Response of organic and conventional apples to freezing and freezing pre-treatments: Focus on polyphenols content and antioxidant activity

Journal Pre-proofs Response of organic and conventional apples to freezing and freezing pre-treatments: focus on polyphenols content and antioxidant activity Santarelli Veronica, Neri Lilia, Sacchetti Giampiero, Carla D. Di Mattia, Dino Mastrocola, Paola Pittia PII: DOI: Reference:

S0308-8146(19)31694-2 https://doi.org/10.1016/j.foodchem.2019.125570 FOCH 125570

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

21 June 2019 17 September 2019 18 September 2019

Please cite this article as: Veronica, S., Lilia, N., Giampiero, S., Di Mattia, C.D., Mastrocola, D., Pittia, P., Response of organic and conventional apples to freezing and freezing pre-treatments: focus on polyphenols content and antioxidant activity, Food Chemistry (2019), doi: https://doi.org/10.1016/j.foodchem.2019.125570

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Response of organic and conventional apples to freezing and freezing pre-treatments: focus on polyphenols content and antioxidant activity

Santarelli Veronica, Neri Lilia*, Sacchetti Giampiero, Carla D. Di Mattia, Dino Mastrocola, Paola Pittia * Faculty of Bioscience and Technologies for Food, Agriculture, and Environment University of Teramo Via Renato Balzarini 1, 64100 Teramo, Italy.

*Corresponding author Tel. +39 0861 266883 Tel. +39 0861 266895 Fax +39 0861 266915 E-mail address: [email protected]; [email protected]

Keywords: apples, organic, freezing, vacuum impregnation, bioactive compounds, antioxidant activity.

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Abstract (150 parole)

The effect of pre-treatment (dipping and vacuum impregnation in lemon juice solution), freezing and frozen storage (FS) on single and total polyphenols (free and conjugated) content and antioxidant activity (AOA) of organic and conventional apples, was investigated. Fresh organic and conventional fruits showed different profiles of free and conjugated polyphenols while their total phenolic content and AOA were similar. Organic and conventional apple tissues showed different physiological responses to physical stresses described by changes in phenolic profiles. Vacuum impregnation in lemon juice affected the polyphenols extractability of both the organic and conventional apples and favored their enrichment with bioactive compounds (AOA=+11.5%). FS decreased the single and conjugated polyphenols content of the pre-treated apple samples and the effect was different between organic and conventional fruits. After FS, not pre-treated organic apples showed a lower AOA reduction than the conventional ones (−13% vs −25%), while no differences were found on pre-treated samples.

1. Introduction The organic farming sector in Europe has rapidly grown in recent years and this growth regards not only the area under organic farming but also the number of holdings and overall organic operators (https://ec.europa.eu/agriculture/organic/eu-policy/data-statistics_en ). Organic agriculture is an alternative cultivation model aimed to respect the biological cycles of the production systems, to maintain and increase soil fertility, to minimise environmental pollution, to avoid the use of synthetic fertilizers and pesticides, to preserve the genetic diversity, and to obtain high quality food by applying sustainable productions (Lima & Vianello, 2011). These features influence the consumers’ perception and their willingness to pay for organic products that are believed also as healthier, more nutritive, better testing and safer compared to conventionally produced foods. 2

Scientific attempts aimed to verify the truthfulness of this "halo effect" have been recently reviewed and discussed by some authors (Bernacchia, Preti, & Vinci, 2016; Paoletti, 2015) who compared data on the composition and nutritional quality of organic and conventional produces. Differences in nutrients and secondary plant metabolites between organic and conventional have been ascribed to different resource availability, soil quality, insect and animal herbivory pressures. In particular, the phenolics accumulation in organically produced plant tissues has been explained as a defense mechanism triggered by biotic and abiotic stresses associated with the absence of synthetic pesticides and the restricted use of fertilizers which are, conversely, commonly used in the conventional production system. Phenolic compounds, indeed, contribute to plant resistance to fungal infections, insect wounds or mechanical damage (Lattanzio, Cardinali, & National, 2017). Although organic production is often associated with fresh products, the increasing worldwide sales of organic processed fruits and vegetable products (Willer, Sorensen, & Yussefi-Menzler, 2008) highlights the significant involvement of downstream operators dealing with processing and that operate within a complex system, which includes small and large food producers and local and global distribution networks. Hence, the need to deepen the knowledge and comprehension of the role of cultivation and farming practices not only on the quality of fresh products but also on their technological properties. To this regards, even though numerous studies have sought to determine whether significant differences in postharvest quality exist between organic and convention plant foods (Mditshwa, Magwaza, Tesfay, & Mbili, 2017), very scarce are the information related to their response to processing (Arlai, Nakkong, Samjamin, & Sitthipaisarnkun, 2012; Neri et al., 2019). Thus, investigations aimed at defining the suitability of organic products at processing, and/or at optimizing the technologies to apply in order to preserve or improve the quality, and, in particular, the nutritional and functional properties of organic products are needed. Freezing is a preservation technology that is widely used by the food industry for highly perishable and seasonal plant foods. It is highly accepted by consumers who retain it to have a low impact on the nutritional quality (Cardello 2003; Sacchetti et al., 2009). Freezing combines the preserving 3

effect of low temperatures with the crystallization of water in the form of ice crystals in such a way that it is not available either as a solvent or reactive component. However, the size and location of the ice crystals may damage cell membranes and break down the physical structure and thereby the final quality of the product upon thawing may be lower than the corresponding fresh product. Moreover, freezing pre-treatments, conventionally applied to preserve the quality of frozen products by enzymatic inactivation, may impair the cell structure; as a result, quality degradation including color changes, drip loss, softening and loss of nutrient and bioactive compounds are unavoidable (Rickman, Barrett, & Bruhn, 2007; Chassagne-Berces et al., 2009) However, some authors (Mullen et al., 2002; Sacchetti et al., 2008; Leong & Oey, 2012) have shown that freezing operations may also exert positive effects on quality and functional properties of plant foods, since after freezing, a release of bioactive compounds as bound phenolic acids and anthocyanins, resulting in an increase of antioxidant activity, can be observed. The aim of this study was, thus, to evaluate the effect of freezing and frozen storage on the content of functional compounds, such as free and conjugated polyphenols, and the antioxidant activity of organic and conventional apples (cv. Golden Delicious). The effect of freezing pre-treatments aimed to improve the quality and stability of frozen products over time, such as dipping and vacuum impregnation with an organic lemon juice solution, was also explored to assess if different responses of plant tissues to process-induced stresses exist between organic and conventional apples.

2. Materials and methods 2.1 Materials Conventional (CONV) and organic (ORG) “extra” category apples (Malus domestica Bork., cv. Golden Delicious), cultivated in Trentino-Alto Adige (Italy), and picked in September 2016, were bought in a local market. All the experiments were carried out by using, for each apple type, a

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single batch of 80 kg. On arrival, fruits were stored at 4 °C in a chilled room with 90% RH and processed within 4 days. A single batch (14 kg) of organic lemons (Citrus limon L.) Burm. cv. Femminello Comune was collected in a local market. After washing and cutting, lemons were manually squeezed to extract the juice and filtered by using a nylon filter. The filtered juice (°Brix= 6.25, citric acid concentration = 4.75 g L-1; pH= 2.50) was immediately portioned in hermetically sealed glass vials, frozen at −40 °C and thawed at 4 °C until use. All chemicals and standards were purchased from Sigma-Aldrich (Steinheim, DE). 2.2 Samples preparation Before treatments, ORG and CONV apples were washed and peeled, and the core was manually removed by a core borer; the remaining pulp was then cut in 1 cm3 cubes by using a cube cutter. Thus, ORG and CONV apple cubes were pre-treated by dipping (DIP) or vacuum impregnation (VI) using as impregnant medium an organic lemon juice solution, which was prepared by diluting the lemon juice with deionized water to achieve a citric acid content of 0.5% w/v. In order to evaluate the effect of dipping and vacuum impregnation in lemon juice solution (VI_L) on the apple tissues, apple cubes obtained from fresh fruits (F) were used as reference sample. Apples vacuum impregnated with deionized water (VI_C) were also produced and used as control in order to discriminate the effects induced on the plant tissue by physical stresses (hydrodynamic mechanisms and deformation and relaxation phenomena) occurring during VI as an effect of the pressure variation.from those ones due to impregnation with lemon juice solution. Dipping was carried out at 20 °C ± 1 °C using a glass cylindrical vessel connected to a thermostatic water bath. Each treatment was performed using 600 g of apple cubes and a 1:4 (w/w) fruit/solution ratio. Apple cubes were dipped and left into the lemon juice solution for 60 sec. In order to remove the surface liquid, after dipping the apple cubes were drained and lightly dabbed with absorbent paper.

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VI was carried out at 20 °C ± 1 °C by exploiting a vacuum laboratory equipment, which was composed of a glass cylindrical chamber linked to a vacuum pump (SC 920, KN ITALIA, Milan, Italy), and to a thermostatic water bath. Each treatment was performed using 600 g of apple cubes and a 1:4 (w/w) fruit/solution ratio. The VI process was conducted using a pressure of 738 mbar for a holding time of 10 s. The total contact time of the apple cubes with the lemon juice solution from the immersion of the apple cubes into the impregnant medium up to the recovery of the atmospheric pressure was of 60 sec. In order to remove the surface liquid, after VI the apple cubes were drained and lightly dabbed with absorbent paper. Fresh (not pre-treated, NP) and pre-treated (DIP, VI_C and VI_L) ORG and CONV apple cubes, immediately after their preparation, were aliquoted, packed in air as single layer in bi-oriented polypropylene (BOPP) bags, and immediately stored at 4 °C until analysis, or frozen in a blast freezer (mod. Forma, Thermo Scientific, Milan, Italy) set at −40 °C to obtain high freezing rate. Thus, the apple samples were stored at −40 °C for 300 days. Before analysis, samples were thawed for 15 h at 4 °C in a chilled room. 2.3 Moisture and starch index Moisture content was measured according to the AOAC Ref 925.10 gravimetric method (AOAC, 1990). The evaluation of the starch index (SI) was carried out by dipping cross-sectional halves of 10 apples for 30 sec in an iodine solution (40 g KI + 10 g I2 L-1). Thus, the color patterns developed after the iodine treatment were compared with color reference charts, which rates SI using the one (all stained) to ten (not stained) Eurofru scale (Ctifl, France). 2.4 PPO extraction and analysis Polyphenoloxidase (PPO) extraction and assay were performed as follow. 3 g of the freeze-dried sample was added to 40 mL of McIlvaine buffer (pH 7.5) containing 0.5% di Triton X100 and 25 6

mM of ascorbic acid. The solution was homogenised at 13500 rpm for 1 min with a lab homogeniser (Ultra-Turrax yellow line DI25 basic, IKA-Werke, Staufen, Germany), stirred under ice for 15 min, centrifuged at 4 °C at 3000 rpm (2012 ×g) for 10 min and then filtered with paper. For the PPO assay, the substrate solution was prepared daily by mixing 20 mM 4-methylcatechol solution with McIlvaine buffer. Enzyme activity was tested in a glass cuvette with 10 mm-pathlength at 25 °C by adding 100 µL of enzyme extract to 2900 µL of substrate solution and monitoring for 4 min the absorbance at 420 nm using a spectrophotometer (Lambda Bio 20, Perkin Elmer, Boston, MA, U.S.). Reaction rate was measured from the slope (ΔA min-1) of the initial linear portion of the plot of absorbance vs. time. One unit (U) was defined as the amount of enzyme necessary to achieve an increase in absorbance of one unit in one min under the assay conditions. PPO inhibition (I %) was computed as [(AA0)/A] × 100, where A is the enzymatic activity after pre-treatments and A0 is the enzymatic activity of the fresh apple. 2.5 Organic acids determination The extraction and the analysis of the organic acids (oxalic acid, citric acid , tartaric acid, ascorbic acid, malic acid and succinic acid) were performed on the lemon juice and on the apple samples before and after DIP and VI pre-treatments as described by Neri et al. (2019). 2.6 Extraction of free and conjugated polyphenols. Free polyphenols extraction was performed on freeze-dried samples according to Mrkìc et al. (2006) with slight modifications. 0.5 g of sample was mixed to 5 mL of acetone/water 70:30 v/v, homogenate at 13500 rpm with Ultra-Turrax for 1 min, stirred under ice for 15 min and centrifuged at 3000 rpm (2012×g for 15 min. The resulting pellet (P1) was used for the extraction of conjugated polyphenols while the supernatant (S1) was filtered with cellulose filters and acetone was evaporated almost to dryness under nitrogen. The residue was dissolved in methanol (0.2 mL), membrane filtered (0.45 m) and used for HPLC analysis. 7

The extraction of conjugated polyphenols was performed on P1 by adding 5 mL of acetone/water (70:30 v/v) acidified with H2SO4 (0.01 N). After homogenization for 1 min with Ultra-Turrax (13500 rpm), stirring for 15 min and centrifugation for 15 min at 3000 rpm (2012 ×g), the supernatant (S2) was filtered with cellulose filters and acetone was evaporated to dryness under nitrogen. The residue was dissolved in methanol (0.2 mL), filtered (0.45 m) and used for HPLC analysis. 2.7 Identification and quantification of polyphenols Chromatographic analyses were performed on a 1200 Agilent Series HPLC (Agilent Technologies, Milano, Italy) equipped with a G1322 degasser, a G1311A quaternary pump, a G136A column thermostat, an autosampler injection system and a diode array detector. The system was controlled with Agilent ChemStation for Windows (Agilent Technologies). Polyphenols determination was carried out according to the chromatographic condition used by Schieber, Keller and Carle (2001), slightly modified to reduce time analysis. The sample (10 L) was injected onto a C18 reversedphase column, Kinetex 5 µm C18 100A 250 × 4.6 mm (Phenomenex, Bologna, Italy). Separation of phenolic compounds was carried out at a flow rate of 1 mL min-1 with a non-linear gradient from A (2% acetic acid solution) to B (acetonitrile:water:acetic acid 50:49.5:0.5). Gradient elution was as follows: from 10% to 20% B from 0 to 10 min, from 20% to 40% B from 10 to 15 min, from 40% to 80% B from 15 to 20 min, from 40% to 80% from 25 to 30 min, from 80% to 40% from 20 to 25 min from 40% to 10% from 25 to 30 min. The DAD acquisition range was set from 200 to 400 nm. Calibration curves were made with epicatechin, catechin, chlorogenic acid, caffeic acid, p-coumaric acid, rutin, phloridzin, coumarin, and the results were expressed as mg 100 g-1 of dry weight apple. 2.8 Total polyphenol content (TPC) Extraction of free polyphenols was performed on lyophilized sample. 0.5 g of sample was added to 5 mL of acetone/water (70:30 v/v), homogenised at 13500 rpm for 1 min by a lab homogeniser (Ultra-Turrax yellow line DI25 basic, IKA-Werke, Staufen, Germany), stirred under ice for 15 min 8

and centrifuged at 3000 rpm (2012 ×g) for 15 min. The total polyphenol content of the free phenolic fractions was determined by using the Folin-Ciocalteau reagent according to Giacintucci, Di Mattia, Sacchetti, Neri and Pittia (2016). 2.9 Radical scavenging activity The radical-scavenging activity was determined on the acetone/water 70:30 v/v polyphenolic extracts (section 2.8) by the ABTS radical cation decolorization assay. ABTS was dissolved in water to a 7 mM concentration; the ABTS radical was formed by reacting ABTS stock solution with 2.45 mM potassium persulphate and allowing the mixture to stand in the dark at room temperature for 12–16 h before use. The reaction was started by the addition of 30 l of sample opportunely diluted to 2970 l of ABTS.+ solution (Abs = 0.7 ± 0.02). The percentage of decoloration after 7 min was used as the measure of antioxidant activity. Antioxidant activity was calculated by the ratio of the regression coefficient of the dose-response curve of the sample and the regression coefficient of the dose-response curve of Trolox and was expressed as moles of Trolox equivalents per g of dry matter. The analysis was carried out on each extract in triplicate. 2.10 Statistical analysis Experimental data were reported as mean and standard deviation and additionally analyzed by multifactorial ANOVA. Significant differences between means were calculated by LSD test at a significance level ≤ 0.05. Data of total and single content in free and conjugated polyphenols were also processed by PLS-DA analysis in an attempt to discriminate apple samples classes. The PLSDA model was validated by full cross validation and the optimal number of extracted factors was calculated by the program. The adequacy of the model was tested by the optimum number of extracted factors, the determination coefficient R2, and the percentage of correct classification. The original variables which were most relevant in explaining the response (Y) matrix and thus having a Variable Importance in Projection (VIP) value equal or higher than 1.0 were used to discuss differences 9

among classes. Data processing was performed by XLSTAT 2016 for Windows (StatSoftTM, Tulsa, OK) software.

3. Results and discussion Fresh organic and conventional apples with equal maturity grade (starch index equal to 9, which accounts for mature fruits) were used to the aim of this study. The polyphenoloxidase activity of apples was also evaluated and both the fruits showed a PPO activity of about 11 U g-1 dm (p>0.05). After pre-treatments in lemon juice solution, a significant (p<0.001) PPO decrease was observed, which was respectively of 19 ± 7 % and 32 ± 6 % for the ORG DIP and VI_L samples, and of 14 ± 4 and 16 ± 2 % (p>0.05) for the CONV DIP and VI_L ones. The reduction of the PPO activity is due to the organic acids contained in the impregnant/dipping solution and, in particular, to the citric acid, which exerts a specific inhibitory effect on PPO (Zhou et al., 2016). In general, the ORG apples showed a decrease of the PPO activity higher than the CONV ones due to their higher citric acid uptake during pre-treatments (3.5 vs 3.1 mg g-1dm in DIP samples and 4.8 vs 4.5 mg g-1 dm in VI_L ones) due to their higher porosity (Neri et al., 2019). Free and conjugated polyphenols content of ORG and CONV apples was determined and results are reported in Tables 1 and 2, respectively. According to other studies (Schieber et al., 2001; Kschonsek, Wolfram, Stöckl, & Böhm, 2018; Vrhovsek, Rigo, Tonon, & Mattivi, 2004), different classes of phenolic compounds including flavanols (catechin and epicatechin), hydroxycinnamates (chlorogenic, p-coumaric, caffeic acid), flavonols (rutin), dihydrochalcones (phlorizin) and coumarins (coumarin) were detected. No peaks related to procyanidins were identified by using retention times and absorption spectra of reference compounds, despite analyses were performed on an acetone/water extracts, which assure procyanidins extraction and PPO denaturation (Vrhovsek et al., 2004), as well as by using the optimized chromatographic conditions proposed by Schieber et al. (2001), which have been shown to be able to separate procyanidins from other polyphenols. 10

The main polyphenols found in Golden Delicious apples were chlorogenic acid, epicatechin, and phlorizin as also observed by other authors on the same cultivar (Napolitano et al., 2004; Wu et al., 2007). ORG and CONV fresh apples showed a similar content (p>0.05) in total free and conjugated polyphenols. In both the fruits the conjugated polyphenols (catechin, epicatechin, phlorizin and chlorogenic acid) represented about 12% of their total content, value similar to that observed by Chu, Sun, Wu, and Liu (2002). As regards the single polyphenols, the highest content in free caffeic acid and catechin was determined in the CONV fruits while conjugated catechin was highest in the ORG ones. It was not possible to compare these results with others reported in literature since, to the author knowledge, other studies aimed to investigate the polyphenols profile in organic and conventional apples (Stracke, Rufer, Weibel, Bub, & Watzl, 2009; Valavanidis, Vlachogianni, Psomas, Zovoili, & Siatis, 2009) analyzed the apple peel or the whole edible portion and did not evaluate the free and conjugated fractions. The pre-treatments did not influence the total content of free and conjugated polyphenols (p>0.05). However, when single polyphenols were considered, a higher content of free catechin in all the pretreated organic samples and of its conjugated form in both ORG and CONV VI samples was determined. This result could be ascribed to the increased extractability of this compound favored by the mechanical stresses related to the deformation phenomena and mass transfers determined by pressure changes on the cell walls, the lamella mediana, and the plasmalemma of the cell structures (Occhino, Hernando, Llorca, Neri, & Pittia, 2011). VI did not impair the composition of the secondary metabolites of the apple samples since no significant (p>0.05) reduction in polyphenols was observed conversely to what observed by Blanda, Cerretani, Cardinali, Bendini, and Lercker (2008) where lisciviation phenomena or leakage of native liquid induced by the gas release during the vacuum step occurred. This is likely due to the milder process conditions, in terms of sub-pressure and processing time, used in this study. The content of free (Table 1) and conjugated polyphenols (Table 2) was evaluated on the investigated samples also after freezing and frozen storage up to 300 days. In order to analyze the 11

effect of freezing and frozen storage on free and conjugated polyphenols of both ORG and CONV apples either pre-treated or not, multifactorial ANOVA was performed. The main and combined effects of freezing and frozen storage, cultivation method and pre-treatment on free and conjugated polyphenols are reported in Table 3. In general, the cultivation method (M) significantly influenced the content in free p-coumaric and coumarin (with ORG>CONV) as well as phlorizin, caffeic acid and the total content of free polyphenols (with CONV>ORG) of differently pre-treated fresh and frozen apples. Conjugated polyphenols were also significantly affected by the cultivation method with ORG>CONV for catechin and epicatechin and CONV>ORG for phlorizin and chlorogenic acid. As regards the percentage of conjugation, a significant effect (p<0.05) of the cultivation method was observed on catechin and epicatechin with ORG samples presenting a higher percentage compared to the CONV ones. Pre-treatments (P) positively influenced the content of free epicatechin (DIP and VI_L >F), coumarin (pre-treated>Fresh) and rutin (DIP>F and VI_L>VI_C) while no effect was evidenced on conjugated polyphenols. Freezing and frozen storage (FS) negatively affected the content of free epicatechin and rutin as well as of all the conjugated polyphenols except catechin. The variations in the content of free and conjugated polyphenols observed on ORG and CONV processed samples upon freezing could be due to hydrolysis reactions of sugar residues (Blanda et al., 2008; Sacchetti et al., 2008), changes in distribution of both polyphenols and PPO within the cells, as well as differences of the PPO activity as a consequence of a series of factors and in particular: i) the different enzyme inhibition induced on ORG and CONV fruits by the impregnation with lemon juice solution; ii) the different affinity between PPO and phenols and iii) the different inhibition exerted by single polyphenols and their oxidation products (Le Bourvellec, Le Quéré, Sanoner, Drilleau, & Guyot, 2004). In this regard, Janovitz-Klapp, Richard, Goupy, and Nicolas (1990) found that among the polyphenols present in apple, chlorogenic acid is the preferential PPO substrate, followed by the monomeric catechins and caffeic acids. Moreover, caffeic acid was shown to react with PPO by forming a caffeoyl-PPO 12

complex, which is very slowly transformed in quinone, leaving a small part of the enzyme available for the binding with other phenol substrates (Janovitz-Klapp et al., 1990). Finally, as reported by Neri et al. (2019), ORG apples showed after processing a higher firmness than CONV ones, which implies differences in mechanical strength between the tissues and, consequently, different cell responses toward physical damages induced by VI and/or freezing and frozen storage. Freezing may determine, in fact, cellular disruption, thus improving the extraction of analytes (Blanda et al., 2008). The significance of the combined effect of cultivation method and processing operations (either pretreatment and freezing) on free and conjugated polyphenols indicates that the response of organic apples’ tissue to mechanical and thermal stresses induced by processing is different from that of conventional apples. Hence the release and oxidation of polyphenols during processing followed different pathways in conventional and organic fruits, possibly due to differences in the cell wall composition and corresponding mechanical resistance of tissues (Neri et al., 2019). Infact, as explained by Bradley, Kiellbom and Lamb (1992), the composition and architecture of cell walls in plants is markedly altered by environmental stimuli. In particular, mechanical wounding, infection, or elicitors, to which organic products are notably more subjected in comparison to those grown by conventional methods, cause the accumulation in the cell wall of specific hydroxy-proline rich glycoproteins and other antimicrobial proteins. Moreover, elicitors cause a rapid H2O2-mediated oxidative cross-linking of cell wall structural proteins which contribute to the final functional architecture of cell walls during development and rapid toughening of cell walls. In order to test if free and conjugated polyphenols allow discriminating organic and conventional samples, the data matrix was processed by PLS-DA (figure 1). Free and conjugated polyphenols were used as predictor variables and four classes, predefined as fresh and pre-treated organic (1) and conventional (2) apples, and frozen organic (3) and conventional apples (4), were used as categorical variables. A data matrix with 12 samples in the calibration set ad 4 samples in the

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validation set were used and a model with 3 components with a R2Y value of 0.760 was obtained. For all the classes the percentage of correct classification was 100%. Figure 1a shows the scores and loadings plot along the first two components of the PLS-DA model. The discrimination between the ORG and CONV apples was mostly due to the first latent variable while the second latent variable mainly contributed to the discrimination of the CONV fresh and pre-treated apples from the corresponding frozen ones. The polyphenols with VIP values greater than 1.0 on the first component (t1) were caffeic acid, p-coumaric, conjugated catechin, conjugated chlorogenic acid, and free chlorogenic acid in descendent order. VIP on the second component (t2) were caffeic acid, conjugated chlorogenic acid, free and conjugated epicatechin, conjugated catechin and p-coumaric in descendent order. Figure 1b shows the scores and loadings plot along the first and third components of the PLS-DA model. The discrimination of ORG and CONV apples was mostly due to the first latent variable while the third latent variable mainly contributed to the discrimination of the ORG fresh and pretreated apples from the corresponding frozen ones. The polyphenols with VIP values greater than 1.0 on the third component (t3) were: conjugated phlorizin, conjugated chlorogenic acid, catechin, caffeic acid, conjugated epicatechin, and free epicatechin, in descending order. Apple samples were evaluated for their total polyphenol content (TPC) also by using the FolinCiocalteu’s reagent (Figure 2a). Data collected on fresh fruits were respectively higher than those reported by Sacchetti et al. (2008) and lower than those reported by Vieira et al. (2011) who determined the TPC content on acetone/water extracts of Golden Delicious apple pulp, similarly to our experimental conditions. ORG fresh apples showed a TPC higher than CONV ones in agreement with the results reported by Weibel, Bickel, Leuthold and Alfoldi (2000) and Stracke et al. (2009) on Golden Delicious apples (peel + flesh). After pre-treatment, ORG and CONV VI_L samples showed a TPC increase (p<0.05) of +8% and +11%, respectively. This result can be mostly attributed to the samples’ enrichment with bioactive compounds present in the impregnation solution such as hesperidin (Xi, Lu, Qun and Jiao, 2017; 14

Tripoli, Guardia, Giammanco, Majo and Giammanco, 2007), flavonoid of which lemon juice is rich, and that was not determined in this study since it is not characteristic of apple fruits. The total polyphenol contents determined on organic and conventional apple fruits by HPLC and TPC analysis are very different among them, this because the method used for ‘TPC’ exclusively measures the capacity of apple extracts to reduce the Folin-Ciocalteu’s reagent and thus is an index that measures the reducing power of the extract (Prior et al., 2005). Freezing and frozen storage determined a reduction of the TPC values on all the investigated samples. This result could be due to the oxidative reactions occurring due to the residual PPO activity despite the low storage temperature. Overall, after 300 days both the not pre-treated ORG and CONV apples showed a decrease of polyphenols of −13% (p>0.05). This loss was higher than that observed on the free polyphenols (Table 1) but similar to that one observed on Stark and Granny apple slices by Blanda et al. (2008). These authors, in particular, determined the total polyphenols content by HPLC analysis, including procyanidins, and ascribed the decrease evidenced after freezing and frozen storage mostly to the loss of this class of compounds. The TPC of ORG pre-treated samples was impaired by freezing and frozen storage to a higher extent than CONV products and this result could be attributed to different causes. In particular, ORG pre-treated samples showed a PPO residual activity lower than the corresponding CONV fruits, while they were characterized by a higher polyphenol content and this could explain their higher TPC decrease upon freezing. On the other hand, ORG pre-treated apples showed after FS a mechanical strength significantly higher than the CONV ones (Neri et al., 2019) and this means that they were less affected by mechanic damages induced by freezing and following frozen storage. This, in turn, could have determined a lower polyphenols extractability from the organic samples compared to the CONV ones. However, after 300 days of frozen storage ORG samples showed in general, TPC values higher (p<0.05) than the CONV fruits.

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When the antioxidant activity was evaluated by the ABTS method (Figure 2b), no differences were observed between organic and conventional fresh fruits despite their different phenolic content. This result, shown also by Stracke et al. (2009), confirms that antioxidant activity of apple is not only correlated to the phenolic content but is also dependent on the polyphenols composition (Tsao, Yang, Young, & Zhu, 2003; Chu, Sun, Wu and Liu, 2002; Chinnici, Bendini, Gaiani, & Riponi, 2004). The vacuum impregnation in lemon juice solution significantly affected (p<0.05) the antioxidant activity (AOA) of apple products leading to a TEAC increase of +10% and +13% in ORG and CONV fruits, respectively. The apples' uptake of lemon juice and its antioxidant compounds (e.g. flavonoids, citric acid) may account for this result. After freezing and frozen storage a significant (p<0.05) decrease of the antioxidant activity as measured by the ABTS assay, was observed on all the samples. Not pre-treated ORG apples showed a lower (p<0.05) TEAC reduction than the CONV ones (−13% vs -25%) despite their similar TPC decrease. Antioxidant activity as measured by the ABTS assay is generally correlated to TPC but TPC is based on a redox reaction whilst in the TEAC assay, the ABTS radical could undergo to reduction by both single electron transfer (SET) and hydrogen atom transfer (HAT), thus presenting a mixed mechanism of action towards the antioxidants present in the extracts (Prior et al., 2005; Apak et al., 2016). Pre-treatments positively influenced the AOA of frozen samples and this effect was more evident on the CONV ones in agreement with the TPC values. Eventually, after 300 days of frozen storage, no differences (p>0.05) in AOA were found between ORG and CONV pre-treated fruits.

4. Conclusions In this study new knowledge on the effect of cultivation practices on the functional properties of fresh Golden Delicious apples and their response towards processing has been gained. Organic and conventional fresh apples could be distinguished based on their different profiles in 16

free and conjugated polyphenols while no difference in total polyphenols measured by HPLC analysis and in antioxidant activity was observed. Vacuum impregnation in lemon juice solution promoted the extractability of catechin and improved the functional properties of the apple products irrespective of the cultivation method. The effect of freezing and frozen storage on the single content of free and conjugated polyphenols was polyphenol-dependent and allowed to discriminate apples according to their cultivation method. In general, both freezing and frozen storage impaired the functional properties of frozen apples with conventional fruits being more affected than the organic ones. However, when pre-treatments were applied before freezing, the differences between the functional properties of organic and conventional frozen products resulted almost negligible. These results show that the response of organic apples' tissue to physical stresses induced by processing is different from that of conventional apples; hence the release and oxidation of polyphenols during processing follows different pathways in conventional and organic fruits. As previously discussed, this is possibly due to differences in cell wall composition and mechanical resistance of tissues.

Acknowledgment The authors wish to thank the Core Organic Plus Program for financial support in the framework of the SusOrganic-Development of quality standards and optimized processing methods for organic produce project (Nr: 2814OE006) 5. References Apak. R., Özyürek, M., Güçlü, K., & Çapanoğlu, E. (2016). Antioxidant Activity/Capacity Measurement. 1. Classification, Physicochemical Principles, Mechanisms, and Electron Transfer (ET)-Based Assays. Journal of Agricultural and Food Chemistry, 64(5), 997-1027. https://doi.org/10.1021/acs.jafc.5b04739. Arlai, A., Nakkong, R., Sajamin, N., Sitthipaisarnkun, B.(2012). The effects of heating on physical and chemical constitutes of organic and conventional Okra. Procedia Engineering, 32, 38-44. 17

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Sacchetti, G., Cocci, E., Pinnavaia, G., Mastrocola, D., & Dalla Rosa, M. (2008). Influence of processing and storage on the antioxidant activity of apple derivatives. International Journal of Food Science & Technology, 43(5), 797-804. https://doi.org/10.1111/j.13652621.2007.01518.x. Sacchetti, G., Chiodo, E., Neri, L., Dimitri, G., & Fantini, A. (2009). Consumers’ liking towards roasted chestnuts from fresh and frozen nuts. Influence of psycho-social factors and involvement with product. Acta Horticulturae, 844: 53-57. Schieber, A., Keller, P., & Carle, R. (2001). Determination of phenolic acids and flavonoids of apple and pear by high-performance liquid chromatography. Journal of Chromatography A, 910, 265–273. https://doi.org/10.1016/s0021-9673(00)01217-6. Stracke, B. A., Rufer, C. E., Weibel, F. P., Bub, A., & Watzl, B. (2009). Three-year comparison of the polyphenol contents and antioxidant capacities in organically and conventionally produced apples (Malus domestica bork. cultivar ’golden delicious’). Journal of Agricultural and Food Chemistry, 57(11), 4598–4605. https://doi.org/10.1021/jf803961f. Tripoli, E., Guardia, M. La, Giammanco, S., Majo, D. Di, & Giammanco, M. (2007). Citrus flavonoids: Molecular structure, biological activity and nutritional properties: A review. Food Chemistry, 104(2), 466–479. https://doi.org/10.1016/j.foodchem.2006.11.054. Tsao, R., Yang, R., Young, J. C., & Zhu, H. (2003). Polyphenolic profiles in eight apple cultivars using high-performance liquid chromatography (HPLC). Journal of Agricultural and Food Chemistry, 51(21), 6347–6353. https://doi.org/10.1021/jf0346298. Valavanidis, A., Vlachogianni, T., Psomas, A., Zovoili, A., & Siatis, V. (2009). Polyphenolic profile and antioxidant activity of five apple cultivars grown under organic and conventional agricultural practices. International Journal of Food Science and Technology, 44(6), 1167– 1175. https://doi.org/10.1111/j.1365-2621.2009.01937.x. Vieira, F. G. K., Borges, G. D. S. C., Copetti, C., Di Pietro, P. F., Nunes, E. da C., & Fett, R. (2011). Phenolic compounds and antioxidant activity of the apple flesh and peel of eleven cultivars grown in Brazil. Scientia Horticulturae, 128(3), 261–266. https://doi.org/10.1016/j.scienta.2011.01.032. Vrhovsek, U., Rigo, A., Tonon, D., & Mattivi, F. (2004). Quantitation of polyphenols in different apple varieties. Journal of Agricultural and Food Chemistry, 52(21), 6532–6538. https://doi.org/10.1021/jf049317z. Weibel, F. P., Bickel, R., Leuthold, S., & Alfoldi, T. (2000). Are organically grown apples tastier and healthier? A comparative field study using conventional and alternative methods to measure fruit quality. Acta Horticulturae, 517, 417–426. https://doi.org/10.17660/ActaHortic.2000.517.53. Willer, H., Sorensen, N., & Yussefi-Menzler, M. (2008). The World of Organic Agriculture 2008: Summary. In Willer, H. & Lernoud, J. (Eds), TheWorld of Organic Agriculture. Statistics and Emerging Trends 2008, Research Institute of Organic Agriculture (FiBL), Frick, and International Federation of Organic Agriculture Movements (IFOAM), Bonn, pp 15–22 https://doi.org/10.4324/9781849775991.

20

P

F

P

F

Wu, J., Gao, H., Zhao, L., Liao, X., Chen, F., Wang, Z., & Hu, X. (2007). Chemical compositional mg 100 g-1 dm Catechin

sd

Epicatechin

sd

P-cumaric

sd

Phloridzin

sd

Coumarin

sd

Chlorogenic Acid

sd

Caffeic Acid

sd

Rutin

1.19d

0.15

19.3ab

2.2

0.941a

0.032

5.35a

1.14

0.328bc

0.007

47.9b

0.1

0.418b

0.008

2.15bc

1.48c

0.10

21.2ab

2.3

0.851ab

0.040

6.11a

0.26

0.336ab

0.001

53.6ab

1.1

0.407b

0.030

2.40b

1.56bc

0.07

19.4ab

1.1

0.958a

0.008

5.31a

0.85

0.321c

0.001

44.1b

0.1

0.408b

0.010

2.09bc

1.40c

0.06

18.1b

0.7

0.931a

0.033

5.67a

0.26

0.330bc

0.005

49.0b

0.1

0.393b

0.002

1.95c

2.18a

0.04

19.6ab

0.5

0.771ab

0.010

5.81a

1.53

0.324bc

0.006

47.1b

3.7

0.566a

0.069

2.05bc

2.02a

0.01

20.0ab

0.2

0.894a

0.042

7.45a

2.59

0.326bc

0.001

64.3ab

4.6

0.557a

0.027

3.59a

1.67b

0.01

19.7ab

0.2

0.622b

0.046

4.81a

0.71

0.320c

0.002

53.9ab

6.6

0.557a

0.044

1.97bc

2.09a

0.12

25.4a

7.7

0.831ab

0.069

6.61a

1.30

0.338ab

0.013

55.5ab

29.7

0.655a

0.113

2.00bc

1.72ab

0.31

19.4a

1.5

1.00a

0.01

5.87ab

2.94

0.318c

0.000

54.7a

0.4

0.397de

0.005 2.17abc

1.69ab

0.04

16.7ab

0.0

0.834abc

0.013

4.26b

0.29

0.335a

0.000

37.6b

11.7 0.361de

0.005

1.85d

2.17a

0.17

18.7a

0.0

1.01a

0.01

3.99b

0.57

0.336a

0.001

37.4b

0.4

0.400d

0.013

2.18ab

1.28b

0.07

18.0a

0.0

0.902ab

0.007

4.93ab

0.86

0.329ab

0.008

52.5a

0.0

0.358e

0.000

1.88cd

1.61ab

0.53

15.0b

0.0

0.729bc

0.012

5.15ab

0.29

0.325bc

0.005

48.1ab

0.3

0.474c

0.004 1.95bcd

1.47b

0.11

16.9ab

0.0

0.720bc

0.028

6.08ab

2.15

0.322bc

0.001

56.0a

3.3

0.551ab

0.008

1.87d

1.27b

0.20

15.2b

0.0

0.707c

0.022

7.67a

0.09

0.323bc

0.002

50.1a

6.1

0.534b

0.002

1.93a

characterization of some apple cultivars. https://doi.org/10.1016/j.foodchem.2006.07.030.

Food

Chemistry,

103(1),

88–93.

Xi, W., Lu, J., Qun, J., & Jiao, B. (2017). Characterization of phenolic profile and antioxidant capacity of different fruit part from lemon (Citrus limon Burm.) cultivars. Journal of Food Science and Technology, 54(5), 1108–1118. https://doi.org/10.1007/s13197-017-2544-5. Zhou, L., Liu, W., Xiong, Z., Zou, L., Chen, J., Liu, J., & Zhong, J. (2016). Different modes of inhibition for organic acids on polyphenoloxidase. Food Chemistry, 199, 439–446. https://doi.org/10.1016/j.foodchem.2015.12.034.

21

1.75ab

0.47

19.0a

0.2

0.850abc

0.227

7.70a

0.84

0.321bc

0.000

52.6a

4.5

0.581a

0.044 2.29bcd

Table 1. Free polyphenols content in fresh and pre-treated apples before (t0) and after freezing and frozen storage (t300). ORG: organic; CONV: conventional; F: fresh (not pre-treated); DIP; dipped in diluted lemon juice; VI_C: vacuum impregnated in water; VI_L: vacuum impregnated in diluted lemon juice; sd: standard deviation. Data on columns with different letters were statistically different al p level <0.05.

Table 2. Content of conjugated polyphenols and corresponding percentage of conjugation in fresh and pre-treated apples before (t0) and after freezing and frozen storage (t300). mg 100 g-1 dm

% conjugation

in

sd

Epicatechin

sd

Phloridzin

sd

Chlorogenic Acid

sd

TOT

sd

Catechin

sd

Epicatechin

sd

Phloridzin

sd

c

0.009

4.23bc

0.11

0.901a

0.138

3.57bc

0.44

9.34bc

1.83

35.2a

3.2

17.9abc

0.6

14.5a

0.8

bc

0.014

3.80d

0.01

0.884a

0.008

2.74c

0.05

8.09c

1.51

31.2bc

1.0

15.2cd

0.4

12.6a

0.4

a

0.041

4.92a

0.23

1.10a

0.23

4.33bc

0.33

11.2ab

2.1

35.3ab

2.1

20.3a

0.7

17.0a

0.7

b

0.028

4.09cd

0.02

0.992a

0.020

3.80bc

0.96

9.59bc

1.80

33.4a

1.9

18.4ab

0.3

14.9a

0.3

d

0.018

4.04cd

0.42

1.08a

0.42

5.06abc

1.80 10.8abc

2.2

21.2e

0.2

17.2abcd

1.7

15.4a

1.7

d

0.018

3.39e

0.15

1.08a

0.15

5.85ab

1.48

10.9ab

2.4

22.3de

0.5

14.5d

2.4

13.1a

2.4

bc

0.023

4.01cd

0.11

0.901a

0.111

5.33ab

1.22

10.9ab

2.3

28.9c

0.9

16.9abcd

3.6

16.0a

3.6

bc

0.009

4.48b

0.00

1.14a

0.00

7.04a

0.58

13.3a

3.0

24.6d

1.3

15.5bcd

2.5

14.9a

2.5

bc

0.005

3.75ab

0.23

0.846abc

0.153

3.02a

0.06 8.31abc

1.54

28.9

3.8

16.2

1.9

13.5

3.9

d

0.000

3.34bc

0.01

0.738bc

0.013

2.85a

0.51

7.57bc

1.41

27.4

0.5

16.7

1.1

14.8

1.1

a

0.051

3.85ab

0.08

0.750abc

0.081

4.08a

1.55

9.46a

1.85

26.5

0.3

17.2

3.3

16.0

3.3

b

0.009

3.96a

0.03

0.516c

0.032

3.59a

0.49

8.78ab

1.84

35.5

1.6

18.1

2.0

9.62

2.04

de

0.037

2.43d

0.11

0.801abc

0.109

3.07a

0.97

6.88c

1.22

27.6

7.9

13.9

0.9

13.4

0.9

e

0.018

3.41bc

0.36

0.971ab

0.357

3.89a

0.70

8.83ab

1.68

27.8

2.2

16.8

0.2

13.7

0.2

de

0.032

2.41d

0.07

1.03ab

0.07

3.46a

0.33

7.49bc

1.31

32.2

4.6

13.7

0.9

11.8

0.9

e

0.000

3.17c

0.05

1.09a

0.05

4.25a

0.01

9.06ab

1.74

24.3

4.9

14.4

0.7

12.5

0.7

22

ORG: organic; CONV: conventional; F: fresh (not-pretreated); DIP; dipped in diluted lemon juice; VI_C: vacuum impregnated in water; VI_L: vacuum impregnated in diluted lemon juice; sd: standard deviation. Data on columns with different letters were Free Catech Epicatechin in

pcoumaric

Phloridzin Coumarin

Chlorogenic Acid

Caffeic Acid

Rutin

TOT

Catechin

F

M

n.s

n.s

16.2***

9.53**

5.63*

9.91**

57.1***

n.s

9.93**

15.1***

F

P

n.s

3.39*

n.s.

n.s.

3.63*

n.s

n.s

4.07*

n.s.

n.s

F

FS

n.s

27.9***

n.s.

n.s.

n.s.

n.s

n.s

10.3**

5.59*

n.s

F

M×P

n.s

5.98**

n.s.

n.s.

n.s.

n.s

n.s

n.s

n.s.

n.s

F

M×FS

8.70**

4.78*

n.s.

n.s.

n.s.

n.s

n.s

n.s

n.s.

n.s

F

P×FS

n.s.

n.s.

n.s.

4.73*

5.77**

n.s

n.s

4.58*

n.s.

n.s

n.s.

n.s.

n.s.

4.26*

n.s.

n.s

n.s

4.17*

n.s.

n.s

F M×P× FS

statistically different al p level <0.05.

Table 3. Multifactorial ANOVA analysis of the individual and interactive effects of cultivation method (M), pre-treatment (P) and freezing and frozen storage (FS) on free and conjugated polyphenols. n.s. not significant. Significance level * p < 0.05; **p < 0.01; ***p < 0.001.

Highlights 1. ORG and CONV apples showed a different profile in free and conjugated polyphenols 2. Total phenolic content and antioxidant activity were similar in ORG and CONV apples 3. VI pre-treatment increased the functional properties of ORG and CONV apples 4. Freezing differently impaired free and conjugated polyphenols of ORG and CONV fruits 5. ORG and CONV apples’ response to process-induced physical stresses was different

23

E