ε-Poly-l-lysine Suppressed Decay Development and Maintained Storage Quality in Guava Fruit by ROS Level Regulation and Antioxidant Ability Enhancement

Abstract

Guava fruit is susceptible to decay, leading to losses in storability and quality. ε-Poly-l-lysine (ε-PL) is a safe antimicrobial polypeptide that has proven to be effective in preserving produce’s quality. In the present research, ε-PL, at multiple concentrations (1, 2 and 4 g/L), was adopted to treat guavas, and the fruit were stored at 25 °C for 15 d. The results indicated that ε-PL retarded the guava storability decline and enhanced its quality. Treated guavas had a better appearance, as well as the lower disease index, relative electrolytic leakage, weight loss, respiration intensity, a* and b* values and reducing sugar content. They also showed higher firmness, commercially acceptable fruit rate, titratable acidity, L* value, total soluble sugar, vitamin C and sucrose levels. The optimal concentration of ε-PL was determined to be 2 g/L. Furthermore, compared to control guavas, fruit treated with 2 g/L ε-PL exhibited lower levels of superoxide anion, hydrogen peroxide and malondialdehyde but higher antioxidant enzyme activities in terms of ascorbate peroxidase, peroxidase, catalase and superoxide dismutase. These findings suggested that ε-PL raised the antioxidant enzyme activities to enhance the fruit’s antioxidant ability. This, in turn, reduced the reactive oxygen species levels and lipid peroxidation, ultimately improving the guava’s quality. Consequently, ε-PL is of practical significance for commercial application as it suppresses decay and stabilizes the quality of guavas, enhancing their postharvest marketability.

1. Introduction

Guava (Psidium guajava L.), which originated in the Americas, is currently widely cultivated across subtropical and tropical zones around the world, such as in China, Thailand and India [1,2,3]. It is famous for its delicious taste and high nutritional and medicinal values. Studies have verified that the guava fruit is a rich source of dietary fiber, amino acids, vitamin C and antioxidants [4,5,6]. Nevertheless, guava, a climacteric fruit, can exhibit symptoms of yellowing, firmness loss and decay during storage, owing to the fruit’s high metabolic level [2,3,7], along with sensitivity to chilling injury, damage, dehydration or microorganism infection-induced brown or black pitting [5,7]. This can result in limited storage quality and postharvest losses in guava fruit. Hence, it is critical to develop feasible handling processes to suppress postharvest losses and improve the quality of guava fruit.

Reactive oxygen species (ROS) are key for the postharvest decay and quality losses in produce during storage [8,9,10]. After harvest, the quick accumulation of ROS may damage cell membranes, reducing the quality of produce [11,12]. Furthermore, ROS can induce the lipid peroxidation of cellular membranes, which leads to the accumulation of malondialdehyde (MDA), a product of lipid peroxidation, ultimately resulting in the reduced quality of produce [12]. It was shown that the cumulative ROS-induced oxidative damage and membrane breakdown were the triggers for aonla fruit decay [8]. Accelerated pulp breakdown and quality loss in longan fruit were attributed to the serious lipid peroxidation and membrane destruction caused by accumulated ROS [12]. Thus, ROS-induced oxidative stress is associated with the decay process in produce. To decrease ROS-induced oxidative damage, plants may scavenge the excessive ROS via the enzymatic antioxidant system [13,14,15]. Antioxidant enzymes, including ascorbate peroxidase (APX), peroxidase (POD), catalase (CAT) and superoxide dismutase (SOD), are vital in enhancing ROS elimination and reducing the oxidative damage due to accumulated ROS [8,12,15]. It has been revealed that the levels of antioxidant enzymes are related to the quality of produce. For instance, Cai et al. [11] showed that melatonin resulted in increased antioxidant enzyme activities, including that of POD, CAT and SOD, which were effective in suppressing senescence and preserving quality in passion fruit. Moreover, Han et al. [16] showed that sodium nitroprusside improved the quality and inhibited disease in apple fruit, owing to the reduced accumulation of ROS achieved by enhancing the antioxidant enzyme activity. Therefore, the antioxidant levels regulated by antioxidant enzymes are related to the ROS levels and quality of produce.

ε-Poly-l-lysine (ε-PL), which is a natural polycationic peptide containing 25–35 L-lysine residues, is secreted by Streptomyces albulus [17,18]. Notably, ε-PL exhibits strong antimicrobial abilities and water solubility and is safe for the human body [17,18,19]. Moreover, it is also known to trigger defense responses, resulting in the better preservation of produce [19,20]. Prior studies have demonstrated that ε-PL can suppress the occurrence of decay, leading to stabilized storage quality in produce such as apples [20] or fresh-cut kiwifruit [21] by boosting the fruits’ antioxidant capacity. Hence, we speculate that ε-PL plays a role in regulating plants’ antioxidant abilities, thereby affecting the quality of produce. Furthermore, in our previous study, we reported that ε-PL might suppress disease and maintain quality in passion fruit [22]. Nevertheless, a detailed investigation of the impacts of ε-PL on the maintenance of storage quality in guavas is lacking. Therefore, the present study was conducted to investigate the influences of various concentrations of ε-PL on the harvest quality of guava fruit, and the optimal ε-PL condition was chosen. Additionally, the impacts of the optimal ε-PL condition on the storage quality of guavas and their relation to the ROS levels, the content of lipid peroxidation product, the antioxidant enzyme activities and the antioxidant ability were investigated. The present research therefore aimed to uncover the mechanism behind the ε-PL-regulated antioxidant ability and ROS accumulation in guava fruit and to develop safe technique to reduce decay symptoms while preserving the fruit quality of guavas.

2. Materials and Methods

2.1. Guava Fruit and Postharvest Treatment

Fruit of guava cv. Xiguahong, from an orchard in Luoxi Town, Fujian, China, was picked during the commercial maturity stage in the summer and shipped to a lab. This orchard was a commercial orchard, and its planting management was in line with commercial and market practices. In the commercial maturity period, the fruit color was bright light green, and the levels of L* and a* were 65.39 ± 0.87 and −14.55 ± 0.73, respectively; the pulp’s total soluble solids content was 12.53 ± 0.09%. Guavas with a consistent size, color and shape, and without defects and diseases, were then chosen. The fruit was cleaned with distilled water and then employed for investigation.

One batch, i.e., 30 samples, was chosen to measure the fruit attributes at 0 d. The other batch (600 fruit) was separated into four groups with 150 fruit each. There were 3 replications in each group, with 50 samples per replication. The fruit were then treated by dipping them into ε-PL at multiple concentrations of 0 (control), 1, 2 and 4 g/L for 20 min. After being air-dried, the treated fruit were sealed in polyethylene film bags (thickness = 0.020 mm) (5 fruits/bag). All packaged fruit were stored for 15 d at 25 °C and 85% relative humidity. During storage, 30 fruit were sampled from each group to evaluate the storage quality every 3 days. Based on the effects of different concentrations of ε-PL on the storage quality of guava fruit, the optimal ε-PL level was then determined, which was 2 g/L. In addition, this study aimed to explore the mechanism by which ε-PL at the optimal concentration suppressed decay development and stabilized the storage quality in guavas through enhancing their antioxidant abilities.

2.2. Fruit Storability Assay

On the basis of the area ratio of disease on the guava’s surface, the fruit were categorized as follows: 0, no disease; 1, 1–24% disease; 2, 25–49% disease; 3, 50–74% disease; 4, ≥75% disease; 5, = 100% disease. Ten guava samples were sampled to determine the disease index, as described via Chen et al. [2]. The disease index was quantified as Σ (disease grade/the highest grade × each grade corresponding to the proportion of guava).

Based on Chen et al. [2], the relative electrolytic leakage (REL), an index used to evaluate the integrity of cell membranes, was determined using thirty fruit discs, each with an area of 0.2 cm², taken from five guavas. The result was calculated as a percentage (%).

Based on Chen et al. [6], the respiration intensity was determined using five guava fruit, and the result was recorded as mg CO₂/(kg·h).

Based on Chen et al. [2], the equatorial planes of 10 guavas were used to determine the firmness using a texture analyzer, namely the TA-XT Plus model (Stable Micro System, UK), with a P2 cylindrical probe with a diameter of 2 mm. The distance and speed of puncture were 10 mm and 2 mm/s, respectively. The result was recorded in Newtons (N).

The Chen et al. [6] method was employed to determine the weight loss rate using one bag of fruit. The result was calculated via comparison with the fruit weight on harvesting day and recorded in %.

The commercially acceptable fruit rate of the guavas was determined according to the method of Chen et al. [2] by calculating the proportion of the surface without browning or disease using 10 samples. The result was recorded in %.

2.3. Determination of Appearance Quality

According to Zhang et al. [13] and Huang et al. [23], five fruits were used to assess the guavas’ appearance quality through calculating the L*, a* and b* values at four equatorial positions using the CHROMA METER CR-400 chromameter (Konica Minolta Inc., Japan).

2.4. Measurement of Nutritional Quality Levels

According to previously described methods [2,6,24], the levels of nutrients in the guavas were measured.

First, 1 g of guava pulp tissue was mixed with 6 mol/L HCl (10 mL) and distilled water (20 mL) and then boiled. After 0.5 h, 6 mol/L NaOH was employed to neutralize the above solution, with phenolphthalein as an indicator. Finally, the supernatant was acquired to measure the total soluble sugar content after centrifugation. Furthermore, the guava pulp tissue (1 g) from 5 fruits was mixed with potassium ferrocyanide (5 mL) and zinc acetate (5 mL). Distilled water was used to obtain a constant volume (100 mL). After centrifugation, the reducing sugar content was measured using the supernatant. Moreover, the sucrose content was calculated via the following formula: sucrose = total soluble sugar – reducing sugar. These results were recorded in %.

Two grams of guava pulp tissue were homogenized with distilled water, and the supernatant was then obtained by filtration. Then, 10 mL of the above supernatant was sampled for the determination of the titratable acidity (TA) via titration. The result was recorded in %.

Then, 10 mL of 15% trichloroacetic acid (TCA) was mixed with 0.5 g of guava pulp tissue. The mixture was then centrifuged to obtain the supernatant. The Vitamin C content was determined using 1 mL of the supernatant. The result was recorded in mg/kg.

2.5. Measurement of ROS and MDA Content

Referring to previous documents [12,13], the content of ROS, such as superoxide anion (O₂^−.) and hydrogen peroxide (H₂O₂), as well as malondialdehyde (MDA), was measured. Two grams of guava pulp were mixed with 10 mL of 50 mmol/L phosphate-buffered saline (PBS) with pH 7.8, containing 1 mmol/L ethylene diamine tetraacetic acid, and centrifuged for the determination of the O₂^−. generation rate. Furthermore, the pulp tissue (two grams) from five guava samples was mixed with 10 mL of acetone. Then, the H₂O₂ value was quantified after centrifugation. Moreover, pulp tissue (2 g) was dissolved in 10 mL of 10% TCA. After centrifugation, the MDA level was quantified. These results were recorded in mmol/(min·kg), mol/kg and μmol/kg, respectively.

2.6. Antioxidant Enzyme Activity Assay

Two grams of guava pulp were mixed with 10 mL of 50 mmol/L PBS (pH 7.0), and the supernatant was collected after centrifugation to determine the APX, SOD and CAT activity. Moreover, 10 mL of 50 mmol/L PBS (pH 5.5), which contained 2% polyvinyl pyrrolidone, was mixed with 2 g of guava pulp. The supernatant was adopted to quantify the POD activity. These results were recorded in U/kg.

2.7. Statistical Analysis

In this research, the indices were measured three times. A one-way analysis of variance was carried out, and Duncan’s test was adopted to compare the differences among the treatments using the SPSS 21.0 software (IBM Corp., Armonk, NY, USA). Significant differences were indicated by p < 0.05 or 0.01.

3. Results

3.1. Changes in Decay Symptoms and Disease Index

As shown in Figure 1A, the guava fruit was bright light green at 0 d. Meanwhile, the fruit was complete in appearance, with an attractive fragrance. With the extension of the storage time, the color of the control guavas gradually changed to yellow and then red. Moreover, the fruit developed decay and tissue softening, accompanied by an unpleasant smell. Further observation revealed that the guavas displayed pronounced disease symptoms, and the disease index increased rapidly from 6 d (Figure 1B). However, all ε-PL-treated guavas presented slower color changes, reduced decay development and fewer disease symptoms (Figure 1A), accompanied by a lower disease index (Figure 1B). Notably, the 2 g/L ε-PL-treated group showed fewer decay symptoms and a notably lower disease index than the control group at 9–15 d. Therefore, ε-PL (particularly at a concentration of 2 g/L) could mitigate decay symptoms and reduce the disease incidence in guava fruit.

3.2. Changes in REL and Respiration Intensity

An increase in REL was seen in all groups at 0–15 d (Figure 2A). However, in the control group, 1 g/L ε-PL-treated group, 2 g/L ε-PL-treated group and 4 g/L ε-PL-treated group, it rose from 18.50% at 0 d to 31.03%, 27.20%, 24.22% and 25.70% at 15 d, respectively, with the optimal condition being 2 g/L. Moreover, at 6–15 d, the 2 g/L ε-PL-treated guavas exhibited a notably lower level than the control guavas.

Figure 2B revealed that the respiration intensity of the control fruit rose from 19.50 mg CO₂/(kg·h) at 0 d to a peak of 30.97 mg CO₂/(kg·h) at 9 d, followed by an overall downward trend after 9 d. However, all ε-PL-treated groups, particularly the group treated with 2 g/L of ε-PL, showed a lower respiratory peak. Furthermore, the 2 g/L ε-PL-treated group had a notably lower level than the control group at 9–15 d.

Therefore, ε-PL (especially at a concentration of 2 g/L) maintained lower REL and respiration intensity values in guava fruit.

3.3. Changes in Firmness, Weight Loss Rate and Commercially Acceptable Fruit Rate

Figure 3A showed that decreased firmness was observed in the four groups at 0–15 d. In the control group, 1 g/L ε-PL-treated group, 2 g/L ε-PL-treated group and 4 g/L ε-PL-treated group, it was reduced from 8.67 N at 0 d to 2.72 N, 3.59 N, 4.86 N and 4.04 N at 15 d, respectively, with the optimal condition being 2 g/L. Moreover, 2 g/L ε-PL-treated fruit exhibited significantly higher firmness than the control fruit at 3–15 d.

Figure 3B showed that an increment in the weight loss rate was observed in the four groups during storage. In the control group, 1 g/L ε-PL-treated group, 2 g/L ε-PL-treated group and 4 g/L ε-PL-treated group, it increased from 0% at 0 d to 6.75%, 3.45%, 2.89% and 3.76% at 15 d, respectively, with the optimal condition being 2 g/L. Moreover, notable differences in the weight loss rate between the 2 g/L ε-PL-treated guavas and control guavas were measured at 3–15 d.

Figure 3C revealed that, at 15 d, the commercially acceptable fruit rate in control samples, 1 g/L ε-PL-treated samples, 2 g/L ε-PL-treated samples and 4 g/L ε-PL-treated samples were 86.67%, 73.33%, 53.33% and 66.67% lower than that at 0 d, respectively. The ε-PL group with 2 g/L experienced the best effect in terms of delaying the decline in the commercially acceptable fruit rate and showed a clearly higher level than the control group at 3–15 d.

Therefore, ε-PL (especially at a concentration of 2 g/L) resulted in higher firmness and commercially acceptable fruit rate and lower weight loss rate in guava fruit.

3.4. Changes in Appearance Quality

Reduction in the value of L* (Figure 4A) and increases in the values of a* (Figure 4B) and b* (Figure 4C) were observed in the guava fruit at 0–15 d. Regarding the value of L*, those in the control group, 1 g/L ε-PL-treated group, 2 g/L ε-PL-treated group and 4 g/L ε-PL-treated group were 15.77%, 13.04%, 8.26% and 11.48% lower at 15 d than at 0 d, respectively. Regarding the value of L*, it rose from −14.55 at 0 d to 3.69, −1.06, 7.69 and −5.18 at 15 d, respectively, in these four groups. Regarding the value of a*, it rose from 36.14 (0 d) to 42.57, 41.39, 39.60 and 40.21 (15 d), respectively. Additionally, compared with the control group, the 2 g/L ε-PL-treated group retained the highest L* value at 0–15 d, with clear differences at 3–15 d, but displayed the lowest a* and b* values at 0–15 d, with prominent differences at 3–15 d.

Therefore, ε-PL (particularly at a concentration of 2 g/L) can maintain a higher L* level and lower a* and b* values in guava fruit.

3.5. Changes in Nutritional Quality Levels

Reductions in the content of total soluble sugar (Figure 5A), sucrose (Figure 5B) and TA (Figure 5D) were seen in the guava fruit at 0–15 d. The ε-PL-treated samples revealed higher values, especially in the ε-PL group with 2 g/L. Compared to the control samples, clearly higher TA, total soluble sugar and sucrose contents were observed in the 2 g/L ε-PL-treated samples at 12–15 d, at 9 and 15 d and at 3–9 d and 15 d, respectively. At 15 d, the levels of the three nutrients in 2 g/L ε-PL-treated fruit were 1.09 times, 1.05 times and 1.06 times those of the control fruit, respectively.

The reducing sugar level of all groups showed a similar trend, exhibiting a dynamic upward trend during storage (Figure 5C). The lowest reducing sugar content was seen in ε-PL-treated guavas at 0–15 d, especially in the group with a ε-PL concentration of 2 g/L. Moreover, the 2 g/L ε-PL-treated samples retained a significantly lower value than the control samples at 3–15 d. At 15 d, the reducing sugar value in 2 g/L ε-PL-treated guavas was 3.40% lower than that in control guavas.

Figure 5E showed that the vitamin C content in the control samples and 1 g/L ε-PL-treated samples decreased rapidly at 0–9 d, followed by a slow increase at 9–15 d. The vitamin C content in the other two ε-PL-treated groups decreased rapidly until 12 d but rose rapidly until 15 d. Vitamin C could be used as an ROS scavenger, and the increased vitamin C content might have been caused by a rapid rise in the ROS levels during the late storage period, thus delaying ROS-induced oxidative damage, retaining the membrane structure and preserving the quality of the guava fruit. The underlying cause of the increment in the vitamin C level should be further studied. Additionally, 2 g/L ε-PL exhibited the best impact in maintaining a higher level of vitamin C in guava fruit during storage, and it exhibited notable differences at 3–15 d. At 15 d, the vitamin C level in the 2 g/L ε-PL-treated group was 1.32 times that of the control group.

Hence, ε-PL (particularly at a concentration of 2 g/L) suppressed the decrease in the levels of total soluble sugar, sucrose, TA and vitamin C but reduced the amount of reducing sugar in the guava fruit.

3.6. Changes in ROS and MDA Amounts

Since 2 g/L ε-PL was identified as the most effective concentration to preserve the guava’s fruit quality, further analyses focused on the mechanism by which this specific ε-PL concentration preserved the storability and quality of the fruit by influencing its antioxidant capacity.

An increment in the O₂^−. generation rate was seen in the guava fruit during storage (Figure 6A). Furthermore, the ε-PL-treated samples had a lower level, with significant differences at 6 d and 12–15 d. Notably, the O₂^−. generation rate in ε-PL-treated guavas was 80.18% of that of control guavas at 15 d.

Figure 6B revealed that the H₂O₂ content in control samples rose rapidly at 0–3 d, followed by a slight change at 3–6 d, and it then rose rapidly again until 15 d. However, in guavas treated with ε-PL, the H₂O₂ content was reduced slightly at 0–3 d, while it rose rapidly after 3 d. Moreover, in contrast to control fruit, ε-PL-treated fruit maintained an obviously lower level at 9–15 d. Notably, at 15 d, the H₂O₂ content of ε-PL-treated fruit was 24.58% lower than that of the control fruit.

Figure 6C revealed that the MDA content of control guavas rose from 0.20 μmol/kg (0 d) to 1.30 μmol/kg (15 d). Meanwhile, in the ε-PL-treated group, the MDA amount increased from 0.20 μmol/kg (0 d) to 0.90 μmol/kg (12 d) but then declined to 0.71 μmol/kg (15 d). Additionally, at 3 d and 9–15 d, the ε-PL-treated guava fruit exhibited markedly lower value than the control guava fruit.

Overall, ε-PL might delay the increases in ROS (O₂^−. and H₂O₂) and MDA contents in guava fruit.

3.7. Changes in Antioxidant Enzyme Activity

Figure 7A showed that the SOD activity in the control fruit increased from 86.29 × 10⁶ U/kg (0 d) to 90.68 × 10⁶ U/kg (6 d) but decreased to 66.35 × 10⁶ U/kg (15 d). Meanwhile, in ε-PL-treated fruit, it rose from 86.29 × 10⁶ U/kg (0 d) to 99.94 × 10⁶ U/kg (6 d) but decreased to 80.80 × 10⁶ U/kg (15 d). Moreover, at 6 d and 12–15 d, the ε-PL-treated groups had significantly higher activity than the control guavas.

Figure 7B showed that the CAT activity in the control samples rose from 22.16 × 10⁶ U/kg (0 d) to 25.60 × 10⁶ U/kg (3 d) before declining to 18.13 × 10⁶ U/kg (15 d). Nevertheless, in ε-PL-treated samples, it rose from 22.16 × 10⁶ U/kg (0 d) to 34.37 × 10⁶ U/kg (6 d) before declining to 20.78 × 10⁶ U/kg (15 d). Additionally, markedly greater activity was revealed in ε-PL-treated samples compared to control guavas at 6–9 d.

Figure 7C showed that the APX activity in the control guavas rose until 6 d; it subsequently dropped from 20.69 × 10⁶ U/kg at 6 d to 15.55 × 10⁶ U/kg at 15 d. In contrast, the APX activity in ε-PL-treated samples rose rapidly at 0–9 d before declining from 27.21 × 10⁶ U/kg at 6 d to 21.64 × 10⁶ U/kg at 15 d. Moreover, compared to control guavas, ε-PL-treated guavas exhibited significantly higher value at 6–15 d.

Figure 7D showed that the POD activity of the control guavas rose from 7.65 × 10⁶ U/kg (0 d) to 8.13 × 10⁶ U/kg (6 d) but dropped to 5.12 × 10⁶ U/kg (15 d). Meanwhile, in ε-PL-treated guavas, it rose from 7.65 × 10⁶ U/kg (0 d) to 11.56 × 10⁶ U/kg (6 d) but declined to 6.49 × 10⁶ U/kg (15 d). In addition, at 6–15 d, ε-PL-treated samples exhibited significantly higher level than the control group.

Consequently, ε-PL could enhance the activities of antioxidant enzymes, like SOD, CAT, APX and POD, in guava fruit.

4. Discussion

As a climacteric fruit, the guava fruit is highly susceptible to postharvest problems including rot, dehydration, disease and softening [2,3,5,7]. This can hinder the maintenance of guava fruit’s quality, thus expediting the development of decay. In recent years, ε-PL, as an antimicrobial polypeptide, has demonstrated potential effectiveness in suppressing quality losses and enhancing the preservation of produce [19,22]. Furthermore, the integrity of the cell membrane structure is key for the physiological activity of organisms [24]. Structural damage to the cell membrane has the potential to trigger the decay of produce [8,24]. REL commonly serves as a parameter in evaluating the membrane damage degree. Moreover, respiration is of pivotal importance in physiological metabolism. A high respiration intensity can lead to the rapid depletion of substrates, expedite the decay process and undermine the commodity value of produce [24,25]. Moreover, weight loss results from the quick respiratory process, which might affect the storability of produce [24]. Additionally, the fruit texture, such as firmness, has a vital impact on the storability of produce after harvest [2]. In this work, compared to control fruit, ε-PL, especially at a concentration of 2 g/L, was found to effectively suppress fruit quality loss (Figure 1A). It also mitigated increases in the disease index (Figure 1B), REL (Figure 2A), respiration intensity (Figure 2B) and weight loss rate (Figure 3B). Moreover, it prevented reductions in the firmness (Figure 3A) and commercially acceptable fruit rate (Figure 3C) of guavas. This indicated that ε-PL, especially at 2 g/L, could be effective in maintaining the postharvest storability of guava fruit. Consistently with this, improved fruit storage performance had been demonstrated in melatonin-treated guavas [6] or 24-epibrassinolide (EBR)-treated table grapes [26]. This enhancement was attributed to the superior maintenance of the postharvest quality of such fruits.

The external color serves as a key attribute that indicates the freshness of produce. Parameters related to color changes are regarded as significant in determining consumers’ acceptability. The a*, L* and b* values are regarded as key indicators of color changes and contribute to appraising the appearance quality of produce [13]. In this work, compared to control samples, ε-PL (especially at 2 g/L) delayed the drop in the L* value (Figure 4A) but slowed down the rise in the a* (Figure 4B) and b* (Figure 4C) values in guava samples. Hence, ε-PL had the potential to retard the changes in the surface color of guava fruit. As a result, it helped to retain the guava’s appearance quality. These findings were consistent with those of Zhang et al. [13], who found that slightly acidic electrolyzed water (SAEW) could prevent the decline in the L*, a* and b* values of litchi pericarp. In this way, it reduced the alterations in the external color, thus effectively suppressing the tissue browning of litchi fruit.

The nutrient levels of produce are related to the TA, sugar and vitamin C content [13,24]. However, these nutrients can be consumed as substrates through respiration, thus reducing their levels in produce [13]. In the present research, compared to control fruit, ε-PL (particularly at a concentration of 2 g/L) could postpone the decreases in the contents of total soluble sugar (Figure 5A), sucrose (Figure 5B), TA (Figure 5C) and vitamin C (Figure 5E), but reduced the rise in the reducing sugar level (Figure 5D). Hence, ε-PL might retard the consumption of nutrients by reducing the respiration intensity, resulting in higher values of vitamin C, total soluble sugar, TA and sucrose, thereby maintaining the guava’s nutrient quality. Similar results were also shown in litchis treated with SAEW [13] or longans treated with chitosan [24]. These findings indicated that such treatments could slow down the decline in the levels of nutrients, including the total soluble sugar, vitamin C and sucrose. As a result, these fruits could maintain higher nutritional quality.

Based on the above results, it might be inferred that ε-PL displays better efficacy in postponing decay and preserving the storage quality in guavas, accompanied by higher levels of fruit storability, appearance quality and nutritional quality. Upon further comparison, the ε-PL concentration of 2 g/L was the most effective in maintaining the guava fruit storage quality. Moreover, 2 g/L ε-PL extended the quality maintenance period of guava fruit by approximately 9 days, as determined by the indices of fruit storability and quality. Similarly, our previous study demonstrated that 100 μmol/L melatonin could extend the storage time by about 28 days in cold-stored guava fruit [6]. Therefore, this concentration of ε-PL was selected for further investigation regarding its capacity to retard decay and retain the storage quality in guavas by boosting the fruit’s antioxidant abilities.

ROS are inevitably generated in fruit, and their production and clearance systems maintain homeostasis. However, this balance is disrupted under external stresses, leading to the accumulation of ROS [27]. ROS and the oxidative damage induced by them are responsible for decay in produce [11,28]. Many researchers have shown that raised ROS levels, including O₂^−. or H₂O₂, can induce serious oxidative damage and reduce the storage performance of produce [12,29]. In addition, the postharvest decay of produce may involve lipid peroxidation. This process leads to membrane integrity loss, as evidenced by elevated levels of MDA and REL [11,29]. In the present research, compared with the control guavas, ε-PL-treated guavas exhibited lower levels of ROS, such as O₂⁻. (Figure 6A) and H₂O₂ (Figure 6B). Additionally, the level of MDA in the ε-PL-treated guavas increased at a slower rate (Figure 6C). Likewise, ε-PL-treated guavas demonstrated delayed decay symptoms. Their storability was enhanced, and they maintained a better appearance as well as higher nutrient quality (Figure 1, Figure 2, Figure 3, Figure 4 and Figure 5). Therefore, these findings suggested that ε-PL was likely to lessen the accumulation of ROS and restrict lipid peroxidation. This led to reduced oxidative damage and a stable membrane structure, thereby delaying the decay process and improving the storage quality of guava fruit. Similarly, Li et al. [26] showed that the EBR-suppressed decay incidence and improved quality of table grapes were due to lower MDA and ROS levels. Furthermore, Wu et al. [30] reported that carboxymethyl chitosan might decrease decay while maintaining quality in grapefruit. This effect was attributed to decreased ROS levels and inhibited oxidative injury.

Scavenging excessive ROS in the host is essential in retaining a steady state of ROS [28]. Antioxidant enzymes are key in eliminating ROS and thus maintaining the balanced production and clearance of ROS [9,28]. Generally, POD, APX, SOD and CAT are significant antioxidant enzymes that reduce the levels of ROS and then relieve the oxidative damage caused by ROS [12,28,31]. O₂^−. is rapidly converted into the more stable H₂O₂ through the action of SOD [15], while CAT, APX and POD can further reduce H₂O₂ [8,12]. It had been shown that higher antioxidant enzyme activity could help to decrease ROS levels, alleviate lipid peroxidation and accordingly slow down decay development in products [8,32,33]. In the present research, the raised activities of SOD (Figure 7A), CAT (Figure 7B), APX (Figure 7C) and POD (Figure 7D) were seen in guava fruit during the early storage period, which might had been due to a stress response to elevated ROS like O₂^−. and H₂O₂ (Figure 6A,B). Meanwhile, the rapid elevation in the ROS levels in guavas during the middle and late storage periods was attributed to the attenuated antioxidant enzyme activity, which led to membrane damage and subsequent quality deterioration in the fruit. Additionally, compared to the control group, the ε-PL-treated group exhibited higher activities of antioxidant enzymes, including SOD (Figure 7A), CAT (Figure 7B), APX (Figure 7C) and POD (Figure 7D), but lower values of ROS (Figure 6A,B) and MDA (Figure 6C). Moreover, ε-PL-treated guavas also displayed suppressed decay, improved storability and a better appearance and nutrient quality than control guavas (Figure 1, Figure 2, Figure 3, Figure 4 and Figure 5). Thus, ε-PL could be effective in eliminating superabundant ROS through improving the antioxidant enzyme activity. We inferred that the increments in the activities of the above enzymes might help to boost the antioxidant ability and inhibit oxidative damage, reducing lipid peroxidation and retaining the membrane structure. As a result, this process could attenuate the guava fruit’s decay and improve its storage quality. Therefore, ε-PL played a pivotal role in enhancing the antioxidant levels of guavas, resulting in good storage quality. Similarly, methyl jasmonate might alleviate the overproduction of ROS by promoting APX, SOD and CAT activities, suppressing chilling injury in prune fruit [34]. Moreover, melatonin promoted the activities of enzymes related to enhancing the antioxidative levels, thus reducing ROS accumulation and maintaining the storage performance in citrus fruit [35].

The mechanism by which ε-PL slows down decay and stabilizes the storage quality in guava fruit, through raising the fruit’s antioxidant ability, is illustrated in Figure 8. Specifically, ε-PL is capable of enhancing the SOD, CAT, POD and APX activities in guava fruit, thereby improving its antioxidant capacity. This, in turn, reduces the accumulation of ROS and oxidative damage induced by ROS, ultimately retarding lipid peroxidation. Collectively, these effects contribute to delaying the decay and stabilizing the storability, appearance quality and nutritional quality of the fruit. However, the potential mechanisms, particularly those at the molecular level, by which ε-PL suppresses decay and preserves the storage quality of guavas remain unclear. Thus, in the future, it is necessary to investigate the molecular mechanisms underlying ε-PL’s enhancement of guava fruit’s quality through omics-based analyses.

5. Conclusions

Therefore, during storage, ε-PL under different conditions exerted impacts in terms of postponing decay occurrence and preserving the storage quality of guava fruit. This was accompanied by better fruit storability, as well as higher appearance quality and nutritional quality. In particular, 2 g/L was the optimal level of ε-PL for guava fruit. Additionally, the guavas treated with 2 g/L ε-PL exhibited higher activities of CAT, APX, POD and SOD, but lower levels of MDA, H₂O₂ and O₂^−. Therefore, ε-PL was able to boost the fruit’s antioxidant abilities, effectively reducing the ROS levels and hindering lipid peroxidation. This led to the protection of the cell membrane’s integrity. As a result, ε-PL significantly delayed the progression of decay and effectively maintained the storage quality of guava fruit. Thus, ε-PL, as a food additive, has good application prospects in terms of stabilizing the quality of produce in the food industry. Moreover, owing to its remarkable safety and high effectiveness, ε-PL can play a pivotal role in preserving the quality of guavas and other subtropical or tropical fruits, and this work provides a valuable reference for the practical production of such fruits. Further research is necessary to analyze the sensory properties of guava fruit and confirm the absence of off-flavors and other defects resulting from treatment with ε-PL.