Active pullulan-based coatings incorporated with Auricularia auricular extracts for preserving potato fresh-cuts

Abstract

In the present study, Auricularia auricular polysaccharides (AAP) and Auricularia auricular proteins (AAPR) obtained from the waste products of Auricularia auricular were incorporated into pullulan (PUL) to obtain active packaging films/coatings. Results showed that incorporating AAP/AAPR into PUL-based films decreased their transparency, but increased the compactness, thermal stability, antioxidant, and antimicrobial properties. Adding 2% PUL films with 10%:10% of AAP/AAPR exhibiting good mechanical properties were applied to fresh-cut potatoes to avoid spoilage during eight days of storage, with significantly decreased in browning index, weight loss, microbial growth prevention and the total soluble solids was maintained. These results substantiated that pullulan containing AAP/AAPR as an active film/coating with antioxidant and antimicrobial properties has significant potential for maintaining safety and quality of fresh-cut potatoes and extending their shelf life.

Introduction

The past decade has witnessed significant scientific progress, leading to the advent of edible films/coatings that can be consumed with packaged foods. Edible films/coatings made of natural polymer biomaterials have attracted wide attention from researchers due to their environmental friendliness, easy degradability and sustainability (Han et al., 2020; Mor et al., 2021). Edible films/coatings can be made from various materials, including polysaccharides and proteins. Among these, pullulan is a water-soluble polysaccharide with excellent physical properties, such as good film formation, high water solubility, effective oxygen barrier properties and degradability (Singh et al., 2020; Tabasum et al., 2018). Accordingly, pullulan films/coatings are suitable for food packaging materials due to their transparent surface, colorless, odorless, nontoxic properties, good oil and oxygen resistance, high thermal stability, and biodegradability (Hassannia-Kolaee et al., 2016). However, the brittleness and lack of active function of pullulan films/coatings limit their broad application. Intrinsic limitations can be overcome by introducing additional active substances, such as essential oils, whey protein, corn starch, mucilage, and chitosan (Chu et al., 2019; Farris et al., 2014; Treviño-Garza et al., 2017). An increasing body of literature suggests that pullulan-based composite films/coatings and the obtained bioactive edible packaging systems can ameliorate food safety and extend their shelf life (Chang et al., 2019; Silva et al., 2018).

Auricularia auricular is a traditional gelatinous fungus commonly used for food and medicine in China, North Korea, South Korea, Mongolia, and Japan (Ma et al., 2018). China is the main producer of A. auricular, with a total output of 7.018 million tons in 2019 (Miao et al., 2020). During the selection and sorting process of A. auricular, a significant portion of the total output, approximately 5%-10%, comprises defective products that do not contain mold. Harnessing these defective products to replace bioactive compounds extracted from A. auricular can reduce production costs and environmental pollution (Wang, 2020). Auricularia auricular polysaccharide (AAP) and Auricularia auricular protein (AAPR) are the main functional component of the fruit bodies. Studies have shown that AAP has antioxidant (Khaskheli et al., 2015), antibacterial (Cai et al., 2015), antitumor (Qiu et al., 2018), anticoagulant (Bian et al., 2020), and immunomodulatory (Zhao et al., 2019) biological activities. AAP is an acidic polysaccharide that possesses numerous intrinsic properties, including low toxicity, high biocompatibility, abundant availability, and excellent biodegradability (Khaskheli et al., 2015). Furthermore, AAPR is a valuable source of eight essential amino acids that are necessary for the human body (Liu et al., 2021). It also boasts high nutritional value, good solubility, water retention, emulsification, foaming and possesses functional properties (An et al., 2021; Yang et al., 2023). Studies have demonstrated that AAPR has many physiological activities, such as antibacterial activity (Oli et al., 2020), immunomodulatory (Agyei and Danquah, 2011), and reducing osteoporosis (Qu et al., 2019). Therefore, incorporating AAP and AAPR, which are additives of natural antioxidant and antimicrobial components, into the pullulan film matrix, may enhance the physiochemical properties of pullulan-based films/coatings. Consumers tend to favor minimally processed potatoes (fresh-cut potatoes) because of their freshness, convenience, nutrition and hygiene. However, the widespread consumption of fresh-cut potatoes is hindered by various factors, including enzymatic browning, water loss, texture deterioration and perishability during processing and storage. These factors ultimately result in a decline in quality and shelf life. As a solution, edible coatings have proven successful in overcoming the limitations of minimal processing (Wu and Chen, 2013).

This study aimed to prepare AAP/AAPR/PUL-based edible films/coatings and characterize their properties, including barrier, mechanical, transparency, antioxidant, antibacterial, microstructure, and thermostability, as well as the interaction between proteins and polysaccharides. Finally, we evaluated the impact of AAP/AAPR incorporated in film-forming solutions to preserve the quality of fresh-cut potatoes following coating. Our assessment focused on several key factors, including appearance, color, microbial activity, weight loss, and soluble solids.

Materials and methods

Chemicals

Pullulan (≥ 99% purity, molecular weight [MW] 1.4–2.7 × 10⁴ Da) was obtained from Yuanye Biotechnology Co., Ltd. (Shanghai, China). A. auricular was obtained from a local market (Nanchang, China). Glycerol (food grade ≥ 99.9% purity) was purchased from Wanbang Chemical Technology Co., Ltd. (Henan China). T-series dextran standards: T-500, T-250, T-150, T-50, T-20, T-5, and glucose were obtained from Kuer Chemistry Technology Co., Ltd. (Beijing, China). Protein standards: bovine serum albumin, ovalbumin, lysozyme, and cytochrome C were procured from Macklin Biochemical Technology Co., Ltd. (Shanghai, China). Dialysis bags (MWCO 8.0–14.0 kDa, Flat Width: 34 mm, Solarbio Science & Technology Co., Ltd. Beijing, China). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis-(3-ethylbanzthiazoline-6-sulfonate acid) (ABTS) were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). All chemicals used for this research were of analytical grade.

Extraction and purification of Auricularia auricular protein (AAPR)

Auricularia auricular was crushed and passed through a 100-mesh sieve, defatted with petroleum ether by reflux and dried to obtain extraction powder. Ultrasound-assisted enzymatic extraction is a state-of-the-art technology implemented in recent years based on the principle that protease can modify and degrade AAPR, leading to peptide chain shortening and reduced molecular weight. In this respect, proteases increase the yield by releasing the protein bound to the polysaccharide matrix (Görgüç et al., 2020). In this study, we conducted ultrasonic-assisted enzymatic extraction of AAPR using the following conditions: solid-to-liquid ratio of 1:88 g/mL, ultrasound power of 180 W, ultrasonic time of 30.5 min, enzymolysis pH of 8.5, enzymolysis temperature of 65.8 °C, enzymolysis time of 1.5 h, as previously described (An et al., 2021). A crude protein solution (2%, w/v) was decolorized with activated carbon (4%, w/v) at 45 °C for 30 min. Then it was concentrated by rotary evaporation under reduced pressure, followed by 48 h dialysis at 4 °C to remove impurities such as monosaccharides, oligosaccharides, and salts. The purified AAPR was obtained by vacuum freeze-drying (Scientz-10N, Scientz Biotechnology Co., Ltd, Ningbo, China).

Extraction and purification of Auricularia auricular polysaccharide (AAP)

AAP was extracted from the residue generated after the extraction of AAPR from A. auricular, using the water extraction method (Yang et al., 2020), with slight modifications. Briefly, the residue was dried, redissolved in water at a solid–liquid ratio of 1:40 g/mL, and then extracted at 90 °C for 2 h. After centrifugation at 3000×g for 10 min (SF-GL-20A, Shanghai Fichal Analytical Instrument Co., Ltd., Shanghai, China), the supernatant was collected. The extraction was repeated twice, and the combined supernatants were concentrated via a rotary evaporator (RE-52AA, Shanghai Yarong Biochemical Instrument Factory, Shanghai, China) at 40 °C. Sevag reagent (chloroform:n-butanol = 4:1, v/v) was added for deproteinization until no precipitate was generated (Khaskheli et al., 2015). The pH was adjusted to 8.0 with 25% ammonia, slowly stirred, 30% hydrogen peroxide (1/5 of the original volume) for decolorization was added, and the mixture was incubated at 50 °C for 2 h. Then, the decolorized polysaccharide solution was dialyzed in deionized water at 4 °C for 48 h. Four volumes of absolute ethanol were added and the mixture was allowed to stand at 4 °C for 12 h. The precipitate was collected by centrifugation and washed with 95% ethanol, acetone, and ether thrice by a vacuum suction filtration device. The obtained AAP was finally subjected to by vacuum freeze-drying (Scientz-10N, Scientz Biotechnology Co., Ltd., Ningbo, China).

Characterization of Auricularia auricular protein (AAPR) and Auricularia auricular polysaccharide (AAP)

The content of AAPR was measured by utilizing a Kjeldahl nitrogen analyzer (Model K-355, BUCHI, Swiss) in accordance with the People's Republic of China (PRC) standard GB5009.5—2016. The content of AAP was analyzed using the phenol–sulfuric acid method, which adhered to the PRC standard NY/T 1676-2008. The regression equation was obtained by generating the standard curve of glucose: $y=10.982x+0.0206,R^2=0.9987.$ The purified AAP solution was scanned by an ultra-micro ultraviolet/visible spectrophotometer (OD1000+, Wuyi Technology Co., Ltd, China) in the 220–500 nm range, and characteristic absorption peaks were observed near 260 nm and 280 nm. The molecular weights of AAPR and AAP were determined using high-performance gel permeation chromatography (HPGPC) (LC-20A, Shimadzu, Japan) with an evaporative light scattering detector (ELSD, Shimadzu, Japan) (Hu et al., 2017). A TSK GEL G4000 PWXL gel column (7.8 × 300 mm, 10 μm, Tosoh Corp, Tokyo, Japan) was used at 35 °C with an injection volume of 20 μL. The separation was operated with ultrapure water at a 0.8 mL/min flow rate. The parameters of the detector were set as follows: the drift tube temperatures were 40 °C for AAPR and 80 °C for AAP, and the nebulizer nitrogen gas pressure was set to 350 kPa.

Preparation of films/coatings

Pullulan-based composite films/coatings containing different ratios of AAP and AAPR were prepared using the casting method with glycerol as a plasticizer (Chu et al., 2019). Pullulan solution (2%, w/v) was obtained by dissolving pullulan powder in distilled water. Glycerol (20%, w/w) was added to the pullulan solution. Pullulan-based composite film solutions were prepared by adding different ratios of AAP and AAPR: 20%:0%, 15%:5%, 10%:10%, 5%:15%, and 0%:20% (based on pullulan content) (Hereafter referred to as P1, P2, P3, P4 and P5, respectively). All film-forming solutions were heated and stirred in a water bath at 40 °C until completely dissolved, and then stirred evenly with a magnetic agitator at 100 rpm for 15 min, followed by ultrasonic degassing and defoaming at 150 W for 10 min. Each film-forming solution (20 mL) was cast over an acrylic plate (5 × 10 × 1 cm) (polymethyl methacrylate, PMMA) tank for film formation. The composite films were dried at 45 °C for 8 h. After cooling, the film was carefully peeled off and placed in a box with constant temperature and humidity (25 °C, 60% RH) to equilibrate for 48 h until evaluation.

Physico-chemical properties of films/coatings

Thickness, moisture content (MC), water vapor permeability (WVP) and solubility

The thickness of the films was measured using a screw micrometer (accuracy 0.001 mm) (measuring and cutting tool factory, Shanghai, China). The average value thickness was calculated based on the measurements obtained. Ten points (one of which is the center point) were randomly selected and evenly dispersed on the specimens.

The MC of films was assessed following the method described by Moghadam et al. (2020). Films were weighed (m₁) and dried in an oven at 105 °C until reaching a constant weight (m₂). MC was determined with Eq. (1):

$$\mathrm{Moisture}\mathrm{content}\left(\%\right)=\frac{m_1-m_2}{m_1}\times 100.$$ 1

The WVP of the films was measured by the gravimetric method as outlined in ASTM (1995), with minor modifications. Films were sealed using a rubber band at the mouth of a 50 mL conical flask. The conical flask was filled with 10 g of allochroic silicagel. Then the conical flask was carefully placed inside a desiccator containing distilled water at 25 °C. The weight of the flask was then measured hourly until a consistent weight was obtained. WVP was determined according to Eq. (2):

$$\mathrm{WVP}\left(\mathrm{g}\mathrm{mm},/,{\mathrm{m}}^2,,\mathrm{h}\mathrm{kPa}\right)=\frac{\mathrm{\Delta }m\times D}{A\times t\times \mathrm{\Delta }P},$$ 2

where Δm is the weight change of the conical flask (g), D is the film thickness (mm), A is the area of permeation (m²), t is the time of permeation (h), and ΔP is the vapor pressure difference across the film (3.168 kPa).

The solubility of films was measured in accordance with the methods previously reported (Antoniou et al., 2015). The films (2 × 2 cm) were dried at 105 °C until they reached a constant weight, which was recorded as m₁. The films were submerged in 30 mL of deionized water and stored at 25 °C for 24 h. After this time, any water on the surface was removed using filter paper. Next, the films were dried at 105 °C until a constant weight was achieved, and then weighed (m₂). The solubility was then calculated using Eq. (3):

$$\text{Solubility (}\%)=\frac{m_1-m_2}{m_1}\times 100$$ 3

Mechanical attributes

The mechanical attributes of films were assessed using a texture analyzer (TA-Xtplus, Stable Micro Systems, UK) as previously documented (ASTM, 2001) with slight adjustments. To obtain the tensile strength (TS) and elongation at break (EAB) of the composite films, the strips (measuring 15 × 80 mm) were cut. The initial grip separation and detector speed were set at 30 mm and 5 mm/s. Equations (4) and (5) were then used to calculate the required values.

$$\mathrm{Tensile}\mathrm{strength}(\mathrm{MPa})=\mathrm{F}/\mathrm{S}$$ 4

$$\mathrm{Elongation}\mathrm{at}\mathrm{break}(\%)=\frac{L-L_1}{L_1}\times 100,$$ 5

where F is the maximum force (N), S is the cross-sectional area of the film (mm²), which is the width (mm) multiplied by the thickness (mm), L₁ is the initial length of the film (mm), and L is the length of the film at the breaking point (mm).

Color and opacity

Color parameters of the film (L*, a*, b*) were evaluated with a colorimeter (Hunterlab, Color Quest XE, USA) (Riaz et al., 2018) on a white background (L = 99.9, a = 0, b = 0.02). The total color difference (ΔE) was obtained according to Eq. (6):

$$\mathrm{\Delta }\text{E}={[{(L^{*}-L)}^2+{(a^{*}-a)}^2+{(b^{*}-b)}^2]}^{0.5}$$ 6

The transmittance of the film was determined using a UV-5200PC spectrophotometer (Yuanxi Instrument Co., Ltd, Shanghai, China). The strips (measuring 40 × 10 mm) were cut. Film thickness (D) was measured and then closely attached to the inner wall of the colorimetric dish. A UV-5200PC spectrophotometer measured the absorbance of composite films at a wavelength of 600 nm (Abs₆₀₀) (Riaz et al., 2018). Opacity (O) was calculated according to Eq. (7):

$$\text{O}={\mathit{ABS}}_{600}/D$$ 7

Structural characterization of films/coatings

Scanning electron microscopy (SEM)

SEM (EVO 18, Carl Zeiss, Germany) was employed to observe the surface and cross-sectional microstructures of the pullulan-based films (Chu et al., 2019). Films were frozen in liquid nitrogen and subsequently fractured to observe the cross-sectional structure. Then the films were affixed to the stage using conductive tape and sprayed with a 10 nm layer of gold. The accelerating voltage was 5 kV. The observation was conducted at 1000× and 800× magnifications.

Fourier transform infrared (FT-IR) spectroscopy

The chemical structure of the pullulan-based composite films was tested using FT-IR spectroscopy (Nicolet-iS5, Thermo Fisher, USA) (Chang et al., 2019). The films and potassium bromide were dried in an oven until the water completely evaporated, mixed (at a mass ratio of 1:100), ground evenly, and tableted. The scanning wavelength range, resolution, and scanning times were 400–5000 cm⁻¹, 4 cm⁻¹, and 32, respectively.

Differential scanning calorimetry (DSC)

DSC (214Polyma, Netzsch, Germany) was conducted to assess the thermal stability of the pullulan-based composite films. The films were weighed (3–5 mg) and carefully positioned in an aluminum crucible. Using an empty crucible was utilized as the reference point. The heating rate was controlled at 10 °C min⁻¹, and the temperature was gradually increased from 25 to 170 °C for DSC analysis.

X-ray diffraction (XRD)

The crystal structure and compatibility of the films were obtained by X-ray diffraction (XRD-7000, Shimadzu, Japan) in the 2θ range of 10°–60° at a 5° min⁻¹ scan rate with a Cu Ka radiation source, 40 kV voltage, and 40 mA current.

Biological activities of films/coatings

Antioxidant activity

DPPH radical scavenging activity was performed as previously described by Moghadam et al. (2020) with slight modifications. 50 mg of the films were immersed in a tube filled with ethanol (5 mL) for 24 h to obtain the film extract solution. Then, the film extract solution (0.1 mL) or absolute ethanol (0.1 mL) (as the control) was added into a 3.9 mL ethanol solution of DPPH (0.2 mM) and shaken evenly. The absorbance was measured at 517 nm after incubation in a dark room at 25 °C for 30 min. The results were obtained according to Eq. (8):

$$\mathrm{DPPH}\mathrm{scavenging}\mathrm{activity}(\%)=\left(\frac{A_0-A_1}{A_0},\times ,1000\right)$$ 8

where A₀ is the absorbance of the control, and A₁ is the absorbance of the film extract solution with DPPH solution.

ABTS radical scavenging activity was tested using the method of Moghadam et al. (2020) with minor modifications. ABTS solution (7.4 mM) was mixed with potassium persulfate (2.6 mM) in a 1:1 ratio and kept away from light for 16 h. The absorbance of the mixture diluted with ethanol at 734 nm was 0.70 ± 0.001. The film extract solution (0.1 mL) or absolute ethanol (0.1 mL) (as the control) was mixed with the ABTS solution (1.0 mL) and incubated in the dark for 10 min. The absorbance of the mixture was measured at 734 nm, and the results were obtained according to Eq. (9):

$$\mathrm{ABTS}\mathrm{scavenging}\mathrm{activity}\left(\%\right)=\left(\frac{A_0-A_1}{A_0},\times ,100\right)$$ 9

where A₀ is the absorbance of the control, and A₁ is the film extract solution with ABTS solution.

Antibacterial activity

Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) are pathogenic microorganisms that can easily cause food-borne diseases. Therefore, it is crucial to prevent the spread of these bacteria in the food production chain. The antibacterial properties of the films were evaluated by the agar disk diffusion method (LB Nutrient Agar), with the diameter of the inhibition zone measured (Zhang et al., 2019a; 2019b). Stock solid cultures of the bacterial species were maintained in a refrigerator at 4 °C. The tested organisms E. coli (G−) and S. aureus (G+) were inoculated into LB broth liquid medium, cultivated and activated at 37 °C for 24 h. The number of microbial cells in bacterial suspensions was obtained by the direct microscopic count method using a continuous zoom stereomicroscope (BIO-600, Aopu Optoelectronic Technology Co., Ltd, Chongqing, China). Bacterial suspensions (0.1 mL) of E. coli (G−) (5 × 10⁶ CFU/mL) and S. aureus (G+) (2 × 10⁶ CFU/mL) were inoculated onto the surface of the agar plates of culture medium and coated evenly. The film samples were cut into discs with a diameter of 10 mm and sterilized under UV light. The discs were laid on the culture medium and incubated at 37 °C for 24 h. The diameter of the inhibition zone was obtained with a Vernier caliper.

Application for preserving the fresh-cut potatoes

Preparation of the fresh-cut potatoes samples

Before use, the slicing knives, trays and other utensils were soaked in a 1% sodium hypochlorite solution for 10 min. Afterward, they were removed from the solution, rinsed multiple times with distilled water, and drained. To ensure quality, we carefully chose fresh potatoes with uniform size and color that were free from any signs of disease, insect damage, or mechanical harm. These potatoes were then washed with distilled water before being soaked in a 1% sodium hypochlorite solution for a period of 10 min. The potatoes were peeled and sliced to a thickness of 4 mm. These slices were then divided into two groups, each weighing 200 g. The division was done randomly. The composite films with AAP/AAPR at a ratio of 10%:10% [based on pullulan content (2%, w/v)] were applied to fresh-cut potatoes according to the physico-chemical properties and structural characteristics. The control group was immediately soaked in distilled water for 2 min. The experimental group was immersed in the AAP/AAPR/PUL-based composite coating solution for 2 min. Next, the fresh-cut potato pieces were withdrawn from the solution and excess water on the surface was drained off with gauze in an ultra-clean workbench at 25 °C. The samples were placed in a fresh-keeping box (PE) (10 cm × 5 cm), covered with plastic wrap (PE, thickness 0.01 mm). The storage conditions involved placing the box in a constant temperature/humidity incubator, where the temperature was set at 4 °C and the relative humidity (RH) was maintained at 90%. The experiment was performed in triplicates.

Browning index (BI)

To determine the browning index, we followed the method outlined by Galus et al. (2021). A colorimeter (Hunterlab, Color Quest XE, USA) was used to evaluate the L*, a* and b* values of fresh-cut potato chips during storage. Ten samples were repeatedly measured in each treatment, one value was measured on the front and back of each sample, and then calculated the average.

$$BI=\frac{100(\mathrm{x}-0.31)}{0.172}$$ 10

where

$$\mathrm{x}=\frac{a^{*}+1.75L^{*}}{5.645L^{*}+a^{*}-3.012b^{*}}$$ 11

Microbial analysis

The conventional plate counting method was used to measure the total bacteria count (TBC), as described in Wu et al. (2021) documentation.

Weight loss

The samples were weighed every 24 h. For each treatment group, 3 random pieces were weighted. To calculate the weight loss rate, we measured the quality of samples before storage (m₀) and after storage (m₁) and took the average value as the experimental result. The formula for weight loss is as follows (12).

$$\mathrm{Weight}\mathrm{loss}(\%)=\frac{m_0-m_1}{m_0}\times 100$$ 12

Total soluble solids

The samples were chopped, ground and mixed evenly, filtered with 4 layers of gauze to obtain filtrate, and the total soluble solids of the samples were evaluated by a refractometer (PAL-1, ATAGO, Japan). Three values were obtained, and the average value was taken.

Statistical analysis

Duncan's new multiple range test was utilized to evaluate the significance of differences (P < 0.05) using SPSS 26.0 software. All experiments were performed in triplicates, and Origin 2018.0 software was employed to visually represent the data.

Results and discussion

Preparation and characterization of AAPR and AAP

The study found that the extraction yield and AAPR content were 4.44 ± 0.02% and 80.13 ± 0.56%, respectively. Additionally, the extraction yield and AAP content were 7.62 ± 0.04% and 90.03 ± 0.61%, respectively. These findings align with the results reported by Xiong et al. (2016) regarding the content of AAP. The UV–vis spectrum did not exhibit any noticeable absorption peaks at 260 nm and 280 nm, indicating that AAP was free of nucleic acids and protein (Xiong et al., 2016). MW determination showed that the MWs of AAPR and AAP ranged from 18 kDa to 41.54 kDa and 153 kDa to 537.14 kDa, respectively.

Physico-chemical properties of films/coatings

Thickness, MC, WVP and water solubility

Thickness, MC, WVP and water solubility of the pullulan-based composite films containing different ratios of AAP and AAPR are depicted in Table 1. It has been established that the thickness of films is a vital indicator reflecting the structural compactness of the films (Li et al., 2020b). The inclusion of AAP/AAPR resulted in a notable increase in the thickness of pullulan-based composite films (P < 0.05). This can be explained by AAP/AAPR filling the gaps between pullulan, resulting in a more compact structure. As the AAP/AAPR ratio decreased, the MC of the films showed an initial increase followed by a decrease, in comparison to the control film. This is because AAP contains many hydroxyl groups and has strong hydrophilicity, which enhances the binding ability of composite films with water. However, since the side chain residues of the compounded AAPR have a certain degree of hydrophobicity, their water-binding ability is weak. The film's water absorption and retention ability were limited, leading to a decrease in MC.

Table 1.

Thickness, MC, water vapor barrier, solubility and mechanical properties of the PUL-based composite films containing different ratios of AAP and AAPR

Sample code	Thickness (mm)	Moisture content (%)	Water vapor permeability (g mm/m2 h kPa)	Solubility (%)	Tensile strength (MPa)	Elongation at break (%)
P0	0.072 ± 0.002b	15.707 ± 0.05b	0.374 ± 0.002d	11.745 ± 0.712d	6.002 ± 0.008b	5.626 ± 0.014f
P1	0.077 ± 0.004a	15.919 ± 0.053a	0.451 ± 0.003a	13.943 ± 0.891d	2.497 ± 0.03e	10.886 ± 0.014d
P2	0.078 ± 0.003a	15.763 ± 0.062b	0.429 ± 0.002b	16.330 ± 1.024 cd	4.077 ± 0.011d	12.461 ± 0.034a
P3	0.077 ± 0.002a	15.702 ± 0.042b	0.396 ± 0.002c	22.472 ± 1.896bc	6.175 ± 0.013a	11.950 ± 0.02b
P4	0.079 ± 0.007a	15.431 ± 0.049c	0.362 ± 0.005e	25.454 ± 1.253b	5.546 ± 0.031c	11.186 ± 0.014c
P5	0.076 ± 0.003a	15.430 ± 0.065c	0.374 ± 0.003d	33.649 ± 1.562a	6.024 ± 0.017b	10.488 ± 0.014e

P0: PUL without active compound, P1: PUL with 20% AAP, P2: PUL with 15% AAP and 5% AAPR, P3: PUL with 10% AAP and 10% AAPR, P4: PUL with 5% AAP and 15% AAPR, P5: PUL with 20% AAPR. Data are means ± SD (n = 3). Different lowercase letters in each column are significantly different (P < 0.05)

Current evidence suggests that the WVP of films is a vital indicator for evaluating the water barrier properties (Chu et al., 2019). A lower WVP is particularly advantageous for preserving the quality of food products over a longer period of time (Riaz et al., 2018). In this study, we found that adding AAP significantly increased the WVP of pullulan-based films (P < 0.05) from P1 (20%:0%) to P3 (10%:10%). This increase can be explained by the high number of hydrogen bonds present in AAP, which increased the hydrophilicity of the films and weakened their water resistance. Pullulan-based composite films experienced a noticeable decrease in WVP (P < 0.05) with a decrease in AAP content and an increase in AAPR content. However, the WVP of P5 films (0%:20%) did not show a significant difference compared to the pullulan control film (P > 0.05) because the intermolecular interaction forces cross-linked AAPR and polysaccharide molecules to form a more compact polymer network structure. The pore size between the film-forming molecules was reduced, which led to a gradual decline in the WVP of the pullulan-based films. The incomplete dissolution of the AAP/AAPR in the pullulan-based films had an impact on the passage of water vapor through the film, resulting in higher WVP values. The microstructures of the films were consistently observed to be the same under SEM analysis. As a result, it was found that most films containing AAP/AAPR were more permeable to water vapor than the corresponding control film consistent with the literature (Hanani et al., 2019).

Water solubility represents an important factor for evaluating the water resistance of films (Riaz et al., 2018). The addition of AAP/AAPR to the pullulan-based composite films resulted in a gradual increase in solubility, which can be explained by the abundance of hydroxide groups and the strong hydrophilicity of AAP. Covalent bonding between hydrophobic groups on the side chains of polysaccharides and protein molecules improved the hydrophilicity of films, thus increasing the solubility of films. Additionally, AAPR is a macromolecule that exhibits hydrophilic properties due to the presence of hydroxyl groups and other similar groups.

Mechanical characteristics

TS and EAB are important mechanical properties of packaging materials, which are critical in ensuring that food is protected from external damage during transportation and storage (Li et al., 2020a). The inclusion of AAP led to a notable reduction in the TS of the pullulan-based films (P < 0.05), as indicated in Table 1. The TS of the pullulan-based films gradually increased as the AAP decreased and AAPR increased from P1 (20%:0%) to P3 (10%:10%). This is because the interaction between polysaccharides reduced the rigidity of the film, making it more malleable and flexible. The EAB of the pullulan-based composite films was significantly higher than that of the pullulan control film (P < 0.05), with the EAB increasing from 86 to 121%. The addition of AAP/AAPR and glycerol improved the flexibility and fluidity of the pullulan-based composite film structure. These results may be attributed to the electrostatic attraction between the polymers, strong hydrogen bond cross-linking, and better blending at the molecular level (Wu et al., 2013). According to Han et al. (2020), the composite films displayed the best mechanical properties when the ratio of egg white to pullulan was 1:1. This was attributed to an increase in the number of amino groups and a strengthening of intermolecular hydrogen bonds. To improve the mechanical properties of pullulan-based films, alternative plasticizers such as sorbitol, glycols, and sugars could be utilized, or multilayer films could be produced (Tyagi et al., 2021).

Color and opacity

Film color is crucial to measure the production of food packaging and consumer acceptance (Chang et al., 2019). Table 2 displays the color and opacity of pullulan-based composite films with varying ratios of AAP and AAPR. As the AAP/AAPR ratio decreased, the L* and a* values of the films significantly decreased from P1 (20%:0%) to P2 (15%:5%) (P < 0.05), while the b* values significantly increased from P1 (20%:0%) to P5 (0%:20%) (P < 0.05). The addition of AAP/AAPR exacerbated the darkness and the green and yellow color of the pullulan-based composite films. This could be due to the fact that decolorized AAP had a slightly yellowish-brown, and AAPR was pale white, resulting in a darker, greener, and yellowish color. The study found that there was no statistically significant variation in the total color difference (ΔE) for P5 (0%:20%) from the control film. However, the other films showed a noticeable increase (P < 0.05). Transparency of films is evaluated based on opacity, with higher opacity indicating lower transparency (Peng and Li, 2014). In comparison with the control film, the opacities of P1 (20%:0%), P2 (15%:5%), and P3 (10%:10%) were significantly increased. However, there was no significant difference observed in the opacities of films P4 (5%:15%) and P5 (0%:20%) (P > 0.05). The addition of AAP/AAPR to composite films resulted in decreased the transparency. However, studies have shown that this alteration in transparency and the altered composition of the composite films do not affect the appearance of fresh-cut fruits and vegetables during use (Zhu et al., 2014). Ju and Song (2020) also indicated that the color properties of Tremella fuciformis polysaccharides films were significantly improved by adding peanut skin extract.

Table 2.

Color parameters and opacity of the PUL-based composite films containing different ratios of AAP and AAPR

Sample code	L*	a*	b*	ΔE	Opacity (A600 mm−1)
P0	97.63 ± 0.03a	− 0.09 ± 0.02a	1.74 ± 0.36a	2.93 ± 0.24c	0.542 ± 0.014c
P1	95.42 ± 0.45c	− 0.30 ± 0.06b	8.02 ± 1.85b	9.23 ± 1.82a	0.904 ± 0.02a
P2	96.50 ± 0.60b	− 0.22 ± 0.07ab	4.70 ± 1.33bc	5.86 ± 1.37b	0.649 ± 0.032b
P3	97.09 ± 0.41ab	− 0.21 ± 0.18ab	3.55 ± 1.61bc	4.61 ± 1.53bc	0.645 ± 0.027b
P4	97.18 ± 0.12a	− 0.19 ± 0.11ab	2.81 ± 0.58c	3.97 ± 0.48bc	0.560 ± 0.013c
P5	97.39 ± 0.08a	− 0.12 ± 0.02ab	2.39 ± 0.07c	3.53 ± 0.01c	0.559 ± 0.008c

P0: PUL without active compound, P1: PUL with 20% AAP, P2: PUL with 15% AAP and 5% AAPR, P3: PUL with 10% AAP and 10% AAPR, P4: PUL with 5% AAP and 15% AAPR, P5: PUL with 20% AAPR. Data are means ± SD (n = 3). Different lowercase letters in each column are significantly different (P < 0.05)

Structural characterization of films/coatings

SEM analysis

SEM is a crucial tool for assessing the compatibility of blends since it can directly monitor the surface morphology of composite films and the compatibility between components (Zhang et al., 2019a; 2019b). Because of the covalent crosslinking and tightly packed molecular arrangement, we discovered that the surface of the films was compact, smooth, and uniform (Fig. 1). The control film's surface was not sufficiently flat with a few folds observed, which may be caused by the uneven spreading of pullulan and the movement of fluid through the membrane during the drying process. These folds were consistently found during cross-sectional observation suggesting that pullulan was brittle. Compared to the control film, the surface of P1 (20%:0%) was improved without folds. Small insoluble particles were observed in the cross-section, which may be caused by insufficient dissolution of AAP. Supplementation of AAPR made the surface of P2 (15%:5%) relatively flat, uniform, and smooth. However, with an increase in the proportion of AAPR, the composite films formed a more compact structure and enhanced the TS of the films. The EAB of the pullulan-based films likewise reduced when the molecular mobility of the film components decreased (Yu et al., 2018). The smooth and uniform surface and cross-section of P3 (10%:10%) suggest that AAP/AAPR/PUL has good mechanical properties and compatibility at this ratio, as there are no visible pores or cracks present. A cross-sectional examination of P4 (5%:15%) showed a smooth and uniform surface structure with small insoluble particles. The surface of P5 (0%:20%) was continuous but not smooth, with various crisscross patterns and elevated areas. The variation observed in microstructure results can be attributed to the degree of glycosylation and the formation of intermolecular hydrogen bonds (Han et al., 2020).

Fig. 1.

Scanning electron microscopy micrographs of the surface (a, 1000×) and cross-section (b, ×800) of the pullulan-based composite films containing different ratios of AAP and AAPR. P0: PUL without active compound, P1: PUL with 20% AAP, P2: PUL with 15% AAP and 5% AAPR, P3: PUL with 10% AAP and 10% AAPR, P4: PUL with 5% AAP and 15% AAPR, P5: PUL with 20% AAPR

FT-IR spectra analysis

FT-IR spectra were used to analyze the functional groups and structural alterations of pullulan-based composite films that contained various ratios of AAP and AAPR (Chentir et al., 2019) (Fig. 2A). The distinctive absorption peak of the control film was 3420 cm⁻¹ (the stretching vibration peak of free O–H stretching overlapped with N–H stretching in amino groups), 2927 cm⁻¹ (CH and CH₂ stretching), 1154 cm⁻¹ (polysaccharide α-(1 → 4) glycosidic bond), 1078 cm⁻¹ (C–OH stretching), and 1023 cm⁻¹ (–C–O–C– in glycosidic linkage). Except for a few shifts, the absorption bands observed in the FT-IR spectra of the control film were generally consistent with those reported in the literature by Moghadam et al. (2020) and Zhu et al. (2014). The slight heterogeneity in the shifts observed in the absorption bands can be explained by different sources and/or treatments of materials. The broadening of the absorption band at 3422 cm⁻¹ and the shift of the absorption peak at 1620 cm⁻¹ observed after adding AAP may be related to the formation of intermolecular hydrogen bonds of pullulan (P1 (20%:0%)) (Wu et al., 2013, 2020). After adding AAPR, the absorption peaks at 3420 cm⁻¹ and 1618 cm⁻¹ also shifted and exhibited different degrees of changes in width. These changes in width could be attributed to the formation of new hydrogen bonds between and within molecules (Wu et al., 2020). The characteristic absorption peaks of AAPR (P2 (15%:5%)) were observed at 1621 cm⁻¹, 1548 cm⁻¹, and 1245 cm⁻¹, corresponding to the amide-I band (C=O stretching coupled with a C–N stretch in plane NH bending and CCN deformation mode), amide–II band (coupling of N–H bending and C–N stretching vibration), and amide-III band (combination peaks between C–N stretching vibrations and N–H deformation from amide linkages), respectively (Chentir et al., 2019; Han et al., 2020; Moghadam et al., 2020). In the composite films of P3 (10%:10%), P4 (5%:15%), and P5 (0%:20%), the maximum absorption peaks of amide-I bands shifted to 1640 cm⁻¹, 1647 cm⁻¹, and 1655 cm⁻¹, respectively. The absorption band was enhanced with increased AAPR, exhibiting a sharper peak shape was sharper, possibly due to hydrogen bonding between AAPR and pullulan (Chentir et al., 2019).

Fig. 2.

Fourier-transform infrared spectroscopy spectra (A), Differential scanning calorimetry thermograms (B), and X-ray diffraction patterns (C) of the pullulan-based composite films containing different ratios of AAP and AAPR. P0: PUL without active compound, P1: PUL with 20% AAP, P2: PUL with 15% AAP and 5% AAPR, P3: PUL with 10% AAP and 10% AAPR, P4: PUL with 5% AAP and 15% AAPR, P5: PUL with 20% AAPR

DSC analysis

DSC was employed to investigate the thermal stability of the pullulan-based composite films containing different ratios of AAP and AAPR (Chentir et al., 2019). The melting peak of the control film was 122.8 °C, and the addition of AAP and AAPR led to a significant improvement in the melting peak of pullulan-based composite films (Fig. 2B). It has been reported that AAP exhibited excellent thermal stability during rheology and DSC analysis (Bao et al., 2016). The increase in peak temperature indicated that supplementing AAP and AAPR changed the structure of the composite films, and hydrogen bonds formed between molecules, thus changing the mechanical properties of pullulan-based films (Luís et al., 2021). SEM revealed that P3 (10%:10%) exhibited the best compatibility and thermal stability with the highest melting peak temperature (146.6 °C). Similar to this study, Ebadi Ghareh Koureh et al. (2023) found that the adding the CEO to the salep-PVA improved its thermal stability.

XRD analysis

XRD can detect the crystal structure and compatibility of biomacromolecules including polysaccharides and proteins (Chang et al., 2019). In our study, a wide diffuse diffraction peak between 2θ = 19.7°–21.1° was observed for all films (Fig. 2C). The control film indicated a characteristic diffraction peak at 2θ = 19.7°, attributed to the amorphous structure of pullulan (Han et al., 2020). A decrease in the AAP/AAPR ratio resulted in varying degrees of right shifts for the diffraction peak of the composite films. The right shift of P3 (10%:10%) was the most obvious (2θ = 21.1°), proving the presence of intramolecular hydrogen bonds between the protein and polysaccharide, and the amorphous structure of the pullulan-based composite films was changed to some extent, accounting for the good dispersion and compatibility. Results from this study are consistent with earlier reports for egg white/pullulan blend film (Han et al., 2020). Similar findings have been documented by Ebadi Ghareh Koureh et al. (2023).

Biological activities of films/coatings

Antioxidant activity

The DPPH method is a reliable and efficient technique for analyzing the free radical scavenging ability of natural compounds, which sets it apart from other methods. The ABTS assay is a valuable tool used to evaluate the antioxidant activity of hydrogen-donating and chain-breaking antioxidants. It is commonly employed to assess the effectiveness of these antioxidants in neutralizing free radicals and preventing oxidative damage (Khaskheli et al., 2015). Natural antioxidants are well-established for their ability to reduce or avoid food spoilage (Silva et al., 2018). The antioxidant activity of pullulan-based films was assessed by measuring DPPH and ABTS free radical-scavenging tests (Fig. 3A). The findings revealed that the inclusion of AAP/AAPR considerably enhanced antioxidant activity of the pullulan-based films (P < 0.05) in contrast to the control film, which exhibited no antioxidant activity, consistent with previous research (Chu et al., 2019; Luís et al., 2021). P2 (15%:5%) yielded the highest antioxidant activity with DPPH and ABTS radical scavenging activities of 61.27% and 72.52%, respectively. Meanwhile, the results of P5 (0%:20%) indicated that AAPR had free radical scavenging activity. The study conducted by Ebadi Ghareh Koureh et al. (2023) demonstrated that the antioxidant activity of salep-PVA nanofibers from 7.2% to 26.88% is probably due to the increased of CEO. Adding bioactive extracts can significantly affect the antioxidant capacity of films (Ansarian et al., 2022; Ju and Song, 2020; Moghadam et al., 2020; Roshani Neshat et al., 2022; Synowiec et al., 2014). Recent research has shown that the antioxidant capacity of AAP is influenced by several factors, including its variety, types of glycosidic bond conformation structure, monosaccharide composition, and MW (Miao et al., 2020; Su and Li, 2020; Zeng et al., 2012).

Fig. 3.

DPPH and ABTS radical scavenging activity (A) and antibacterial activities (B) of the pullulan-based composite films containing different ratios of AAP and AAPR. P0: PUL without active compound, P1: PUL with 20% AAP, P2: PUL with 15% AAP and 5% AAPR, P3: PUL with 10% AAP and 10% AAPR, P4: PUL with 5% AAP and 15% AAPR, P5: PUL with 20% AAPR. Data are means ± SD (n = 3). Different lowercase letters above the same (white or grey) bar are significantly different (P < 0.05). a, b Agar Well Diffusion Assay of against E. coli and S. aureus, c diameter of the inhibition zone (mm) against E. coli and S. aureus

Antibacterial activity

Figure 3B shows the antibacterial activities of films against E. coli (G−) and S. aureus (G+). The absence of an inhibition zone around the control film in Fig. 3Ba and b confirms that the pullulan film does not possess any antibacterial activity (Synowiec et al., 2014). The study findings suggest that all composite films, except for the control film, exhibited antibacterial activity. As illustrated in Fig. 3Bc, the P3 (10%:10%) solution exhibited the most potent antibacterial activity against E. coli (G−) with a bacteriostatic zone diameter of 16.11 mm. P5 (0%:20%) demonstrated the strongest antibacterial activity against S. aureus (G+) with a bacteriostatic zone diameter of 15.89 mm. The results of both P1 (20%:0%) and P5 (0%:20%) indicated that AAPR had higher antibacterial activity compared to AAP. There is an increasing consensus that AAP and AAPR have antibacterial properties against E. coli and S. aureus (Cai et al., 2015; Gebreyohannes et al., 2019; Oli et al., 2020). Further exploration is warranted to determine the antibacterial mechanism of AAP and AAPR. Our results substantiated that the antimicrobial activity of films could be significantly enhanced by adding AAP/AAPR.

Determination of the optimal ratio of films/coatings

The results of physico-chemical properties and structural characterization showed that compared with other AAP/AAPR/PUL formulations, after adding AAP/AAPR with a ratio of 10%:10%, the pullulan-based composite films/coatings showed improved stretchability and superior tensile strength. The antioxidant activity of the pullulan-based films was significantly improved by the addition of AAP/AAPR (10%:10%). Moreover, P3 (10%:10%) demonstrated the strongest antibacterial activity against E. coli (G−) (Fig. 3B). Meanwhile, the thermal stability of the pullulan-based films/coatings was significantly improved, and the intermolecular cross-linking formed a dense network structure. Antioxidant and antimicrobial activities of pullulan-based films/coatings play a crucial role in preventing browning, inhibiting tissue softening and ensuring microbial safety to prolonging the shelf life of products and maintain their freshness (Manzoor et al., 2021). Therefore, the AAP/AAPR/PUL formulations with AAP/AAPR at a ratio of 10%:10% [based on pullulan content (2%, w/v)] were applied as the coating of fresh-cut potatoes.

Application for preserving fresh-cut potatoes

The development of brown color in fresh-cut vegetables is a significant factor that affects their shelf life and marketability. To characterize the extent of browning in minimally processed vegetables, the browning index is commonly used (Teoh et al., 2016). During the whole storage process, compared with the control, the fresh-cut potatoes treated with AAP/AAPR/PUL-based composite coating exhibited a significant anti-browning effect (Fig. 4A). The BI value exhibited an upward trend, and the AAP/AAPR/PUL-based composite coating treatment was significantly lower than the control from days 6–8 (P < 0.05) (Fig. 4B). The study demonstrated that the AAP/AAPR/PUL-based composite coating effectively delayed the browning in fresh-cut potatoes. Similar to this study, Wu (2019) reported that 1%ODP-based edible coatings effectively inhibited the browning process in fresh-cut potatoes.

Fig. 4.

Browning index (A, B), total bacteria count (C), weight loss (D) and total soluble solids (E) of composite coated and uncoated fresh-cut potatoes. Uncoated fresh-cut potatoes (control), coated fresh-cut potatoes (AAP/AAPR/PUL). Data are means ± SD (n = 3). Different lowercase letters are significantly different (P < 0.05)

It has been established that the quality of fresh-cut product is degraded due to contamination and proliferation of microorganisms following tissue damage and loss of the protection provided by the epidermis due to slicing (Ali et al., 2021). According to Fig. 4C, the total number of colonies of fresh-cut potatoes exhibited a gradual increase over the course of storage. During the 8 days of storage, the composite coating treatment using AAP/AAPR/PUL resulted in significantly lower colony numbers compared to the control group (P < 0.05). Specifically, on day 8, the composite coating treatment group had a significantly lower total number of colonies in comparison with the control group (5.20 vs. 5.62 Log CFU/g, P < 0.05). The strong inhibitory effect on the growth of microorganisms observed in the composite coating can be attributed to the presence of AAP and AAPR (Cai et al., 2015; Oli et al., 2020). The results of the antibacterial experiments support this claim. As a result, the spoilage of fresh-cut potatoes was delayed to some extent. Similar findings have been documented by Treviño-Garza, et al. (2017).

The decline of the physiological quality of fresh-cut product during storage can be explained to water transpiration (Kumar et al., 2021b). As shown in Fig. 4D, a gradual increase in the weight loss rate of fresh-cut potatoes was observed. In the initial storage phase, there was no noticeable distinction between the coating treatment and the control (P > 0.05). On day 8 of storage, the application of AAP/AAPR/PUL-based composite coating treatment resulted in significantly lower water loss and quality deterioration compared to controls (P < 0.05), substantiating the efficacy of the composite coating treatment in preserving the quality of fresh-cut potatoes (Ali et al., 2021). Zhang et al. (2019a; 2019b) observed that the use of pullulan/chitosan coatings resulted in a reduction in papaya weight loss and effectively maintained its post-harvest quality. Kumar et al. (2021a) reported a similar effect in their study on green bell pepper coated with a chitosan-pullulan coating that contained pomegranate peel extract.

Total soluble solids is an important biological parameter of fruit and vegetable products (Ali et al., 2021). During storage, the total soluble solids of fresh-cut potatoes exhibited a pattern of initial decrease, followed by an increase, and then a final decrease (Fig. 4E). The AAP/AAPR/PUL-based composite coating treatment group was no statistically significant variation of the control group, except on day 4 (P > 0.05). At other times, however, there were noticeable variations (P < 0.05). The decrease could be due to the damage of potato tissue after cutting, the destruction of the epidermis and the acceleration of tissue aging. The respiration rate of potatoes steadily increased, and the decomposition of many organic substances into sugar, acid and minerals resulted in a rise in soluble solids (Yin et al., 2019). After cutting, the enzymatic activity and metabolic rate of potatoes increased, leading to the decomposition of numerous nutrients. This phenomenon resulted in a decline in the total soluble solids. The group treated with composite coating using AAP/AAPR/PUL showed significantly higher levels compared to controls (P < 0.05). Compound coatings treatment is beneficial to slow down the loss of soluble solids in fresh-cut potatoes, delay tissue senescence and prolong the storage period attributed to the coating's low oxygen and high carbon dioxide environment reducing fresh-cut potatoes' metabolic activity, resulting in slower degradation of polysaccharides and thus lowering the total soluble solids (Yin et al., 2019). According to a study by Kumar et al. (2021a; 2021b), a chitosan-pullulan coating, infused with pomegranate peel extract, effectively preserved the total soluble solids of coated mango fruits.

Declarations

Competing interests

The authors have declared no conflict of interest.