Synergistic Utilization of ε -Polylysine and p-Coumaric Acid as Natural Preservatives for Enhancing the Shelf Life of Fresh-Cut Green Bell Peppers

Te utilization of natural preservatives presents a promising avenue for mitigating the spoilage of fresh-cut fruits and vegetables induced by microorganisms, enzymatic browning, and water loss. We have developed an innovative method for preserving fresh-cut green peppers using the combined efects of ε -polylysine ( ε -PL) and p-Coumaric acid (p-CA). Trough concentration screening experiments, we determined that the optimal concentrations of ε -PL and p-CA were 25mg/L and 10mg/L, respectively ( ε -p-CA). Treatment with ε -p-CA signifcantly improved the quality of fresh-cut green peppers. It efectively reduced hardness and weight loss, preserving the texture and appearance of the peppers. Furthermore, ε -p-CA treatment delayed the increase in respiratory rate, electrolyte leakage, and ethylene production, thereby maintaining the structural integrity. Meanwhile, ε -p-CA treatment efectively inhibited the malondialdehyde (MDA) content increase and maintained DPPH radical scavenging activity. Te microbial analysis demonstrated the ε -p-CA-treated peppers also showed lower total bacterial, mold, and yeast counts, which prolonged the freshness of fresh-cut peppers. In addition, ε -p-CA treatment improved the retention of phenolics and vitamin C without signifcantly afecting the color and soluble sugar content of


Introduction
With the rapid pace and increasing diversity of society, there is a rising demand for precut fruits and vegetables, which are gaining popularity due to their convenience and nutritional benefts [1].Nevertheless, the fragile cellular structure of freshly cut fruits and vegetables, microbial proliferation, enzymatic browning, and moisture loss render them more susceptible to putrefaction and deterioration, thereby curtailing their shelf life.Tis has engendered substantial agricultural wastage and economic losses [2].Henceforth, the development of efcacious and secure preservation techniques for freshly cut agricultural products has emerged as a pivotal quandary within the realm of the food industry [3].Although conventional methods for preserving fruits and vegetables involve the use of chemical preservatives like hydrogen sulfde and sodium benzoate, their efectiveness is limited due to concerns related to food safety, environmental impact, and preservation efcacy [4].In recent years, the pursuit of developing novel methods for preserving freshly cut fruits and vegetables has emerged as the foremost endeavor of researchers in the relevant scientifc community.Meng et al. employed pressurized argon gas treatment on freshly cut cucumbers to prolong their shelf life [5].Similarly, Elias et al. investigated the application of electron beam irradiation as a preservation technique for fresh raspberries, demonstrating its efectiveness in maintaining quality and extending storage life [6].However, the implementation of these physical preservation techniques often involves substantial expenses and presents hurdles in terms of promotion and widespread adoption [7].
In recent times, there has been a growing focus on approaches that amalgamate natural preservatives with other preservation methodologies.FM Pintos et al. efectively employed ribofavin to prolong the shelf life of fresh-cut green peppers under specifc conditions of 94% relative humidity and a temperature of 4 °C [8].Ali et al. successfully utilized Lcysteine to retard the enzymatic browning and decay of lychee fruits under controlled conditions of 90 ± 5% relative humidity and a temperature of 5 ± 1 °C [9].Concurrently, we have observed the potential of ε-PL and p-CA as natural compounds with antibacterial and antioxidative properties.ε-PL, a poly-L-lysine polymer, exhibits a broad-spectrum antibacterial activity against bacteria, yeast, and fungi and has received approvals from regulatory agencies such as the FDA and EFSA as a recognized safe and edible natural preservative [10].Lan et al. demonstrated the remarkable ability of ε-PL to efectively impede the disruption and impairment of cellular membranes caused by Shewanella putrefaciens, indicating its potent membrane-stabilizing properties [11].p-CA, a natural phenolic compound found in plants, has a variety of biological activities such as antioxidant, antibacterial, anticancer, and anti-infammatory [12,13].Ismail et al. demonstrated the robust efcacy of the p-CA in efectively scavenging free radicals, making it a valuable tool for mitigating or inhibiting lipid oxidation in food matrices and preventing the formation of detrimental oxidation byproducts [14].Despite ε-PL being acknowledged as a natural antimicrobial agent and p-CA possessing potent antioxidant characteristics, there is a scarcity of literature addressing the synergistic utilization of these two components for the preservation of fresh-cut fruits and vegetables.
In the present investigation, fresh-cut bell peppers were subjected to a treatment involving the combined application of ε-PL and p-CA.It is widely recognized that the storage limit for freshly cut green peppers is 7 days at 7 °C [15].Meanwhile, storing fresh-cut bell peppers at temperatures below 7 °C can induce physiological and metabolic perturbations, compromising membrane integrity and impairing disease resistance capabilities [16].In order to counteract the adverse efects of low-temperature-induced cold injury and excessive moisture loss on the quality of fresh-cut bell peppers, we implemented storing them under controlled conditions of 8 ± 1 °C temperature and 92 ± 2% relative humidity.Te preservation efcacy of bell peppers was investigated with regard to their physicochemical attributes, nutrient profle, and microbial population, aiming to ofer a practical guideline for preserving the freshness of fresh-cut bell peppers.

Concentration Optimization of ε-PL.
To explore the impact of varying concentrations of ε-PL on the freshness of fresh-cut green bell peppers, the peppers were subjected to sequential processes of deionized water washing, gentle drying, and immersion in a sodium hypochlorite solution of 100 mg/L for a duration of 1 min.Subsequently, the embryos and seeds of the peppers were removed, and the remaining fesh was sliced longitudinally with a sharp knife into strips measuring 2.5 × 1.5 cm.From each experimental group, 30 slices of green bell pepper (about 5-7 grams per slice) were chosen and immersed in ε-PL solutions (25 °C) with concentrations of 0, 5, 10, 15, 20, and 25 mg/L for 5 min.Following that, the samples were gently air-blown for drying and then arranged in white polypropylene trays (L × W × H � 335 × 255 × 4 mm).To mitigate excessive carbon dioxide buildup, a total of 20 evenly spaced 2 mm diameter air permeation holes were punctured above the cling flm-covered trays.Finally, the trays were placed in a refrigerated cabinet maintained at a controlled temperature of 12 ± 1 °C and relative humidity of 92 ± 2% for a duration of 10 days (Figure 1(a)), during which the incidence of decay was meticulously documented.Te incidence of decay was evaluated using a hedonic four-level (0-3) intensity scale (0 � healthy individuals; 1 � the presence of brown spots less than or equal to 2; 2 � the number of brown spots greater than 2; 3 � the presence of obvious mold spots with visible mycelial growth).Te incidence of decay was calculated by the formula  � (2, 3 number)/total number, and the experiment was repeated three times.

Concentration Optimization of ε-p-CA.
To further examine the infuence of varying concentrations of p-CA on the freshness of fresh-cut bell peppers, the optimal ε-PL concentration, as determined in Section 2.2, was employed.Fresh-cut peppers were immersed in a composite solution containing p-CA and ε-PL at concentrations of 0, 5, 10, 15, 20, and 25 mg/L, respectively, for a duration of 5 min.Subsequently, the samples were subjected to gentle air blowing for drying and stored in a freezer maintained at a temperature of 12 ± 1 °C and a relative humidity of 92 ± 2% for a duration of 10 days (Figure 1(a)).Troughout this storage period, the incidence of decay was recorded.

Determination.
Based on the results of the preliminary screening, the formulation consisting of an ε-PL concentration of 25 mg/L and a p-CA concentration of 10 mg/L (pH � 6.2) was selected as the optimal combination.Te samples were placed in polypropylene trays and covered with perforated cling flm, ensuring proper ventilation.Subsequently, they were stored under controlled conditions of 8 ± 1 °C and 92 ± 2% relative humidity (Figure 1(b)).Sampling was conducted on days 0, 3, 6, 9, and 12 to evaluate various physical and chemical properties including weight loss, hardness, color variation, electrolyte leakage, malondialdehyde content, respiration rate,

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Journal of Food Biochemistry ethylene content, and antioxidant capacity.In addition, the nutrient composition including soluble sugars and Vc was analyzed.Finally, microbial enumeration was performed to evaluate the presence of bacteria, mold, and yeast.

Weight Loss.
To explore the impact of ε-PL and p-CA on the water loss of fresh-cut green bell peppers, weight loss experiments were performed.Te trays containing the freshcut green bell peppers were weighed at specifc time points, including days 0, 3, 6, 9, and 12. Te rate of weight loss was then calculated using the following equation: where m 1 denotes the initial weight of the sample, m t denotes the weight of the sample on days 0, 3, 6, 9, and 12, and m 0 denotes the weight of the tray.Tree replicate measurements were performed for each group of samples.

Hardness.
To examine the efect of ε-PL and p-CA on the tissue properties of fresh-cut green bell peppers, the hardness was measured using a texture analyzer (TA.XT.plusSMS UK).Puncture testing was carried out on the pepper's peel using a P2 probe at a controlled speed of 1 mm/s and a strain setting of 80%.A total of 30 measurements per sample group were taken, with fve random measurements per pepper.

Color.
To study the infuence of ε-PL and p-CA on the hue of freshly sliced green bell peppers, a random evaluation of the color of the pepper samples was conducted employing the colorimeter (NH310, TreeNH Technology, Shenzhen, China) according to the Yildiz et al. method.A total of 30 measurements were carried out for each group.L * is defned as luminosity, a * (±red-green) signifes the redness, and b * (±yellow-blue) conveys the yellowness [17].Te overall color variation (ΔE) is computed using the following equation: where ΔL, Δa, and Δb are the color parameter diferences between samples and standard samples in each group.

Electrolyte Leakage (EL).
To explore the impacts of ε-PL and p-CA on the permeability of cell membranes in fresh-cut green bell pepper, the measurement of electrolyte leakage was carried out using a conductivity meter (DDS-307, Yidian Science, Shanghai, China).With slight modifcations based on the approach by Ali et al., a beaker was flled with 50 mL of distilled water, followed by the addition of 25 g of green pepper slices [18].Te mixture was then incubated at 25 °C for 20 min.After incubation, the initial solution conductivity E 0 was measured.Subsequently, the beaker containing the sample was boiled for 15 min, allowed to cool down to 25 °C, and the fnal solution conductivity E 1 was measured.Each experimental group consisted of three replicates.Te electrolyte leakage was determined using the subsequent equation: 2.3.5.Malondialdehyde (MDA) Content.Malondialdehyde (MDA) is a commonly used biomarker for lipid peroxidation, providing a means to assess the extent of oxidative damage to cell membranes in various biological systems [19].Following the methodology described by Ali et al. [18], Journal of Food Biochemistry Initially, 10 g of green bell pepper samples were obtained and homogenized using a tissue mashing homogenizer (JJ-2, Guohua Electric, Changzhou, China) after the addition of 50 mL of trichloroacetic acid solution with a concentration of 0.1 g/mL.Te resultant mixture was then centrifugation at 10, 000 × g for a duration of 20 min at a temperature of 4 °C.Subsequently, 4 mL of the supernatant was combined with an equal volume of thiobarbituric acid solution at a concentration of 6.7 g/L.Te mixture was incubated in a boiling water bath for 20 min, followed by cooling and another round of centrifugation for 10 minutes.Te absorbance of the supernatant was measured at 450 nm, 532 nm, and 600 nm using a double-beam UV-visible spectrophotometer (TU-1901, Beijing Puxi General Instrument Co., China), and the measured absorbance values were recorded.Te MDA content was calculated using the following formula: where V t represents the total volume of the extract, V s denotes the volume of the extract utilized for the reaction, and m signifes the mass of the sample.Each sample group underwent three replicates.

Respiration Rate and Ethylene Content.
Te efects of ε-PL and p-CA on the freshness of fresh-cut bell peppers were evaluated by measuring the respiratory rate and ethylene production.Following the methodology described by Prasad et al. [20], approximately 80 g of pepper samples were placed into a 500 mL beaker, and sealed with cling flm.Te beaker was then incubated at 25 °C for 20 min.Subsequently, 30 mL of gas was accurately extracted from the beaker using a C 2 H 4 /O 2 /CO 2 analyzer (F-940, Yangguangyishida, Beijing, China) to determine the CO 2 content.Te obtained results were expressed as the mass of CO 2 generated per kilogram of fresh weight within a 1 h timeframe (mg).Afterward, the C 2 H 4 /O 2 /CO 2 analyzer was calibrated using an ethylene tube, and the same procedure was repeated to determine the amount of ethylene in the beaker.Te ethylene content was expressed as the volume fraction (μL) of ethylene produced per kg of fresh weight in 1 h.Tree replicate measurements were performed for each group of samples.

DPPH (2,2-Diphenyl-1-Picrylhydrazyl) Free Radical
Scavenging Activity.Te efects of ε-PL and p-CA on the antioxidant activity of fresh-cut bell peppers were evaluated using the DPPH assay [1]. 10 g of samples were acquired and ground.Te resulting homogenate was then centrifugation at 16000 × g for 30 min at 4 °C.Subsequently, 0.02 mL of the supernatant was combined with 7.8 mL of a methanol solution containing DPPH at a concentration of 25 mg/L.Te mixture was thoroughly shaken and allowed to incubate for 30 min.Te absorbance of the reaction solution was measured at 517 nm.Te DPPH radical scavenging rate was determined using the subsequent equation: where A DPPH represents the absorbance of the DPPH-methanol solution, while A s denotes the absorbance of the DPPHmethanol solution when mixed with the green pepper solution.

Total Phenolic Content (TPC).
To examine the efect of ε-PL and p-CA on the antinutritional and antioxidant properties of fresh-cut bell peppers, the total phenolic content was assessed utilizing the Folin-Ciocalteu reagent method [21].A 5 g sample of green peppers frozen in liquid nitrogen was taken and ground.Incorporate this fne powder into a solution consisting of 30 mL of 95% ethanol, extract it for a duration of 5 min, and then adjust the volume to 50 mL.Subsequently, the obtained extract was centrifugation at 15000 × g for 10 min.A volume of 0.6 mL of the resulting supernatant was combined with 4.5 mL of Folin-Ciocalteu solution with a concentration of 1 mol/L and incubated for 5 min.Following this, 0.6 mL of Na 2 CO 3 solution with a concentration of 10% was added and allowed to react for 60 min under light-protected conditions.Te absorbance at a wavelength of 765 nm was measured, and the total phenolic content was calculated using the gallic acid standard curve: where V T is the total volume of the extract, C 1 is the total phenol content of the standard curve check, V s is the volume of the supernatant taken, and F w is the sample mass.Each group of samples was repeated three times.

Soluble Sugar Content (SSC).
To assess the impact of ε-PL and p-CA on the soluble sugar content in fresh-cut bell peppers, the phenol-sulfuric acid method was employed for its determination [22].Approximately 10 g of green pepper samples were frozen in liquid nitrogen and subsequently ground into a fne powder.Precisely 1 g of the green pepper powder was weighed and combined with 10 mL of water, followed by extraction in boiling water for a duration of 30 min.Extract 0.5 mL of the sample solution and mix it with 1 mL of phenol solution with a concentration of 0.9 g/L and 5 mL of concentrated sulfuric acid.Allow the mixture to stand at room temperature for a duration of 30 min, following which the absorbance of the resulting solution is measured at a wavelength of 485 nm.Calculation of soluble sugar content using the sucrose standard curve: where m ′ is the mass of sucrose (μg) from the standard curve, V is the total volume of sample extract (mL), N is the dilution multiple, V s is the volume of extract (mL) taken for the determination, and m is the mass of sample (g)

Vitamin C Content (Vc).
To study the impact of ε-PL and p-CA on the vitamin C (Vc) content in fresh-cut bell peppers, the 2,6-dichlorophenol indophenol method was employed for its quantifcation [23].A quantity of 10 g of green pepper was taken and combined with 10 mL of a 2% oxalic acid homogenate, which was then diluted to a fnal volume of 50 mL.Subsequently, 5 mL of the resulting extract was transferred into a conical fask, and the 2,6-dichlorophenol indophenol extract was added dropwise from a microburette until a persistent red color emerged without any further color change within a duration of 15 seconds.Te volume of the extract required for the titration was recorded.Te Vc content was calculated using the subsequent equation: where K represents the titration factor, V 1 denotes the volume of 2,6-dichlorophenol indophenol solution consumed during the titration of the sample (mL), V 0 signifes the volume of 2,6-dichlorophenol indophenol solution consumed during the blank titration (mL), V s represents the volume of the sample liquid used in the titration (mL), and m signifes the mass of the sample (g).Each sample group underwent three replicate measurements.

Microbiological Analysis.
Microbial analysis serves as a crucial indicator in assessing the quality of freshly sliced green bell peppers.To assess the impact of ε-PL and p-CA treatment on the microbial quality of fresh-cut green bell peppers, a quantitative analysis of bacterial, mold, and yeast populations was conducted using the plate count method, following the National Standard of the People's Republic of China [24].25 g of the sample within aseptic pouches, infusing them with 225 mL of sterile physiological saline solution, boasting a concentration of 0.85 g/100 mL, and agitate the mixture for a duration of 3 min.Subsequently, enact a tenfold serial dilution of the solution, aspirating 0.1 mL of the diluted solution and introducing it into beef extract peptone agar medium (for aerobic bacteria) and chloramphenicol yeast glucose agar medium (for molds and yeast).Tese cultures shall then be incubated at 37 °C for 3 days and 25 °C for 5 days, respectively.Repeat three times for each set of samples.
2.6.Statistical Analysis.Data analysis was conducted using SPSS Statistics 25 software, and the results were presented as the mean ± standard deviation.Subsequently, the data were subjected to a one-way analysis of variance (ANOVA), followed by post hoc analysis using Duncan's multiple range test.Statistical signifcance was considered at a threshold of P < 0.05.

Results and Discussion
3.1.Impact of ε-PL and p-CA Concentrations on the Quality of Green Bell Pepper.To explore the infuence of diferent concentrations of ε-PL on the preservation efcacy of fresh-cut green bell peppers, screening experiments were carried out (Figure 2(a)).Treatment groups with ε-PL concentrations of 0, 5, 10, 15, 20, and 25 mg/L were used to observe statistics on the incidence of decay in fresh-cut bell peppers.Over the course of day 10, the decay rate of green bell pepper slices exhibited a progressive decline in tandem with the elevation of ε-PL concentration.In the control group, the incidence of decay reached a staggering 43%.On the cross-sections of freshly sliced green bell peppers, a profuse leakage of nutrients occurred, thereby providing the essential sustenance for microbial proliferation and consequently hastening the deterioration of the pepper's quality.Simultaneously, we also observed that even when the concentration of ε-PL was between 5 mg/L and 10 mg/L, a notably high decay rate persisted, with no discernible diferences compared to the control group.As the concentration of ε-PL is further augmented, the incidence of decay undergoes a pronounced reduction.Te preservation outcome was found to be the most favorable at a concentration of 25 mg/L, leading to a remarkable decay rate of only 20%.Owing to its expansive spectrum of bacteriostatic properties, ε-PL profciently suppresses enzyme activities associated with both bacterial respiratory and cellular metabolism.Tis dual inhibition not only curtails bacterial cellular growth but also heightens cellular membrane permeability, ultimately culminating in cellular demise [11].Te fndings indicated that the ε-PL concentration of 25 mg/L exerted a notable efect in mitigating the decay of fresh-cut green bell pepper, thus enhancing its preservation of freshness.Te infuence of p-CA concentration on fresh-cut green bell peppers was studied while maintaining a constant ε-PL concentration of 25 mg/L (Figure 2(b)).Te incidence of decay within each treatment group was 20%, 16.7%, 11%, 12%, 15.7%, and 22% with the addition of p-CA at 0, 5, 10, 15, 20, and 25 mg/L.Te results suggested that as the p-CA concentration increased, the incidence of decay in fresh-cut green bell pepper initially exhibited a decline and subsequently displayed an upward trend.Upon reaching a concentration of 10 mg/L, p-CA demonstrated the lowest incidence of decay.Tis phenomenon can be attributed to the capacity of an optimal p-CA concentration to efectively counteract the generation of free radicals during metabolic processes and delay the formation of harmful oxidation products [13].Concurrently, the presence of appropriate ε-PL inhibited the growth and proliferation of microorganisms, contributing to the preservation of freshness [25].Nevertheless, an elevation in p-CA concentration beyond the optimal level triggered an interaction between polyphenol oxidase (PPO) present on the surface of the peppers and p-CA, leading to the generation of black pigments in the Journal of Food Biochemistry presence of oxygen.Tis undesirable consequence intensifed the browning process in fresh-cut bell peppers, ultimately compromising their overall freshness [26].

Freshness Preservation
Performance.Te quality of fresh-cut green bell peppers depends on their own oxidative respiration, microbial infection, and moisture loss [2].As depicted in Figure 3, during the entire storage duration, the three groups of samples exhibited no conspicuous disparities in their outward appearance of the preceding day 6, closely correlated with suitable storage temperatures and elevated relative humidity.However, the control group began manifesting evident alterations in appearance on day 9, displaying browning lesions on the cut surfaces and extensive rot and fungal growth on day 12.In the absence of any treatment, the fragile cellular structure of the crosssections of fresh-cut bell peppers led to the leakage of nutrients.Concurrently, microbial attachment and proliferation further weakened the pepper's innate defenses, resulting in a rapid decline in quality during the later stages of storage.Previous studies have reported a maximum shelf life of day 7 for freshly sliced green bell peppers, consistent with the fndings of this study [15].Notably, the ε-PL group exhibited a certain degree of lesion development on day 12, whereas the ε-p-CA group displayed only minor blemishes.Tus, it is visually apparent that the combined treatment of ε-PL and p-CA signifcantly mitigates the deterioration of the quality of freshly sliced green bell peppers.

Physical and Chemical Properties
3.3.1.Weight Loss. Figure 4(a) illustrates the weight loss rate of green bell pepper slices throughout the storage period.On day 3, exhibited no signifcant disparity among the three groups exhibited no signifcant disparity among the three groups.As time elapsed, starting from day 6, the weight loss rate in the control group demonstrates a noteworthy disparity when compared to the ε-PL group (2.71%) and the ε-p-CA group (2.56%).Tis considerable increase in weight loss signifes a rapid deterioration in the quality of green pepper slices.On day 9, the weight loss rate in the control group had reached 6.39%, exceeding the reported acceptable limit of 5% for fresh-cut green pepper consumers [15], while the ε-p-CA group maintained a weight loss rate of 4.32%, well within the acceptable range for consumers.On day 12, the ε-p-CA group displayed the lowest weight loss rate of 7.39%, which was signifcantly diferent from the weight loss rates of 9.14% in the ε-PL group and 10.58% in the control group (P < 0.05).Te fndings reveal that all experimental groups exhibited a decline in weight during the storage period, primarily attributed to intrinsic oxidative metabolism and transpiration dehydration processes.However, the ε-p-CA group demonstrated the minimal weight loss, likely attributed to p-CA's role in scavenging reactive oxygen species on the surface of fresh-cut green bell pepper, thereby mitigating metabolic reactions [27].Concurrently, ε-PL efectively inhibits microbial attachment and growth, thus retarding the consumption of nutrients [25].4(b), throughout the entire storage period, the hardness of the control group gradually diminished, with a more pronounced decline commencing on day 3.In contrast, both the ε-PL and ε-p-CA groups exhibited a slight decrease in hardness during the storage period.On day 6, the hardness of the control group measured (4.55 ± 0.24) N, while the ε-p-CA group recorded (4.84 ± 0.19) N, signifying a signifcant disparity, akin to the observations made by Ranjeet Singh in his research [23].Te swift decline in hardness within the control group can be attributed, on the one hand, to the substantial consumption of its intrinsic nutrients, leading to the disintegration of its tissue  structure.On the other hand, the lack of inherent immunity, coupled with microbial intrusion in the pepper slices, further expedited the consumption of nutrients and the evaporation of moisture.On day 12, the control group's hardness had declined by 21.2%, whereas the ε-PL and ε-p-CA groups had decreased by 7.6% and 6.0%, respectively.Chen et al. used chitosan-catechin coating to treat satsuma oranges and also observed a progressive decrease in the hardness of sweet oranges during storage [28].Tis is likely due to the attachment of ε-PL and ε-p-CA to the surface of the bell peppers, retarding the oxidative respiration process responsible for the depletion of intrinsic nutrients and aligning with the measured weight loss indicators.Te foregoing results underscore the potential of ε-PL and ε-p-CA as preservatives for fresh-cut bell peppers, serving as additives for food preservation.

Color.
Te color values of green pepper slices throughout the storage period are presented in Table 1.Regarding the brightness (L * ) parameter, no signifcant disparity was observed between the control group and the treatment groups at the commencement of storage.A slight elevation in brightness was discernible on day 3, followed by consistent maintenance of this level throughout the storage period.Te ε-p-CA group showed a slight decrease of 1.25 units on day 6.On day 12, no signifcant distinction in brightness was observed among the three sample groups (P > 0.05).Regarding the red-greenness (a * ) parameter, all three sample groups fuctuated slightly during the storage period, maintaining values within acceptable ranges.Notably, the ε-p-CA group displayed an increase of 0.81 units on day 6, indicating a lesser decline in green color intensity.Similarly, Pintos et al., in their study using ribofavin in the treatment of fresh-cut green peppers, also found that the color diference  of green pepper slices did not change signifcantly during the entire storage period [8].Tis outcome can be attributed to the degradation of chlorophyll resulting from factors such as oxygen presence and chlorophyllase enzymes.Te reduction in enzyme activity at lower temperatures contributes to the delayed alteration in color [29].Regarding the yellow-blue (b * * ), the control group slightly decreased throughout the storage period, while the treatment group showed an increase followed by a decrease.However, no signifcant distinction was observed between the treatment and control groups on day 12 of storage (P > 0.05).Te total color diference (ΔE) gradually increased overall.Notably, a signifcant rise was observed in the ε-p-CA group on the sixth day, which remained relatively stable thereafter.Te ε-p-CA group had less efect on the color of the peppers, and their color remained within acceptable parameters throughout the storage period.

Electrolyte Leakage.
Electrolyte leakage serves as a quantitative parameter that refects the extent of cellular damage in plants and exhibits a correlation with the quality of fruits and vegetables throughout the storage duration [18].As depicted in Figure 5(a), the electrolyte leakage was marginally higher in the ε-p-CA group (5.95 ± 0.21) % compared to the control group (5.77 ± 0.24) % at the commencement of the storage period.Tis discrepancy could potentially be attributed to the presence of residual substances on the surface of the peppers subsequent to the combined treatment of the two compounds, although the disparity observed was not statistically signifcant (P > 0.05).On day 6, there was a notable increase in electrolyte leakage in the control group, reaching (8.16 ± 0.14) %, which was signifcantly higher compared to the ε-p-CA group, where the electrolyte leakage was (7.22 ± 0.16) %.It is manifest that the control group evinces an augmentation in cellular membrane permeability, culminating in the substantial efux of intracellular substances, thereby hastening the deterioration of the quality of bell pepper slices.On day 12, the ε-p-CA group exhibited a 33.6% reduction in electrolyte leakage compared to the ε-PL group and a remarkable 92.8% reduction compared to the control group.Tis indicates that the combined treatment of the two drugs is more efective.Pintos et al. similarly reported an elevation in electrolyte leakage in fresh-cut green bell peppers as the duration of storage increased [8].Higher values of electrolyte leakage indicate compromised cell integrity.Te fndings demonstrated that ε-p-CA exhibited efective inhibition of the cell membrane permeation rate in fresh-cut peppers, thereby preserving the tissue integrity of the peppers more efectively.

MDA Content.
MDA is one of the fnal products of lipid peroxidation in cell membranes, possesses direct cytotoxicity, and contributes to the exacerbation of cell membrane damage.It serves as a crucial indicator for evaluating the extent of oxidative stress in green pepper samples [19].As shown in Figure 5(b), the MDA content exhibited an upward trajectory in all three sample groups throughout the storage period, and a similar trend was observed for fresh-cut green bell peppers by Chen et al. [30].At the onset of storage, the MDA content in all three sample groups was approximately (0.19 ± 0.015) μmoL/g.On day 3, there was a substantial surge in the MDA content of the control group to (0.36 ± 0.015) μmoL/g, which exhibited a signifcant disparity when compared to the ε-p-CA group's MDA content of (0.27 ± 0.013) μmoL/g (P < 0.05).In the cross-section of freshly sliced green peppers, the accumulation of free radicals and reactive oxygen species leads to the escalating oxidation of cellular membrane lipids.On day 9, there was a notable escalation in MDA levels within the ε-PL group, displaying a signifcant distinction from the ε-p-CA group (P < 0.05).On day 12 of the storage period, the MDA content in the control group surged to (0.98 ± 0.02) μmoL/g, indicative of severe peroxidative damage to cell membrane lipids.In contrast, the ε-PL and ε-p-CA groups exhibited MDA contents of (0.58 ± 0.018) μmoL/g and (0.39 ± 0.011) μmoL/g.In comparison to the ε-p-CA group, the MDA content in the control group was 2.5 times higher, while in the ε-PL group, it was 1.49 times higher.Increased MDA content is also linked to the impairment of membrane Vertical bars show the standard error of the means and diferent letters at the same storage time are signifcantly diferent at P < 0.05.

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Journal of Food Biochemistry permeability and the accumulation of reactive oxygen species [15].Tese fndings suggested that the synergistic application of ε-p-CA could efectively suppress the buildup of MDA and mitigate the extent of membrane peroxidation damage in green bell pepper fruits throughout the storage period.

Respiratory Rate.
Respiration rate, as an indicator of metabolic activity, refects changes in the physiological metabolism of the sample [31].As depicted in Figure 6(a), the respiration rate of the three groups of samples exhibited a progressive elevation throughout the storage period.Tis efect can be attributed to the induced stress resulting from the mechanical injury incurred during the cutting process, which subsequently triggers an accelerated respiratory activity in fresh-cut peppers [30].Respiratory rates were generally consistent between control and treated groups during the initial day 0 of storage.However, commencing from day 6, there was a sharp increase in respiratory rate in the control group and a mild increase in the ε-p-CA group.On day 12 of storage, there was a substantial surge of 163% in the respiration rate of the control group, accompanied by a 140% increase in the ε-PL group, both in comparison to the initial storage period.In contrast, the ε-p-CA group exhibited a noteworthy reduction of 56.4% compared to the control group.Te fndings suggested that ε-p-CA has a signifcant impact on modulating the respiration rate in fresh-cut peppers, leading to a reduction in oxidative metabolism.Simultaneously, the application of ε-PL and p-CA on the surface of chili exhibits a signifcant inhibitory efect on microbial respiration and cellular metabolism, retarding microbial intrusion into the pepper slices and preserving tissue integrity [11].

Ethylene Content.
Fresh-cutting procedures induce the generation of wound ethylene, and wound ethylene can accelerate the ripening and aging of fruits and vegetables [32].Figure 6(b) illustrates the variations in ethylene content in fresh-cut green bell pepper throughout the 12-day storage duration.During the initial day 6 of storage, both the treated and control groups displayed a moderate increase in ethylene content, and there was no signifcant diference observed between them (P < 0.05).On day 9, there was a substantial rise in ethylene content across all three groups, with the control group exhibiting (12.79 ± 0.70) μL/kg/h, the ε-PL group displaying (10.82 ± 0.69) μL/kg/h, and the ε-p-CA group demonstrating the lowest level at (9.84 ± 0.70) μL/kg/h.On the 12th day of storage, the ethylene content of the control group exhibited a signifcant increase to (18.95 ± 0.61) μL/kg/h, which was 4.76 μL/kg/h higher than that of the ε-PL group and 6.23 μL/kg/h higher than that of the ε-p-CA group.Tese fndings indicate that the treatment of fresh-cut peppers with ε-p-CA efectively suppresses ethylene production, leading to better preservation of the quality of fresh-cut peppers.Tis may be attributed to the efective suppression of the metabolic activity of ethylene synthase by ε-p-CA, resulting in restricted ethylene biosynthesis pathways [33].

DPPH Scavenging Activity.
Free radicals within cells induce harm and disruption to macromolecules such as nucleic acids and proteins, while the accumulation of free radicals promotes membrane lipid oxidation and hastens cellular senescence [34].As depicted in Figure 7(a), prior to the commencement of storage, the control, ε-PL, and ε-p-CA groups exhibited free radical scavenging capacities of 83.20 ± 1.67%, 83.40 ± 0.88%, and 83.13 ± 1.29%, respectively, with no statistically signifcant disparity observed among the three groups.Nevertheless, on day 9, the free radical scavenging capacity of the control group exhibited a rapid decline and displayed a signifcant disparity compared to the ε-p-CA group.On day 12 of the storage period, the ε-p-CA group maintained a notably high free radical scavenging capacity of 78.03%, whereas the free radical scavenging capacity of the ε-PL group decreased to 71.70%, and that of the control group reached a mere 65.10%.Tis phenomenon can be attributed to the interaction between p-CA and the free radical (R * ), leading to the transfer of a hydrogen atom from p-CA to the free radical by disrupting the O-H bond.Consequently, this process stabilizes or deactivates the free radical, resulting in the maintenance of a high free radical scavenging capacity within the sample [35].10 Journal of Food Biochemistry 3.3.9.TPC.Phenolic compounds, as signifcant antioxidant constituents, possess numerous functional and active efects on the quality attributes of fruits and vegetables [3].Te TPC of the ε-p-CA group was signifcantly higher than the other two groups at the beginning of storage, owing to the inclusion of p-CA (Figure 7(b)).On day 3 of storage, the TPC experienced a general increase and reached its maximum level in all three sample groups, followed by a subsequent decline.Tese changes are mainly due to the mechanical damage caused by cutting green pepper, which cause an increase in phenylalanine aminolysis enzyme activity, which contributes to a signifcant accumulation of phenolics [36].Starting from day 6, the TPC of the ε-p-CA group exhibited stability, whereas the control and ε-PL groups demonstrated a declining pattern.On day 12, the ε-p-CA group maintained a TPC of (55.80 ± 1.79) mg/100 g, the ε-PL group had (41.43 ± 1.88) mg/100 g, and the control group displayed a signifcantly lower content of (26.07 ± 2.21) mg/100 g (P < 0.05).Tese results indicated that the ε-p-CA treatment efectively preserved a higher TPC in fresh-cut green bell peppers.

Nutritional Composition
3.4.1.SSC.Te alterations in the soluble sugar content of green bell pepper slices are depicted in Figure 8(a).During the initial stages of storage, there was no signifcant disparity in soluble sugar content between the control and experimental groups.However, on day 6, the soluble sugar content in the control group reached its zenith at (2.99 ± 0.05) g/100 g, displaying a pronounced diferentiation from the experimental group.Over the course of time, the soluble sugar content in the control group commenced a decline, while the ε-PL group, after attaining its peak on day 9 at (2.98 ± 0.08) g/100 g, also embarked on a descending trajectory.Tis phenomenon could potentially be attributed to the environmental stress experienced during the storage of fresh-cut bell peppers, which triggers the breakdown of endogenous macromolecules, particularly proteins.Tis process leads to an increased production of soluble sugars [37].However, as the storage duration progressed, the substantial depletion of soluble sugars was observed, attributable to the accelerated respiration rate and heightened electrolyte leakage.It is noteworthy that the ε-p-CA group did not show a decrease in soluble sugar content at the later stages of storage.Tis observation can be attributed to the combined treatment of the ε-PL and p-CA, which exhibited a retarding efect on the activity of cellular metabolic enzymes.Tis efect was closely associated with a lower respiration rate and a reduction in electrolyte leakage [38].

Vc Content.
Te assessment of Vc content stands as a pivotal criterion for appraising the quality of freshly cut green bell peppers.Green bell peppers, favored by consumers for their elevated Vc content, are nonetheless susceptible to oxidation in the presence of oxygen [1].Consequently, Vc content serves as an indicator of the degree of oxidation in freshly cut green bell peppers.As illustrated in Figure 8(b), throughout the entire storage period, the Vc content in the ε-p-CA group exhibits a gradual descent, while the control group experiences a more rapid decline.Te ε-p-CA group displays the most minimal Vc loss at a mere 12.41%, in contrast to the ε-PL group at 17.36% and the control group at 32.23%.Te decline in Vc content not only profoundly impacts the nutritional composition of green pepper slices but also indirectly signifes a weakening of their inherent defensive capabilities.Tis is similar to the trend of Vc content measured in fresh-cut kiwifruit treated by high-intensity sonication in Yıldız [39].In the initial phase of storage, the Vc levels among the three groups measured (82.40 ± 1.12) mg/ 100 g, with no conspicuous disparities.On day 6, the Vc content in the control group swiftly declined, displaying a signifcant contrast when compared to the ε-p-CA group.On day 12 arrived, the ε-p-CA group continued to maintain a certain level of Vc content at (72.21 ± 1.66) mg/100 g, while the control group had only (55.86 ± 1.19) mg/100 g.Te research outcomes signify that ε-p-CA efectively curbs the depletion of Vc in freshly cut green peppers.

Microbiological Analysis
3.5.1.Aerobic Bacteria.In this study, the alterations in the population of aerobic bacteria in freshly cut green bell pepper samples are depicted in Figure 9(a).At the onset of storage, the aerobic bacterial plate count stood at (3.36 ± 0.1) log CFU/g, signifying the exceptional quality of the samples.Following the combined treatment with ε-PL and ε-p-CA, no noticeable alterations in bacterial counts were observed immediately, and all three sample groups showed a gradual increase in aerobic bacterial counts throughout the storage period.Te control group increased sharply from day 6, reaching (4.88 ± 0.05) log CFU/g, whereas the ε-PL and ε-p-CA groups demonstrated a slower increase at (4.25 ± 0.01) log CFU/g and (4.11 ± 0.03) log CFU/g, respectively.Tis fnding is consistent with the results reported by Ranjitha, who observed that the microbial population in freshly cut green bell peppers remained within acceptable limits during storage at 8 °C for day 6 [40].On day 9, the aerobic bacterial count was (6.04 ± 0.04) log CFU/g in the control group, while the ε-PL and ε-p-CA groups had counts of (4.91 ± 0.04) log CFU/g and (4.73 ± 0.07) log CFU/g, respectively.It is worth noting that a limit of 5.0 log CFU/g is commonly used as a tolerance level for evaluating the microbiological quality of ready-to-eat or ready-to-eat fresh-cut green peppers [15].Figure 3, the images of fresh-cut green pepper samples from diferent treatment groups during storage also visually refect the increase in the total number of microorganisms.Te results demonstrated that the treatment with ε-PL and ε-p-CA exhibited signifcant inhibitory efects on the growth and proliferation of aerobic bacteria, resulting in an extended shelf life for fresh-cut green peppers.

Molds and Yeasts.
Te preservation efect of ε-PL and p-CA treatments on fresh-cut green bell peppers was investigated by quantifying mold and yeast populations (Figure 9(b)).Te initial mold and yeast counts in the control group were (3.36 ± 0.04) log CFU/g, which did not exhibit a signifcant diference when compared to the ε-PL group (3.41 ± 0.06) log CFU/g and the ε-p-CA group

Conclusion
Tis study investigated the combined efects of ε-polylysine (ε-PL) and p-Coumaric acid (p-CA) on the preservation of fresh-cut green bell peppers.Trough experiments, the optimal concentration of ε-PL was determined to be 25 mg/L, while a concentration of 10 mg/L of p-CA signifcantly reduced decay and improved preservation efectiveness.Various physical, chemical, and microbiological properties of the peppers were evaluated during a 12-day storage period.Te results demonstrated that the application of ε-p-CA efectively reduced the decay rate of fresh-cut green bell peppers and minimized weight loss throughout the storage period, thereby maintaining the peppers' hardness.Moreover, it efciently inhibited electrolyte leakage and MDA content, suppressing respiration rate and ethylene production associated with aging and deterioration.In terms of nutritional components, ε-p-CA treatments preserved the Vc content and inhibited soluble sugar loss.In addition, they increased the TPC, contributing to antioxidant activity.Microbiological analysis indicated that the combined treatment signifcantly reduced the populations of aerobic bacteria, molds, and yeasts, suggesting antibacterial properties.In conclusion, the combined treatment of ε-p-CA exhibited favorable efects on maintaining the quality and extending the shelf life of freshcut green peppers.Further research is required to fully understand the potential mechanisms of this preservation method and its potential application in other fresh-cut agricultural products.

Figure 1 :
Figure 1: Experimental design and quality change diagram.

Figure 2 :
Figure 2: Efect of ε-polylysine and p-Coumaric acid concentrations on the quality of bell pepper.Efect of ε-polylysine concentration of 0, 5, 10, 15, 20, and 25 mg/L on fresh-cut green pepper fresh preservation (a); efect of ε-polylysine concentration of 25 mg/L and p-CA concentration of 0, 5, 10, 15, 20, and 25 mg/L on fresh-cut green pepper fresh preservation (b).Vertical bars show the standard error of the means and diferent letters at the same storage time are signifcantly diferent at P < 0.05.

Figure 3 :
Figure 3: Photographs of the untreated green pepper slices and ε-polylysine alone and in combination with p-Coumaric acid treated green bell pepper slices during 12 days storage period at 8 °C.

Figure 4 :
Figure 4: During a 12-day storage period at 8 °C, weight loss (a) and hardness (b) changes in green bell pepper slices treated with ε-polylysine alone and in combination with p-Coumaric acid.Vertical bars show the standard error of the means and diferent letters at the same storage time are signifcantly diferent at P < 0.05.

Figure 5 :
Figure 5: During a 12-day storage period at 8 °C, electrolyte leakage (a) and MDA content (b) changes in green bell pepper slices treated with ε-polylysine alone and in combination with p-Coumaric acid.Vertical bars show the standard error of the means and diferent letters at the same storage time are signifcantly diferent at P < 0.05.

Figure 6 :
Figure 6: During a 12-day storage period at 8 °C, respiratory rate (a) and ethylene content (b) changes in green bell pepper slices treated with ε-polylysine alone and in combination with p-Coumaric acid.Vertical bars show the standard error of the means and diferent letters at the same storage time are signifcantly diferent at P < 0.05.

Figure 7 :
Figure 7: During a 12-day storage period at 8 °C, DPPH scavenging activity (a) and total phenolics (b) changes in green bell pepper slices treated with ε-polylysine alone and in combination with p-Coumaric acid.Vertical bars show the standard error of the means and diferent letters at the same storage time are signifcantly diferent at P < 0.05.

Table 1 :
During a 12-day storage period at 8 °C, color (L * , a * , b * , ΔE) changes of green bell pepper slices treated with ε-polylysine alone and in combination with p-Coumaric acid.