Anti-Browning and Oxidative Enzyme Activity of Rice Bran Extract Treatment on Freshly Cut ‘Fuji’ Apple

: Preserving the quality of freshly cut fruits is essential for food marketing. This study aimed to evaluate the anti-browning effect of rice bran extract (RE) added to a vitamin C mixture (VCM), which is widely used as an anti-browning agent. Freshly cut apples were prepared using the following processes: raw material selection, washing, cutting, soaking, and packaging. A comparison of cut apples soaked with 3% VCM combined with 1% RE (VR) and cut apples treated with 4% VCM showed that the L* and BI values had similar levels. After 12 days of storage, the amount of yeast and mold in cut apples after 4% VCM and VR treatment was 6.15 × 10 4 ± 0.48 CFU/mL and 4.25 × 10 3 ± 0.15 CFU/mL, respectively, and the degree of bacterial growth was reduced by VR treatment. On day 12 of storage, the activities of polyphenol oxidase (PPO) and peroxidase (POD) were similar in the 4% VCM and VR groups. The polyphenol content was signiﬁcantly higher in the VR group (121.0 ± 2.2) than in the 4% VCM group (76.9 ± 2.2). These results indicate that VR treatment for freshly cut apples is a potential alternative to 4% VCM treatment, with an effective anti-browning capacity and improved polyphenol content.


Introduction
Recently, the industry for supplying fresh fruits and vegetables has grown rapidly as consumer preference and demand for fresh and healthy foods have increased [1]. Research on efficient preservation methods for freshly cut apples is drawing attention for its potential to reduce the processing costs in the fresh produce industry [2]. Freshly cut apples deteriorate rapidly as a result of enzymatic browning [3], tissue softening [4], and microbial growth [5]. In particular, fleshly cut apples may undergo browning during the production process such as washing, peeling, coring, cutting, and storage, which affects the flavor, texture, and microbial growth of the product [3]. Freshly cut products are, therefore, susceptible to microbial growth, which ultimately shortens the shelf life [6]. Therefore, extending shelf life by preventing surface browning has been a focus in the freshly cut industry in recent decades [7].
There have been many studies on antioxidants such as ascorbic acid, calcium ascorbate [8], sodium chloride [9], and citric acid [10] in relation to browning inhibition. Among them, ascorbic acid was known as a substance with excellent antioxidant activity and is often used to control discoloration [11]. Therefore, commercially anti-browning agents loss, which is essential for assessing the quality of apples [25]. The weight loss value was determined by the moisture loss of all the tested treatment materials. The weights of the apples were individually monitored with an analytical balance on days 0, 3, 6, 9, and 12 during the storage period. The weight loss ratio W (%) was calculated as follows: where mi is the initial weight and mt is the weight at the end of the storage period.

Total Soluble Solid Content and Titratable Acidity
The apple slice per group for total soluble solid content (SSC) and acidity was measured every 3 days for 12 days for the group of DW-, 4% VCM-, and VR-coated apple slices (n = 3). Each SSC was estimated using a digital refractometer (PAL-1; ATAGO, Tokyo, Japan) from apple juice obtained by squeezing apple slices. Each value represents the average of the six independent replicates. SSC measurements were performed on the same fruit portion and expressed in • Brix, and pH was measured with a pH meter (Orion Star A211 pH benchtop meter; Thermo Scientific™; Waltham, MA, USA). The titratable acidity was determined by titrating apple juice with 0.1 M NaOH to pH 8.2. Titratable acidity was calculated using the following equation: Titratable acidity (%) = (E × V_NaOH × 100)/W where E is the equivalent of malic acid, V is the volume of titrant used (NaOH), and W is the sample weight (g).

Total Phenolic Content
The total phenolic content (TPC) for the apple slices in each group was measured every 3 days for 12 days. The TPC of DW-, 4% VCM-, and 3% VCM combined with 1.0% RE (VR)coated apple slices were measured (n = 3). After pressing 15 g of apples, supernatant was filtered using a 0.45µm filter (Z672661; Whatman, Maidstone, UK). TPC was determined as previously described [26]. Folin-Ciocalteu reagent (1 mL) was added to the diluted supernatant (1:9 v/v in distilled water). After 5 min, sodium carbonate (10 mL, 70 g/L) was added, and distilled water was added to make a final volume of 25 mL. The mixture was shaken and left for 2 h in the dark at approximately 20 • C. The phenolic content was calculated from the absorbance at 760 nm using a spectrophotometer (UV-1800; Shimadzu Corp., Kyoto, Japan). The absorbance was compared to a standard curve prepared using gallic acid. The results were obtained in triplicate and expressed as gallic acid equivalent (GAE)/kg fresh weight of ground apples.

Total Protein and Oxidative Enzyme Activity Assays
The apple slices in each group were measured for their total protein and enzyme activity every 3 days for 12 days. The group of DW-, 4% VCM-, and VR-coated apple slices were measured (n = 3). Total protein was analyzed to adjust the protein concentration. PPO and POD activities were tested using a previously reported method [27,28]. Lysis buffer was prepared using a 0.2 M sodium phosphate buffer (SPB) at pH 6.5 containing 4% (w/w) polyvinylpolypyrrolidone and 1% (w/w) Triton X-100. Then, 20 mL of lysis buffer was added to 10 g of homogenized apples for 3 min. After centrifugation at 12,000× g for 20 min, the supernatant was filtered and used as an apple enzyme extract. The protein concentration of the apple enzyme extracts was determined using the Bradford assay [29]. The changes in the PPO and POD activities in the absorbance/min/g FW of the sample were expressed as the activity of the apple enzyme extract. All analyses were performed in triplicate, and a freshly prepared substrate solution was used for each enzyme activity analysis. The substrate solution contained 0.07 M catechol in 3 mL of 0.05 M sodium phosphate buffer (pH 6.5) solution. First, 0.2 mL of the apple enzyme extract was mixed with 1.5 mL of 40 mM/L catechol and 2.3 mL of 0.2 M SPB solution. The absorbance of Agronomy 2022, 12, 86 4 of 12 the mixture was measured immediately at 420 nm for 10 min using a spectrophotometer in the kinetic mode for PPO activity. POD activity was measured using an assay mixture containing 0.5 mL of apple enzyme extract, 2.3 mL of 0.2 M SPB solution, 0.15 mL of 98% Guaiacol, and 0.05 mL of 9.8 M H 2 O 2 . The absorbance of the mixture was measured immediately at 470 nm for 10 min using a spectrophotometer (UV-1800; Shimadzu Corp., Kyoto, Japan) in kinetic mode for POD activity.

Microbial Counts
The microbial counts for the apple slices in each group was measured every 3 days for 12 days. The group of DW-, 4% VCM-, 1% RE-, and VR-coated apple slices were measured (n = 3). To calculate the microbial counts, methods described in previous studies were used [30,31]. Briefly, 10 g of each sample was combined with 90 mL of sterile peptone water in a filter bag (PX0020P; ELMEX, Nakano, Japan). The apples were homogenized in a stomacher for 3 min. Then, 1 mL of the sample solution was placed on an appropriate dried medium. The sample solution was spread on a film (3M Petri film E. coli/Coliform Count Plates; 3M, Saint Paul, MN, USA) and incubated at 35 • C. The surviving E. coli cell population (log CFU/g) was counted after 24 h. The growth of yeasts and molds was determined 3 days after inoculation into the films at 25 • C.

Sensory Test
The sensory quality of the coated freshly cut apples was tested on 34 people aged from 20 to 50 years. Preference evaluation was conducted in five categories: overall acceptability, color, texture, sweet taste, and sour taste. Scores for each quality were graded in a preference range from 1-9. Apples were treated with 4% VCM and 3% VCM + 1.0% RE solutions and stored in a refrigerator for 24 h. Freshly cut apples were marked with random numbers to exclude bias in the evaluation of preference, and the order of each apple was also randomly arranged and evaluated.

Statistical Analysis
The experiment had a dual-factor (treatment × storage period) design with an analysis of variance (ANOVA) using SPSS software (Version 25, IBM, NY, USA). Statistical significance was assessed at p = 0.05, and means were separated using Duncan's multiple range test. Correlation analysis was performed using a two-tailed Pearson's correlation test [32].

Effects of Anti-Browning Reagent Mixture on Browning Index
In general, commercially available VCM is applied at 4% (w/w) for apple processing to prevent browning, and the shelf life of freshly cut apples is known to be up to 7 days. Since the initial color values of apples differ even within the same variety, the browning index (BI) was calculated as delta BI and used for comparison ( Figure 1). When freshly cut apples were treated with RE alone, their acidities were high (Table 1). Therefore, freshly cut apples were treated with a mixture of VCM and RE to control pH. In the case of the DW treatment group, the initial BI value increased within a day. There were no significant differences between 3% VCM and 4% VCM treatment. All of the mixtures of VCM and RE showed a similar degree of protection against browning as 4% VCM (Figure 1). VCM can eliminate oxygen, an electronic donor in the anti-browning mechanism.
VCM is composed of calcium chloride, potassium citrate, and ascorbic acid. By containing salt, the pH may be lowered compared to the conventional treatment with ascorbic acid alone [33]. When mixing according to the concentration for the combination of VCM and RE, salt was formed when 3% VCM and 0.5% RE were mixed (data not shown). The reason for the formation of salts is that the pH decreases when VCM was combined with RE. At this time, if the pH is not sufficiently reduced, calcium and potassium metal cations in VCM combined with hydroxide to form a salt [34]. Therefore, the experiments were conducted with 0.8%, 1%, and 1.2% RE (w/w). According to the data of the last test day, a tendency to inhibit browning was observed with 4% VCM in all groups except the DW group.
Agronomy 2022, 12, x FOR PEER REVIEW 5 of 12 Figure 1. Evaluation of browning index over storage period. Freshly cut apples were treated with anti-browning agent for 1 min, followed by measurement of the ΔBI values for 12 days. Statistical significance was determined by one-way ANOVA (mean ± SE, n = 4). VCM is composed of calcium chloride, potassium citrate, and ascorbic acid. By containing salt, the pH may be lowered compared to the conventional treatment with ascorbic acid alone [33]. When mixing according to the concentration for the combination of VCM and RE, salt was formed when 3% VCM and 0.5% RE were mixed (data not shown). The reason for the formation of salts is that the pH decreases when VCM was combined with RE. At this time, if the pH is not sufficiently reduced, calcium and potassium metal cations in VCM combined with hydroxide to form a salt [34]. Therefore, the experiments were conducted with 0.8%, 1%, and 1.2% RE (w/w). According to the data of the last test day, a tendency to inhibit browning was observed with 4% VCM in all groups except the DW group.
Of the RE added at concentrations of 0.8%, 1%, and 1.2%, the group of 3% VCM + 1% RE was the most similar to the pH of apple juice (pH 4.3). Thus, 1% RE added to 3% VCM, which is effective for browning and has the most similar acidity to that of apple juice, was used as the optimal concentration in subsequent experiments. According to apple browning studies, delta BI values were corrected to the initial apple color value [35]. As browning progresses, the L* values for brightness decrease, and a* for red and b* for yellow increase [36].
Thus, we evaluated the ΔBI, L* (brightness), a* (green to red), and b* (blue to yellow) of the freshly cut apples ( Figure 2). The color of the DW group was the darkest among the samples and there was no significant difference between the 4% VCM and VR groups. Between day 1 and 12, the a* values of the DW, VCM, and VR samples changed from −5.57 Figure 1. Evaluation of browning index over storage period. Freshly cut apples were treated with anti-browning agent for 1 min, followed by measurement of the ∆BI values for 12 days. Statistical significance was determined by one-way ANOVA (mean ± SE, n = 4). Of the RE added at concentrations of 0.8%, 1%, and 1.2%, the group of 3% VCM + 1% RE was the most similar to the pH of apple juice (pH 4.3). Thus, 1% RE added to 3% VCM, which is effective for browning and has the most similar acidity to that of apple juice, was used as the optimal concentration in subsequent experiments. According to apple browning studies, delta BI values were corrected to the initial apple color value [35]. As browning progresses, the L* values for brightness decrease, and a* for red and b* for yellow increase [36].
Thus, we evaluated the ∆BI, L* (brightness), a* (green to red), and b* (blue to yellow) of the freshly cut apples (Figure 2). The color of the DW group was the darkest among the samples and there was no significant difference between the 4% VCM and VR groups. Between day 1 and 12, the a* values of the DW, VCM, and VR samples changed from −5.57 to −1.20, −5.68 to −4.50, and −5.78 to −4.14, respectively. Similarly, the b* values for the DW, VCM, and VR samples changed from 23 VR group maintained a level similar to that of the 4% VCM group.
A previous study reported that decreasing L* values and increasing a* and b* values could be used as indicators of the browning reaction of freshly cut apples during storage [37]. The result of the DW group for the L*, a*, and b* values were similar to those of Chen et al., 2016 [38]. Both the 4% VCM and the VR group exhibited an anti-browning effect as indicated by the reduction in the degree of the variations in L*, a*, and b* values.

Effects of Anti-Browning Reagents on the Weight and Appearance of Apples
The weight of coated freshly cut apples was measured to analyze the efficiency of the treatment effect to inhibit water loss during the experiment (Figure 3a). In previous studies, weight loss in apples was mainly due to a decrease in water content, and the decrease in other ingredients had a minor effect on weight loss [39]. A previous study reported that decreasing L* values and increasing a* and b* values could be used as indicators of the browning reaction of freshly cut apples during storage [37]. The result of the DW group for the L*, a*, and b* values were similar to those of Chen et al., 2016 [38]. Both the 4% VCM and the VR group exhibited an anti-browning effect as indicated by the reduction in the degree of the variations in L*, a*, and b* values.

Effects of Anti-Browning Reagents on the Weight and Appearance of Apples
The weight of coated freshly cut apples was measured to analyze the efficiency of the treatment effect to inhibit water loss during the experiment (Figure 3a). In previous studies, weight loss in apples was mainly due to a decrease in water content, and the decrease in other ingredients had a minor effect on weight loss [39].
In all the test groups, the weight of the apple slices gradually decreased over time. In particular, apples coated with 4% VCM and VR showed reduced water loss compared to the DW group. The difference in weight loss results between the DW group and the VR group started to appear from day 9, at which time the DW, 4% VCM, and VR groups showed a weight loss of 1.85 ± 0.19%, 1.21 ± 0.18%, and 0.65 ± 0.29%, respectively. The lowest weight loss during the experiment was observed in the VR group. These results indicate that treated apples with a VR solution could prevent the loss of volatile apple components, including moisture. The picture shown in Figure 3b was taken 3 days after the processing of the freshly cut apples, and the 4% VCM and VR groups showed visual similarity. In all the test groups, the weight of the apple slices gradually decreased over t particular, apples coated with 4% VCM and VR showed reduced water loss compa the DW group. The difference in weight loss results between the DW group and t group started to appear from day 9, at which time the DW, 4% VCM, and VR g showed a weight loss of 1.85 ± 0.19%, 1.21 ± 0.18%, and 0.65 ± 0.29%, respectivel lowest weight loss during the experiment was observed in the VR group. These indicate that treated apples with a VR solution could prevent the loss of volatile components, including moisture. The picture shown in Figure 3b was taken 3 day the processing of the freshly cut apples, and the 4% VCM and VR groups showed similarity.
In addition, the pH, Brix, and acidity values of the DW, 4% VCM, and VR g were similar between day 0 and day 12 (Table 2). Therefore, none of the anti-bro agents tested in this study significantly affected the overall quality of apples.

Effects of Anti-Browning Treatments on Total Polyphenol Content and Phenol Oxida zyme Activity
Polyphenols are the main substrates with which enzymatic browning reacts a precursors to brown pigmentation. Total polyphenol content (TPC) and the acti polyphenol oxidase (PPO) and peroxidase (POD) were measured. TPC was signif higher in the VR group (121.0 ± 2.2) compared to both DW (56.9 ± 2.3) and 4% VCM In addition, the pH, Brix, and acidity values of the DW, 4% VCM, and VR groups were similar between day 0 and day 12 (Table 2). Therefore, none of the anti-browning agents tested in this study significantly affected the overall quality of apples.

Effects of Anti-Browning Treatments on Total Polyphenol Content and Phenol Oxidase Enzyme Activity
Polyphenols are the main substrates with which enzymatic browning reacts and are precursors to brown pigmentation. Total polyphenol content (TPC) and the activity of polyphenol oxidase (PPO) and peroxidase (POD) were measured. TPC was significantly higher in the VR group (121.0 ± 2.2) compared to both DW (56.9 ± 2.3) and 4% VCM (76.9 ± 2.2) on day 12. In addition, as in the DW, the TPC was positively correlated with the PPO and POD with r values at 0.7444 and 0.7776, respectively (Table S1; Supplementary Materials).
Ascorbic acid, a major component of 4% VCM, acts as a chemical competitor and is bound to both PPO and POD active sites, inhibiting enzymatic activities [40], further contributing to reduced enzyme activity as a result of a decreased pH [41]. In the case of RE, phytic acid may be responsible for the mechanism of suppression. In addition, phytic acid is known to inhibit enzymes, such as PPO and POD [42]. These results suggest that the inhibition of both enzyme activities could result in the improvement of browning of both 4% VCM and VR. PPO and POD, the phenolic oxidase enzymes, may affect browning in apples [43]. PPO and POD catalyzed the oxidation of monophenols to quinones which are browning pigments [44]. In this context, the activities in the 4% VCM and VR groups tended to decrease over time compared to the DW group. The activities tended to decrease from day 6 in the 4% VCM group and from day 3 in the VR group (Figure 4b,c). The results of the correlation between oxidase and TPC show that the initial decrease range in VCM was lower than DW and the initial decrease range in VR is lower than in VCM. (Figure 4d,e). As a result, PPO activity was inhibited by phytic acid, the main component of RE, and the results were similar to those of previous studies [42].
Ascorbic acid, a major component of 4% VCM, acts as a chemical competitor and is bound to both PPO and POD active sites, inhibiting enzymatic activities [40], further contributing to reduced enzyme activity as a result of a decreased pH [41]. In the case of RE, phytic acid may be responsible for the mechanism of suppression. In addition, phytic acid is known to inhibit enzymes, such as PPO and POD [42]. These results suggest that the inhibition of both enzyme activities could result in the improvement of browning of both 4% VCM and VR.
PPO and POD, the phenolic oxidase enzymes, may affect browning in apples [43]. PPO and POD catalyzed the oxidation of monophenols to quinones which are browning pigments [44]. In this context, the activities in the 4% VCM and VR groups tended to decrease over time compared to the DW group. The activities tended to decrease from day 6 in the 4% VCM group and from day 3 in the VR group (Figure 4b,c). The results of the correlation between oxidase and TPC show that the initial decrease range in VCM was lower than DW and the initial decrease range in VR is lower than in VCM. (Figure 4d,e). As a result, PPO activity was inhibited by phytic acid, the main component of RE, and the results were similar to those of previous studies [42].  The content of polyphenols in apples is an important contributo browning of freshly cut apples [45]. Browning enzymes convert polyph quinones and reduce the total polyphenols. Eventually, browning reduce as polyphenols, which can accordingly reduce oxidative enzyme activ shows that VR inhibits PPO and POD and prevents the loss of total polyph a previous study reported that the levels of total polyphenol, proantho The content of polyphenols in apples is an important contributor to the surface browning of freshly cut apples [45]. Browning enzymes convert polyphenols of fruit to quinones and reduce the total polyphenols. Eventually, browning reduces substrates such as polyphenols, which can accordingly reduce oxidative enzyme activity [46]. Figure 4 shows that VR inhibits PPO and POD and prevents the loss of total polyphenols. Similarly, a previous study reported that the levels of total polyphenol, proanthocyanidin, chlorogenic acid, and catechin in apples were negatively correlated with PPO activity [47].

Effects of Anti-Browning Reagent on Microbial Growth in Apples
Freshly cut fruit is a fertile environment for the growth of microorganisms due to the large amounts of water and energy sources [48]. The anti-browning treatment of freshly cut fruits can be used to inhibit degenerative and pathogenic microbial growth [39].
As shown in Figure 5, treatment with RE (4.39 × 10 4 ± 0.32) was more effective for microbial growth inhibition than 3% VCM (6.15 × 10 4 ± 0.4). However, the best strategy to inhibit microbial growth in freshly cut apple slices was through a combined (VR) application (4.25 × 10 3 ± 0.15). In addition, E. coli was not detected in any of the treated groups (data not shown).
(e) Figure 4. Analysis of TPC and phenolic oxidase activity in freshly cut apples. Differences in TPC, PPO, and POD activity changes in freshly cut apples soaked on days 0, 3, 6, 9, and 12. (a) TPC, (b) PPO activity, and (c) POD activity. Statistical significance was determined by one-way ANOVA (mean ± SE, n = 3). Pearson's correlation coefficient TPC and PPO (d), TPC and POD (e).
The content of polyphenols in apples is an important contributor to the surface browning of freshly cut apples [45]. Browning enzymes convert polyphenols of fruit to quinones and reduce the total polyphenols. Eventually, browning reduces substrates such as polyphenols, which can accordingly reduce oxidative enzyme activity [46]. Figure 4 shows that VR inhibits PPO and POD and prevents the loss of total polyphenols. Similarly, a previous study reported that the levels of total polyphenol, proanthocyanidin, chlorogenic acid, and catechin in apples were negatively correlated with PPO activity [47].

Effects of Anti-Browning Reagent on Microbial Growth in Apples
Freshly cut fruit is a fertile environment for the growth of microorganisms due to the large amounts of water and energy sources [48]. The anti-browning treatment of freshly cut fruits can be used to inhibit degenerative and pathogenic microbial growth [39].
As shown in Figure 5, treatment with RE (4.39 × 10 4 ± 0.32) was more effective for microbial growth inhibition than 3% VCM (6.15 × 10 4 ± 0.4). However, the best strategy to inhibit microbial growth in freshly cut apple slices was through a combined (VR) application (4.25 × 10 3 ± 0.15). In addition, E. coli was not detected in any of the treated groups (data not shown). Ascorbic acid, a major component of VCM, has been reported to be an effective bactericidal agent [49] and has shown inhibitory effects on the growth of E. coli and Streptococcus aureus [50]. RE is a rich source of phytic acid, which can interfere with bacterial Ascorbic acid, a major component of VCM, has been reported to be an effective bactericidal agent [49] and has shown inhibitory effects on the growth of E. coli and Streptococcus aureus [50]. RE is a rich source of phytic acid, which can interfere with bacterial membrane integrity and effectively degrade the membrane of gram-positive bacteria [51]. The six reactive phosphate groups of phytic acid are known to break down the extracellular membrane by chelation of the divalent cations of lipopolysaccharides [52]. As a result of the experiment, the growth of microorganisms in all treatment groups was inhibited at a higher degree compared to that of DW. The microbial growth of freshly cut apples was more effectively inhibited after VR treatment compared to 3% VCM treatment and 1% RE treatment. In particular, VR treatment reduced the number of microorganisms approximately 10-fold compared to 1% RE treatment ( Figure 5).

Sensory Evaluation with VCM and RE Combination
Sensory evaluation was conducted for the overall acceptability, sweetness, and sourness of the treated freshly cut apples. The sensory evaluation was carried out using previously optimized concentrations of 4% VCM and VR.
As shown in Figure 6, the VR group scored higher in overall preference than the 4% VCM group. In particular, the VR group showed significantly higher scores for sweetness and sourness than the 4% VCM group. These results could be attributed to the inhibition of moisture diffusion by anti-browning agents [53]. Therefore, coated freshly cut apples treated with VR appeared to have improved palatability.
viously optimized concentrations of 4% VCM and VR.
As shown in Figure 6, the VR group scored higher in overall preference than the 4% VCM group. In particular, the VR group showed significantly higher scores for sweetness and sourness than the 4% VCM group. These results could be attributed to the inhibition of moisture diffusion by anti-browning agents [53]. Therefore, coated freshly cut apples treated with VR appeared to have improved palatability. Figure 6. Analysis of sensory evaluation characteristics of apples by anti-browning reagents. Sensory analysis results for apple treated with 4% VCM and VR. Statistical significance compared with the 4% VCM and VR group was determined by the Student's t-test (* p < 0.05).

Conclusions
In conclusion, the combination of RE with VCM was confirmed to be a synergistic anti-browning agent, compared to VCM alone, a widely used commercial anti-browning reagent. VR suppressed the browning reaction in freshly cut apples without significant changes in the Brix, pH, and sensory preference indicators. In addition, the inhibition of PPO activity in freshly cut apples prevented the browning reaction and reduced the total polyphenol content. It was also confirmed that RE could increase the shelf life by inhibiting the growth of microorganisms. Taken together, the results presented in this study suggest that the synergistic combination of VCM and RE maximizes the anti-browning effect in freshly cut apples which provides a practical application protocol. Moreover, the utilization of RE, which is an agricultural by-product, might have potential economic benefits.
Supplementary Materials: The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Table S1: Pearson's correlation coefficients among selected quality parameters like TPC and PPO, TPC and POD values.

Conclusions
In conclusion, the combination of RE with VCM was confirmed to be a synergistic anti-browning agent, compared to VCM alone, a widely used commercial anti-browning reagent. VR suppressed the browning reaction in freshly cut apples without significant changes in the Brix, pH, and sensory preference indicators. In addition, the inhibition of PPO activity in freshly cut apples prevented the browning reaction and reduced the total polyphenol content. It was also confirmed that RE could increase the shelf life by inhibiting the growth of microorganisms. Taken together, the results presented in this study suggest that the synergistic combination of VCM and RE maximizes the anti-browning effect in freshly cut apples which provides a practical application protocol. Moreover, the utilization of RE, which is an agricultural by-product, might have potential economic benefits.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/agronomy12010086/s1, Table S1: Pearson's correlation coefficients among selected quality parameters like TPC and PPO, TPC and POD values.