%). The addition of EBSCs significantly reduced the tensile strength of the CS film (p < 0.05) but had no significant effect on the elongation at break in the CS film (p > 0.05). In addition, the tensile strength and elongation at the break of the CS-EBSC films decreased with the increasing addition of EBSC. Compared with the CS film, the tensile strength and elongation at break of the CS-EBSC III film decreased by 34.81% and 17.75%, respectively. Notably, there was no significant difference in tensile strength between the CS-EBSC II and CS-EBSC III films (p > 0.05). The decrease in tensile strength might be due to the interaction between anthocyanins and CS polymers, which reduced the molecular interactions between CS chains (Riaz et al., 2020b; Tan et al., 2015). These changes would lead to an increase in the interchain distance between the CS-EBSC film network, resulting in a decrease in the tensile strength and elongation at the break of the CS-EBSC films (Riaz et al., 2020b). It was reported that the mechanical properties of CS films could be changed by excessive protocatechuic acid (Liu et al., 2017a). Therefore, the mechanical properties of the CS-EBSC films were not significantly different, which was related to the amount of EBSC. Adilah et al. (2018) reduced the ability of mango peel extracts to improve the elongation at break of gelatin films to a certain extent but proposed the tensile strength. This phenomenon was attributed to the different types of biological substrates.

Structural characterization of CS-EBSC films

SEM

The distribution of EBSCs and the morphology of CS-based films are depicted in Table 1. The CS film was smooth and complete without pores and cracks. The addition of EBSC did not destroy the structural integrity of the CS film and dispersed uniformly in the CS film. However, when the addition of EBSC increased from 1–5% wt, the aggregates in CS-EBSC films increased.

The microstructure of the CS composite film was further observed by SEM. The results showed that the surface and cross-section of the CS film were smooth and continuous (Fig. 1). The incorporation of EBSCs did not significantly change the microstructure of the CS film. With the increase in EBSC content, the white spots on the surface of CS-EBSC films increased, which was the reason for the increase in aggregates in CS-EBSC films. Aggregations were mainly caused by the accumulation of anthocyanins (Qin et al., 2019). Furthermore, the cross-section of CS-EBSC films was compact and continuous, and no pores or gaps were observed. However, banded floccules appeared on the upper side of the CS-EBSC films (the lower side in the G and H diagrams corresponded to the actual upper side of the CS-EBSC films). The band widened with the increase in EBSC content. This was also the reason for the appearance of white spots on the surface of CS-EBSC films because some floccules formed protrusions due to gathering. Meng et al. (2020) also observed this phenomenon, and the extract from Flos Sophorae Immaturus formed protrusions in cassia gum film. Qin et al. (2019) and Riaz et al. (2020a) attributed this aggregation to the hydrophilicity of polyphenols.

Fig. 1.

SEM micrographs of the surface and cross-section of films. (A) surface and (E) cross-section of CS film; (B) surface and (F) cross-section of CS-EBSC I film; (C) surface and (G) cross-section of CS-EBSC II film; (D) surface and (H) cross-section of CS-EBSC III film

FT-IR spectra

FT-IR spectroscopy was used to research the interaction between EBSC and CS film, and its spectra are presented in Fig. 2(A). The CS film exhibited a wide band near 3272 cm⁻¹, representing O–H and N–H stretching (Yong et al., 2019b). Several bands at 2925, 2877, and 1407 cm⁻¹ were attributed to symmetric and asymmetric C-H vibrations (Liu et al., 2017a). Two bands at 1636 and 1549 cm⁻¹ were found that belonged to the C = O and N–H bending, vibrations and corresponded to the amide I band and amide II band, respectively (Liu et al., 2016; Riaz et al., 2018). Bands at 1065 and 1024 cm⁻¹ were associated with skeletal stretching of C–O, which was characteristic of a saccharide structure (Liu et al., 2017a). In addition, a wide band (3700–3000 cm⁻¹) presented by EBSC was assigned to the O–H stretching vibration (Yong et al., 2019b). 1603 and 1280 cm⁻¹ corresponded to the C = C stretching and C = O stretching of aromatic rings, respectively (Bi et al., 2019). At the same time, the bands at 1440 and 1060 cm⁻¹ were characteristic of C-H deformation of aromatic rings (Bi et al., 2019; Wang et al., 2018). Notably, the addition of EBSC changed the position of the band and peak intensity in the CS film to some extent. When the content of EBSC increased from 1 to 5% wt, the band of CS-EBSC films gradually widened at 3272 cm⁻¹ and shifted from 3272 to 3201 cm⁻¹. Moreover, the amide I band (from 1636 to 1632 cm⁻¹) and amide II band (from 1549 to 1541 cm⁻¹) of CS-EBSC films also shifted due to the presence of EBSC. This might be due to the formation of intermolecular hydrogen bonds between hydroxyl groups of anthocyanins and hydroxyl or amino groups in CS chains (Yong et al., 2019a). Changes in the band position and peak intensity were also observed after proanthocyanidins and purple, and black eggplant extracts were added to the CS film.

Fig. 2.

(A) FT-IR spectra and (B) XRD patterns of CS film, CS-EBSC I film, CS-EBSC II film, CS-EBSC III film and EBSC

XRD patterns

To analyze the crystallinity and molecular structure of the biopolymer, the CS-based films were characterized by XRD (Cui et al., 2020). The XRD patterns of the CS film and CS-EBSC films are shown in Fig. 2(B). A broader and single peak (approximately 21.5°) was present in the XRD pattern of the CS film, indicating that the CS film had an amorphous structure. The spectrum of EBSC exhibited a broad peak at approximately 22.0°, corresponding to the amorphous state of EBSC. In addition, the CS-EBSC films showed the same diffraction peaks as the CS film, but the diffraction peaks of the CS film became sharp with the addition of EBSC. Meanwhile, the diffraction peak intensities of the CS-EBSC films gradually increased as the amount of EBSC increased. This indicated that EBSC could improve the crystallinity of CS. The results were consistent with the research of Yuan et al. (2019), in which it was observed that the crystallinity of the biodegradable film increased after incorporating black soybean seed coat extract into shrimp shell waste protein and CS composite film. Zhang et al. (2020) suggested that the increase in crystallinity was related to the aggregation of additives, such as the aggregates of mangosteen rind powder exhibiting stronger diffraction ability than mangosteen rind powder alone in CS film. In contrast, the crystallinity of the CS film with Chinese chive root and purple-fleshed sweet potato extract decreased, which was attributed to the newly formed intermolecular hydrogen bond between the CS chain and polyphenols (Riaz et al., 2020a; Yong et al., 2019a). These different results indicated that the crystalline properties of CS-based films rich in polyphenols were affected by the content and composition of polyphenols in the extract (Yuan et al., 2019).

Antioxidant and antibacterial activity of CS-EBSC films

Antioxidant activity

The active food packaging film should have free radical scavenging ability because the existence of free radicals is an important inducement of food oxidation and deterioration (Zhang et al., 2020). The polyphenols in EBSC are mainly anthocyanins, and their polyhydroxy structure makes anthocyanins have strong hydrogen supplying ability (Meng et al., 2020; Wang and Xie, 2019). Therefore, EBSCs were incorporated into CS film to prepare active food packaging films to protect food from oxidative damage and prolong food shelf life. The free radical scavenging activity of CS-based films was evaluated by DPPH and ABTS radical scavenging rates.

As presented in Fig. 3, the radical scavenging activity of DPPH and ABTS by the CS film were the lowest, which were 12.62% and 15.91%, respectively. The free radical scavenging ability of the CS film was enhanced by incorporating EBSCs and was positively correlated with the amount of EBSCs (p < 0.05). However, the ABTS radical scavenging activity of the CS-EBSC I film was not significantly different from that of the CS film (p > 0.05), which might be due to the small amount of EBSCs. When the addition of EBSC increased from 0–5% wt, the DPPH and ABTS radical scavenging activity of the CS-EBSC III film increased by 10.99% and 50.67%, respectively. The improvement in the antioxidant ability of CS-EBSC films was mainly attributed to the effect of EBSC. Anthocyanins rich in EBSC can donate phenolic hydrogen to capture free radicals, thus compensating for the deficiency of the weak free radical scavenging ability of free amino groups at the C-2 position of CS chains (Zhang et al., 2019;2020). Furthermore, the DPPH radical scavenging rate was significantly positively correlated with the ABTS radical scavenging activity (r = 0.98, p < 0.05). In previous studies, apple peel polyphenols, curcumin, and protocatechuic acid all enhanced the antioxidant capacity of CS films (Liu et al., 2016, 2017a; Riaz et al., 2018).

Fig. 3.

Scavenging activities of the CS-based films on DPPH and ABTS radicals

POV of lard

Oxidation and microbial contamination are two vital factors of food spoilage. In previous studies, CS-polyphenol films were applied to walnut and bread packaging to research the antioxidant and antibacterial ability of active food packaging films (Crizel et al., 2018; Tan et al., 2015). As an active food packaging film, its excellent antioxidant properties are particularly important. Thus, lard was selected as a food model to evaluate the antioxidant ability of CS-based films.

The POV measurement results of the control and experimental groups are shown in Fig. 4. With prolonged heating time, the POV of lard increased gradually, but the CS film combined with EBSC could effectively reduce the POV. On the first day, there was no significant difference in POV among all groups (p > 0.05). On the 7th day, the POV in the control reached the maximum (1.75 mmol/kg), and the POV of lard packaged by CS-EBSC II film decreased the most (42.29%). At the same time, the order for POV of lard in all groups was control > CS-EBSC III group > CS group > CS-EBSC I group > CS-EBSC II group. The antioxidant ability of the CS film was attributed to the fact that hydrogen on its free amino group could scavenge some free radicals (Zhang et al., 2019), which was consistent with the conclusion of Fig. 3. The addition of EBSC significantly improved the antioxidant activity of the CS film, which was related to the strong free radical scavenging ability of anthocyanins rich in EBSC (Wang et al., 2018). On the other hand, the CS film and CS-EBSC films could also act as oxygen barriers to block partial oxygen, thus slowing down the oxidative deterioration of lard (Bi et al., 2019). Notably, when the EBSC addition increased to 5 wt%, the POV of the CS-EBSC group increased significantly after day 6 but was still lower than that of the control. Sun et al. (2019) also obtained a similar conclusion that the POV of lard packaged by κC/HMx/Pm₁₆ film (к-carrageenan and hydroxypropyl methylcellulose composite film with 16% wt Prunus maackii juice) was significantly higher than that of κC/HMx/Pm₈ film. This indicated that excessive EBSC could increase the oxygen permeation of the film, leading to lard oxidation, but the presence of anthocyanins would reduce the degree of oxidation (Meng et al., 2020). Zhang et al. (2020) applied CS-mangosteen rind powder film to soybean oil packaging, which also effectively reduced the POV of oil. These results show that biodegradable films combined with natural active substances have great application prospects in the field of food packaging.

Fig. 4.

POV of lard packed by CS-based films with different concentrations of EBSCs

Antimicrobial activity

Foodborne pathogens are important hidden dangers that affect food quality and threaten human health (Zhang et al., 2019). Therefore, active food packaging films should have an antibacterial activity to protect food from microbial contamination. Table 2 shows that all the films exhibited inhibitory effects on Bacillus subtilis, Escherichia coli, and Staphylococcus aureus. The smallest diameter of inhibition zones of CS film represented the lowest inhibition ability, but the incorporation of EBSC improved the inhibition ability of CS film. With the increase in EBSC content, the diameter of the inhibition zones of CS-EBSC films increased gradually to Bacillus subtilis and Staphylococcus aureus. Compared with the CS film, the inhibition zone diameter of the CS-EBSC III film against the three foodborne pathogens increased significantly (p < 0.05). The antibacterial properties of the CS film were related to the fact that CS was a kind of cationic polysaccharide (Zhang et al., 2020). The free amino group of the CS film interacted with cell membranes, resulting in the exudation of cell fluid and cell death (Qin et al., 2019). The presence of polyphenols in EBSCs could not only increase cell membrane permeability but also interfere with the synthesis of microbial genetic material (such as binding with DNA/RNA) and inhibit microbial growth (Zhang et al., 2020). In addition, the difference in inhibition zone diameter between the CS film and CS-EBSC I film was not significant (p > 0.05), which might be related to the lower addition of EBSCs. In general, the CS-EBSC III film inhibited Bacillus subtilis, Escherichia coli, and Staphylococcus aureus. Liu et al. (2020) also reached a similar conclusion when studying the inhibitory effect of CS/polyvinyl alcohol/graphene oxide nanofibrous film loaded with allicin on the three foodborne pathogens.