**1. Introduction**

Browning is associated with deterioration. Its prevention, either by using additives or choosing resistant cultivars, has taken a great part in horticultural and food research. The conventional method to inhibit browning in fruit has been to utilize sulfites [1]. However, due to health concerns, alternative means of controlling enzymatic browning are required [2]. In addition to numerous food processing techniques for prevention of browning, the initial decision on cultivar selection is crucial for all further steps. We now have several known cultivars with a smaller rate or lack of browning, such as arctic apples, 'Ambrosia', 'Eden', 'Aori27', etc. [3–5]. The mechanism behind browning is oxidation of polyphenols. Oxidation occurs when tissues are damaged, either by improper handling causing bruising or by processing, cutting, peeling or grinding. Due to damaged cells, the phenolics come into contact with polyphenol oxidase (PPO). In intact cells, PPO seems to have little activity towards phenolics [6]. In addition to the phenolic content, the activity of PPO is the reason for the development of browning [7]. PPO interacts with phenolic substrates and molecular oxygen, since it is a bi-metalloenzyme with two copper-binding domains [8]. The primary reaction is initiated by PPO accumulated in plastids, and not by *de novo* formed enzyme, although high activation occurs in time after a cell-damaging event. Furthermore, phenolic concentration increases in time after wounding [9]. What are the main mechanisms behind non-browning cultivars? Arctic apple cultivars were genetically

**Citation:** Cebulj, A.; Vanzo, A.; Hladnik, J.; Kastelec, D.; Vrhovsek, U. Apple (*Malus domestica* Borkh.) Cultivar 'Majda', a Naturally Non-Browning Cultivar: An Assessment of Its Qualities. *Plants* **2021**, *10*, 1402. https://doi.org/ 10.3390/plants10071402

Academic Editor: Ivo Vaz de Oliveira

Received: 22 June 2021 Accepted: 6 July 2021 Published: 9 July 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

engineered with a transgene that produces specific RNAs to silence PPO genes [5]. Cultivar 'Eden' has a low phenolic content [3]. In addition to the low phenolic content, a low PPO activity is also thought to be behind a lack of browning in cultivar 'Aori 27' [4]. The lack of browning for cultivar 'Ambrosia' is explained by the lower activity of PPO enzyme [10]. [5]. Cultivar 'Eden' has a low phenolic content [3]. In addition to the low phenolic content, a low PPO activity is also thought to be behind a lack of browning in cultivar 'Aori 27' [4]. The lack of browning for cultivar 'Ambrosia' is explained by the lower activity of PPO enzyme [10].

are the main mechanisms behind non-browning cultivars? Arctic apple cultivars were genetically engineered with a transgene that produces specific RNAs to silence PPO genes

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Another important quality of a cultivar and its part of defense metabolism are two major low-molecular-weight antioxidants, ascorbic acid (Vitamin C) and glutathione (GSH) [11,12]. Ascorbic acid has an ability to reduce quinones back to phenolic compounds prior to their subsequent reaction to form pigments. While GSH is directly linked to cellular ascorbic acid metabolism through the ascorbate-glutathione cycle, GSH is used as a source of reducing power for the enzymatic regeneration of oxidized ascorbic acid [13]. Furthermore, glutathionyl-chlorogenic acid conjugate was reported in apple juice [14]. Glutathionyl conjugates of hydroxycinnamic acids are known for limiting the browning of grape juice, where GSH interferes by trapping the caftaric acid quinones produced by oxidation in the form of 2-s-glutathionylcaftaric acid [15]. In addition, GSH has a role in biosynthetic pathways, detoxification, antioxidant biochemistry, and redox homeostasis [16]. Another important quality of a cultivar and its part of defense metabolism are two major low-molecular-weight antioxidants, ascorbic acid (Vitamin C) and glutathione (GSH) [11,12]. Ascorbic acid has an ability to reduce quinones back to phenolic compounds prior to their subsequent reaction to form pigments. While GSH is directly linked to cellular ascorbic acid metabolism through the ascorbate-glutathione cycle, GSH is used as a source of reducing power for the enzymatic regeneration of oxidized ascorbic acid [13]. Furthermore, glutathionyl-chlorogenic acid conjugate was reported in apple juice [14]. Glutathionyl conjugates of hydroxycinnamic acids are known for limiting the browning of grape juice, where GSH interferes by trapping the caftaric acid quinones produced by oxidation in the form of 2-s-glutathionylcaftaric acid [15]. In addition, GSH has a role in biosynthetic pathways, detoxification, antioxidant biochemistry, and redox homeostasis [16].

All the above-mentioned metabolites have different preserving abilities during storage that depend firstly on cultivar and pre- and post-harvest parameters. According to Awad and Jager [17], total phenolics are relatively stable during storage. A good stability of the main antioxidants (including GSH and ascorbic acid) was also reported [18]. Davey and Keulemans [19] reported on the increased GSH content after 3 months of cold storage of several apple cultivars, as well as increased vitamin C content. The increase, and in some cases the decrease, of GSH and vitamin C mainly depended on the cultivar. However, a weak correlation to the harvest time was implicated, as well. All the above-mentioned metabolites have different preserving abilities during storage that depend firstly on cultivar and pre- and post-harvest parameters. According to Awad and Jager [17], total phenolics are relatively stable during storage. A good stability of the main antioxidants (including GSH and ascorbic acid) was also reported [18]. Davey and Keulemans [19] reported on the increased GSH content after 3 months of cold storage of several apple cultivars, as well as increased vitamin C content. The increase, and in some cases the decrease, of GSH and vitamin C mainly depended on the cultivar. However, a weak correlation to the harvest time was implicated, as well.

Cultivar 'Majda' was confirmed as a variety in 1986 and was made from the cross of 'Jonatan' and 'Golden Noble' [20]. The apple has a dark green basic color with a dark red top color (Figure 1). Even though its non-browning characteristics were described when it was introduced, it is not a well-known or a widely used cultivar. Only a few growers have cv. 'Majda' planted in orchards. One of the reasons is probably the color of fruit, which is not as appealing as the color of modern apple cultivars. It has high acidity and is therefore mainly known for its use in processing. Cultivar 'Golden Delicious' is a well-known cultivar and in numerous countries, the time of harvest and basic characteristic of cultivars are compared to this cultivar. With respect to this, cultivar 'Golden Delicious' was chosen as a comparison to cultivar 'Majda'. The first phenolic analysis of cultivar 'Majda' were made by Persic et al. [21], where they compared several cultivars in terms of phenolic content and browning. They have correlated a stronger oxidation to the high total phenolic content in apples. Their results urged us to focus on this cultivar, to further explore the phenomenon of non-browning of cultivar 'Majda'. Cultivar 'Majda' was confirmed as a variety in 1986 and was made from the cross of 'Jonatan' and 'Golden Noble' [20]. The apple has a dark green basic color with a dark red top color (Figure 1). Even though its non-browning characteristics were described when it was introduced, it is not a well-known or a widely used cultivar. Only a few growers have cv. 'Majda' planted in orchards. One of the reasons is probably the color of fruit, which is not as appealing as the color of modern apple cultivars. It has high acidity and is therefore mainly known for its use in processing. Cultivar 'Golden Delicious' is a wellknown cultivar and in numerous countries, the time of harvest and basic characteristic of cultivars are compared to this cultivar. With respect to this, cultivar 'Golden Delicious' was chosen as a comparison to cultivar 'Majda'. The first phenolic analysis of cultivar 'Majda' were made by Persic et al. [21], where they compared several cultivars in terms of phenolic content and browning. They have correlated a stronger oxidation to the high total phenolic content in apples. Their results urged us to focus on this cultivar, to further explore the phenomenon of non-browning of cultivar 'Majda'.

**Figure 1. Figure 1.** 'Majda' apples. 'Majda' apples.

The aim of this study was to establish the main parameters of inner quality of cultivar 'Majda' in comparison to 'Golden Delicious'. We have determined the content of sugars, organic acids, vitamin C, glutathione (GSH and GSSG), its precursors cysteine, methionine, and phenolics in apple flesh, with the addition of phenolic content in apple peel. Additionally, to establish if the low phenolic content also reflects in leaves, the content of phenolics in leaves was determined. Furthermore, we wanted to decipher the main reasons for its low susceptibility to browning. The change in color in halves and pomace was also measured and the activity of peroxidase and polyphenol oxidase were determined. Moreover, we have repeated the analysis on flesh post-storage to determine if the trait persists after storage. nine, and phenolics in apple flesh, with the addition of phenolic content in apple peel. Additionally, to establish if the low phenolic content also reflects in leaves, the content of phenolics in leaves was determined. Furthermore, we wanted to decipher the main reasons for its low susceptibility to browning. The change in color in halves and pomace was also measured and the activity of peroxidase and polyphenol oxidase were determined. Moreover, we have repeated the analysis on flesh post-storage to determine if the trait persists after storage. **2. Results** 

The aim of this study was to establish the main parameters of inner quality of cultivar 'Majda' in comparison to 'Golden Delicious'. We have determined the content of sugars, organic acids, vitamin C, glutathione (GSH and GSSG), its precursors cysteine, methio-

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#### **2. Results** *2.1. Sugars*

#### *2.1. Sugars* The results of total sugars and organic acids are presented in Figure 2 and their sta-

The results of total sugars and organic acids are presented in Figure 2 and their statistical analysis in Table 1. All three factors (cultivar, time, and location) have a statistically significant influence on sugar content. Moreover, the interactions between the cultivar and time as well as the cultivar and location are significant. The contrast analysis showed that there are statistically significant differences in the mean total sugar content between 'Golden Delicious' and 'Majda' at L2, whereas there were no statistically significant differences between cultivars at L1. However, when also looking at confidence interval, one can see that it is also close to a statistically significant difference at location L1 (Table S1). Following storage, there are statistically significant differences between L1 and L2 with the 'Golden Delicious' cultivar. Time had a statistically significant influence on the content of total sugars in 'Majda' at both locations. The composition of individual sugars also differs between cultivars (Figure S1), with 'Majda' having a higher content of sucrose and sorbitol and a lower content of glucose and fructose. tistical analysis in Table 1. All three factors (cultivar, time, and location) have a statistically significant influence on sugar content. Moreover, the interactions between the cultivar and time as well as the cultivar and location are significant. The contrast analysis showed that there are statistically significant differences in the mean total sugar content between 'Golden Delicious' and 'Majda' at L2, whereas there were no statistically significant differences between cultivars at L1. However, when also looking at confidence interval, one can see that it is also close to a statistically significant difference at location L1 (Table S1). Following storage, there are statistically significant differences between L1 and L2 with the 'Golden Delicious' cultivar. Time had a statistically significant influence on the content of total sugars in 'Majda' at both locations. The composition of individual sugars also differs between cultivars (Figure S1), with 'Majda' having a higher content of sucrose and sorbitol and a lower content of glucose and fructose.

**Figure 2.** Content of total sugars (g kg<sup>−</sup>1 FW; mean ± SE) and total organic acids (g kg−1 FW; mean ± SE) of cultivars 'Golden Delicious' and 'Majda' at two locations (L1 and L2) at harvest and following storage. **Figure 2.** Content of total sugars (g kg−<sup>1</sup> FW; mean <sup>±</sup> SE) and total organic acids (g kg−<sup>1</sup> FW; mean ± SE) of cultivars 'Golden Delicious' and 'Majda' at two locations (L1 and L2) at harvest and following storage.


**Table 1.** Three-factor ANOVA for cultivar (Cul: 'Golden Delicious' and 'Majda'), location (Loc: L1 and L2), and time (T: harvest and storage), as well as their interactions (*p* < 0.05). Total sugars \*\* . \*\*\* NS \*\* . NS Total acids \*\*\* NS \*\*\* \*\* NS \*\* \*\*\*

 **Cul Loc T Cul:Loc T:Cul T:Loc T:Cul:Loc** 

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., Statistically significant differences at *p* < 0.1; \*, statistically significant differences at *p* < 0.05; \*\*, statistically significant differences at *p* < 0.01; \*\*\*, statistically significant differences at *p* < 0.001; NS: Not significant. *2.2. Organic Acids* 

#### *2.2. Organic Acids* Malic and citric acid were quantified among organic acids (Table S2). Fumaric acid

harvest and storage), as well as their interactions (*p* < 0.05).

Malic and citric acid were quantified among organic acids (Table S2). Fumaric acid was also determined, but its amount was exceptionally low and thereby not quantified. Malic acid is the prevalent acid in apples, which is why its content has a decisive influence on the total acid content in apple. When analyzing all the factors, we can see that there is a statistically significant interaction between all of them. After the contrast analysis was made, statistically significant differences were confirmed between cultivars within locations at harvest and at location L1 following storage. Significant differences in the total acid content following storage in 'Golden Delicious' at both locations were noted and in cv. 'Majda' at location L2. was also determined, but its amount was exceptionally low and thereby not quantified. Malic acid is the prevalent acid in apples, which is why its content has a decisive influence on the total acid content in apple. When analyzing all the factors, we can see that there is a statistically significant interaction between all of them. After the contrast analysis was made, statistically significant differences were confirmed between cultivars within locations at harvest and at location L1 following storage. Significant differences in the total acid content following storage in 'Golden Delicious' at both locations were noted and in cv. 'Majda' at location L2.

#### *2.3. pH 2.3. pH*  The results for apple juice mean that the pH values (± SE) are presented in Figure 3

The results for apple juice mean that the pH values (±SE) are presented in Figure 3 and statistical analysis in Table 1. The cultivar and time have a statistically significant influence on pH as well as their interaction. Apple juice pH is not influenced by location. The analysis of contrasts (Table S3) confirmed statistically significant differences between cultivars. and statistical analysis in Table 1. The cultivar and time have a statistically significant influence on pH as well as their interaction. Apple juice pH is not influenced by location. The analysis of contrasts (Table S3) confirmed statistically significant differences between cultivars.

**Figure 3.** Content of apple juice pH (mean ± SE) of cultivars 'Golden Delicious' and 'Majda' at two locations (L1 and L2) at harvest and following storage.
