Chemical composition of apple fruit, juice and pomace and the correlation between phenolic content, enzymatic activity and browning

Accepted Manuscript Chemical composition of apple fruit, juice and pomace and the correlation between phenolic content, enzymatic activity and browning Martina Persic, Maja Mikulic-Petkovsek, Ana Slatnar, Robert Veberic PII:

S0023-6438(17)30236-0

DOI:

10.1016/j.lwt.2017.04.017

Reference:

YFSTL 6154

To appear in:

LWT - Food Science and Technology

Received Date: 20 July 2016 Revised Date:

3 April 2017

Accepted Date: 6 April 2017

Please cite this article as: Persic, M., Mikulic-Petkovsek, M., Slatnar, A., Veberic, R., Chemical composition of apple fruit, juice and pomace and the correlation between phenolic content, enzymatic activity and browning, LWT - Food Science and Technology (2017), doi: 10.1016/j.lwt.2017.04.017. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Chemical composition of apple fruit, juice and pomace and the correlation

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between phenolic content, enzymatic activity and browning

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Martina Persica,*, Maja Mikulic-Petkovsek a, 1, Ana Slatnar a, 2, Robert Veberic 3, a

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a

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Wine and Vegetable Growing, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia

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* Corresponding author: [email protected]; Tel. +386 1320110; Fax: +386

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14231088

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[email protected]

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[email protected]

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[email protected]

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University of Ljubljana, Biotechnical faculty, Department of Agronomy, Chair for Fruit,

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ACCEPTED MANUSCRIPT ABSTRACT:

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Primary and secondary metabolites were evaluated in apple fruit, juice and pomace of scab

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resistant and leading European apple cultivars. The primary goal was to study the chemical

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composition of apple fruit fractions in correlation with their enzymatic browning. Additional

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goal was to assess the suitability of apple pomace for the extraction of phenolic compounds.

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Furthermore, the cultivars were grouped according to their suitability for fresh-cut fruit slices

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by measuring their enzymatic activity, total phenolic content (TPC) and the rate of browning.

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Highest TPC was determined in ‘Granny Smith’ pomace suggesting its optimal suitability for

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phenolic extraction among the analyzed apple cultivars. ‘Majda’ fruit (investigated for the

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first time) can be offered to the market as fresh-cut slices as almost no changes in pulp color

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have been detected due to oxidation. The correlation test showed that oxidation of apple pulp

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is highly dependent on TPC and weakly correlated to the activity of polyphenol oxidase.

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Contrary, no significant correlation has been determined between the rate of oxidation and the

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activity of peroxidase.

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KEY WORDS: phenolic content, enzymatic activity, enzymatic browning, fresh-cut fruit,

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apple pomace

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1. Introduction

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In recent years consumers have become more aware of diverse health benefits of non-

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processed or low processed fruit (Rico, Martin-Diana, Barat, & Barry-Ryan, 2007). In

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addition to a fast growing market for locally produced apple fruit and juice, an increasing

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interest for fresh-cut apple slices or cubes has been recorded as consumers are in search for

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fresh and nutritious snacks. However, apple slices often develop unattractive brownish color

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due to oxidative processes in the fruit. Genetically modified apple cultivars that do not change 2

ACCEPTED MANUSCRIPT color due to oxidation (Prakash, 2014) are not suitable for European markets due to the ban of

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genetically modified organisms in most European countries. Therefore, this trait must be

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ascertained in existing and resistant apple cultivars, which have been introduced into modern

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apple orchards. These cultivars are often organically cultivated and contain high levels of

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nutrients and phenolic compounds (Mikulic-Petkovsek, Slatnar, Stampar, & Veberic, 2010;

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Mikulic-Petkovsek, Stampar, & Veberic, 2007). The later are involved in natural defensive

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reactions of plants against herbivores and plant pathogens (Korkina, 2007). The content of

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phenolic compound is highly dependent on the apple cultivar and various cultivation practices

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(Mikulic-Petkovsek, Slatnar, Stampar, & Veberic, 2010; Slatnar, Mikulic-Petkovsek,

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Halbirth, Stampar, Stich, & Veberic 2010; van der Sluis, Dekker, de Jager, & Jongen, 2001;

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Veberic, Trobec, Herbinger, Hofer, Grill, & Stampar 2005; Zupan, Mikulic-Petkovsek, Cunja,

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Stampar, & Veberic, 2013). On the other hand, phenolics are also partially responsible for

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deterioration of fresh-cut apple fruit.

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The main setback in fresh-cut industry is rapid product deterioration due to enzymatic

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browning. This does not only affect flavor and nutrient content, but also reduces visual quality

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of the product (Quevedo, Jaramillo, Diaz, Pedreschi, & Aguilera, 2009). Browning of fresh-

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cut fruit can be ascribed to oxidative reactions of enzymes and phenolic compounds, while

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non-enzymatic browning usually occurs in heat-processed products. Polyphenolics are

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oxidized mainly by polyphenol oxidase and to a lesser extent by peroxidase. Goupy, Amiot,

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Richard-Forget, Duprat, Aubert, & Nicolas (1995) correlated the rate of browning with

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substrate content and enzyme activity. Contrary, Robards, Prenzler, Tucker, Swatsitang, and

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Glover (1999) summed up opposing results on this subject and reported high cultivar

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dependency of phenolic profile and enzymatic activity.

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Phenolic compounds have been well studied in apple and apple juice, yet on the other hand

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leftover pulp or pomace of apple juice production has not been sufficiently tested for potential

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ACCEPTED MANUSCRIPT extraction of phenolic compounds (Candrawinata, Golding, Roach, & Stathopoulos, 2013).

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Van der Sluis, Dekker, Skrede, and Jongen (2002) suggested a second-stage extraction of

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polyphenols from apple pomace and subsequent addition of phenolics to juice in order to

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make “enhanced apple juice” with high marketable value. Moreover, phenolic extracts from

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apple pomace are not only interesting as an addition to the juice but could commercially be

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used as nutritional supplements or added to other products and functional foods (Lu & Foo,

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2000). Phenolic extraction of apple pomace could therefore be considered as added

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commercial value of the juice-making process.

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In the present research traditional, native, resistant and susceptible apple cultivars have been

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evaluated for their suitability for extraction of phenolics from pomace and their use as fresh-

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cut fruit. Phenolic profiles and the content of organic acids and sugars have been compared

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among cultivars in fresh apple fruit, juice and pomace. Additionally, enzymatic activity of

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polyphenol oxidase and peroxidase have been investigated in apple pulp, peel and juice to

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determine the link between total phenolic content, enzymatic activity and the rate of browning

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in fresh-cut apples. To our knowledge, the fruit of ‘Majda’ cultivar has not been chemically

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characterized until now.

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2.

Materials and methods

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2.1.

Plant material and sample preparation

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The research was carried out on eight apple cultivars: three widespread European cultivars -

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‘Jonagold’ (JG), ‘Golden Delicious’ (GD) and ‘Granny Smith’ (GS); two traditional cultivars

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- ‘Boskoop’ (BO) and ‘Kronprinz Rudolf’ (KR), two scab resistant cultivars - ‘Topaz’ (TO)

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and ‘Florina’ (FL) and a local Slovenian cultivar - ‘Majda’ (MA). All apple cultivars were

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harvested at technological maturity and were grown according to guidelines for integrated

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production. Apples of all examined cultivars were classified by size and 20 medium-sized

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ACCEPTED MANUSCRIPT apples were used for measurements and further analysis. The measurements were carried out

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at room temperature (22±2°C) and 50-60% relative humidity.

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Ten apples per cultivar were washed and cut in half. One half of each apple was used for juice

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extraction and the other half was peeled with a ceramic fruit peeler. Pulp and peel were

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weighted separately to calculate the peel to pulp ratio of each studied cultivar.

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The apple juice was prepared using automatic juice extractor AE 3150 (Clatronic, Kempen,

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Germany). The juice was immediately filtrated into vials and frozen at -20°C until further

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analyzes of phenolic compounds, organic acids and sugar content. The pomace was retrieved

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from the juicer and analyzed according to the same procedure as apple pulp.

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The rest of the apples (10 per cultivar) were used for measurements of enzymatic activity and

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browning. Each apple was cut in half and one half was used for measuring the enzymatic

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activity of peel and pulp. Juice was extracted from the other half and immediately analyzed

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for enzymatic activity.

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2.2. Extraction and determination of sugars and organic acids

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Sugars and organic acids were extracted from apple pulp according to the method described

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by Mikulic-Petkovsek, Stampar, and Veberic (2007). For the extraction of sugars and organic

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acid, 25 g of fresh apple pulp (in five repetitions per cultivar) was homogenized with an Ultra-

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Turrax T-25 (Ika-Labortechnik, Stauden, Germany) in 25 mL of double distilled water. The

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extracts were left for 30 minutes at room temperature with continuous stirring. After the

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extraction, samples were centrifuged and filtered through 0.20 µm cellulose mixed ester filters

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(Macherey-Nagel; Düren, Germany), into vials. Sugars and organic acids from the pomace

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were extracted in the same way as in fresh apple pulp. Samples were further analyzed for the

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ACCEPTED MANUSCRIPT content of individual sugars and acids using the Thermo Finnigan Surveyor HPLC system

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(Thermo Scientific, San Jose, CA). Chromatographic conditions were as described by

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Mikulic-Petkovsek, Schmitzer, Slatnar, Stampar & Veberic (2012). The content of individual

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sugars and organic acids were calculated from calibration curves of corresponding standards.

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Sugar/acid (S/A) ratio was calculated from the obtained results.

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2.3. Extraction and determination of individual and total phenolics

Phenolic compounds were extracted from apple pulp, peel, juice and pomace according to the

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method described by Mikulic-Petkovsek, Schmitzer, Slatnar, Stampar, & Veberic (2015) with

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slight modifications. For determination of individual phenolic compounds and total phenolic

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content (TPC), 10 g of pulp, 10 g of pomace and 5 g of peel was extracted with 10 mL of

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methanol containing 3% of formic acid. The extraction lasted one hour and was facilitated

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with ultrasonic waves. Subsequently, samples were centrifuged and filtered through 0.20 µm

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Chromafil AO-20/25 polyamide filters (Macherey-Nagel, Düren, Germany) into vials.

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Identification of individual phenolic compounds was carried out using Thermo Scientific

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Dionex UltiMate 3000 Series UHPLC+ (Thermo Scientific, San Jose, Calif., U.S.A.) under

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conditions as described by Wang, Zheng, & Galletta (2002). Concentration of individual

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phenolic compounds was calculated from corresponding calibration curves. Individual

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phenolic compounds were grouped into corresponding phenolic classes (flavanols, flavonols,

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dihydrochalcones and hydroxycinnamic acids) and their levels were calculated from the sum

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of all identified compounds. Phenolics were also identified in fresh apple juice, which was

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filtrated directly into vials and frozen until further analysis. TPC measurements were carried

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out according to the method described by Singleton, Orthofer, & Lamuela-Raventos (1999)

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and modified by Mikulic-Petkovsek, Stampar and Veberic (2007). The content of individual

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phenolic group (i.e. flavanols, dihydrochalcones, hydroxycinnamic acids and flavanols) in the

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entire apple fruit ( ) was calculated using the following formula = mg⁄kg) ×

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%) + 6

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mg⁄kg) ×

%) ; where

is the content of individual phenolic group in apple peel,

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the content of individual phenolic group in apple pulp,

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the percentage of pulp in the entire apple fruit.

is

2.4. Enzymatic activity and browning

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is the percentage of peel and

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Activity of the polyphenol oxidase (PPO) and peroxidase (POX) enzymes was measured

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spectrophotometrically. The activity of PPO was assessed as described in Worthington

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manual (Worthington Enzyme Manual, 1972) and the activity of POX was measured

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according to the method described by Halbwirth, Kampan, Stich, Fischer, Meisel, Forkmann,

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& Rademacher (2002). For measurements of enzymatic browning, approx. 1 cm thick

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longitudinal slices (from stem to calyx) of apple fruit were cut with a ceramic knife. The color

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of the pulp was immediately recorded with a portable colorimeter (CR-10 Chroma; Minolta,

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Japan) and the slices were left to oxidize on plastic plates for one hour. Subsequently, the

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color was measured again. a* (redness), b* (yellowness) and L* (lightness) parameters were

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obtained from the first and second measurement and ∆E parameter was calculated according

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to the color difference formula:

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∆E =

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The color change was expressed in color difference units ∆E (CIE, 2004), which represent the

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sum of differences among absolute values of parameters a*, b* and L* measured immediately

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after slicing (T0) and after one hour of oxidation (T1). ∆L Parameter was also calculated to

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indicate the specific change in color lightness.

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2.5. Statistical analysis

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Statistical analysis was carried out using Statgraphics Plus 4.0 (Manugistics, Rockville, Md.,

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U.S.A.). For differences among fractions of apple fruit, one-way analysis of variance 7

ACCEPTED MANUSCRIPT (ANOVA) was calculated and Duncan test was used to distinguish among fractions. p-Values

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lower or equal to 0.05 were considered statistically significant. For multiple variable analyses

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Pearson’s correlation coefficient (r) was used. Ward’s method based on square Euclidian

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distance was used for hierarchical cluster analysis and grouping of cultivars according to their

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PPO and POX activity and total phenolic content. Results are presented as average of five

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repetitions ± standard error.

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3. Results

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3.1. The content of sugars and organic acids and S/A ratio

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The content of individual sugars (a), organic acids (b) and their ratio (c) is represented in

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Figure 1. The highest content of sugars and organic acids per kg was recorded in freshly

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extracted apple juice in comparison to pulp and leftover pomace irrespective of the cultivar

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(Fig. 1a and Fig. 1b). TO, KR, FL and BO cultivars were characterized by a significantly

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higher content of sugar in pulp compared to pomace (Fig. 1a). Contrary, no significant

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differences in sugar content were detected between pulp and pomace of GD, JG, GS and MA

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cultivars (Fig. 1a). A comparable amount of organic acids was measured in pulp and pomace

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of all cultivars (Fig 1b.). Higher S/A ratios were measured in KR, FL, BO, GD and GS

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pomace in comparison to the ratio of corresponding juice (Fig. 1c). Lowest S/A was recorded

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in juice of all cultivars, except TO, JG and MA. Cultivars TO and JG were characterized by a

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similar S/A ratio of juice, pulp and pomace, the cultivar MA showed a higher S/A ratio in

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pulp and juice than in pomace (Fig. 1c).

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3.2. Phenolic composition and total phenolic content (TPC) of apple fruit, apple juice and leftover pomace

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The content of individual phenolics in analyzed apple fractions has previously been reported

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by many authors and therefore, these data is added as supplement material (Suppl. Table S18

ACCEPTED MANUSCRIPT S4) and merely reported in in the first part of the following text. The point of interest was the

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proportion of a specific phenolic group in apple fruit, juice and pomace of an individual

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cultivar. Nevertheless, the content of phenolic groups varied among the analyzed apple

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fractions and the first paragraph of the results shortly represents these differences.

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The content of hydroxycinnamic acids in apple pulp ranged from 2.52 ± 0.34 mg/kg in JG to

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93.61 ± 13.11 mg/kg in GS (Suppl. Table S1). The highest content of flavanols in pulp was

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measured in BO (398.30 ± 2.86 mg/kg) and the lowest in MA pulp (15.31 ± 1.64 mg/kg). The

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content of dihydrochalcones ranged from 1.28 ± 0.17 mg/kg in FL pulp to 22.29 ± 5.51 mg/kg

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in KR fruit. The peel of all analyzed cultivars had the highest content of flavanols and the

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most divergent flavanol profile in comparison with other studied fractions (Suppl. Table S2).

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Eighteen flavanols were identified in apple peel of analyzed cultivars with a content of total

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flavanols ranging from 282.95 ± 41.77 mg/kg in MA peel to 1281.48 ± 212.01 mg/kg in BO

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peel. Procyanidin trimer was the only flavanol identified in the juice of MA cultivar (Suppl.

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Table S3). Generally, the pomace of all studied cultivars had the lowest content of identified

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phenolics (Suppl. Table S4).

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The ratios of specific phenolic groups in apple fractions of an individual cultivar are further

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described in the second paragraph of the results and in Figure 2. Generally, flavanols have

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most abundant share in identified phenolics in fruit, juice and pomace of most of the analyzed

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apple cultivars (Fig.2). In FL (76 %), BO (79 %) and JG (77 %) cultivars, flavanols

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represented more than ¾ of all identified phenolics in apple fruit (Fig. 2a). In comparison to

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other cultivars, BO had the highest share of flavanols in fruit, FL cultivar had the highest

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share of flavanols in juice, and cultivar TO had the highest share of flavanols in pomace

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(statistical data not shown). In terms of apple fractions (fruit, juice and pomace), only TO and

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GS had comparable amounts of flavanols in fruit and pomace, while in all other studied

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ACCEPTED MANUSCRIPT cultivars, flavanols had a higher share in fruit than in pomace (Fig. 2a). The share of flavanols

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in apple juice ranged from 2 % in MA to 76 % in juice of the FL cultivar.

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The juice of KR, BO, JG and GS had a higher share of hydroxycinnamic acids in comparison

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to fruit and pomace of the corresponding cultivar (Fig. 1b). The content of hydroxycinnamic

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acids in fruit ranged between 11 % (JG) and 26 % (GS), between 5 % (FL and MA) and 56 %

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(KR) in juice and between 1% (GS) and 18 % (JG) in pomace (Fig. 2b).

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Among analyzed cultivars, juice of the GD cultivar can be considered to be a good source of

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dihydrochalcones (Fig. 2c). This group of phenolic compounds in GD juice accounted for

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more than 60 % (Fig. 2c) of total analyzed phenolic compounds or 298.94 ± 37.75 mg/kg

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(Supp. Table 3).

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The share of flavonols in pomace of all cultivars was significantly higher than in fruit and

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juice (Fig. 2d). The pomace of MA cultivar had the highest share of flavonols in fruit (22 %),

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juice (38 %) and pomace (55 %) in comparison to other cultivars. Interestingly, the juice of all

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other varieties contained less than 3% of flavonols.

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KR and BO fruit and juice were characterized by significantly higher content of total

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phenolics than the pomace of the corresponding cultivar (Fig. 3). No significant differences in

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TPC have been recorded among apple fruit, juice and pomace of TO, GD, JG and GS

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cultivars. TPC content of MA apple fruit, juice and pomace was significantly lower compared

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to other analyzed apple cultivars (statistical data not shown).

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3.3. Browning of the apple slices

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Significant differences in ∆E relate to various levels of color changes among the analyzed

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apple cultivars (Fig. 4). The highest ∆E was observed in BO and GS apple slices indicating

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high level of enzymatic browning. Contrary, lowest ∆E was recorded in MA cultivar. TO,

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KR, FL, GD and JG were moderately susceptible to enzymatic browning. Extreme change in

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brightness (∆L), which is the best indicator of browning, was observed in BO cultivar (Fig. 4). 10

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Contrary, L* parameter of MA apple slices remained constant even after one hour of

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oxidation. Apart from BO and MA cultivars, a similar pattern of ∆L was recorded for other

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cultivars (Fig. 4). 3.4. PPO and POX activity in correlation to TPC and enzymatic browning

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The activity of PPO and POX enzymes and TPC of peel, pulp and juice is depicted in Figure

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5. Dendrograms in the right corner represent the grouping of cultivars in relation to enzymatic

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activity and a distinct clustering has been detected in all apple fractions. Enzymatic activity of

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BO and KR peel was most dissimilar to the activity in peel of other cultivars analyzed in the

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present study (Fig. 5a). ‘Boskoop’ peel was characterized by extremely high PPO activity and

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KR peel by particularly low activity of PPO and POX enzymes. In regard to the enzymatic

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activity of apple pulp, GS cultivar diverges with high PPO activity (Fig. 5b). Other cultivars

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were distributed into two clear groups. The first group included KR, MA, JG and GD

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cultivars, which were characterized by low activity of both enzymes. The second group

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contained BO, FL and TO cultivars, which had a distinct pattern of high POX and low PPO

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activity in the pulp. High POX and low PPO activity was recorded in TO and BO juice, which

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formed a distinctive cluster (Fig 5c). In the second group, KR juice diverges from the rest of

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the cultivars by its high PPO activity. Highest enzymatic activity (PPO and POX) has been

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recorded in apple peel of BO, FL and GD cultivars compared to the activity in pulp and

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pomace. Similar differences in POX activity have been detected among peel, pulp and juice of

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FL and JG cultivars, but no differences in PPO activity were recorded among apple fractions

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of these cultivars (statistics data not shown). A comparable activity of both enzymes was

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recorded in peel, pulp and juice of KR. POX activity of MA remained unchanged in pulp, peel

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and juice. Contrary, significant differences in PPO activity were detected between MA pulp

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and peel.

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ACCEPTED MANUSCRIPT The correlation between enzymatic activity and TPC, ∆L and ∆E and TPC and ∆L and ∆E is

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shown in Table 1. The results indicate a tight correlation between enzymatic activity of both

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enzymes and TPC. A similarly strong correlation has been recorded between TPC and ∆L

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(r=0.78). Interestingly, the activity of POX and PPO enzymes has no impact on the changes in

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apple pulp lightness (∆L). On the other hand, PPO activity moderately affected ∆E (r=0.32).

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No correlation between TPC and ∆E could be confirmed. 4. Discussion

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Sugar and organic acid content in apple is highly cultivar dependent (Mikulic-Petkovsek et

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al., 2007; Vieira, Borges, Copetti, Amboni, Denardi, & Fett, 2009). Their content and

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especially their ratio greatly affects the desirability of apple for direct consumption and their

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suitability for processing. For direct consumption (as well as for processing) a high sugar/acid

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ratio is desired. Traditional cultivars like KR, BO and MA are thus mostly used for

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processing. Highest sugar and organic acid content has been measured in juice of all cultivars

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analyzed in the present study. Cell wall rupture during juice making process releases sugars

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and organic acids (and several other components) into the juice, which explains a higher

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content of these compounds in juice in comparison to pulp and pomace. Sugar content of

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analyzed juice samples was approximately 2-2.5 fold higher than sugar content of apple pulp.

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However, despite fairly equal proportions of sugar released from the pulp into the juice, not

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all cultivars retain the same sugar/acid ratio of pulp and juice. This could be ascribed to a

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higher release of organic acids from the pulp and peel into the juice. Organic acid content of

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BO, KR and GS juices was more than threefold higher than that of the pulp. Possibly a higher

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extraction of organic acids from the peel is the reason for increased amounts of these

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compounds in the juice (Cebulj, Cunja, Mikulic-Petkovsek & Veberic, 2017).

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ACCEPTED MANUSCRIPT Several authors have previously described the differences in phenolic composition of apple

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pulp, juice and pomace (Le Bourvellec, Bouzerzour, Ginies, Regis, Plé, & Renard, 2011; van

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der Sluis, Dekker & van Boekel, 2005). The composition of the entire apple fruit was

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therefore compared to the phenolic profile of apple juice and pomace of each cultivar to get

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an accurate overview of phenolic components in non-processed and processed apple products.

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Flavanols were the main phenolic constituents of fresh apple fruit and juice in most cultivars

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analyzed. Similar findings were reported by Le Bourvellec et al. (2011). Higher or lower

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relative content of flavanols in juice in comparison to whole apple fruit greatly depends on the

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cultivar as half of the cultivars analyzed in the present study contained more/less flavanols in

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juice in comparison to the entire apple fruit. On the other hand, pomace represents a valuable

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source of flavonols and flavanols. The most distinctive phenolic profile has been recorded

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definitely for MA cultivar. This traditional and local Slovenian cultivar was characterized by

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the high share of flavonols and dihydrochalcones in the entire apple fruit, juice and pomace.

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Interestingly, extremely low content of flavanols has been identified in MA’s apple fractions.

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Apple peel and seeds generally contain the highest content of phenolic compounds in apple

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fruit (Robards et al., 1999). During apple juice production only a fraction of these phenolics

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transfers into the juice, while the rest is retained in the pomace (Lu & Foo, 2000; Oszmiański,

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Wojdyło, & Kolniak, 2011; van der Sluis et al., 2005), which makes it a valuable side-product

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in apple juice making. Highest TPC was recorded in GS pomace, suggesting its optimal

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suitability for extraction of phenolic compounds among the cultivars analyzed in the present

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research. GS’s pomace mostly consists of flavanols (184.15 ± 32.18 mg/kg; 56 %) and

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flavonols (126.13 ± 14.75 mg/kg; 40 %), and to a smaller extent of dihydrochalcones (11.11 ±

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2.55 mg/kg; 3 %) and hydroxycinnamic acids (1.55 ± 0.2 mg/kg; 1 %).

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∆E and ∆L parameters were employed to describe the rate of enzymatic browning. ∆E

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parameter was previously utilized by Holderbaum, Kon, Kudo, and Guerra (2010) to quantify

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ACCEPTED MANUSCRIPT enzymatic browning during apple fruit development, by Lee, Seo, Rhee, and Kim (2016) to

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evaluate the effect of onion addition on apple juice browning and by Murata, Noda, and

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Seiichi (1995) to describe the relationship between browning, polyphenol content and PPO

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activity. ∆E encompasses a*, b* and L* parameters, i.e. the change in redness, yellowness and

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lightness of the sample. Contrary, ∆L describes the change in lightness and may therefore

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accurately express color changes related to enzymatic browning. Quevedo, Pedreschi, Bastias,

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and Díaz (2016) linked the decrease of L* parameter with the production of dark pigments as

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a result of enzymatic reactions in pear and apple. The data of the present study singles out two

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extremes: BO cultivar, which was characterized by highest ∆L and ∆E parameters, and MA

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cultivar with minimal changes in brightness and the lowest ∆E parameter. No correlation

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between TPC and ∆E has been recorded among the cultivars analyzed. Contrary, ∆E was in

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moderate correlation with PPO and ∆L correlated with TPC. No correlation between ∆L and

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PPO activity corresponds with the results obtained by Murata et al. (1995). ∆E and ∆L

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parameters were also not correlated with the activity of POX enzyme. Lack of significant

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correlation between the rate of oxidation and activity of oxidizing enzymes could be

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explained by other mechanisms of oxidation. Browning can additionally occur as a result of

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polyphenolic auto-oxidation in the presence of metal ions (Mellican, Li, Mehansho, &

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Nielsen, 2003). Recently, Le Deun et al. (2015) demonstrated that flavanol monomers,

317

hydroxycinnamic acids and dihydrochalcones are the main phenolic classes containing color

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precursors in apple juice. According to our results, we suggest that overall low TPC is the

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main reason for the weak change of color (browning) of the ‘Majda’ cultivar. In all cultivars,

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an overall higher enzymatic activity of PPO and POX, as well as higher TPC content were

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recorded in apple peel. This can be explained by the protective role of external plant organs

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(Passardi, Cosio, Penel, & Dunand, 2005; Szalay, Hegedûs, & Stefanovitis-Banyai, 2005;

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Wolfe, Wu, & Liu, 2003) and the accumulation of plant protective compounds in these

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PPO and TPC is not surprising. Contrary, POX oxidizes a broad range of phenolic

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compounds, lignin precursors, auxin and other secondary metabolites (Hiraga, Sasaki, Ito,

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Ohashi, & Matsui, 2001). For this reason, the strong correlation between POX and TPC is

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rather unexpected.

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5. Conclusion:

Phenolic profiles of eight apple cultivars and several fruit fractions were determined. The

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results indicate high cultivar dependency of fruit phenolic composition (TPC). Surprisingly,

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cultivars with high levels of phenolic compounds in the fruit are not necessarily best suited for

333

the extraction of phenolic compounds from the leftover pomace. However, TPC of apple fruit

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is strongly correlated to the rate of enzymatic browning. Additionally, the activity of PPO and

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POX enzymes was in moderate correlation with the susceptibility of apple slices to enzymatic

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browning. The first report on chemical composition of ‘Majda’ cultivar suggests a high

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potential of this local Slovenian cultivar for fresh-cut fruit and further studies are in progress

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to determine the variability of primary and secondary metabolites in this cultivar.

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Acknowledgements

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This work is part of the program Horticulture P4-0013-0481, supported by the Slovenian

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Research Agency (ARRS). Authors would like to thank Anka Zupan for providing plant

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material of ‘Majda’ cultivar and for valuable comments on an earlier version of the

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manuscript.

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ACCEPTED MANUSCRIPT Table 1. Correlation (r) between enzymatic activity of polyphenol oxidase (PPO), peroxidase (POX), total phenolic content (TPC), change of color (∆E) and change of brightness (∆L). Pearson’s correlation factor was calculated from combined data of all cultivars. p-Value less than 0.05 was considered statistically significant.

PPO-TPC

POX-∆L

PPO-∆L

TPC-∆L

POX-∆E

PPO-∆E

TPC-∆E

r

0.6126

0.5262

0.0023

0.3581

0.7746

-0.1100

0.3231

0.06397

p-value

0.000

0.000

0.9914

0.0858

0.000

0.4965

0.04195

0.6949

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POX-TPC

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Fig. 1. (a) The content of individual sugars ( fructose, glucose, sucrose and sorbitol); (b) organic acids ( malic, citric, shikimic, fumaric acid); (c) sugar/acid ratio in pulp, juice and pomace of eight apple cultivars; ‘Topaz’ (TO), ‘Kronprinz Rudolf’ (KR), ‘Florina’ (FL), ‘Boskoop’ (BO), ‘Golden Delicious’ (GD), ‘Jonagold’ (JG), ‘Granny Smith’ (GS) and ‘Majda’ (MA). Significant differences in total sugars and total organic acids were calculated among three apple fractions, separately for each cultivar by Duncan test (p<0.05). Data is presented as average value of five repetitions.

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Fig. 2. The proportion (%) of flavanols (a), HCA (b), DHH (c) and flavonols (d) in total analyzed phenolics from apple fruit, juice and pomace of apple cultivars ‘Topaz’ (TO), ‘Kronprinz Rudolf’ (KR), ‘Florina’ (FL), ‘Boskoop’ (BO), ‘Golden Delicious’ (GD), ‘Jonagold’ (JG), ‘Granny Smith’ (GS) and ‘Majda’ (MA). Significant differences were calculated among three apple fractions, separately for each cultivar by Duncan test (p<0.05). Data is presented as average value of five repetitions.

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Fig. 3. Total phenolic content (TPC) of apple fruit, juice and pomace of leading European cultivars (‘Jonagold’ (JG), ‘Golden Delicious’ (GD) and ‘Granny Smith’ (GS)), traditional cultivars (‘Boskoop’ (BO) and ‘Kronprinz Rudolf’ (KR)), scab resistant cultivars (‘Topaz’ (TO) and ‘Florina’ (FL)) and a local cultivar (‘Majda’ (MA)). Significant differences in TPC were calculated among three apple fractions, separately for each cultivar by Duncan test (p<0.05). Dendogram on the right depicts the grouping of cultivars using Ward’s method based on square Euclidian distance based on TPC content of juce, pomace and apple fruit. Data is presented as average value of five repetitions, error bars represent standard error.

Fig. 4. Changes in color ( ∆E) and brightness (L*) of eight apple cultivars (‘Topaz’ ( ), ‘Kronprinz Rudolf’ (KR), ‘Florina’ ( ), ‘Boskoop’ ( ), ‘Golden Delicious’ ( ), ‘Jonagold’ ( ), ‘Granny Smith’ ( ) and ‘Majda’ ( ) after one hour of enzymatic oxidation at room temperature. Different letters indicate significant differences among cultivars by Duncan test (p<0.05). Data is presented as average value of five repetitions, error bars represent standard error.

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Fig. 5. PPO and POX activity in apple peel (a), pulp (b) and pomace (c) of eight apple cultivars (‘Topaz’ (TO), ‘Kronprinz Rudolf’ (KR), ‘Florina’ (FL), ‘Boskoop’ (BO), ‘Golden Delicious’ (GD), ‘Jonagold’ (JG), ‘Granny Smith’ (GS) and ‘Majda’ (MA)). The size of the circle corresponds to TPC of each cultivar. Dendogram in the upper right corner depicts the grouping of cultivars using Ward’s method based on square Euclidian distance combining activity of PPO and POX. Data is presented as average value of five repetitions.

ACCEPTED MANUSCRIPT Highlights: Apple fruit, pomace and juice characterized by different phenolic groups

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Enzymatic browning is strongly correlated to total phenolic content

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Enzymatic browning is moderately correlated to enzymatic activity

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High potential of local ‘Majda’ cultivar for fresh cut apple fruit

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