*2.6. Weight Loss*

Owing to respiration and transpiration, strawberries are highly susceptible to the rapid loss of water. In our studies, weight loss was tracked over the storage period of six days. Figure 3a shows that all strawberries experience progressive weight loss during the storage period. The packaged samples experience significantly less weight loss than non-packaged samples after two days (*p* < 0.05). This is because the major cause of weight loss among non-packaged samples is the migration of water from the fruit to the environment [33], and the packaging materials serve as semipermeable barriers that block oxygen, carbon dioxide, and moisture. This reduces respiration, water loss, and oxidation [34]. Since PVA has the lowest water vapor permeability, strawberries packaged with it exhibit the lowest weight loss. PVA/CH-2.5 exhibits the lowest weight loss among the PVA/CH bilayer films. This is because PVA/CH-2.5 has higher oxygen permeability, and the hydrophilic CH interacts with water molecules to increase the transport of water vapor. Hence, the packaging of strawberries in bilayer films is clearly effective in providing a physical barrier to moisture loss, and therefore in retarding respiration and fruit shriveling [35].

**Figure 3.** Effect of unpackaged PVA and different PVA/CH films on the (**a**) weight loss, (**b**) decay, (**c**) firmness, (**d**) titratable acid, (**e**) total soluble solids and (**f**) ascorbic acid quality parameters of strawberries during storage times.

#### *2.7. Decay Percentage*

Strawberries are highly perishable fruits with high postharvest physiological activities that limit their shelf lives. Generally, the infected areas increase gradually with storage time, as shown in Figure 3b. The decay percentages of the packaged strawberries range from 22.7% ± 3.2% to 52.9% ± 5.7%, but the non-packaged strawberries experience a decay of 85.1% ± 7.4% after 6 days. The packaging significantly reduces strawberry decay during the six days test (*p* < 0.05). PVA/CH films and pure PVA film can decrease the rate at which the fruit rots as well. Some studies have indicated that CH appears to have multiple functions, as it interferes directly with fungal growth and activates several biological processes in plant tissues [36]. PVA/CH-2.5 reduces the degree of decay better than other packaging materials throughout the storage period. This may be because of its WVP and OP, which may affect the growth environment for bacteria.

#### *2.8. Firmness*

Loss of texture is one of the main factors that limits quality and the postharvest shelf lives of fruit and vegetables. Therefore, texture is an important strawberry quality parameter. During ripening, strawberries soften considerably due to degradation of the middle lamella of the cell wall. Cortical parenchyma cells, cell wall strengths, cell-to-cell contact [37], and cellular turgor can also influence firmness [38]. The changes in strawberry firmness are shown in Figure 3c. The firmness of all strawberries was significantly lower among the non-packaged samples than among the packaged fruit (*p* < 0.05). This leads to increased water loss and fungal infections among strawberries that lack packaging protection, which leads to more pronounced tissue senescence and broken cell walls [39]. After four days, the firmness curve exhibits a turning point. The firmness of all packaged strawberries begins to decrease significantly due to senescence, which softens the fruit via pectin hydrolysis, and depolymerization degradation of the cell wall [40]. After six days, the loss of firmness is approximately 48% among non-packaged fruit. The losses are 31.5% ± 9.7%, 16.2% ± 4.7%, 10.4% ± 2.4%, and 20.6% ± 5.1% in PVA, PVA/CH-2, PVA/CH-2.5 and PVA/CH-3 packaged fruit samples, respectively. The PVA/CH-2.5 samples do not experience significant surface softening when compared to the fresh strawberries, and the film is effective with regard to firmness retention. It also has been reported that chitosan coatings and other biopolymers are selective O2 and CO2 barriers, and thus can modify the internal atmosphere and slow the respiration rates of fresh fruits and vegetables [41].

#### *2.9. Titrable Acidity (TA)*

Organic acids are among the most important components of the flavor of a strawberry. Changes in acidity are significantly affected by the rate of metabolism, especially respiration. Respiration consumes organic acid, and therefore acidity declines during storage. This is also the main cause of fruit senescence [10]. The effect of packaging on the TA of strawberries is shown in Figure 3d. At the end of the storage period, the TA of non-packaged strawberries decreases significantly faster (*p* < 0.05) than packaged samples. This is because the packaging can modify the internal atmosphere around the strawberries, and may therefore delay the utilization of organic acids. The decrease in the TA of the PVA-packaged fruit is less (23.4%) than that of the unpackaged fruit (36.5%), but more than that of fruit protected by PVA/CH films (from 17.6% to 19.8%). This suggests that bilayer films are better than pure PVA materials at delaying decreases in TA during storage. The PVA/CH-2.5 bilayer film is the most effective at maintaining higher TA levels after six days.

#### *2.10. Total Soluble Solids (TSS)*

Total soluble solids (TSS) is an important parameter that affects fruit quality and consumer acceptability. Changes in the TSSs of strawberry samples with storage time are shown in Figure 3e. The TSS increases significantly during four days of storage. Starting on the fifth day, the TSS begins to decrease rapidly in PVA-packaged samples (from 8.7% to 7.3%) due to hydrolysis. Experiments also

show that adding CH to the coating formulation helps to maintain higher TSS accumulation at the end of the storage period. After six days, the PVA/CH bilayer film slows the conversion that reduces sugar levels in the strawberries. The PVA/CH-2.5 sample retains the highest TSS in the current work, which can be explained by the considerable loss of water experienced by strawberries during storage at room temperature. The PVA/CH-2.5 bilayer film offers the most suitable environment for strawberry preservation, as previously noted with regard to weight loss.

#### *2.11. Ascorbic Acid*

Obviously, the ascorbic acid content gradually declines with postharvest elongation in both packaged and non-packaged fruits (Figure 3f). All packaging materials inhibited ascorbic acid loss in packaged fruit. With the PVA/CH-2.5 film, the ascorbic acid content decreased from 66.2 ± 4.8 mg/100 g (one day) to 57.9 ± 3.6 mg/100 g (six days), while the unpackaged samples showed significantly (*p* < 0.05) lower amounts of ascorbic acid (42.4 ± 2.9 mg/100 g, six days). Some studies have also shown that the incorporation of CH can slow the deteriorative oxidation of ascorbic acid in fruit [10]. During storage, strawberry decay and the low CH content increase ascorbic acid degradation.

#### *2.12. Sensory*

Sensory acceptance scores, including those for appearance, color, odor, flavor, texture, and overall acceptability were measured after six days and are shown in Figure 4. The appearance acceptability scores of the control strawberries are significantly different from those of the packaged strawberries after two days. The control and PVA-packaged strawberries have unacceptable scores after four days of storage, while the PVA/CH films maintain acceptable scores (greater than five) until five days. The color acceptability scores of the packaged strawberries do not change significantly until after three days of storage. After three days, the color acceptability of samples other than PVA/CH-2.5 decrease significantly. The odor acceptability scores of the control strawberries fail consumer acceptance after four days. The odor acceptability scores of the PVA/CH-2 and PVA/CH-3 samples are almost equal, with the former having the highest scores. The control has the lowest odor acceptability score after six days. The flavor acceptability scores of all strawberries decrease significantly during the six days. The scores of PVA-packaged strawberries are greater than those of the control strawberries, and are the highest among all of the samples after two days. Both control and PVA-packaged strawberries are unacceptable after five days. Although the flavor scores of PVA/CH film-packaged strawberries decrease during the 6 days of testing, they remain acceptable. The overall acceptance scores of the control strawberries decrease during storage. The PVA and PVA/CH-2-packaged strawberries are not acceptable after five days. However, the PVA/CH-2.5 and PVA/CH-3 samples are still acceptable at the end of the storage period. This finding agrees with those of Sangsuwan et al., who reported that CH beads loaded with lavender essential oil can extend the mold-free storage lives of strawberries stored at 7 ◦C from two days (control) to eight days with acceptable overall sensory scores [43].

The result of sensory evaluation was similar to the appearance changes of strawberries. As shown in Figure 5, both packed and unpacked strawberries have begun to decay after four days except the group packed with PVA-CH-2.5 film. The decay of the strawberries could have been prevented by PVA-CH-2.5 film until the 6th day. The mold and yeast counts during the 6 days storage also proved the similar conclusion.

As shown in Figure 6, the strawberries packaged with PVA/CH films presented a significantly lower amount of mold and yeast growth than the uncoated strawberries (*p* < 0.05). Moreover, the mold and yeast reduction was more evident in the strawberries that were packaged with high concentration CH due to the antimicrobial capacity of CH, especially against the fungi and yeast spoilage of strawberries [44]. CH potentially causes severe cellular damage in mold and yeast by altering the synthesis of fungal enzymes [45], inducing morphological changes, and causing structural alterations and molecular disorganization in fungal cells [46]. Similar results were observed by Valenzuela et al. [44]. The results achieved here also indicated that PVA/CH-2.5 film can not only maintain the nutritive and organoleptic properties of strawberries, it could also reduce the amount of mold and yeast during storage times. The high effectiveness of PVA/CH-2.5 is due to the ionic and hydrophilic interaction between PVA and CH, which increased the availability of the amino groups of the CH to the antimicrobial properties [44].

**Figure 4.** The sensory scores of (**a**) appearance acceptability; (**b**) color acceptability; (**c**) odor acceptability; (**d**) flavor acceptability; (**e**) texture acceptability and (**f**) overall acceptability of strawberries during storage times.

**Figure 5.** Digital images of the appearance and inner changes of strawberries during storage times.

**Figure 6.** Evolution of mold and yeast counts of unpackaged and packaged strawberries with different PVA/CH films during storage times.

#### **3. Materials and Methods**

#### *3.1. Materials*

CH (food grade, 90% degree of deacetylation, molecular mass of 165,000 Da, low viscosity) was obtained from Shangdong Aokang Biological Technology (Jinan, Shangdong, China). PVA with molecular weight of 124,000–186,000 was purchased from Sigma Aldrich (98%, St. Louis, MO, USA). Acetic acid (glacial 100%, water solution) was obtained from Guoling Instrument Inc. (Dongguan, China) All other chemical reagents used were of analytical grade and obtained from Chengdu Kelong Reagent Co. (Chengdu, China), unless otherwise indicated. Strawberries were harvested from an orchard in Shuangliu, Sichuan. The selected fruit was of uniform size and color and free of physical damage and fungal infection. They were washed with 1% sodium hypochlorite for 1 min, then rinsed with distilled water and allowed to dry. Packaging experiments were carried out on the same day.

#### *3.2. PVA/CH Film Preparation*

PVA was dissolved in boiling water at 100 ◦C and stirred for 8 h to produce a clear, 10 wt % solution. It was degassed in a vacuum desiccator and poured into a Teflon pan (20 cm × 20 cm). The solution was dried in an oven at 50 ◦C for four days to ensure the removal of residual solvents, and the film was peeled from the pan and kept in a vacuum oven until use. To produce a completely homogenized CH solution, various CH mass ratios were dissolved in 0.8% acetic acid and stirred overnight. Briefly, a CH solution in acetic acid/water/ethanol was loaded into a syringe equipped with a metal capillary. A rotating metal plate collector was placed approximately 5 cm from the capillary tip. The solution was extruded from the needle tip at a constant flow rate of 10 μL/min using a precision pump (Zhejiang University Medical Instrument Company, Hangzhou, China). The applied voltage was set to approximately 15 kV using a high-voltage statitron (Tianjing High Voltage Power Supply Company, Tianjing, China). Low-humidity conditions were created by using a dehumidifier during the electrospray process. After CH deposited on both surfaces, the films were dried, first in a vacuum at 50 ◦C for 24 h and then at 18 ± 2 ◦C.

#### *3.3. Film Characterization*

The pure PVA, PVA/CH-2, PVA/CH-2.5, and PVA/CH-3 bilayer films' thickness were measured with a ZUS-4 micrometer (Yue Ming Small Machine Co., Changchun, China). The thickness measurements were taken at 10 random positions on each film, and the mean was calculated. The mean value for the thickness was used in calculating the film oxygen permeability (OP) and mechanical properties. The morphologies of the films were examined using SEM (FEI Quanta 200, Eindhoven, The Netherlands) equipped with a field-emission gun and Robinson detector, after the samples were vacuum-coated with thin layers of gold to minimize the charging effect. Attenuated total reflectance–Fourier transform infrared (ATR-FTIR) spectrometry (ATR-FTIR, Nicolet 5700, Thermo Nicolet Instrument Corp., Madison, WI, USA) was used to identify the chemical structures of the PVA/CH composite films and the possible interactions between their components. A small section cut from each composite film was used. The samples were analyzed with a resolution of 4 cm−1, an aperture setting of 6 mm, a scanner velocity of 2.2 kHz, a background scan time of 32 s, a sample scan time of 32 s, and a total of 100 scans per sample, in the range of 400 to 4000 cm<sup>−</sup>1. The composite films were cut into rectangles of 50 mm in length and 5 mm in width for PVA/CH fibers. Tensile testing was performed using a universal testing machine (UTM, Instron 5583, Norwood, MA, USA) with the crosshead speed of 5 mm/min and a 30 mm gauge length. The average value from five measured samples is reported for subsequent analysis.

#### *3.4. Strawberries Preparation and Packaging*

The strawberries were randomly divided into four groups and conducted with three replicates, with 10 strawberries in each treatment. The weight ratio of PVA:CH of 80:20, 75:25, and 70:30 were

denoted as PVA/CH-2, PVA/CH-2.5, and PVA/CH-3, respectively. Ten strawberries (250 ± 10 g) were packed in sealed PVA or PVA/CH bags (18 cm × 18 cm). These samples were placed in conditions of 18 ± 2 ◦C and 60 ± 5% RH. The quality of both packaged and control strawberries were evaluated after one day, two days, three days, four days, five days, and six days of storage.
