plants Article Apple (Malus domestica Borkh.) Cultivar ‘Majda’, a Naturally Non-Browning Cultivar: An Assessment of Its Qualities Anka Cebulj 1,* , Andreja Vanzo 1 , Joze Hladnik 1, Damijana Kastelec 2 and Urska Vrhovsek 3 1 Agricultural Institute of Slovenia, Department of Fruit Growing, Viticulture and Oenology, Hacquetova ulica 17, 1000 Ljubljana, Slovenia; [email protected] (A.V.); [email protected] (J.H.) 2 Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia; [email protected] 3 Research and Innovation Centre, Fondazione Edmund Mach, Via E. Mach 1, 38010 San Michele all’Adige, Italy; [email protected] * Correspondence: [email protected] Abstract: Browning of apple and apple products has been a topic of numerous research and there is a great number of methods available for browning prevention. However, one of the most efficient ways, and the one most acceptable for the consumers, is the selection of a non-browning cultivar. Cultivar ‘Majda’ is a Slovenian cultivar, a cross between ‘Jonatan’ and ‘Golden Noble’. In this study, it was thoroughly examined and compared to the well-known cultivar ‘Golden Delicious’ with the aim to decipher the reason for non-browning. We have determined the content of sugars, organic acids, vitamin C, glutathione and phenolics in apple flesh, with the addition of phenolic content in apple peel and leaves. The change in color in halves and pomace was also measured and the activity of peroxidase (POX) and polyphenol oxidase (PPO) were determined. Additionally, the analyses of flesh were repeated post-storage. The most prominent results were high acidity (malic acid), low phenol Citation: Cebulj, A.; Vanzo, A.; content, especially hydroxycinnamic acid and flavan-3-ol content of cultivar ‘Majda’ in comparison Hladnik, J.; Kastelec, D.; Vrhovsek, U. Apple (Malus domestica Borkh.) to ‘Golden Delicious’, and no difference in PPO activity between cultivars. After the overview of the Cultivar ‘Majda’, a Naturally results, we believe that both low phenol content and high reduced glutathione content impact the Non-Browning Cultivar: An non-browning characteristics of cultivar ‘Majda’. Assessment of Its Qualities. Plants 2021, 10, 1402. https://doi.org/ Keywords: non-browning; polyphenol oxidase; phenolics; vitamin C; glutathione 10.3390/plants10071402 Academic Editor: Ivo Vaz de Oliveira 1. Introduction Received: 22 June 2021 Browning is associated with deterioration. Its prevention, either by using additives Accepted: 6 July 2021 or choosing resistant cultivars, has taken a great part in horticultural and food research. Published: 9 July 2021 The conventional method to inhibit browning in fruit has been to utilize sulfites [1]. How- ever, due to health concerns, alternative means of controlling enzymatic browning are Publisher’s Note: MDPI stays neutral required [2]. In addition to numerous food processing techniques for prevention of brown- with regard to jurisdictional claims in ing, the initial decision on cultivar selection is crucial for all further steps. We now have published maps and institutional affil- iations. 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 Copyright: © 2021 by the authors. have little activity towards phenolics [6]. In addition to the phenolic content, the activity Licensee MDPI, Basel, Switzerland. of PPO is the reason for the development of browning [7]. PPO interacts with phenolic This article is an open access article substrates and molecular oxygen, since it is a bi-metalloenzyme with two copper-binding distributed under the terms and conditions of the Creative Commons domains [8]. The primary reaction is initiated by PPO accumulated in plastids, and not by Attribution (CC BY) license (https:// de novo formed enzyme, although high activation occurs in time after a cell-damaging event. creativecommons.org/licenses/by/ Furthermore, phenolic concentration increases in time after wounding [9]. What are the 4.0/). main mechanisms behind non-browning cultivars? Arctic apple cultivars were genetically Plants 2021, 10, 1402. https://doi.org/10.3390/plants10071402 https://www.mdpi.com/journal/plants Plants 2021, 10, x FOR PEER REVIEW 2 of 17 Plants 2021, 10, 1402 2 of 17 are the main mechanisms behind non-browning cultivars? Arctic apple cultivars were ge- netically 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, engineered with a transgene that produces specific RNAs to silence PPO genes [5]. Cultivar a low PPO activity is also thought to be behind a lack of browning in cultivar ‘Aori 27’ [4]. ‘Eden’ has a low phenolic content [3]. In addition to the low phenolic content, a low PPO The lack of browning for cultivar ‘Ambrosia’ is explained by the lower activity of PPO activity is also thought to be behind a lack of browning in cultivar ‘Aori 27’ [4]. The lack of enzyme [10]. browning for cultivar ‘Ambrosia’ is explained by the lower activity of PPO enzyme [10]. Another important quality of a cultivar and its part of defense metabolism are two 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 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 com- (GSH) [11,12]. Ascorbic acid has an ability to reduce quinones back to phenolic com- poundspounds priorprior toto theirtheir subsequentsubsequent reactionreaction toto formform pigments.pigments. WhileWhile GSHGSH isis directlydirectly linkedlinked toto cellularcellular ascorbicascorbic acidacid metabolismmetabolism throughthrough thethe ascorbate-glutathioneascorbate-glutathione cycle,cycle, GSHGSH isis usedused asas aa sourcesource of of reducing reducing power power for for the the enzymatic enzymatic regeneration regeneration of oxidized of oxidized ascorbic ascorbic acid [acid13]. Furthermore,[13]. Furthermore, glutathionyl-chlorogenic glutathionyl-chlorogenic acid conjugateacid conjugate was reportedwas reported in apple in apple juice juice [14]. Glutathionyl[14]. Glutathionyl conjugates conjugates of hydroxycinnamic of hydroxycinnamic acids acids are knownare known for limitingfor limiting the the browning brown- ofing grape of grape juice, juice, where where GSH GSH interferes interferes by trapping by trapping the caftaricthe caftaric acid acid quinones quinones produced produced by oxidationby oxidation in thein the form form of 2-s-glutathionylcaftaricof 2-s-glutathionylcaftaric acid acid [15 [15].]. In In addition, addition, GSH GSH hashas aa rolerole inin biosyntheticbiosynthetic pathways,pathways, detoxification,detoxification, antioxidantantioxidant biochemistry, andand redoxredox homeosta-homeosta- sissis [[16].16]. AllAll thethe above-mentionedabove-mentioned metabolites metabolites have have different different preserving preserving abilities abilities during during storage stor- thatage dependthat depend firstly firstly on cultivar on cultivar and pre- and and pre- post-harvest and post-harvest parameters. parameters. According According to Awad to andAwad Jager and [17 Jager], total [17], phenolics total phenolics are relatively are relatively stable duringstable during storage. storage. A good A stability good stability of the mainof the antioxidants main antioxidants (including (including GSH andGSH ascorbic and ascorbic acid) wasacid) also was reported also reported [18]. Davey[18]. Davey and Keulemansand Keulemans [19] reported[19] reported on the on increasedthe increased GSH GSH content content after after 3 months 3 months of cold of cold storage storage of severalof several apple apple cultivars, cultivars, as well as well as increased as increased vitamin vitamin C content. C content. The increase, The increase, and in and some in casessome thecases decrease, the decrease, of GSH of and GSH vitamin and vitamin C mainly C mainly depended depended on the on cultivar. the cultivar. However, How- a weakever, a correlation weak correlation to the harvest to the harvest time was time implicated, was implicated, as well. as well. CultivarCultivar ‘Majda’ was confirmed confirmed as as a a variety variety in in 1986 1986 and and was was made made from from the the cross cross of of‘Jonatan’ ‘Jonatan’ and and ‘Golden ‘Golden Noble’ Noble’ [20]. [20 The]. The apple apple has has a dark a dark green green basic basic color color with with a dark a dark red redtop topcolor color (Figure (Figure 1). Even1). Even though though its non-br its non-browningowning characteristics characteristics were weredescribed described when whenit was itintroduced, was introduced, it is not it a is well-known not a well-known or a widely or a widelyused cultivar. used cultivar. Only a few Only growers a few growershave cv. have‘Majda’ cv. ‘Majda’planted plantedin orchards. in orchards. One of Onethe reasons of the reasons is probably is probably the color the colorof fruit, of fruit,which which is not isas not appealing as appealing as the as color