Effect of ε-Polylysine Addition on Pullulan Biodegradable Films for Blueberry Surface Coating

Abstract

A biodegradable film was successfully prepared using pullulan and ε-polylysine (ε-PL). The impact of ε-PL contents on biodegradable film was explored through mechanical properties, water vapor permeability, and an FTIR spectroscopy test. Amongst all the prepared films, the 0.5% ε-PL had the most desirable results in film performance. The TS, EAB, and WVP values were 5.63 MPa, 21.5%, and 0.27 × 10⁻⁶ g m/Pa h m², respectively. The blueberries coated with 0.5% ε-PL and pullulan were stored at 20 °C for 12 days. The effect of edible coatings on the performance of blueberries was evaluated by detecting the physicochemical properties and the contents of antioxidants. The experimental results displayed that ε-PL and a pullulan blend film could decrease the weight loss and maintain the hardness, polyphenol, anthocyanin, and antioxidant capacity of the blueberry preservation. The results indicated that the biodegradable film is a potential fruit-coating material.

1. Introduction

Blueberries, a very favored fruit, possess nutritional characteristics because of the richness of bioactive substances, such as anthocyanins, chlorogenic acids, flavonoids, and α-linolenic acid [1,2,3]. Nevertheless, blueberries have a short shelf life and are prone to spoilage [4,5]. But consumers demand that packaged blueberries are fresh and rich in health-promoting compounds without quality damage. Numerous methods, such as physical, chemical, and biological methods, have been exploited to preserve the quality of blueberries according to the aim of preservation [6,7,8]. Among them, edible coatings are a low-cost, green, and environmentally friendly preservation method that could effectively improve the storage quality of blueberry [9,10,11].

Edible coatings are always used to maintain the quality of fruits and vegetables by controlling their physiological, biochemical, or oxidation processes [12,13]. Finding adequate compositions of their formulations is essential to improve the efficiency and stability of edible coatings. In the recent years, biodegradable natural polymers, such as polysaccharides and protein, have been widely used as edible films. Edible coatings carrying bioactive and functional compounds, for instance, antiseptics, could improve the nutritive value and stability of the product during shelf-life [14,15].

Pullulan films have attracted much attention due to their many advantages, such as high water solubility, colorlessness, odorlessness, transparency, flexibility, low oil permeability, oxygen permeability, and heat sealability. Many studies aim to synthesize pullulan films by blending them with other biopolymers to ameliorate the physical and functional properties of pullulan films [16,17,18]. In recent years, there has been growing interest in developing materials with film-forming ability and antimicrobial activity to improve food safety and shelf life [19].

ε-Polylysine (ε-PL) is a homo-polymeric amino acid linked by peptide bonds between amino and carboxyl groups, which is water-soluble, biodegradable, and biocompatible [20]. ε-PL has been shown to be effective against a variety of bacteria, molds, yeasts and viruses [21,22]. Nowadays, ε-PL is commonly used as a safety material and food preservative in the food industry [23].

To our knowledge, Limited research has been performed on extending the shelf life of blueberries by applying the combination of pullulan and ε-PL as active packaged ingredients. Therefore, this study aimed to develop active pullulan films reinforced with ε-PL, the physical, barrier, and mechanical properties of which were identified. In addition, the effects of the prepared films on the physico-chemical properties of blueberries during storage at 20 °C for 12 days were explored.

2. Materials and Methods

2.1. Materials

Pullulan was obtained from Hayashibara. (Shanghai, China). ε-PL was from Bomei Bio-Engineering Co. (Hefei, China). Other reagents were purchased from Sinopharm. (Shanghai, China).

The complete methodology was showed in Scheme 1.

2.2. Preparation of Blend Films

Pullulan film-forming solutions were prepared by dissolving 3 g of pullulan into 100 mL of distilled water. Then, 1.0 g of glycerol was added, and different amounts of ε-PL (1, 3, 5, 7, and 9 g) were added to the solution. In order to remove the bubbles in the film- forming solution, it was degassed in a vacuum oven and further cast onto flat glass plates. Then, the film-forming solution was dried in an environmental chamber at 50 °C and 50% RH for 20 h. The resulting film was peeled off the glass plate and further tested at 20 °C and 50% RH [24].

2.3. Physico-Chemical Characterization of the Film

2.3.1. Mechanical Properties

Prior to the test, films were cut into 150 mm × 15 mm strips. The thickness of the specimens was measured using a micrometer (Wenzhou Weidu Electronics Co., Ltd., Wenzhou, China). The tensile strength (TS) and elongation at break (EAB) of each film strip were determined using a texture analyzer (TA, XT) on the basis of the method reported Guo et al., 2020 [25]. The samples were tested at a speed of 100 mm/min with an initial distance of 100 mm. The results were the average of eight samples.

2.3.2. Water Vapor Permeability (WVP)

The WVP of films was measured using the gravimetric cup method according to ASTM 1653 (2004). The film was sealed on the top of a glass permeating cup containing distilled water (100% RH; 2337 Pa vapor pressure at 20 °C), which was placed in a desiccator at 20 °C and 0% RH containing silica gel (0 Pa water vapor pressure). The cups were weighed every 12 h for 1 week. The amount of water permeating through the films was determined from the weight loss of the cups. The results were the average of three samples.

2.3.3. Fourier Transform Infrared (FTIR) Analysis

FTIR spectra of the nanocomposites were obtained using a PerkinElmer spectrometer (Spectrum 100, USA). Each spectrum resulted from 16 scans in the wavenumber range of 400–4000 cm⁻¹. Signal averages were obtained at a resolution of 4 cm⁻¹.

2.3.4. X-ray Diffraction (XRD)

XRD pattern analysis of the AGM nanocomposite films was performed using an X-ray diffractometer (Rigaku D/Max, Tokyo, Japan). The XRD spectra were recorded using Cu Kα radiation as the incident X-ray source (40 kV, 200 mA) and at room temperature in the range of 2θ = 10°–80° with a scan rate of 2 °/min [16].

2.3.5. Scanning Electron Microscopy (SEM)

The representative areas of film cross-sections were examined using an SEM (Model S-4800 SEM, Hitachi Ltd., Tokyo, Japan). All samples were frozen and fractured in liquid nitrogen to reveal the morphology of the film. Double-sided tape was used to install the broken film on a bronze short column, and then it underwent sputter coating with a gold layer (10 nm). The gold film was scanned with an accelerating beam voltage of 1.0 kV.

2.4. Preservation Experiment of Blueberries

A batch of fresh blueberries (purchased from Hefei Blueberry Base, Hefei, China) was delivered via cold chain transportation, and the diseased blueberries were removed. The moderate-sized blueberries and those with analogous maturity blueberries were selected, soaked in 10 g/L sodium hypochlorite solution for 3 min, washed in pure water, and dried at room temperature. The blueberries was allocated randomly into two groups (200 each). The first group was soaked in the pullulan and ε-PL (5 g) coating solutions prepared in Section 2.2 for 3 min, while the other group was treated as a control group.

All samples were stored at 20 °C and 75% RH for 12 days. The preservation index was detected every 48 h in each group.

2.5. Determination of Blueberry Properties

2.5.1. Mass Loss (ML)

The weight of blueberries was measured every 2 days, and the weight loss ratio was calculated using Equation (1):

$$\mathrm{W}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \mathrm{l}\mathrm{o}\mathrm{s}\mathrm{s} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\left(\%\right)=\frac{W_0-W_t}{W_t}$$

(1)

where W₀ is the initial weight of blueberries (g) and W_t is the sample weight after storage (g) [13]. The results were the average of twenty samples.

2.5.2. Texture

Hardness evaluation was performed using a penetration test using a TA (Stable Micro Systems, Surrey, Godalming, UK) [26]. A standard flat probe (SMS P/50) was used for the 50% compression test. The probe’s measurement speed was 1 mm/s, and the automatic triggering force was 5 g at a testing distance of 30 mm. The highest peak was hardness. The results were the average of ten samples.

2.5.3. Measurement of Blueberry Properties

The blueberries were cut into small pieces and ground in a mortar to obtain a homogeneous pulp. The measurement of total polyphenol content (TPC) and total anthocyanin content (TAC) was carried out according to the method of Tahir et al., 2013 [27], and Rodriguez et al., 2016 [28]. DPPH analysis was conducted following the method described by Yang et al., 2019 [29]. The results were the average of five samples.

2.6. Statistical Analysis

Significant differences in the results were evaluated through analysis of variance (ANOVA) and the Duncan test at 5% level of significance using SPSS24.0 software (SPSS Inc., Chicago, IL, USA). Values are given as the means ± standard deviation.

3. Results and Discussion

3.1. Characterization of Films

3.1.1. Mechanical Properties

The TS and EAB of the ε-PL–pullulan blend films were determined. The data are detailed in

Table 1. Mechanical properties and WVP of ε-PL–pullulan blend films.

Content of ε-PL (g/100 mL)	TS (MPa)	EAB (%)	WVP × 10−6 (g m/Pa h m2)
0	3.95 ± 0.26 b	12.2 ± 1.20 e	0.20 ± 0.05 c
0.1	4.02 ± 0.24 b	13.0 ± 0.95 e	0.21 ± 0.01 c
0.3	4.79 ± 0.11 ab	17.5 ± 1.02 d	0.24 ± 0.02 bc
0.5	5.63 ± 0.57 a	21.5 ± 1.23 c	0.27 ± 0.01 b
0.7	4.80 ± 0.52 ab	25.0 ± 0.81 b	0.40 ± 0.03 a
0.9	4.10 ± 0.66 b	30.0 ± 1.50 a	0.41 ± 0.01 a

Values are means ± SD, and the values that are in the same row and share the same subscript letters are not statistically significant (p > 0.05).

. Compared with the mechanical properties of pure pullulan film, those of blend films with ε-PL added were enhanced significantly. The blend film containing 0.5 g/100 mL of additional ε-PL had the highest TS of 5.63 MPa, which was 42.53% higher than that of the pure pullulan film. The enhancement of TS in the composite films was due to the intermolecular interaction between ε-PL and pullulan and the combination of carboxyl groups and hydroxyl groups on the molecules to form hydrogen bonds [30,31]. As the ε-PL content increased by 0–0.9 g/100 mL, the EAB of the blend films increased from 12.2% to 30.0%. The results indicated that the addition of the ε-PL changed the flexibility of the pullulan film slightly.

3.1.2. WVP

Preventing or reducing moisture transfer between food and the surrounding environment is an essential requirement for films used as food packaging to extend the shelf life of food [32]. The WVP values of ε-PL–pullulan blend films are shown in Table 1. The pure pullulan film had the lowest WVP (0.20 × 10⁻⁶ g m/Pa h m²), which was comparable to the previously reported result of pullulan films [16]. Adding ε-PL caused an increase in the WVP of the blend films, especially the addition of more than 0.7 g/100 mL ε-PL. When 0.7 g/100 mL of ε-PL was added, the WVP was 0.40 × 10⁻⁶ g m/Pa h m². Similar results were obtained in chitosan biofilm incorporated with ε-PL [31]. The WVP of the films increased gradually with the increase in ε-PL content. This phenomenon could contribute to the great hydrophilicity of ε-PL. ε-PL is a hydrophilic cationic polymer with a large number of polar amino acids, which have strong hydrophilicity, and ε-PL molecules are dispersed in the middle of pullulan molecules, enhancing the permeability of water [32].

3.1.3. FTIR

Figure 1 shows the FTIR spectra of ε-PL, pullulan film, and ε-PL–pullulan blend films. For the FTIR of ε-PL (Figure 1a), the peak located around 3226 cm⁻¹ was due to the NH₂ asymmetric stretching. The characteristic of the C–H stretching vibration was detected at 2927 cm⁻¹ [33], while the two characteristic absorption peaks at 1643 cm⁻¹ and 1245 cm⁻¹ represent the stretching vibration of the carbonyl group (amide I) and the C-N stretching of the amide band, respectively. These features were in good agreement with a previous report [34].

In the FTIR spectrum of the pullulan film (Figure 1g), the broad peak at 3276 cm⁻¹ indicates the stretching vibration of hydroxyl groups. The peak at 2927 cm⁻¹ represents the stretching vibration of C–H, while the bands at 1644 cm⁻¹ and 1413 cm⁻¹ are associated with the O–H bending of water and CH₂, respectively. The assignment of these absorption bands was consistent with the reported pullulan films [16,35].

The bands from 763 cm⁻¹ to 1158 cm⁻¹ were due to the stretching vibration of the C–O. When two or more substances are mixed, the change in the FTIR characteristics’ spectrum peak may be used to reflect the relationship between substances, whether they are physical mixtures or chemical interactions [33,36]. The spectra of ε-PL–pullulan composite films are detailed in Figure 1b–f. The NH bending (amide II) peak is missing, and the stretching vibration of the carbonyl band (amide I) is shifted from 1644 cm⁻¹ to 1666 cm⁻¹ plus ε-PL. In addition, the characteristic peak of the C-H stretching vibration at 2927 cm⁻¹ is shifted to 2921 cm⁻¹. This result suggests that there was an interaction between the hydroxyl groups of pullulan and the amino groups of ε-PL [37]. Lin et al. [38] also reported a similar relationship between chitosan and ε-PL.

3.1.4. XRD

XRD is customarily used for the characterization of the apparent crystallite size of polymers, confirmation of crystallization process and kinetic parameters, the assessment of the fine structure of materials, and the exploration of molecular interactions [39]. The XRD spectra of blend films are shown in Figure 2. The crystallization peaks were detected at around 20° for all edible films. Theis result is in agreement with previous studies. Zhu et al. reported that a very broad peak of pullulan film could be recognized at around 19.5°. If there is no weak interaction between the ε-PL and pullulan, in other words, the ε-PL and pullulan are completely incompatible polymers in the blend systems, two separate crystalline regions would be displayed on the XRD figure. XRD patterns are represented as the proportional diffraction spectra for each component. The results demonstrate the amorphous structure of the film and reveal that a dense network was formed between the molecules [40,41]. This finding was in good agreement with the analysis of FTIR.

3.1.5. SEM

The microstructure of ε-PL–pullulan blend films was observed using SEM (Figure 3). As shown in Figure 3g, the cross-section of the pullulan film is relatively smooth and uniform and forms a continuous matrix without any pores or cracks. The film has good structural integrity, which was a similar result to that reported by other researchers [16,37]. In the absence of any sign of phase separation, a continuous and homogeneous phase in the blend films was observed due to the good interaction between the polymers in the blend films. The micrographs of the ε-PL–pullulan blend films were smooth. This finding indicates that ε-PL and pullulan have high compatibility. Similar results have been obtained by other groups, such as Bonilla et al., 2016 [42], who reported that the gelatin-chitosan edible film mixed with plant ethanolic extracts possessed a compact, uniform, and dense structure and a homogenous appearance. However, the blend films showed some small particles with ε-PL addition. The ε-PL molecules acted as the matrix, changing the force of the secondary bonds between different substances [34,35].

3.2. Preservation Experiment of Blueberries

3.2.1. Photographs of Blueberries in Storage

Photographs of blueberries in storage are shown in Figure 4. The appearance of coated blueberries was brighter than that of uncoated blueberries. On day 0, the surface of all the samples was bright and shiny. During the preservation process, blueberries gradually lose water and the fruit shrinks. These changes were especially apparent in the uncoated group. The uncoated group shrank gradually after 4 days of storage, while the coated group shrank slightly after day 6. After 12 days, the fruit was still relatively plump after coating.

3.2.2. Mass Loss

Mass loss is one of the main factors that affect the storage quality of blueberries [42,43]. Studies have shown that the mass loss of blueberry fruits is mainly caused by the loss of water by transpiration. In addition, the aerobic respiration of the fruits converts a part of the organic matter into CO₂ and H₂O, resulting in a certain mass loss [44].

As shown in Figure 5, all blueberry samples gradually lost weight during the 12-day storage period. The coated samples did not have any significant difference in weight loss during 4 days of storage compared to the control group. After 4 days, the increase in weight loss of the uncoated group was significantly higher than that of the coated group. For example, on the 6th day, the weight losses of the uncoated group and the coated group were 10.55% and 5.01%, respectively. Therefore, compared to the uncoated group, the coating treatment reduced the rate of fruit quality loss. The edible coating on the surface of blueberry fruit formed the protective barrier, which resisted the decomposition effect of microorganisms and effectively inhibited metabolic activities related to respiration. The blueberry maintained good storage quality in the coated group.

3.2.3. Texture

Fruit hardness is an important indicator of storability, and softening is an important characteristic of fruit ripeness. When the fruit becomes soft, the dissolution of the pectin is the most significant change in the cell wall, accompanied by the dissolution of the glue layer in the cell wall and the destruction of the main wall [8]. As shown in Figure 6, the texture characteristics of blueberries in the coated group and the uncoated group maintained the same trend during the whole storage period. The hardness of blueberry samples in the coated group was higher than that in the uncoated group, but the difference was not significant. At 6 days of storage, the hardness of the uncoated group was 984.96 g, which is 88% of the hardness of the coated group. However, on the 4th day of storage, the hardness of all samples was highest due to the loss of moisture, but the fruit had not yet entered decay. The higher hardness indicates that the ε-PL–pullulan blend films could inhibit the decline of blueberry texture and help to maintain the change in texture during blueberry during storage. Kraśniewska et al. [10] studied the effect of pullulan coating on postharvest quality and the shelf life of highbush blueberry. After 28 days of storage at 16 °C, fruit weight losses were found to be 14% and 22% for coated and uncoated samples, respectively. Overall, the edible coating could effectively improve the storage performance of blueberries.

3.2.4. TPC, TAC and DPPH

As shown in Figure 7a, the TPC of all blueberry samples increased and then decreased during storage. The TPC of the uncoated group reached the maximum (281.03 mg/100 g) at day 8 and then rapidly decreased, but the coated group had a value of 257.51 mg/100 g at day 12. The maximum TPC of the coated group appeared on day 10 and then slowly decreased, with a value of 278.61 mg/100 g at day 12. In the literature, similar results were obtained by Zou et al. (2019), where blueberries were coated with gum arabic and white roselle extract. The reduction in TPC may be due to the consumption of phenolic substances as PPO substrates in the browning reaction [45]. Therefore, the higher TPC of the coated group could be attributed to the effective suppression of PPO activity, which slows down the consumption rate of phenolic substances, thereby maintaining a higher TPC in the late storage period.

Figure 7b shows the TAC of blueberries at different sampling intervals during 12 days of storage. The TAC of all blueberries increased during the first 4 storage days, was maintained thereafter, and then dropped. The loss of TAC during storage may be caused by oxidation and/or condensation reactions with other phenolic compounds [46,47]. However, the reduction in TAC in uncoated fruits was significantly higher than that in coated fruits. At the end of the storage time, the TAC (106.14 mg/100 g) of the coated fruit was increased compared with the uncoated fruit (96.47 mg/100 g). Faria et al. [48] reported that the degradation of TAC in blueberries was caused by POD enzymes. Therefore, this result could be because of the composite coating film inhibiting the increase in POD activity by controlling the exchange of oxygen and delaying the consumption of TAC.

The antioxidant capacity of blueberries increased and then decreased during the whole storage period (Figure 7c). The increase in antioxidant capacity may be due to the increase in antioxidant substances in blueberries [49], which leads to improved antioxidant activity. In the present study, the coated group exhibited a significantly higher DPPH radical scavenging ratio after 12 days of storage than the uncoated group. This finding could be attributed to the fact that the coating film effectively maintains a higher content of antioxidant substances (TAC and TPC) in the fruit at the end of storage, thereby imparting higher antioxidant activity to the fruit. Kraśniewska et al. [10] suggested that the coatings create an additional barrier around the skin of the coated blueberry, thereby slowing down the intensity of the transpiration processes and reducing the degradation of phenolic compounds in the skin of blueberry.

4. Conclusions

Composite films containing pullulan and different concentrations of ε-PL were successfully developed in this study. The mechanical properties and WVP of the composite films were enhanced with the increase in ε-PL content in the film. Compared with the control group, the results suggested that the pullulan–ε-PL coating group could better delay the loss of quality, texture, and bioactive substances of blueberries. The blueberries maintained the quality and sensory indexes with the pullulan–ε-PL coating. Thus, for example, the weight and firmness loss of blueberry fruits were significantly reduced compared to uncoated fruit during storage, which helped maintain a better visual appearance. The antioxidant and antibacterial activities of the composite films showed that pullulan and ε-PL had a synergistic effect in combination. Due to its non-toxic and biodegradable properties, pullulan–ε-PL composite film would be the best choice for food-packaging applications. This study concludes that the pullulan and ε-PL composite film is expected to be used in food packaging because of its easy film formation, excellent performance, and biodegradability. In addition, pullulan and ε-PL can effectively prolong the shelf life of blueberries. Therefore, the pullulan polysaccharide and ε-PL composite film can be considered as a natural substitute for fruit preservation.