Effect of the Combined Application of Aqueous Cabbage Seed Extract and Chitosan Solutions on the Shelf Life of Fresh-Cut Apple Cubes

Abstract

Enzymatic browning is the negative color effect of polyphenol oxidase activity in cut fresh fruit products, which reduces their quality, shelf life, and marketability. To preserve the color after cutting, apple cubes were treated with aqueous cabbage seed extracts (ACEs) at 5–10% w:v seed–water ratios, adjusted to pH 4.0 and 6.0 and 1% chitosan added to the ACE before preservation at 7 °C for 0–10 days. Chromatometric readings (L*, a*, and b*) and visual color score were used for shelf life calculation. The ACE total phenolics and glucosinolate levels showed differences among the 5–10% and control groups. Based on color score, uncoated or coated (chitosan or ACE combined with chitosan) apple cubes reached marketing limit levels (score > 3/5) on day one, but apple cubes treated with 5 or 10% ACE alone did so on day four, which was considered the effective shelf life. These findings were further supported by FT-IR analysis. ACE modification to pH 6.0 was more effective at keeping the natural cut apple color than pH 4.0. ACE treatment (at 5 or 10%) without coating is regarded as a very promising natural agent for extending the shelf life of fresh-cut apples, which is a key attribute in their marketing.

1. Introduction

Fresh-cut fruit processing can reduce its shelf life by inducing weight loss, softness, and microbial development in addition to browning [1,2].

The color of fresh-cut fruit is the main determinant of customer choice [3], and polyphenol oxidase can adversely impact this factor. The substrate of polyphenol oxidase is fruit polyphenols which are oxidized to quinones that exert browning on cut produce surfaces [4,5,6]. As polyphenol oxidase inhibitors, ascorbic acid, its derivatives, and sulfites have been the most successfully used compounds in the processing of fresh-cut fruit [7]. However, sulfites have been demonstrated to cause allergies that negatively impact human health, while ascorbic acid’s effects are temporary during preservation [8,9].

Alternatively, for sliced apples, which are favored worldwide due to their distinct flavor [10,11] and health advantages [12], a wide range of natural compounds (extracts of various plant parts) from different plants have been explored over the past 20 years in order to prevent their browning [13,14,15,16,17,18,19,20,21,22,23]. Natural products originated from Cruciferae plants have also been tested. The initiative to investigate Cruciferae plant was based on the observation that the induced polyphenol oxidase activity of cut products is considerably less than that of other produce [24]. Previous studies have demonstrated that Cruciferous leaf extracts inhibited commercial and polyphenol oxidases from grapes [25] and, when combined with allium extracts, they stabilized avocado pulp during refrigerated preservation [26]. In other studies, Cruciferous seed extracts inhibited the browning of cut lettuce leaves [27] or fresh-cut apple [28]. Seeds are considered a favored material due to their year-round availability.

Further, plant extracts or plant material can be combined with edible coating agents, such as chitosan [29], in order to synergistically enhance properties that prolong the shelf life of produce. Among edible coating agents [30], chitosan is a promising material primarily due to its antimicrobial activity and oxygen barrier formation [8,31]. Chitosan has been used as a coating agent for cut apples as a potential shelf life inducer [8,10,11,31].

Extending the shelf life of fresh-cut apples is a contemporary research subject. The purpose of this work was to evaluate the potential for inhibiting enzymic browning utilizing aqueous cabbage seed extracts alone or in combination with chitosan edible coatings, for which there have been no previous findings. In addition to year-round seed availability, aqueous seed extracts are simple to produce and apply immediately to cut apples after processing. Both aqueous cabbage seed extract and/or chitosan coatings were subjected to the necessary pH changes prior to application. The shelf life of cut apple cubes was determined based on measurement of the incidence of browning on their surfaces during preservation, using both subjective (visual) and objective (chromatometric) indices.

2. Materials and Methods

2.1. Chemicals and Reagents

Chitosan (MW of 190,000–310,000 g/mol) with a degree of deacetylation greater than 90% was obtained from Siveele (Breda, The Netherlands). Glycerol (99%) and hydrochloric acid (37% reagent grade) were purchased from Scharlab (Barcelona, Spain). Acetic acid (glacial 99–100%), methanol (HPLC Grade), methanol (Analytical reagent), sodium carbonate (Na₂CO₃, Anhydrous), sinigrin, Folin–Ciocâlteu’s phenol reagent, and palladium (57%) were obtained from Chem-Lab (Zedelgen, Belgium). Sodium hydroxide (NaOH), gallic acid, and DPPH (2,2-diphenyl-1-picrylhydrazyl) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Maxi Clean C-18 (300 mg) and SPE cartridges were obtained from Sepachrom Srl (Rho-Milan, Italy).

2.2. Plant Material

The apples (Malus domestica Borkh. cv. Starking Delicious) used in this study were produced by Zagorin (Zagora, Greece) and were large in size, spherical and elongated in shape, and their flesh was firm and white. The fruits were harvested in September and had been stored for 3–4 months when they were purchased from the local market. The seed of the domestic variety of cabbage (Brassica oleracea L. cv. Kilkis) used in this study was obtained from General Phytotechnic Athens (Athens, Greece). It is a mid–early variety of cabbage (maturing in about 90 days) that produces soft and white-green heads.

2.3. Preparation of Aqueous Seed Extracts, Chitosan-Based Coatings, and pH Adjustment

Based on earlier studies [27], the cabbage seeds were chosen to yield aqueous extracts (ACEs) that prevent freshly cut apple cubes from browning. Pre-experimentation tests were used to define the maximum and intermediate values of the cabbage seed–water ratio (w:v), which were 5 and 10%, respectively. After that, these combinations were homogenized in a water ice bath (5 °C) for two min at 10,000–12,000 rpm using a PT 10–35 GT (Kinematica, Malters, Switzerland) homogenizer. After passing through a 0.5 mm plastic screen, the homogenate was centrifuged using a Universal 320 (Hettich, Tuttlingen, Germany) at 7000 g for 15 min at 25 °C. This produced aqueous seed extracts (ACEs), which were subsequently employed. In addition to the ACE, distilled water was used as a control.

The produced ACEs were then divided in half, with half of them incorporated into 1% chitosan edible coatings (w:v), and the mixture was stirred with a magnetic stirrer until the chitosan was entirely dissolved. During the stirring process, 1 mL of acetic acid (1% v/v) and 0.3 g of glycerol as a plasticizer (30% w:w based on chitosan) were added, according to Nair et al. [32] and Peng et al. [33]. Deionized water was employed as a control coating instead of the ACE.

Solutions used for the uncoated and coated treatments were subjected to two different pH modifications using an HI 221 laboratory pH meter (Hanna Instruments, Woonsocket, RI, USA); the first was adjusted to pH 4.0, which was approximately equal to the initial pH of the ACE, using acetic acid, and the second was adjusted to pH 6.0 using NaOH.

2.4. Sample Preparation and Application of Treatments

Apples of uniform size, appearance, and ripeness were selected. The apples were cleaned with tap water to eliminate any foreign matter, dried, sliced in half, cored, and then cut into 12 mm³ cubes using a multi-cutter (Westmark GmbH, Lennestadt-Elspe, Germany) and a sharp knife. Any cubes containing a portion of the peel were removed, and those judged inappropriate were discarded.

The apple cubes were then immersed in the ACE solutions (no coating and coating) for 10 min without being rinsed before or after immersion. After immersion, the apple cubes with the chitosan coating were allowed to dry naturally for 30 min at room temperature, until the excess coating was removed.

Each treatment consisted of six cubes from a different apple, all immersed in a 20 mL solution; following immersion, they were weighed and placed in a Petri dish; each Petri dish was considered to be one replication. Three dish replications were used for each of the twelve treatments (3 ACE, 2 coatings, and 2 pH treatments). The Petri dishes were kept at 7 °C for 0, 1, 3, 6, and 10 days and used to obtain CIE Lab coordinates and weight loss. Additional apple cubes were also processed and preserved in a similar manner in order to assess hardness, phenolic compounds, and antioxidant activity.

2.5. Quality Assessment Parameters

2.5.1. Determination of Phenolics, Antioxidant Capacity, and Total Glucosinolates

The pure ACE solutions of 5 and 10% (w:v), comprising three batch replications per treatment, were produced and utilized as anti-browning treatments for flesh-cut apple cubes, and analyzed for their phenolic compound content, antioxidant capacity, and total glucosinolates.

Apple cubes from each treatment were homogenized with 1:4 methanol (w:v) using a PT 10–35 GT (Kinematica, Malters, Switzerland) homogenizer and then centrifuged at 7000 rpm for 15 min at 25 °C using a Universal 320 (Hettich, Tuttlingen, Germany). The supernatant was collected to be used for the determination of phenolic compounds and DPPH radical scavenging activity [23].

The total phenolic compounds (TPCs) of the ACE, as well as of the cut apple cubes, were determined using Folin–Ciocalteu reagent according to AOCS [34] and Scalbert et al. [35], with some modifications. ACE or apple cube methanolic extracts, 0.5 mL, were mixed with 1.5 mL Folin–Ciocalteu reagent and, after 3 min with 6 mL of Na₂CO₃ (10% w:v), mixed and kept in a dark environment at room temperature (at 20 °C) for 90 min. The absorbance of the solution was monitored at 765 nm using a Genesys 80 UV-VIS (Thermo Fisher Scientific, Waltham, MA, USA) spectrophotometer. To the control, 0.5 mL of water or methanol was added, for the ACE or apple cube methanolic extracts, respectively. TPC of ACE were determined from the linear regression equation of a standard curve (y = 0.025x − 0.0707, R² = 0.995), and the results are expressed as mg of gallic acid equivalent (GAE) per L of ACE.

The antioxidant activity of the samples was also evaluated using DPPH as a free radical, according to Brand-Williams et al. [36] and Nenadis et al. [37]. In a test tube, 2960 µL of DPPH methanolic solution (100 µM) was mixed with 0.6 mL of ACE, 0.5 mL of apple cube methanol extract, and methanol (control). The tubes were then vortexed and kept in a dark location at room temperature (20 °C) for 30 min. The absorbance of the solution was then measured at 517 nm using a Genesys 80 UV-VIS spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The antioxidant activity of the ACE or apple cube extract (%) values (%RSA) were obtained using the formula below, following adjustment with appropriate blanks.

$$\mathit{RSA}(\%) = {\displaystyle \frac{Abs515(t=0)-Abs515(t=0)}{Abs515(t=0)}}\times 100$$

(1)

The total glucosinolates (GSLs) of the pure ACE 5% and 10% solutions were measured following sample preparation as follows: ACE was filtered, and its hydrophobic compounds (such as phenolics) were excluded using a Maxi-Clean SPE (300 mg) C18 cartridge (Sepachrom Srl, Rho-Milano, Italy). The column was activated with 5 mL of acidified methanol (HPLC grade + acetic acid) and washed with 5 mL of deionized water. ACE samples, 0.5 mL, were run through the column, followed by 0.5 mL of deionized water. GSLs were measured in the 1 mL column, which included the sample plus the deionized water eluent. The determination of the GSLs was carried out according to the method of Ishida et al. [38]; PdCl₂ solution was produced from 60.17 mg PdCl₂, 168 µL hydrochloric acid, and 100 mL deionized water. A mixture of 200 µL of GSL extract, 300 µL of deionized water, and 3 mL of PdCl₂ solution was prepared. The mixture was stirred and left at room temperature (25 °C) for 1 h before the measurement of absorbance was measured at 425 nm using a Genesys180 UV-VIS spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). For the control, 500 µL of deionized water was combined with 3 mL of PdCl₂. The operation was repeated two times. The sinigrin equivalents were calculated using the linear regression equation of a calibration curve (y = 0.0006x − 0.0315, R² = 0.999) and results were expressed in mg/g of cabbage seed.

2.5.2. Chromatometric Color Measurement and Weight Loss

At days 0, 1, 3, 6, and 10 of preservation, the chromatometric color, and the weight loss were monitored. Two color measurements were made for each apple cube, with six cubes per Petri dish replication, and three replications per treatment.

A chromameter (Minolta CR-400, Minolta, Osaka, Japan) with an 8 mm measuring head and a D65 illuminant was used to test the surface color of the cut apple cubes. Before taking any measurements, the device was calibrated using a white reference tile (L* = 97.52, a* = −5.06, b* = 3.57). The CIELAB coordinate color space system was used to determine the L* (0 = black, 100 = white), a* (+red, −green), and b* (+yellow, −blue) color coordinates [39].

The hue angle (h°) and color saturation (C), which are the second derivative parameters of the L*, a*, and b*, were also determined, as shown below.

$$h^o=180+{tan}^{-1}{\displaystyle \frac{b^{\ast }}{a^{\ast }}}$$

(2)

When a* < 0 and b* ≥ 0: 90° < Hue < 180°;

When a* > 0 and b* ≥ 0: 0° < Hue < 90°.

$$C=\sqrt{a^{\ast 2}+b^{\ast 2}}$$

(3)

Additionally, the browning index (BI) was calculated as described below [40]:

$$\mathit{BI} = 100{\displaystyle \frac{x-0.31}{0.17}}$$

(4)

where [41]

$$x={\displaystyle \frac{a^{\ast }+1.75L^{\ast }}{{5.64L}^{\ast }+a^{\ast }-{0.012b}^{\ast }}}$$

(5)

Weight loss (WL) was measured in the same apple cubes used for chromatometric readings; three Petri dish replications for each treatment were weighed at each sampling interval during preservation. Weight loss was determined using the equation

$$\mathit{WL} (\%)={\displaystyle \frac{mt-m0}{m0}}·100$$

(6)

where m0 is the initial weight of the freshly cut apple cubes and mt is the weight of the apple cubes at time t of preservation [42].

2.5.3. Hardness Determination

For hardness (HNS) measurement, 9 individual typical cubes were utilized for each treatment on each preservation day. Individual apple cubes were picked at random and placed on the bottom of a single-chip setup (TA.XT plus C, Stable Micro Systems Ltd., Surrey, UK). Each cube was penetrated with a 2 mm diameter cylindrical probe at a crosshead speed of 0.8 mm/s. The texture analysis parameter employed was the absolute force required to penetrate the apple cube, which was measured in grams. After being punctured, they were frozen (−20 °C) for use in determining total phenolics and antioxidant capacity.

2.5.4. FTIR FT-IR Spectroscopy

To help explain the browning development of cut apple cubes that were uncoated and those that were coated with chitosan, FTIR analysis was utilized. FTIR analysis is a flexible method for classifying and identifying materials according to their molecular structure. It can be useful for fingerprinting coating interactions, comprehending the chemistry of browning in cut apples, or for evaluating their overall quality.

A 6700 IR (Jasco, Essex, UK) spectrometer with a DLaTGS detector, a high-throughput Single Re-flection ATR with diamond crystal, and the Spectra Manager software (Jas-co, Essex, UK) were used to obtain attenuated total reflection (ATR) FT-IR spectra. Ten scans were collected for each spectrum in the reflectance mode, with a resolution of 4 cm⁻¹ and 32 scans per sample, spanning from 4000 to 400 cm⁻¹.

Spectra were obtained for both uncoated and chitosan-coated cut apple cubes; the ACE of 0, 5, and 10% adjusted to pH 4.0 was utilized. Before any additional pre-processing was performed, 10 spectra from each sample were recorded and averaged to provide the corresponding overall spectrum. Spectra Manager software (V.2.15.01, JASCO, Great Dunmow, UK) was used to adjust the original spectra.

2.5.5. Visual Evaluation of Browning

A three-person trained panel evaluated the degree of browning on the cut apple cube surface using a scale of 1 to 5, where 1 indicated no browning, 3 was the moderate-limit of marketability, and 5 indicated severe browning. A standard sensory evaluation room with separate booths furnished with neutral hues and lighting was used for the visual examination.

Table 1. Visual color evaluation scores of browning and acceptability of cut apple cubes.

Degree of Browning Score	Acceptability/Browning Degree	Color Range
1	Absolutely acceptable No sign of browning
2	Accepted Evidence of browning beyond doubt
3	Borderline acceptable/unacceptable Noticeable browning
4	Unacceptable Obvious time wear—inappropriate
5	Unacceptable Decomposition image

shows in photographs the range of the five scales used. The scores of each replication (three replications per treatment) were averaged to produce a visual score index (VCS) for each treatment at days 0, 1, 3, 6, and 10 of preservation.

2.5.6. Shelf Life Determination

The shelf life was defined by the period of time it took for a unit of replication to reach a score of 3 (Table 1), which indicated that browning had proceeded to an unsatisfactory level for marketing. Three Petri dish replications (each containing 6 apple cubes) were utilized to assess shelf life, and the average shelf life for each treatment was computed for VCS. VCS was additionally correlated with the BI to create a threshold value that corresponded to a score of 3 (unacceptable degree for marketing). This value was used to determine shelf life according to BI.

2.6. Statistical Analysis

The design of the statistical analysis was completely randomized. Eleven dependent variables (L*, a*, b*, h° C, BI, VCS, HNS, WL, TPC, and DPPH) and three factors (ACE, pH adjustment and preservation period) were used in a three-way ANOVA with three replications per treatment. Analysis was performed on the main effects of the factors and their interactions. Additionally, principal component analysis (PCA) and Pearson’s correlation coefficient were also used to determine the interactions of variables. Data mean separation was performed using Duncan’s multiple range test. Statistical analyses were performed using the SPSS statistical software for Windows (version 29).

3. Results and Discussion

3.1. Total Phenolics, Antioxidant Capacity, and Total Glucosinolates of Cabbage Seeds Aqueous Extracts

Table 2. Means ± standard deviation (n = 3) of total phenolics (TPCs), antioxidant capacity (DPPH), and total glucosinolates (GSLs) of aqueous extracts of cabbage seeds (ACEs) as well as of coating solutions using ACE and chitosan.

ACE % Coating	TPC			DPPH			GSL
	(mg GA/L)			(%Inh)			(mg Sin/g Seed FW *)
ACE 5%	799.8	±	15.6C **	51.55	±	1.38C	5.41	±	0.27
ACE 10%	1497.0	±	9.6A	78.43	±	2.03B	7.24	±	0.84
Chitosan	n.d. ***		n.d.	11.35	±	1.97D	n.d.		n.d.
Chitosan/ACE 5%	792.6	±	3.6C	54.75	±	1.68C	n.d.		n.d.
Chitosan/ACE 10%	1443.0	±	13.2B	82.97	±	2.36A	n.d.		n.d.

* FW: fresh weight basis, ** Different letters within each factor represent statistically significant difference according to Duncan’s multiple range test, *** n.d.: not determined.

presents the concentrations of TPC, DPPH radical scavenging activity, and GSL in various levels of ACE solutions (no coating—control) and coating solutions after chitosan addition, before pH adjustment. The TPC of 5% ACE was 799.8 mg gallic acid/L and those of 10% were about double, and this difference was maintained after developing the coating solution with chitosan (1% w:w), with 1% (v/v) acetic acid, and glycerol (0.3 g/g chitosan). The same pattern was observed for DPPH radical scavenging activity. It has been reported that all natural preservation agents with anti-browning capabilities (and their aqueous extracts) have elevated TPC and antioxidant activity [43,44]. TPC and DPPH values were equivalent to those determined by Androudis et al. [27] when investigating 10 and 20% aqueous extracts of cabbage seeds, and Wessels et al. [28] while analyzing broccoli seed supercritical CO₂ extract solutions.

GSL concentrations of 5% and 10% ACE were 5.41 and 7.24 mg sinigrin/g seed on a fresh weight basis, respectively. Cabbage seeds contain various glucosinolates, but the exact concentrations vary significantly between cultivars, developmental stages, and even different parts of the plant. The GSL content of ACE was within the reported range of 2.1–8.3 mg/g of cabbage seed on dry mass basis [45,46], even though the reported values were determined by different methodologies. GSL of the chitosan combinations with ACE were not detected due to the inability of chitosan to pass through the C18 cartridge. Pre-experiments demonstrated that the sample preparation phase in which phenolic compounds were retained using a C18 cartridge was necessary for determining GSL. It was evident that the phenolic compounds interfered with the determination of the GSL in ACE using the method of Ishida et al. [38]. Additionally, the effect of pH adjustment on any of the variables was found to be within the standard deviation range and is not presented.

3.2. Analysis of Variance

The study’s data (dependent variables) were colorimetric, based on CIE L*a*b* coordinate color model; organoleptic, based on visual evaluation (VCS); and physicochemical, based on the changes in weight loss or flesh hardness, as well as in phenolic component content and antioxidant activity. Enzymatic browning is commonly assessed using as variables the polyphenol oxidase activity [47,48] and/or the surface color of cut tissue; Eissa et al. [49], in order to assess enzymatic browning in apples, used the L*, a*, and b* color model, their second derivatives C and h°, and the BI, which is also calculated from the L*, a*, and b* model. Decreased L* shows darker tones associated with tissue browning, whereas positive and high a* or b* values represent red or yellow colors, both linked with enhanced tissue browning. BI is calculated using the CIE L*a*b* color space coordinates (L*, a*, and b*) determined on cut apple cubes. A higher BI value suggests a greater level of browning. Incardona et al. [50] and Luo et al. [51] investigating the effects of cutting tool quality on the main physical, chemical, and microstructural properties of fresh apples used VCS in addition to the L*, a*, and b* color model (which includes C and h° and the BI). On the other hand, Moreira et al. [52] solely employed the color parameters L* and h^o for fresh-cut apple browning and indicated that brightness (L*) was considered the most significant indicator of enzymatic fruit and vegetable browning.

Table 3. Main effects and means for the variables L*, a*, b*, hue angle (h°), saturation (C), as well as indices for browning (BI), visual color score (VCS), hardness (HNS), weight loss (WL), phenolic compounds (TPCs, mg GAE/kg FW), and antioxidant capacity (DPPH) obtained during the 10 day/7 °C preservation of cut apple cubes; the cubes were previously exposed to pH-adjusted aqueous extracts of cabbage seeds (ACEs) before coating.

MAIN EFFECTS	DF	L*	a*	b*	h°	C	BI	VCS	HNS	WL	TPC	DPPH
ACE	Water/control	68.70B	2.06A	31.40A	90.70A	31.71A	63.75A	3.77A	681.5A	4.47A	344.5B	33.85A
	5%	72.11A	0.35B	28.27B	90.72A	28.56B	49.96B	3.20B	703.8A	4.08A	521.6A	46.46B
	10%	72.02A	0.22B	27.80B	90.35A	28.10B	50.91B	3.18B	709.5A	4.53A	445.1A	36.56B
pH	pH 4.0	69.63Β	2.57A	30.70A	86.94B	31.13A	63.21A	3.60A	697.8A	4.18A	434.3A	35.33A
	pH 6.0	72.25A	−0.81B	27.61B	94.29A	27.78B	46.53B	3.16B	698.7A	4.54A	439.8A	42.58B
Coating	Non-coated/control	73.67A	−1.10B	27.35B	93.09A	27.44B	44.41B	2.99B	639.9B	4.41A	500.4A	42.47A
	Coated, 1% chitosan	68.22B	2.86A	30.97A	88.09B	31.47A	65.33A	3.78A	756.6A	4.31A	373.7B	35.44B
Preservation (days)	0	74.56A	−1.90E	25.52D	94.78A	25.65E	38.76E	1.75E	788.6A	0.00C	-	-
	1	74.09A	−0.89D	27.82C	92.81A	27.93D	45.27D	3.01D	735.9A	2.36B	459.7A	40.98A
	3	71.28B	0.76C	29.77B	90.01A	29.95C	54.77C	3.70C	661.7B	5.84A	-	-
	6	67.95C	2.47B	31.10A	86.67A	31.46B	64.85B	4.10B	651.1B	6.60A	414.3A	36.93A
	10	66.83D	3.95A	31.59A	88.68A	32.28A	70.71A	4.34A	653.9B	7.01A	-	-

Different letters within each factor represent statistically significant differences according to Duncan’s multiple range test.

displays the main effects as a function of ACE level in the presence or absence of coating, adjusted at two different pH levels and preservation times. The color of the water (control, uncoated fruit)-treated cubes differed significantly in L*, a*, b*, BI, C, and VCS when compared to apple cubes treated with ACE; however, there was no difference between the 5% and 10% ACE levels (Table 3). Apple cubes treated with ACE exhibited less browning, indicating that their quality had been maintained. Cruciferous plant part extracts were tested for their ability to reduce enzymatic browning in cut apples and other fruits or vegetables. Eissa et al. [49] prepared aqueous extracts from white cabbage leaves (Brassica oleracea L.) at levels of 5, 10, 15, 20, and 25% to evaluate the enzymatic browning of apples; the assessed inhibitory activity increased when the concentration was raised from 5% to 15%, but gradually decreased when the concentration was raised above this point. Fresh-cut apples treated with 1% broccoli seed extract were shown to have partial anti-browning properties [26]. Furthermore, treatment with aqueous extracts of cabbage seeds at 10% (w:v) was as effective as 20% or close to the bisulfate control in demonstrating anti-browning activity in lettuce leaf mid-rib segments [27]. According to Bustos et al. [26], the great efficacy of cruciferous vegetable extracts such as cauliflower and Brussels sprouts as polyphenol oxidase inhibitors increased the shelf life of chilled avocado pulp. Furthermore, cruciferous vegetables include glucosinolates [53,54], which are considered to be responsible for the effective inhibition of PPO, while the particular mechanism of action is unknown.

Most of the variables were impacted by the ACE pH level (Table 3). ACE adjusted to pH 6.0 resulted in increased L* (indicating less browning), compared to pH 4.0; other color attributes such as a*, b*, BI, C, and VCS decreased while h^o increased (also indicating less browning). This indicates that adjustment of ACE to pH 6.0 was superior overall in keeping enzymatic browning low (Table 3). The optimal pH for polyphenol oxidase has been reported to be directly influenced by the type of phenolic substrate [55]. The impact of pH was also investigated by Karaibrahimoglu et al. [56], however, on apple slices treated with calcium ascorbate. Attributes a* and b* were higher and L* was lower at pH 4.0 than at pH 6.0; the pH decrease between 4.5 and 3.5 resulted in more browning on the apple’s surface, but 4.5 is generally a crucial pH for preserving apple quality. The reported conclusions on how different pH values may impact the responses of cut apple cubes as reflected by colorimetric variables are supported by the outcomes of the current experiment (Table 3).

The factors ACE strength and pH had no significant effect on the tissue HNS and WL parameters; however, the addition of a coating maintained the hardness without decreasing the weight loss when compared to uncoated samples (Table 3).

In regard to coating application, the immersion time for freshly cut apples in chitosan coatings varies from 2 min [57,58] to 30 min [49,59,60]. In the present experiment, an immersion time of 10 min was chosen for both the chitosan and ACE solutions and the coated apple cubes were left to dry for 30 min, as recommended by Karagöz & Demirdöven [59] and Demircan & Velioglu [31].

The anti-browning capacity of ACE was more effective in the absence of a coating than in its presence (1% chitosan), as evidenced by higher L* values and lower a*, BI, C, and VCS values (Table 3). The chitosan-coated apple cubes had a surprisingly lower L* and higher BI than the uncoated water-treated control, implying that coating them with chitosan might not have been sufficient to properly protect the color of freshly cut apples (Table 3). Opposite to the results of this study, apple enzymatic browning has been effectively inhibited by a 1% chitosan coating [42,58]. Luo et al. [51] postulated that the acidic nature of the chitosan solution most likely corroded the apple surface and accelerated enzymatic browning reactions by promoting the release of metal ions. This in turn facilitated reactions with enzymes or polyphenolic compounds found in apple flesh, and exacerbating oxidative processes. On the other hand, according to Batkan et al. [61], Starking Delicious apples had a pH of 3.77, making them marginally more acidic than the 4.0 of the liquids in which they were submerged. Ackah et al. [62] suggested that the application of chitosan speeds up enzymatic browning in injured apples by encouraging the phenylpropanoid pathway to produce more phenolic compounds and thereby increasing the activity of polyphenol oxidase to stimulate the oxidation of phenols to quinones. The findings of the current study, which show an increase in a*, b*, and C, and a decrease in L* and h° (Table 3), may confirm this hypothesis.

Many investigations emphasize the effects of chitosan coating on hardness and weight loss during preservation. Apples treated with chitosan reduced tissue softening, preserving hardness when compared to the control (Table 3), which is consistent with previous published works [57,63]. A chitosan coating has been reported to maintain the uniformity [51] and stability [64] of fresh-cut apples during preservation. Tissue stress after cutting might lead to loss of cohesion and texture changes, primarily due to enzymatic hydrolysis of cell wall agents caused by the action of pectinolytic enzymes such as polygalacturonase, cellulose crystallinity reduction, and cell wall thinning [57].

Another important factor influencing fresh fruit quality is the rate at which it loses weight [65]. When cut apple tissue is subjected to low relative humidity, it loses weight. Weight loss in fresh-cut fruits is associated with transpiration and a loss of storage carbon as a result of CO₂ generation by respiration [51,66]. In this study apple cubes coated with chitosan lost a comparable weight to the other treatments (Table 3) and did not prove to be effective as water vapor barriers for the entire preservation duration, which is consistent with the findings of Xiao et al. [63] and Qi et al. [57]. Furthermore, due to its hygroscopic character, chitosan as a hydrocolloid does not function as an effective water vapor barrier in food substrates with high water activity. Humidity, which determines the degree of adherence of both hydrophilic and hydrophobic coating materials, could explain the fruit’s inability to generate a homogeneous coating [57]. In addition, alginate-coated apple cubes had higher relative enzyme activity values than untreated samples in the Fernades et al. [67] study; when compared to untreated samples, the coating may result in increased water activity, trapping moisture within the coated apple cubes. Because enzyme activity increases with water activity, the coating’s application was ineffective in preventing oxidation during the dehydration process.

As expected, preservation time was the most critical determinant in the development of browning in cut apple cubes (Table 3). Studies on the kinetics of enzymatic browning, a catalytic reaction that changes with time, are prevalent [68]. In the total analysis of variance, the time of preservation accumulated the highest variability as reflected by a high eta squared (Table S1). The preservation factor reduced the variable L* while raising all other parameters, including considerable changes in a*, b*, BI, C, and VCS. Regardless of the preservation conditions (low temperature and lack of light), the apple cubes’ color changes toward browning increased over time, making it the most important factor impacting their quality (Table 3). Preservation had an inversely proportionate effect on the average values of two variables, HNS and WL, decreasing HNS but increasing WL over time (Table 3).

TPC values for Delicious apple cultivars ranged from 340 to 500 mg GAE/kg FW, which was within the previously reported range of 161.9–882.4 mg GAE/kg FW, with variations attributed to factors such as cultivar, processing method, and storage conditions [69,70]. Only between the water-treated control and the ACE-treated apple cubes (5 or 10%) did the ACE level have an impact on the variables TPC and DPPH. By preventing the generation of free radicals and triggering the intracellular antioxidant defense system, TPC is known to increase antioxidant capacity [71,72]. The TPC variable was unaffected by pH adjustment, while greater DPPH values were noted at pH 6.0. The antioxidant profile and ability of chitosan to prevent fruit browning and reduce the loss of phenolic compounds has been shown to help preserve TPC and DPPH [73,74], but the uncoated samples exhibited higher TPC and DPPH values than the coated samples (Table 3). In a study of blue honeysuckle and chitosan coating, Qiao et al. [75] found that the TPC of the control samples was significantly higher than that of the chitosan-coated samples. The TPC and DPPH parameters, which were only tested on days 1 and 6, showed a tendency to decrease with the duration of preservation (Table 3).

3.3. Pearson Linear Correlation and Principal Component Analysis

To establish safe ground regarding the perception of enzymic browning by consumers, Pearson’s linear correlation coefficient was used to correlate chromatometric variable(s) with visual color as well as with physicochemical parameters.

Table 4. Pearson linear correlation coefficients of the variables L*, a*, b* hue angle (h°), saturation (C), browning index (BI), visual color score (VCS), hardness (HNS), weight loss (WL), phenolic compounds (TPCs), and antioxidant capacity (DPPH).

	L*	a*	b*	ho	C	BI	VCS	HNS	WL	TPC	DPPH
L*	--
a*	−0.939 ***	--
b*	−0.776 ***	0.846 ***	--
ho	0.712 ***	−0.76 ***	−0.71 ***	--
C	−0.823 ***	0.888 ***	0.996 ***	−0.733 ***	--
BI	−0.953 ***	0.977 ***	0.902 ***	−0.758 ***	0.937 ***	--
VCS	−0.74 ***	0.782 ***	0.82 ***	−0.663 ***	0.824 ***	0.792 ***	--
HNS	−0.038	0.043	−0.008	−0.056	0.001	0.021	−0.065	--
WL	−0.345 ***	0.371 ***	0.375 ***	−0.302 **	0.375 ***	0.365 ***	0.601 ***	−0.34	--
TPC	0.554 ***	−0.5 ***	−0.505 ***	0.319	−0.514 ***	−0.531 ***	−0.495 ***	−0.205	−0.034	--
DPPH	0.458 ***	−0.469 ***	−0.4 ***	0.345 **	−0.419 ***	−0.468 ***	−0.401 ***	−0.174	0.006	0.797 ***	--

** Correlation is significant at the 0.01 level; *** correlation is significant at the 0.001 level.

shows the analysis of Pearson coefficients; significant strong correlations are shown among color variables (L*, a*, and b*), their second derivatives, as well as the calculated indexes (BI) and VCS (p < 0.05). HNS did not correlate with any of the other variables, indicating that enzymatic browning is rather independent of tissue softening; cut apples may be firm yet brown due to browning processes, or they may soften but not brown substantially if enzymatic browning is inhibited. TPC and DPPH were moderately correlated with all chromatometric variables except h° and VCS, but not with WL or HNS. As expected, TPC and DPPH were strongly correlated with one another.

The visual evaluation was considered the most representative indicator of browning, reflected in the variable VCS; the Pearson linear correlation coefficient of the VCS with the other variables revealed that VCS had a very high correlation with b* and C and a high correlation with L*, a*, h°, and BI, with Pearson correlation values of 0.82 and 0.79–0.66, respectively (Table 4); however, VCS showed a moderately negative association with TPC and DPPH, whereas with HNS it showed virtually no correlation (0.065).

To support the differences between treatments with respect to the variables examined (L*, a*, b*, h°, C, BI, VCS, HNS, WL, TPC, and DPPH), PCA was conducted (Figure 1). Two main components, which together accounted for 73.8% of the total variability, were extracted from the data gathered for the treatments. A PCA biplot displays the data matrix pertaining to variables (vectors) and treatments (symbols).

Potential connections between the variables themselves are also highlighted by the loading plot of PC1 vs. PC2 (Figure 1A), which displays the primary relationship between variables and principal components [76]. Vectors with similar directions indicate positively correlated variables, whereas opposing directions suggest negative correlations. The loading plots show a possible relationship between browning development and discriminative treatment aspects. As previously indicated in Table 4, the vectors of L*, h°, TPC, and DPPH point in the opposite direction to those of BI, a*, b*, h°, C, and VCS, suggesting negative connections.

Component 1, which explains 59.6% of parameter variability, was negatively influenced by variables L*, h°, TPC, and DPPH, and positively by variables a*, b*, C, BI, and VCS; component 2, which explains 14.2% of the variability, was primarily influenced by the variables WL and HNS (Figure 1A). This reveals that browning is predominantly associated with reductions in L*, h°, TPC, and DPPH, as well as increases in a*, b*, C, BI, and VCS.

As previously shown in Table 4, highly correlated groups, including L* and h° (R² = 0712), and TPC and DPPH (R² = 0.797), were linked on the PC1′s negative side, whereas the groups of a*, b*, C, BI, and VCS (R² = 0.902–0.782) were coupled on the PC1′s positive side.

Day 1 and 6 of preservation were clearly separated on the PCA score plot (Figure 1B). Chitosan-coated samples were placed in the second quadrant, suggesting that the coating treatments were inferior to the control because of causing enzymatic browning. Coated and uncoated treatments were also distinguished within each preservation day (Figure 1B). The first component’s positive side (quadrants 2 and 4) showed more browning, whereas its negative side (quadrants 1 and 3) showed less. Control coating treatments found in the first quadrant exhibited the least browning on the first day of preservation, but the chitosan-treated cubes were split between the first and second quadrants. Day 6 of preservation shows similar pattern to day 1; however, only the uncoated treatments exposed to 5 or 10% ACE were found in the third quadrant, indicating the occurrence of a very low level of browning (Figure 1B).

3.4. FTIR

FT-IR (Fourier transform infrared) spectroscopy was used to investigate changes in the structural properties and identify the major functional groups involved in the reactions occurring in tissue browning. The FT-IR spectra of non-coated (water control plus ACE) and coated (chitosan control plus chitosan combined with ACE) apple cubes obtained throughout preservation for 0 or 10 days are shown in Figure 2. All spectra exhibited a noticeable decrease in both the intensity and broadness of the –OH stretching vibrations within the range of 3500–3000 cm⁻¹ after 10 days of preservation. This change may indicate the degradation of phenolic compounds and the appearance of browning on the apple surface due to polyphenol oxidase activity, which is consistent with both visual and colorimetric measurements (Table 3). More distinct changes in the -OH band of apple cubes coated with chitosan were observed. This demonstrated the low anti-browning impact of chitosan coatings compared to uncoated ACE treatment.

As previously reported, polyphenol oxidase catalyzes the oxidation of phenolic compounds, leading to the formation of quinones [2,3,4]. Notably, in water-treated apple cubes, new peaks appeared at 2920 and 2852 cm⁻¹ after 10 days of preservation, assigned to the asymmetric and symmetric stretching vibrations of –CH₂– groups, respectively [77]. Similarly to the –OH reflectance band, changes were observed at 1637 cm⁻¹, corresponding to the C=C and C–C stretching vibrations within the aromatic ring of phenolic compounds [78,79]. In addition, significant alterations were detected at 1673 cm⁻¹, attributed to C–H bending vibrations in aromatic ring structures for all apple cube samples. The peaks at 1146 cm⁻¹ and 1034 cm⁻¹, associated with C–OH and C–O–C stretching vibrations, are characteristic of alcohols, carboxylic acids, and esters. The peak at 1044 cm⁻¹ is attributed to C–C and C–O stretches in carbohydrate structures as well as C–O bonds in phenolic compounds. The proportionate reduction in the intensity of these peaks, which are associated with phenolic compounds, supports the finding that the ACE had an effective anti-browning impact on the apple cubes. Furthermore, it confirms that chitosan had limited anti-browning efficiency when compared to ACE. These observations are in agreement with findings reported by Irchad et al. [80] and Hssaini et al. [81].

3.5. Visual Color Score, Browning Index, and Shelf Life

Figure 3A,B and

Table 5. Appearance changes in control or (1%) chitosan-treated cut apple cubes over 10 min following preservation for 10 days at 7 °C. Both uncoated and coated (chitosan) treatments were prepared by using pH-adjusted aqueous extracts of cabbage seeds (ACEs).

	UNCOATED (No Chitosan)						COATED (1% Chitosan)
Preservation	ACE 0%		ACE 5%		ACE 10%		ACE 0%		ACE 5%		ACE 10%
(days)	pH 4.0	pH 6.0	pH 4.0	pH 6.0	pH 4.0	pH 6.0	pH 4.0	pH 6.0	pH 4.0	pH 6.0	pH 4.0	pH 6.0
0
3
6
10

show the development of browning, expressed as VCS, for uncoated and chitosan-coated apple cubes, respectively, during preservation for 10 days at 7 °C. All treatments showed an increase in VCS which continued to increase until the end of the preservation period, but the treatments with chitosan showed a greater increase. The changes in the appearance of fresh-cut apple cubes reflecting visual color during preservation are shown in Table 5.

The pH adjustment of the ACE employed in the uncoated treatments had no significant effect on the change in VCS of apple cubes over time; however, treatments adjusted to pH 4.0 reached VCS values beyond the acceptable limit of 3/5 more quickly than pH 6.0 (Figure 3, Table 5). The VCS of uncoated apple cubes treated with water-control (0% ACE) reached the acceptable sales level in one day, whereas those treated with 5 or 10% ACE took about four days; the exception was the treatment with 10% ACE adjusted to pH 4, which took 3 days.

Throughout the preservation period, chitosan-coated apple cubes adjusted to pH 4.0 had significantly higher VCS values than those adjusted to pH 6.0 (Table 5). This is demonstrated by the fact that applying ACE adjusted to pH 4.0 to the coating resulted in VCS values above the accepted sales limit of 3/5 upon treatment (day 0), whereas adjustment to pH 6.0 kept the VCS within acceptable limits for just one day (Figure 3, Table 5). These findings highlight that adjusting ACE to pH 6.0 was more effective in delaying visible browning and preserving marketability.

The demand for fresh-cut fruit reflects consumer preferences towards appearance and freshness at the time of purchase as being among the main quality criteria [1]. Browning is another challenge for fresh-cut apple preservation, which significantly affects consumer perceptions among other sensory parameters [48,82].

Correlation of VCS with BI (R² = 0.837, p < 0.05), using the curve-fitting model of exponential growth, a single third parameter, indicates that the acceptable limit (a VCS score of 3/5) corresponds to a BI value of 41.85 (Figure S1). Androudis et al. [27], who used aqueous cabbage extracts on freshly cut lettuce stem sections, discovered an approximate threshold acceptable value for BI of 40. Figure 4A,B depict the development of browning, indicated as BI, in ACE-treated apple cubes (coated and uncoated) preserved for 10 days at 7 °C. In general, BI rose during preservation, whereas chitosan treatments caused a substantially larger rise.

Apple cubes coated with ACE adjusted to pH 4.0 showed maximum BI values from the beginning (>40) to the end (>100) of preservation duration (Figure 4B), followed by the uncoated controls (0% ACE) adjusted to either pH 4.0 or 6.0 (Figure 4A). Apple cubes subjected to coating with ACE plus chitosan adjusted to pH 6.0 showed intermediate BI (values of ~60 at the end of preservation), while minimum BI was noted in treatments of 5 or 10% ACE without coating (Figure 4A).

BI was more sensitive than VCS for the majority of the treatments. However, under the treatment of 5% ACE adjusted to pH 6.0, BI values remained below 41.85 for the whole preservation duration (Figure 4A), despite the fact that VCS had risen over 3 by day 4 (Figure 3A) and the average color had altered to darker brownish tones between days 3 and 6 (Table 5). This was probably due to perceptual sensitivity differences between human scoring and instrumental color change. Similar was the case for h^o (Figures S1 and S2) The decrease in BI values correlated with the increase in L values during preservation. Karseno et al. [83] used BI to describe the browning of fresh-cut apples.

The shelf life was determined as the total time it took for apple cubes to reach the VCS threshold of 3/5, with the corresponding value for BI being 41.85, indicating that browning had proceeded to an undesirable level. VCS was acquired from three Petri dishes, three replicates per treatment (6 apple cubes per replication) and shelf life was calculated using the average shelf life of each Petri dish (three replicates) per treatment. For cases when the sample did not approach the value 3 on a given day but instead proceeded right to a higher value, the Acceptance Rate vs. Preservation Time curve was used to estimate the day when it passed from a “threshold of 3” on a scale of 1–5. Following the analysis of variance, the means were compared using the least significant difference (LSD) method at a 95% confidence level; treatments marked with different letters (A, B, and C) differ significantly (p < 0.05) (Figure 5).

The treatments with the best performance were uncoated 5% and 10% ACE adjusted to pH = 6.0, or to pH = 4.0, which provided 4.0 and 4.2, or 4 and 3.6 days of effective acceptable brown color levels based on VCS (Figure 5A). On the other hand, the addition of chitosan did not result in an extension of shelf life, compared to uncoated water control (Figure 5A). The combination of ACE and chitosan failed to extend shelf life, most likely due to metal ion permeability issues intensifying oxidative processes [51] or stimulating oxidation via the production of phenol substrates [62], as previously discussed.

BI was found to be more sensitive for calculating shelf life (Figure 5B). Uncoated apples treated with 5% and 10% ACE at pH 6.0 had a shelf life extension of 7–8 days, compared to only 4–4.5 days at pH 4.0 (p < 0.05). Chitosan coatings had a similar shelf life for both VCS and BI calculations (Figure 5B).

According to Rojas-Graü et al. [84], fresh-cut fruit’s browning may be delayed by natural sulfur compounds. Glucosinolates, which are naturally occurring sulfur- and nitrogen-containing chemicals that have been discovered to be present in cruciferous vegetables, and may be responsible for the anti-browning characteristics of ACEs. To determine the impact of one or both kinds of phytochemicals on the enzymatic browning of fresh-cut apple more research and examination of the interrelationship between phenolic and glucosinolate profile and ACE may be required.

4. Conclusions

In this study, the ACEs were found to have a high phenolic and glucosinolate content, with the 10% level significantly higher than the 5%. Untreated cut apples began browning to unsatisfactory levels (based on VCS assessment) on day one, whereas 5 or 10% ACE treatment delayed it until day four of preservation, which was regarded as the effective shelf life. When BI was used to calculate shelf life, ACE treatments were found to delay browning until day 8 of preservation. This indicated that BI has a different sensitivity to browning than VCS. However, both were regarded as valid indices of enzymatic browning; to balance sensitivities, the VCS scale might be based on a 1–9 range.

Furthermore, ACE modified to pH 6.0 was more efficient than that at pH 4.0 in delaying the browning of cut apples. However, the pH issue may require further investigation to maximize the shelf life extension of cut apples.

After applying chitosan coatings (without or with the addition of ACE) to apple cubes, they developed browning to unsatisfactory levels as early as day one, as did the uncoated water control. These findings were supported by FT-IR analysis during preservation. Considering these results, chitosan coatings are not recommended for Starking Delicious cut apples. Further research is required to clearly show the mechanism of chitosan action.

This study shows that the compounds present in cabbage seeds may inhibit browning reactions in a cut apple cube model. However, the possible anti-browning capabilities of the ACE must be assigned to specific chemicals in future research, which will employ more advanced analysis techniques (HPLC, LC-MS/MS) as well as validation across various cut fruits and vegetables.