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Article

A Film of Chitosan Blended with Ginseng Residue Polysaccharides as an Antioxidant Packaging for Prolonging the Shelf Life of Fresh-Cut Melon

National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing 100083, China
*
Author to whom correspondence should be addressed.
Coatings 2022, 12(4), 468; https://doi.org/10.3390/coatings12040468
Submission received: 7 March 2022 / Revised: 24 March 2022 / Accepted: 28 March 2022 / Published: 30 March 2022

Abstract

:
Ginseng residue polysaccharides (GRP) at three levels were excellently blended into chitosan to form antioxidant composite films, which exhibited higher density, opacity and moisture, as well as lower water vapor permeability, tensile strength and elongation ratio than those of neat chitosan film. Thermogravimetry evidenced no difference in stability, and SEM and AFM revealed smooth and dense surfaces with no cracks and micropores, whereas structural analyses disclosed slight changes in films’ structures after adding GRP. A chitosan film containing 0.5% GRP (Chitosan + GRP) was then employed for a fruit preservation study. Fresh-cut melon covered with Chitosan + GRP displayed delayed deteriorating compared with other groups. A possible antioxidant mechanism in fruit preservation was then suggested, and PCA and correlation analyses supported these findings. The results demonstrated that our antioxidant chitosan films incorporated with GRP are quite promising for enabling the food industry to produce eco-friendly and sustainable packaging.

1. Introduction

Melon (Cucumis melo L.) is widely consumed globally due to its richness in sweet flavors and nutrients such as vitamin C and beta-carotene [1,2]. Fresh-cut products such as fruit or vegetable offer convenience and add value for meeting the growing demand, and their convenience is a particularly desirable feature for fruit like melon that is too large to eat and requires a certain degree of preparation before eating, along with the associated disposal of residues such as its skin and seeds. However, firmness, sweetness, respiration rate, and the level of aroma volatiles of fresh-cut product, which are key indicators affecting quality, deteriorate easily, resulting in its limited shelf-life [2,3].
Conventional plastic used in food packaging, which is mainly synthesized from fossil fuels, protects food from external condition [4]. Owing to the non-biodegradable nature of most plastic, it is necessary to find other materials that may replace plastics to reduce environmental pollution and avoid making our planet a “plastic Earth”. Biopolymers, such as polysaccharides and proteins used in food packages, are considered as the ideal material for replacing the non-biodegradable polymers [5]. Chitosan, a high-molecular-weight polysaccharide, is produced commercially by partial deacetylation of chitin, which is the most abundant constituent of the exoskeleton of crustaceans, fungal cell walls, and other biological materials [6,7]. Among the natural and renewable resources, chitosan is one of the essential components of films because of its low cost, availability, and biodegradability, and developing non-toxic and biodegradable films is considered to be the most promising alternative to conventional plastics [8,9]. Chitosan films had been successfully examined experimentally on several fresh and dairy products and found to increase their shelf life and quality [10,11]. However, the film structure formed by chitosan alone is usually insufficient to meet the practical application, and the composite film formed by mixing different additives to chitosan can usually improve its application performance.
The concept of active antioxidant packaging is attracting interest. By adding antioxidants, active antioxidant packaging can remarkably improve the quality attributes of the packaged food [12]. Traditionally, synthetic antioxidants include organophosphate, thioester, and polyphenol compounds [13]. Nevertheless, the use of synthetic antioxidants in packaging is doubted due to the possible toxicity derived from their migration into food products. An alternative approach that is being studied widely is using natural antioxidants from herbs and fruits. Muley and Singhal [14] reported a composite film formed by combining chitosan with whey protein isolate and demonstrated that it had high antioxidant activity, prevented strawberries from spoilage and prolonged their shelf life compared with neat chitosan film. A composite film composed of pectin, tea polyphenols, and chitosan, as reported by Gao et al. [15], exhibited an excellent ability to prevent food deterioration and inhibit meat discoloration. Jancikova et al. [16] studied that the films based on κ-carrageenan, chitosan, and the natural extract from red cabbage had preservation ability for fresh-cut apple pieces as intelligent packaging and the active packaging. However, few studies have explored natural products recovered from waste as antioxidants mixed with chitosan to prolong the shelf-life of fruits and vegetables.
Ginseng residue polysaccharide (GRP), which was derived by means of hot water extraction and ethanol precipitation from the waste of extraction of ginsenosides from ginseng root, can be applied as a polysaccharide-based coating to prolong the shelf lives of strawberry and fresh-cut apple due to its various advantages, such as safety, edibility, and antioxidant activity (data unpublished). Since making full use of GRP can avoid resource waste and reduce environmental pollution, it is necessary to expand the scope and intensity of its use. However, to our knowledge, GRP has not been reported as a natural antioxidant mixed with chitosan to form the antioxidant composite film and improve fruit preservation performance. In order to test the hypothesis above and develop an environment-friendly functional polysaccharide film suitable for the food industry, we studied the effects of incorporating GRP as an active ingredient and glycerol as a plasticizer on the performance of chitosan-based films for food packaging. We also examined the physicochemical and functional properties of the composite films and compared with those of the neat chitosan film. Finally, by evaluating quality parameters and antioxidant indexes of fresh-cut melon, we determined the possibility of using film as a packaging material for fresh-cut fruit.

2. Materials and Methods

2.1. Materials and Reagents

Ginseng root was purchased from Dandong, Liaoning province, China, during the harvest season of field ginseng. Plastic film, which is mainly composed of polyethylene with hydrophobic properties and without any antioxidants and other additives, and melon (Cucumis melo L.) were purchased from a local branch of a popular supermarket (MerryMart, Beijing, China) on the same day as performing the experiments.
Glycerol, chitosan with a degree of deacetylation of 90%, and the diagnostic kits for assaying catalase, ascorbic acid, and malondialdehyde were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Furthermore, 2,2-Diphenyl-1-picrylhydrazyl (DPPH•) and Folin–Ciocalteu were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Other reagents of analytical grade were bought from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Ultrapure water was prepared using a Milli-Q50 SP Reagent Water System (Millipore Corporation, Billerica, MA, USA).

2.2. Preparations of Ginseng Residue Polysaccharides and Films

Ginseng residue after extraction of ginsenosides was further extracted to obtain the ginseng residue polysaccharide (GRP) using distilled water (1:15, g/mL) at 80 °C for 30 min with ultrasonic cleaner for three times. After centrifugation at 4000× g and 4 °C for 10 min, the three supernatants were combined and concentrated in a rotary evaporator under reduced pressure, and a four-fold volume of ethanol was added to the concentrate which was then kept at 4 °C overnight to precipitate the polysaccharides. The precipitate was collected and lyophilized to obtain GRP.
Figure 1 is a schematic diagram of preparing the antioxidant films. Briefly, chitosan was added into acetic acid solution (1%, v/v) to the ratio of 1:25 (w/v). Glycerol was then slowly added to the chitosan solution to the ratio of 1:10 (v/v). Finally, GRP was added to the mixture, after stirring at 60 °C for 30 min, to reach the final concentration of 0% (CF), 0.5% (CF + L-GRP), or 2.0% (CF + H-GRP; w/v based on the dry weight of GRP in mixture). The mixture was stirred at 60 °C for another 10 min and poured into the culture dishes with a radius of 4.5 cm (10 mL mixture per plate). Subsequently, all plates were incubated at 50 °C for 12 h in an air-dry oven (GXZ-9240 MBE, Shanghai Boxun Industrial Co., Ltd., Shanghai, China), and then all films were peeled off from the plates and preserved in an incubator (Life Technology Co., Ltd., Ningbo, China) set at 25 °C and 55% relative humidity for further analysis.

2.3. Determination of Physical Properties of Films

2.3.1. Thickness, Density, and Opacity

Ten different positions were randomly measured using a digital micrometer with an accuracy of 0.001 mm for thickness of an individual film. For its density, a film was roughly cut into six squares (1 cm × 1 cm), and their lengths, widths, and weights were measured accurately. The density of the film was calculated as follows: p = m/v, where m and v are the weight in gram and volume in cm3 (thickness × length × width).
Film opacity was measured as reported by Kalaycıoğlu et al. [17] with slight modifications. In brief, a film was cut into rectangular pieces and they were placed inside the test cell of the microplate reader (Bio-Rad xMarkTM Microplate Absorbance Spectrophotometer, Bio-Rad Technology Co., Ltd., Hercules, CA, USA). The absorbance of the film was recorded at 600 nm and the opacity was calculated as follows: O (A/mm) = A600/d, where A600 is the absorbance of the film at 600 nm and d is the film thickness in mm.

2.3.2. Moisture Content and Water Vapor Permeability

Moisture content was measured according to the method of Pérez Córdoba and Sobral [18] with slight modifications. Briefly, a film was cut into 2 cm × 2 cm square pieces and weighed (M1), and then kept in an oven set at 105 °C until reaching its constant weight (M2). The moisture was calculated as follows: Moisture (%) = (M1M2)/M1 × 100.
Film water vapor permeability was determined using the gravimetry method [19] with slight modifications. In brief, the mouth of a glass jar filled with completely dried silica gel was sealed with a film. Weight of the film sealing the jar was recorded as M1 and the sealed jar was placed in a constant incubator set at 25 °C and 75% relative humidity. To ensure a steady-state permeation, changes in the jar weight were monitored at an interval of 12 for 7 consecutive days, and the final weight was recorded as M2. Water vapor permeability was calculated as follows: Water vapor permeability (g·mm·kPa−1·day−1·m−2) = (w/t) × (d/A × ΔP), where w in gram equals M2M1, t in days is the time required to reach equilibrium, d in mm is the film thickness, A in m2 is the permeation area, and ΔP is saturation vapor pressure of water (3.1671 kPa at 25 °C).

2.3.3. Mechanical Properties

The mechanical properties of the films were determined using a universal mechanical testing machine (INSTRON 5982, INSTRON Co., Ltd., Shanghai, China) with software for data processing. A film was cut into squares of 100 mm in width and 100 mm in gauge length, and tests were carried out at room temperature and at a moving crosshead speed of 50 mm/min [20]. Three tests were performed for each film. Data of a single test are shown and those of the other two tests are presented as Supplementary Materials (Figure S1).

2.4. Assays of Thermal Properties of Films

Thermal stability of films was evaluated as reported by Muley et al. [14] with slight modifications. Briefly, CF, CF + L-GRP, and CF + H-GRP were cut into a square of a size of 1 cm × 1 cm. Then, each prepared film was weighed and heated in the air from 30 °C to 800 °C at a constant heating rate of 10 °C/min, in an inner atmosphere of N2 with STA 449F5 thermogravimetric analyzer, (STA 449F5, NETZSCH Group, Shanghai, China). The change of weight of each group with temperature was recorded. Three tests were performed for each film. Data of a single test are shown and those of the other two tests are presented as Supplementary Materials (Figure S2).

2.5. Assays of Structural Properties of Films

The variations in crystallinity patterns of CF, CF + L-GRP, and CF + H-GRP were studied on a continuous scan mode X-ray diffractometer with CuK α-radiation (Bruker D8 Advance, Bruker Science & Technology Co., Ltd., Billerica, MA, USA) and 2θ data collected between 5° and 75° at a speed of 1°/min with a current of 40 mA and a voltage of 40 kV. FT-IR was used to analyze surface functional groups of the CF, CF + L-GRP, and CF + H-GRP at a resolution ratio of 4 cm−1, the scan time of 32 s−1, and measurement range of 4000–600 cm−1 with a Fourier transformed infrared spectrophotometer (Perkin Elmer Frontier, PerkinElmer Management Co., Ltd., Waltham, MA, USA).

2.6. Assays of Microstructure of Films

The microstructure of the films was analyzed using scanning electron microscopy (SEM) and atomic force microscopy (AFM). Films were coated with a conductive layer of platinum to increase its conductivity before SEM observation. SEM images were recorded on a ZEISS Gemini 300 microscope (Zeiss Group, Heidenheim, Germany). The acceleration voltage was set at 0.02–30 kV and continuously adjustable 10 V stepping.
AFM analyses of CF, CF + L-GRP, and CF + H-GRP were performed using a cypher ES microscope (Oxford Instruments Group, Oxford, UK). Small pieces of each film were cut and glued to AFM pucks with double-sided tape. The AFM tip used for AFM topography imaging (tapping mode) has a curvature radius of typically less than 2 nm. The images were scanned for each sample and the signal corresponding to sample topography and phase shift were collected to obtain the two-dimensional and three-dimensional of the films. After the test was completed, the root mean square roughness (Rq) was obtained from the root mean square deviation of the average data plane height deviation.

2.7. Evaluations of Preservation Performance of Fresh-Cut Melon with Films

2.7.1. Fresh-Cut Melon Preparation

According to the results of the above film performance experiments and based on the concept of sustainable development, the chitosan film with a low concentration of GRP was selected in the following fresh-cut melon preservation experiment. Melons were cleaned by immersion in deionized water and dried with filter paper before cut into 3 cm × 3 cm × 3 cm. All the fresh-cut pieces were randomly divided into three groups: plastic film (PF), chitosan film (CF), chitosan film with GRP (CF + GRP). Four pieces of fresh-cut melons were placed in each sterile polypropylene tray (7.5 cm × 7.5 cm × 4 cm) and covered with the different films. The trays and their films were stuck with a double-sided adhesive so that the fruit was unable to have direct contact with the environment outside the film. Each group of samples was packaged at the same time under the same natural conditions and stored at the same temperature (25 ± 1 °C) and relative humidity (55 ± 1%) for 4 days in a controlled growth chamber (Life Technology Co., Ltd., Ningbo, China). The above process ensured the concentrations of O2 and CO2 were basically the same in the internal atmosphere of films. In other words, the variation of parameters is unlikely to be affected by changes in gas composition and/or concentration, or is negligible if any. The experiments of the three groups were completed in the same “4 days”, and each treatment group was repeated three times in three consecutive “4 days”.

2.7.2. Determination of Quality Parameters and Antioxidant Parameters of Fresh-Cut Melon

The weight loss percentage of fresh-cut melon was determined by weighing the samples at specific time intervals compared to initial weight. Six fresh-cut melons in each group were weighed. The weight loss rate was calculated according to the equation: Weight loss rate (%) = (W0W1)/W0 × 100, where W0 is the weight of samples at 0 days and W1 is the weight of samples at the corresponding days.
Firmness was measured using a fruit hardness tester (GY-5A, Aipu Measuring Instruments Co., Ltd., Hengzhou, Hunan, China), which was pressed into three different points in the central zone of each fresh-cut fruit for a depth of 10 mm. Soluble solids in fresh-cut melon pulp were determined by an Abbe Refractometer (2WAJ, Shanghai Optical Instrument Co., Ltd., Shanghai, China) and expressed as percentages. Titratable acidity of fresh-cut melon was determined by adjusting the pH of the titrated filtrate to neutral with 0.1 mol/L NaOH [21].
Total phenols of fresh-cut melon were determined by the Folin–Ciocalteu colorimetric method using gallic acid as a standard [22]. Malondialdehyde can be condensed with thiobarbituric acid under acidic and high-temperature conditions to form 3,5,5-Trimethyloxazolidine-2,4-dione, and its maximum absorption wavelength is 532 nm. Therefore, it was assayed with the thiobarbituric acid method according to the manufacturer’s instructions of the commercial kit. Briefly, fresh-cut melon pulp (0.1 g) was homogenized in 1 mL trichloroacetic acid, and the homogenate was centrifuged at 8000× g for 10 min. The supernatant obtained was used for assay of malondialdehyde, and the result was expressed as nmol/g on a fresh weight basis.
Ascorbic acid was assayed with the 2 6-dichlorophenol indophenol method according to manufacturer’s instructions of the commercial kit. Briefly, a 0.1 g sample of pulp was blended in 1 mL oxalate to extract ascorbic acid. The mixture was centrifuged at 8000× g and 4 °C for 10 min, and then the supernatant was taken for ascorbic acid assay. Ascorbic acid was expressed as nmol/g on a fresh-weight basis.
Fresh-cut melon was homogenized in 1 mL of a 0.1-M Na2HPO4-NaH2PO4 buffer (pH 7.0), and then was centrifuged at 8000× g for 10 min at room temperature. The supernatant was used for assays of the catalase activities, which were assayed according to manufacturer’s instructions of the commercial kits. H2O2 decomposition was measured at 240 nm. Catalase activity unit is defined as the ability of each gram of tissue to catalyze 1 μmol of H2O2 per minute in the reaction system.
Fruit pulp of 2 g was homogenized in 20 mL aqueous ethanol (80%, v/v) as ethanol extract solution and the filtrated solution were used to DPPH• and OH• scavenging experiments. DPPH• scavenging activity was determined as reported by Muley et al. [14] and calculated by the following formula: Scavenging activity (%) = (A0A1)/A0 × 100, where A1 is the absorbance of each sample, and A0 is that of a control prepared without adding the sample. OH• scavenging activity was determined as reported by Liu et al. [23]. The scavenging rate was calculated by the following formula: Scavenging rate (%) = [1 − (A0A1)/A2] × 100, where A0 is the absorbance of the reaction systems with various sample, A1 is that when salicylic acid was replaced with distilled water, and A2 is that of blank control in which the sample was replaced by distilled water.

2.8. Statistical Analysis

Three parallel experiments were performed for each treatment in this study, and data were shown as means ± standard deviations. The statistical significance of one-way analysis of variance (ANOVA) was tested using SPSS software (ver. 20.0; SPSS Inc., Chicago, IL, USA). Different letters indicate significance at p < 0.05 based on Tukey test. Principle component analysis (PCA) was operated by the Origin 2021 software (ver. 2021, OriginLab Corporation, Northampton, MA, USA).

3. Results and Discussion

3.1. Effects of GRP on Physical Properties of the Polysaccharide Films

Homogenous free-standing films were obtained by heating and drying chitosan or mixture solution of chitosan and GRP. Photographs (Figure 2A–C) demonstrate that the obtained films (CF, CF + L-GRP, and CF + H-GRP) were all homogeneous, flexible, and transparent, except CF + H-GRP was translucent due to its relatively high level of GRP. The high flexibility of the three films was attributed to glycerol plasticizer added, and as shown in Figure 2D, their thickness exhibited no difference (p < 0.05). Figure 2E shows that the opacity of CF + H-GRP was higher (p < 0.05) than those of CF and CF + L-GRP with no significant difference between them. Values of density (Figure 2F), moisture (Figure 2G), and water vapor permeability (Figure 2H) all possessed significant differences among the three films (p < 0.05), with the former two properties increasing and the latter two decreasing in a GRP-dose-dependent manner.
The water resistance performance of chitosan is weak because it contains hydrophilic groups such as free hydroxyl and amine groups [24]. However, most of these hydrophilic groups might be consumed when cross-linked with GRP (Supplementary Figure S3), leading to excellent mixing effect between them. This excellent mixing could ensure the formation of a densely cross-linked structure, which in turn enhanced the adhesion between the cross-linked structure and the water molecules within the polysaccharide molecules, thereby reducing the film’s absorption of water molecules from the environment. Our results that the composite films of chitosan mixed with GRP had higher density and moisture and lower water vapor permeability compared with CF (Figure 2) support the above arguments. The results are also consistent with those obtained with the polysaccharide film formed by pectin, chitosan, and tea polyphenols [15].
Thermal stability of CF, CF + L-GRP, or CF + H-GRP was determined under nitrogen at temperatures up to 800 °C by the thermogravimetry assay and further evaluated by the derivative thermogravimetry analysis. From Figure 3A, it is obvious that the films showed two stages of weight loss throughout the thermogravimetry assay. The first stage, up to 120 °C, was attributed to evaporations of water and volatile compounds such as acetic acid. The second stage, between 120 and 320 °C, was related to thermal degradation of glycerol, chitosan, and even GRP. Uranga et al. [25] also reported a two-stage degradation pattern with the fish gelatin–chitosan films incorporated with citric acid. Figure 3B presents the second derivative of the data shown in Figure 3A, which can more intuitively reflect which temperature has the maximum weight loss rate. The reversed peaks of the derivative thermogravimetry correspond to the maximum change rate of weight loss [26]. As shown in Figure 3B, for both CF, CF + L-GRP, and CF + H-GRP, there is only one reversed peak in the first stage, but two in the second stage. The temperatures leading to maximum weight loss rates of CF, CF + L-GRP, and CF + H-GRP were 208.65, 212.74, and 207.10 °C, respectively. The results allowed to reach a solid conclusion that addition of GRP had no influence on thermal stability of the films and the films were unable to degrade even under nature’s normal extreme maximum temperatures.

3.2. Effects of GRP on Mechanical Property and Chemical Structure of the Polysaccharide Films

The mechanical properties of the films were reflected by the relationship between tensile strength and elongation percentage (i.e., tensile strain), which represent the film’s ability to stretch and the flexibility prior to breakage. As shown in Figure 4A, CF had the highest tensile strength and elongation at break among all the groups. Further increase of GRP concentration decreased the mechanical properties of the complex film, which was consistent with a report by Don et al. [27]. A similar phenomenon had also been reported in chitosan films with banana peel extract [28]. The results were probably related to the addition of GRP, causing a slight change in the structure of the film. In addition, this might also be due to the solid particles on its surface, which was also confirmed by subsequent SEM analysis. Although the tensile strength of the three films was only about 1 MPa, it is sufficient for certain less demanding packages such as those for fruits and vegetables.
According to their X-ray diffraction patterns, CF, CF + L-GRP, and CF + H-GRP were all semi-crystalline in nature (Figure 4B), with broad peaks at around 21° (2θ), consistent with those reported by Rubilar et al. [29]. The intensity of the diffraction peak was obviously weaker, and the width of the peak was wider (see also the upper built-in Figure of Figure 4B), indicating that crystallinity of the films decreased with the increase of GRP levels, which might be attributed to the good compatibility between GRP and chitosan. Moreover, as amplified in the lower built-in Figure of Figure 4B, CF + H-GRP possessed a small and sharp diffraction peak at 28.62°, which might be caused by the high level of GRP.
Figure 4C showed FT-IR spectra of CF, CF + L-GRP, and CF + H-GRP. He absorption band from 3320 to 3250 cm−1 was assigned to the -OH and -NH stretching vibrations [30], and that from 2940 to 2883 cm−1 attributed to the C-H stretching in the films. Absorption peaks at 1646 and 1565 cm−1 were due to the C=O stretching of acetyl group (amide-I) and -NH bending and stretching (amide-II), respectively [31]. A peak at 1409 cm−1 associated with C-H bending vibration, and those in the range from 1110 to 1015 cm−1 were assigned to C-O-C, suggesting that the sugar rings were pyranose rings [32]. In addition, weak absorption bands in the region between 922 and 850 cm−1 implied that α- and β-glycosidic linkages existed simultaneously [33]. Obviously, compared with CF, the main functional groups of CF + L-GRP and CF + H-GRP featured no changes after the incorporation of GRP. However, a slight shift of the peak position at 1646 cm−1 of CF could be observed, which moved to 1640 and 1634 cm−1 after adding to it a low or high level of GRP, respectively, indicating that GRP interacted with the amide-I bond of chitosan.

3.3. Effects of GRP on Microstructure of the Polysaccharide Films

SEM and AFM are the effective assays to investigate the surface morphology and structure of films [34]. Surface morphologies of films analyzed by SEM (Figure 5A–C) showed that the films were homogenous and dense, with no cracks or large pores. Nevertheless, tiny solid particles were observed on the surface of the two composite films (Figure 5B,C) and the number of particles increased with the increase of the GRP level. Obviously, the viscosity of the film solution was enhanced along with the addition of GRP in a dose-dependent manner, resulting in poor fluidity of the film solution. Thus, the agglomeration phenomenon occurred after drying of the films. This agglomeration led to the passage of water molecules through film becoming tortuous, which was probably one of the reasons for the decline of water vapor permeability (Figure 5I).
AFM images of 2D topography present the roughness of CF (Figure 5D), CF + L-GRP (Figure 5E), and CF + H-GRP (Figure 5F). The values of Rq revealing the roughness of CF, CF + L-GRP, and CF + H-GRP were 1.231 ± 0.147, 1.311 ± 0.048, and 1.522 ± 0.198 nm, respectively, with no difference (p < 0.05) in roughness between each pair of them. The 3D topographic images for the films (Figure 5G–I) show well-distributed hills and valleys, similar for CF, CF + L-GRP, and CF + H-GRP.
According to the results of the above studies on the CF, CF + L-GRP, and CF + H-GRP and the principle of saving resources, CF + L-GRP had better properties and used less GRP, so it was selected for the next verification test of fresh-cut fruit.

3.4. Effects of the Composite Film on Preservation of Fresh-Cut Melon

3.4.1. Effects on Appearance and Four Quality Parameters of the Fresh-Cut Melon

For assessing the preservation effect, three groups, including PF, CF, and CF + GRP (i.e., CF + L-GRP as defined in Section 2.7.1), were employed to cover polypropylene trays containing the fresh-cut melon. Figure 6 shows the appearances of fresh-cut melon protected by the three films for four days compared with those before protection. Obviously, three groups of fresh-cut melon exhibited different degrees of decay and wilting, and the degree of deterioration and wilting was PF > CF > CF + GRP, implying that polysaccharide films possessed preservation effect on fresh-cut melon and adding of GRP to CF enhanced the preservation effect.
The weight loss of fruit is an important index that reflects respiration rate and moisture evaporation of the fruit tissue. Firmness is a visible change that occurs during fruit maturation, storage, and distribution as a consequence of metabolic changes and water loss [35]. Titratable acidity reflects the changes of organic acid in fresh fruit during storage. Soluble solids are one of the most important parameters affecting fruit quality and consumer acceptability, and a higher soluble solids value is preferred [36]. Weight loss, firmness, titratable acidity, and soluble solids were used to evaluate the qualities of fresh-cut fruit in this study. The weight (Figure 7A), firmness (Figure 7B), titratable acidity (Figure 7C), and soluble solids (Figure 7D) of all the three groups (i.e., PF, CF, and CF + GRP) showed similar trends of gradual decrease during the storage of fresh-cut melon. Notably, the values of soluble solids of the three samples were kept around 11%, this may be attributed to the fact that melon belongs to non-respiratory climacteric fruit and its soluble solids were remarkably unchangeable during storage [37]. Importantly, compared to PF, both CF and CF + GRP improved or retained better all the four indexes of the fresh-cut melon for extending its shelf life (p < 0.05), and CF + GRP exhibited even better effectiveness than CF (p < 0.05).

3.4.2. Effects on Six Antioxidant Indexes of Fresh-Cut Melon

Malondialdehyde formation is one of the important indexes to evaluate the integrity of the membrane after oxidative stress [38]. Malondialdehydes of the three groups (Figure 7E) steadily increased during storage. Nevertheless, compared to PF, both CF and CF + GRP suppressed (p < 0.05) its formation, with no significant differences observed between the three groups. Ascorbic acid is a powerful antioxidant which can prevent or attenuate the damage caused by reactive oxygen species in fruit and vegetables, and its level could be used as the index of the capability of organisms to combat aging [39]. Ascorbic acid levels (Figure 7F) of all the fresh-cut melon of the three groups started to decrease (p < 0.05) from the 1st, 2nd, and 3rd day, respectively. Moreover, compared to CF, CF + GRP reduced the decline more rigorously from the 3rd day. Phenolic compounds are plant secondary metabolites synthesized by the phenylpropanoid metabolism pathway and are associated with postharvest disease resistance [40]. Total phenols (Figure 7G) of the three groups were firstly unchanged then increased abruptly (p < 0.05) from the 3rd day. Nevertheless, there was no significant difference among the three groups.
Higher catalase activity and DPPH• and OH• scavenging abilities are beneficial for alleviating oxidative stress of fruit during storage [41]. As shown in Figure 7H, catalase activity in fresh-cut melon decreased continuously during storage. CF and CF + GRP could reduce their decreases from the 2nd day (p < 0.05), with CF + GRP showing even more rigorous effects than that of CF from the 3rd day (p < 0.05). Figure 7I shows the DPPH• scavenging activities of fresh-cut melon that remained unchanged up to the 2nd day for CF and the 3rd day for CF + GRP, then the scavenging rates were decreased (p < 0.05). Nevertheless, the effect of PF also decreased continuously during storage, and compared to CF, the impact of CF + GRP became significant on the 4th day (p < 0.05). The OH• scavenging activities (Figure 7J) of PF and CF + GRP decreased (p < 0.05) from the 2nd day, whereas that of the CF decreased (p < 0.05) from the 3rd day, and the effect of CF + GRP was higher than those of PF and CF from the 3rd day (p < 0.05). These findings suggested that compared to PF and CF, CF + GRP covering the fresh-cut melon can retain its good antioxidant status for a longer time, and GRP played an essential role in this antioxidant activity.

3.4.3. Possible Mechanisms and PCA and Correlation Analyses

In the pre-experiment, we determined the antibacterial activity of GRP with different concentrations (5, 10, and 20 mg/mL) against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 by using the Oxford cup method. By observing the experimental results, we found that there was no bacteriostatic circle in each group, indicating that GRP had no significant antibacterial activity (data not shown). Therefore, this study mainly focused on how the composite films were formed by the mixture of GRP as an antioxidant and chitosan affect the fresh-cut melon.
Figure 8A outlines a possible model for the antioxidant system in film-protected fresh-cut melon. Phenylalanine ammonia-lyase was activated and then the phenylpropanoids pathway was initiated to form phenolic compounds after cell membrane damage by cutting [42]. At the same time, a large number of reactive oxygen species was also produced, leading to formation of malondialdehyde by membrane lipid peroxidation [43], inhibition of catalase activity, and reduction of ascorbic acid [44], and then causing deterioration of the fresh-cut melon. In contrast, when covered with polysaccharide films, the fresh-cut melon maintained catalase activity, the malondialdehyde level was reduced, and the decline in ascorbic acid was alleviated, therefore creating an excellent antioxidant environment for the fresh-cut melon and thus prolonging its shelf-life. Following evidence reported by Yu et al. [45] and Don et al. [27], we speculate that a certain number of GRP may be sustainably released as antioxidants from the composite film to scavenge the formed free radicals, and/or the formed free radicals may be absorbed and scavenged by the GRP in the film. Another reason for the delayed decay may be that the film density after GRP mixing was higher (Figure 2F), which made it more difficult for external microorganisms to invade the film while encountering antioxidation resistance. On the other hand, microorganisms left over from cleaning inside the package also cause the packaged fruits and vegetables to not stay fresh all the time.
Subsequently, PCA was applied to evaluate the effectiveness of PF, CF, and CF + GRP on the fresh-cut melon during storage. As shown in Figure 8B,C, multivariate treatments of the data obtained for all the samples allowed these variables to be mainly reduced to two principal components that explained 96.5% of the total variability of the fresh-cut melon, of which PC1 accounts for 90.1% and PC2 for 6.4%. The proximity of different samples in the score plot indicates similar behaviors of their effects on the fruit properties [46]. The differences between PF and CF and between PF and CF + GRP were appeared from the 1st day (Figure 8B), due to the scores between them being different on the score plot. In addition, the scores of CF and CF + GRP were similar, indicating that the two films exhibited no significant difference in the preservation effect on fresh-cut melons at the 1st day and the 2nd day. Specially, the PF on the 2nd day and CF + GRP at the 3rd day, and the CF at the 3rd day and the CF + GRP at the 4th day were located in positions with similar scores. These results confirm that CF + GRP showed moderate preservation for fresh-cut melon, compared with PF and CF. Figure 8C shows the loading plots of principal components analyses of the four quality parameters and six antioxidant parameters in effects of films on fresh-cut melon during storage. The longer arrow of the variance means the PCA can explain more variance information [47]. Obviously, the length of each arrow was similar, indicating that each of the four parameters and six indexes had a similar contribution to PC1 and PC2.
To further evaluate relations among the four quality parameters as well as the six antioxidant indexes of the fresh-cut melon, a correlation-based approach using the Pearson coefficient was adopted. As shown in Figure 8D, three of the four quality parameters (e.g., firmness, titratable acidity, and soluble solids) were pairwise positively correlated (p < 0.01), and they all negatively correlated (p < 0.01) with weight loss (the other quality parameter). For the six antioxidant indexes, malondialdehyde was positively correlated (p < 0.01) with total phenols and they were both negatively correlated (p < 0.01) with the other four antioxidant indexes (e.g., ascorbic acid, catalase, DPPH•, and OH•) which were pairwise positively correlated (p < 0.01). Finally, the quality parameter weight loss correlated (p < 0.01) positively with two antioxidant indexes (malondialdehyde and total phenols) and negatively with the other four antioxidant indexes (ascorbic acid, catalase, DPPH•, and OH•) which in turn positively correlated (p < 0.01) with the other three quality parameters (firmness, titratable acidity, and soluble solids). These correlations clearly show that weight loss and malondialdehyde increase led to the decline of fresh-cut fruit quality, and maintaining firmness, titratable acid, soluble solids, ascorbic acid, and catalase levels, as well as DPPH• and OH• scavenging capacities reflected the better quality of fruit that can be associated with better taste, richer flavor, and longer shelf-life. It must be pointed out that the increase of total phenols was due to the activation of phenylpropane metabolism caused by cutting, which was unable to prove that total phenol is a negative index in the preservation of fresh-cut fruits.

4. Conclusions

In this study, GRP at three different concentrations was excellently cross-linked with chitosan to form antioxidant composite films that exhibited higher density, opacity, and moisture, as well as lower water vapor permeability, tensile strength, and elongation than those of the neat chitosan film, and these phenomena became even more dramatic with the increase of the GRP level in the composite films (Figure 2 and Figure S3). Thermogravimetric analysis evidenced that thermal stability of the films showed no significant change, whereas X-ray diffraction and FT-IR analyses disclosed a slight change in the structure of the films after adding GRP (Figure 3 and Figure 4). Furthermore, SEM and AFM tests revealed smooth and dense surfaces of the films with no cracks or macropores on them (Figure 5). Considering the above properties of the films and the principle of saving resources, chitosan film with a low concentration of GRP, namely, CF + GRP, was used to study the preservation of fresh-cut melon. Fresh-cut melon covered with CF + GRP visibly deteriorated more slowly compared with those covered with PF and CF (Figure 6), and these facts were firmly supported by the results from measurements of four quality parameters and six antioxidant indexes (Figure 7). Possible antioxidant mechanisms of the composite film in fruits preservation were then suggested (Figure 8A), and PCA and correlation analyses supported these findings (Figure 8B–D). This study suggests that GRP as a natural antioxidant added to chitosan-based films can prolong the shelf-life of fresh-cut fruit and minimize the environmental impacts caused by the use of conventional (synthetic) materials. It can also provide valuable academic reference for the future application of plant waste in the development of organic or green agricultural products.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/coatings12040468/s1, Figure S1: Mechanical properties of the other two repeated tests of three polysaccharide films. Black, red and blue curves represent those of CF, CF + L-GRP and CF + H-GRP, respectively. CF, CF + L-GRP, and CF + H-GRP, chitosan films with 0%, 0.5%, and 2.0% GRP, respectively; GRP, ginsenosides-extracting residue polysaccharides; Figure S2: Thermogravimetric analysis (A and C) and derivative thermogravimetry analysis (B and D) of the other two repeated tests of three polysaccharide films. Black, red and black curves represent those of CF, CF + L-GRP and CF + H-GRP, respectively. CF, CF + L-GRP, and CF + H-GRP, chitosan films with 0%, 0.5%, and 2.0% GRP, respectively; GRP, ginsenosides-extracting residue polysac-charides; Figure S3: Cross-linked simulation images between chitosan and GRP. GRP, ginsenosides extracting residue polysaccharides (Based on our previous study).

Author Contributions

J.S.: Conceptualization, Methodology, Investigation, Writing—original draft. Y.L. (Yuanhang Li): Investigation, Software. X.C.: Investigation. F.Y.: Software. L.S.: Validation. Y.L. (Yujun Liu): Conceptualization, Writing—review & editing, Supervision. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by [Forestry Public Welfare Scientific Research Projects] grant number [No. 201404718], China.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

This work was financially supported by the special funds for Forestry Public Welfare Scientific Research Projects [No. 201404718], China.

Conflicts of Interest

The authors state that there is no conflict of interest.

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Figure 1. The schematic diagram of preparing the antioxidant films.
Figure 1. The schematic diagram of preparing the antioxidant films.
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Figure 2. Appearance images and physical properties of three polysaccharide films. (AC) images of CF, CF + L-GRP, and CF + H-GRP, respectively. (DH) thickness, opacity, density, moisture content, and WVP of the three films, respectively. CF, CF + L-GRP, and CF + H-GRP, chitosan films with 0%, 0.5%, and 2.0% GRP, respectively; GRP, ginsenosides-extracting residue polysaccharides; WVP, water vapor permeability. Results are presented as mean ± SD of three independent experiments (n = 3). Different lower-case letters indicate significant differences at p < 0.05 based on Tukey test.
Figure 2. Appearance images and physical properties of three polysaccharide films. (AC) images of CF, CF + L-GRP, and CF + H-GRP, respectively. (DH) thickness, opacity, density, moisture content, and WVP of the three films, respectively. CF, CF + L-GRP, and CF + H-GRP, chitosan films with 0%, 0.5%, and 2.0% GRP, respectively; GRP, ginsenosides-extracting residue polysaccharides; WVP, water vapor permeability. Results are presented as mean ± SD of three independent experiments (n = 3). Different lower-case letters indicate significant differences at p < 0.05 based on Tukey test.
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Figure 3. Thermogravimetric analysis (A) and derivative thermogravimetry analysis (B) of three polysaccharide films. CF, CF + L-GRP, and CF + H-GRP, chitosan films with 0%, 0.5%, and 2.0% GRP, respectively; GRP, ginsenosides-extracting residue polysaccharides.
Figure 3. Thermogravimetric analysis (A) and derivative thermogravimetry analysis (B) of three polysaccharide films. CF, CF + L-GRP, and CF + H-GRP, chitosan films with 0%, 0.5%, and 2.0% GRP, respectively; GRP, ginsenosides-extracting residue polysaccharides.
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Figure 4. Mechanical properties (A), X-ray diffraction (B), and Fourier-transform infrared spectroscopy (C) of three polysaccharide films. The upper and lower built-in figures in (B) were the y-axis offset stacking diagram and the partial enlarged view from top to bottom, respectively. CF, CF + L-GRP, and CF + H-GRP, chitosan films with 0%, 0.5%, and 2.0% GRP, respectively; GRP, ginsensides-extracting residue polysaccharides.
Figure 4. Mechanical properties (A), X-ray diffraction (B), and Fourier-transform infrared spectroscopy (C) of three polysaccharide films. The upper and lower built-in figures in (B) were the y-axis offset stacking diagram and the partial enlarged view from top to bottom, respectively. CF, CF + L-GRP, and CF + H-GRP, chitosan films with 0%, 0.5%, and 2.0% GRP, respectively; GRP, ginsensides-extracting residue polysaccharides.
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Figure 5. Surface morphologies of the three films CF (A,D,G), CF + L-GRP (B,E,H), and CF + H-GRP (C,F,I). (AC) scanning electron microscopes; (DF) atomic force electron microscope images of 2 D topography; and (GI) atomic force electron microscope images of 3D topography. CF, CF + L-GRP, and CF + H-GRP, chitosan films with 0%, 0.5%, and 2.0% GRP, respectively; GRP, ginsenosides-extracting residue polysaccharides.
Figure 5. Surface morphologies of the three films CF (A,D,G), CF + L-GRP (B,E,H), and CF + H-GRP (C,F,I). (AC) scanning electron microscopes; (DF) atomic force electron microscope images of 2 D topography; and (GI) atomic force electron microscope images of 3D topography. CF, CF + L-GRP, and CF + H-GRP, chitosan films with 0%, 0.5%, and 2.0% GRP, respectively; GRP, ginsenosides-extracting residue polysaccharides.
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Figure 6. Appearance images of fresh-cut melon covered with different films during four days of storage. (AC), fresh-cut melons covered with PF, CF, and CF + GRP, respectively, and their appearance images at the beginning and the end of the 4th day. PF, plastic film; CF and CF + GRP, chitosan films with 0% and 0.5% GRP, respectively; GRP, ginsenosides-extracting residue polysaccharides.
Figure 6. Appearance images of fresh-cut melon covered with different films during four days of storage. (AC), fresh-cut melons covered with PF, CF, and CF + GRP, respectively, and their appearance images at the beginning and the end of the 4th day. PF, plastic film; CF and CF + GRP, chitosan films with 0% and 0.5% GRP, respectively; GRP, ginsenosides-extracting residue polysaccharides.
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Figure 7. Changes of four quality parameters and six antioxidant indexes of fresh-cut melon covered with different films during four days of storage. (AJ), weight loss, firmness, titratable acidity, soluble solids, malondialdehyde, ascorbic acid, total phenols, catalase, DPPH• scavenging activity, and OH• scavenging activity, respectively, of fresh-cut melon during storage at 25 ± 1 °C under a relative humidity of 55 ± 1%. PF, plastic film; CF and CF + GRP, chitosan films with 0% and 0.5% GRP, respectively; TA, titratable acidity; SS, soluble solids; GRP, ginsenosides-extracting residue polysaccharides. The depth of the legend color to the right sides of (AD) represents the level of the corresponding value. Results are presented as mean ± SD of three independent experiments (n = 3). Different lower-case and upper-case letters indicate significant differences between and within groups, respectively, at p < 0.05 based on Tukey test.
Figure 7. Changes of four quality parameters and six antioxidant indexes of fresh-cut melon covered with different films during four days of storage. (AJ), weight loss, firmness, titratable acidity, soluble solids, malondialdehyde, ascorbic acid, total phenols, catalase, DPPH• scavenging activity, and OH• scavenging activity, respectively, of fresh-cut melon during storage at 25 ± 1 °C under a relative humidity of 55 ± 1%. PF, plastic film; CF and CF + GRP, chitosan films with 0% and 0.5% GRP, respectively; TA, titratable acidity; SS, soluble solids; GRP, ginsenosides-extracting residue polysaccharides. The depth of the legend color to the right sides of (AD) represents the level of the corresponding value. Results are presented as mean ± SD of three independent experiments (n = 3). Different lower-case and upper-case letters indicate significant differences between and within groups, respectively, at p < 0.05 based on Tukey test.
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Figure 8. Possible mechanisms (A) for the antioxidant system, score (B), and loading (C) plots of principal components analyses, and correlation analyses (D) in fresh-cut melon covered with different films. (A) Green and red boxes indicate inhibition and boost, respectively. Solid lines imply strong and dotted lines weak effects. PAL, phenylalanine ammonia lyase; ROS, reactive oxygen species; MDA, malondialdehyde; CAT, catalase; AsA, ascorbic acid; TA, titratable acidity; SS, soluble solids; DPPH•, DPPH radical scavenging activity; OH•, hydroxyl radical scavenging activity. (B) Circles of different colors represent different treatments of fresh-cut melon for storage. (C) Lengths of arrows indicate information drawn from a variance. (D) Orange and blue colors represent positive and negative correlations, respectively. PF, plastic film; CF and CF + GRP, chitosan films with 0% and 0.5% GRP, respectively; GRP, ginsenosides-extracting residue polysaccharides. ** indicates significant correlations at p < 0.01.
Figure 8. Possible mechanisms (A) for the antioxidant system, score (B), and loading (C) plots of principal components analyses, and correlation analyses (D) in fresh-cut melon covered with different films. (A) Green and red boxes indicate inhibition and boost, respectively. Solid lines imply strong and dotted lines weak effects. PAL, phenylalanine ammonia lyase; ROS, reactive oxygen species; MDA, malondialdehyde; CAT, catalase; AsA, ascorbic acid; TA, titratable acidity; SS, soluble solids; DPPH•, DPPH radical scavenging activity; OH•, hydroxyl radical scavenging activity. (B) Circles of different colors represent different treatments of fresh-cut melon for storage. (C) Lengths of arrows indicate information drawn from a variance. (D) Orange and blue colors represent positive and negative correlations, respectively. PF, plastic film; CF and CF + GRP, chitosan films with 0% and 0.5% GRP, respectively; GRP, ginsenosides-extracting residue polysaccharides. ** indicates significant correlations at p < 0.01.
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Sun, J.; Li, Y.; Cao, X.; Yao, F.; Shi, L.; Liu, Y. A Film of Chitosan Blended with Ginseng Residue Polysaccharides as an Antioxidant Packaging for Prolonging the Shelf Life of Fresh-Cut Melon. Coatings 2022, 12, 468. https://doi.org/10.3390/coatings12040468

AMA Style

Sun J, Li Y, Cao X, Yao F, Shi L, Liu Y. A Film of Chitosan Blended with Ginseng Residue Polysaccharides as an Antioxidant Packaging for Prolonging the Shelf Life of Fresh-Cut Melon. Coatings. 2022; 12(4):468. https://doi.org/10.3390/coatings12040468

Chicago/Turabian Style

Sun, Jing, Yuanhang Li, Xinxin Cao, Fan Yao, Lingling Shi, and Yujun Liu. 2022. "A Film of Chitosan Blended with Ginseng Residue Polysaccharides as an Antioxidant Packaging for Prolonging the Shelf Life of Fresh-Cut Melon" Coatings 12, no. 4: 468. https://doi.org/10.3390/coatings12040468

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