Effects of Sugar Addition on Total Polyphenol Content and Antioxidant

Article pubs.acs.org/JAFC

Effects of Sugar Addition on Total Polyphenol Content and Antioxidant Activity of Frozen and Freeze-Dried Apple Purée Ante Loncaric,*,† Krunoslav Dugalic,§ Ines Mihaljevic,§ Lidija Jakobek,† and Vlasta Pilizota† †

Department of Food Technologies, Faculty of Food Technology Osijek, Osijek 31000, Croatia Department for Fruit-Growing, Agricultural institute Osijek, Osijek 31000, Croatia

§

ABSTRACT: The objective of this study was to investigate the influence of simple sugar addition including (glucose, G; fructose, F; sucrose, S; and trehalose, T) on the total polyphenol content (TPC) and antioxidant activity (AOA) of apple purée processed by freezing and freeze-drying and stored for 6 months. The apple polyphenol profile was mostly preserved in the freeze-dried samples with sugar addition during 6 months of storage, whereas the polyphenol profile in frozen samples consists only of quercetin glycosides, of which rutin had the largest share. After 6 months, the highest level of polyphenols was detected in frozen ‘Idared’ and ‘Fuji’ apple purée with addition of T 5% (12.2 and 16.7 mg/100 g FW, respectively), whereas in freeze-dried apple purée the highest TPC was in ‘Idared’ and ‘Fuji’ with addition of T 1% (16.3 and 13.6 mg/100 g FW, respectively). Results indicate that sugar addition before processing could have potential for enhancing product quality. KEYWORDS: polyphenol preservation, antioxidative activity, sugar addition, storage, freezing, freeze-drying

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conditions.13−15 Golding et al. reported that phenolic metabolism in apple peel is relatively stable, and the health benefits of phenolics in apple peel should be maintained during long-term storage.16 Another study conducted on apple peel showed an increase of total polyphenol content (TPC) in the skin of ‘Golden Delicious’ apples in the first 60 days. After 100 days, the concentration of TPC starts to decrease, but even after 200 days in storage, the total phenolics (TP) were similar to those at the time of harvest.17−19 To improve the quality of fruit products, a common approach is to apply less invasive processes or to use specific additives. Freezing is used extensively to preserve foods such as fruits and vegetables and others. Fruits and purées preserved in this way maintain their nutritional value at a great measure, as well as their appearance and characteristic sensory attributes.20 However, especially in the case of fruit, ice crystal formation resulting in vacuole rupture causes loss of turgor pressure and structural damage of cells and cell walls, which can lead to polyphenol loss due to enzymatic browning and oxidation. Freeze-drying is generally considered to be the superior process of dehydration, because the biological value of the material is mainly retained in the product. In addition, there is little or partial loss in sensory qualities of the product. However, the dried product is highly hygroscopic and it reconstitutes easily.21,22 The food and agricultural product processing industries generate substantial quantities of phenolic-rich byproducts, which could be valuable natural sources of antioxidants. Some of these byproducts (such as apple peel) have been the subject of investigations and have proven to be effective sources of phenolic antioxidants. For instance, peels from apples, peaches,

INTRODUCTION Plant foods such as fruits are a major source of phenolic compounds in the human diet. Apples are a significant part of the human diet, and they are ranked in the top five consumed fruits in the world.1 They have been identified as one of the main dietary sources of food antioxidants, mainly phenolic compounds such as flavonols, flavan-3-ols, phenolic acids, and dihydrochalcones, and they also possess high antioxidant capacity.2−4 These apple constituents are consistently associated with reduced risk of cancer, heart disease, asthma, and type II diabetes when compared to other fruits and vegetables and other sources of flavonoids. Apple consumption was also positively associated with increased lung function and weight loss.3,5,6 The polyphenol content of fruits and vegetables depends on a number of intrinsic (genus, species, and cultivar) and extrinsic (agronomic, environmental, handling, and storage) factors.7,8 Many researchers have found out that the antioxidant activity (AOA) and concentration of total polyphenols are much greater in the peel of apples than in flesh or in the whole fruit.2,5 During processing, polyphenols may be lost or altered, for different reasons. Reductions or losses of phenolic compounds have been reported in commercial juices, and these have been attributed to commercial processing procedures.9,10 Processing and storage may have various impacts on different phenolic compounds, as seen in berry processing where myricetin and kaempferol were found to be more prone to losses than quercetin.11 Markowski and Płocharski reported that during the production of juices the level of every class of phenolic substances significantly decreased.12 They also reported that during applesauce production the contents of dihydrochalcones and phenolic acid are decreased, whereas quercetin glycosides were increased.12 Storage has little to no effect on apple phytochemicals. Some authors reported that quercetin glycosides, phloridzin, and anthocyanin contents of five apple varieties were not affected by long-term storage in controlled atmospheric © 2014 American Chemical Society

Received: Revised: Accepted: Published: 1674

November 7, 2013 January 24, 2014 January 28, 2014 January 28, 2014 dx.doi.org/10.1021/jf405003u | J. Agric. Food Chem. 2014, 62, 1674−1682

Journal of Agricultural and Food Chemistry

Article

Table 1. Physicochemical Parameters of ‘Idared’ and ‘Fuji’ Apple Varieties, na = 20

and pears were found to contain twice the amount of total phenolics as found in the peeled fruits.5,23 Major advances have been achieved in recent years in understanding the absorption and metabolism of most other classes of phenolic compounds. These studies have focused on anthocyanins, flavanones, catechins, proanthocyanidins, and the hydroxybenzoic and hydroxycinnamic acids.24 Some additives that can preserve the natural properties of foods are simple sugars. A number of mechanisms have been suggested for the explanation of sugar’s effectiveness on preservation. These include sugar’s ability to form glass, to lock in compounds, to mimic the hydrogen-bonding character of water, to increase the surface tension of the bulk solvent, to prevent thermotropic phase separations in lipid bilayers, and to prevent the fusion of membranes.25,26 The objective of this study was to investigate the influence of several simple sugar additions at levels of 1 and 5% on the polyphenol content and antioxidative activity of apple purée processed by freezing and freeze-drying and stored for 6 months.

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parameter

‘Idared’

av fruit wt (g) 209.24 ± 24.760 hardness (kg/cm2) 8.60 ± 0.423 moisture (%) 83.76 ± 0.133 soluble solids (°Brix) 13.27 ± 0.152 L-ascorbic acid (mg/100 g) 5.58 ± 0.230 acids (g/100 g of malic acid) 0.25 ± 0.020 pH 3.43 ± 0.006 sugars reducing 8.40 ± 0.091 total 11.64 ± 0.057 color parameters L* 39.65 ± 0.990 a* 3.4 ± 0.053 b* 15.48 ± 0.100 total polyphenols content (g GAE/kg) peel 5.72 ± 0.182 peel + flesh 1.16 ± 0.009 flesh 1.06 ± 0.027 antioxidant activity ABTS assay (mmol trolox/100 mL) peel 15.75 ± 0.338 peel + flesh 12.72 ± 0.558 flesh 3.22 ± 0.250 DPPH assay (mmol trolox/100 mL) peel 6.02 ± 0.104 peel + flesh 2.46 ± 0.060 flesh 1.67 ± 0.044

MATERIALS AND METHODS

Chemicals. Folin−Ciocalteu reagent was purchased from Kemika (Zagreb, Croatia); 2,6-dichlorophenol indophenols, phloretin, catechin, epicatechin, rutin, quercetin, chlorogenic acid, caffeic acid were obtained from Sigma Chemical Co. (St. Louis, MO, USA); 2,2′-azinobis(3ethylbenzothiazoline-6-sulfonate), 2,2-diphenyl-1-picrylhydrazyl, and procyanidin B2 were from Fluka (St. Louis, MO, USA); phlorizin was from Aldrich (St. Louis, MO, USA); and methanol (HPLC grade) and orthophosphoric acid (85%) were obatined from Panareac (Barcelona, Spain). Apple Cultivars Used for Experiment. Actual apple cultivars ‘Idared’ and ‘Fuji’ were purchased from the Agricultural Institute (Osijek, Croatia). Both apple varieties were stored under regular atmosphere (RA) at 2−6 °C before they were purchased for study. During apple purée preparation the core area was removed and two opposite cuts of each fruit were pureed and mixed with simple sugars (S, sucrose; G, glucose; F, fructose; and T, trehalose). Sixteen variants of mixed purees were prepared: 1 and 5% of each sugar were mixed with both apple cultivars. Prepared samples were used for processing, freezing, and freeze-drying. Physical and Chemical Analysis. Apples were held at room temperatures for ca. 1 h before preparation for analysis. Before the apple fruits were disintegrated using a Braun Multiquick Professional 600 W Turbo for the analysis, the core was removed. The content of soluble solids of apples was measured with a tabletop Abbe refractometer and given in Brix (°Brix). Acids were measured by titration with 0.1 M NaOH and phenolphthalein as an indicator and given as grams per 100 g of malic acid. Reducing and total sugars were determined by Luff Schoorl’s method,27 and vitamin C (L-ascorbic acid) was determined by a volumetric method: titration using dichlorophenol indophenol (DCPIP).28 Freezing and Freeze-Drying. Apple purée samples used for freezing were placed in plastic bags and frozen in a laboratory freezer. Samples were maintained at −18 °C. Apple purée samples used for freeze-drying were placed into plastic bags and frozen at −18 °C for 12 h before freeze-drying in a laboratory freeze-dryer (Christ FreezeDryer, Gamma 2-20, Germany). Drying conditions were as follows: freezing temperature, −55 °C; temperature of sublimation, −35 to 0 °C; vacuum level, 0.220 mbar. The temperature of isothermal desorption varied from 0 to 22 °C under the vacuum of 0.060 mbar. Freeze-drying lasted until the total solids content was 94−98%, which was about 48 h. Extraction of Phenolics. The extraction of polyphenols for the determination of TPC and AOA was performed as follows: disintegrated apple parts (peel, peel + flesh, flesh), freeze-dried, and frozen apple purée were homogenized with methanol containing

a

‘Fuji’ 193.25 ± 29.310 9.96 ± 0.185 84.46 ± 0.550 13.10 ± 0.173 6.16 ± 0.230 0.15 ± 0.001 3.76 ± 0.017 9.10 ± 0.144 12.35 ± 0.075 42.96 ± 0.134 −2.74 ± 0.020 15.77 ± 0.210 3.28 ± 0.082 0.85 ± 0.022 0.62 ± 0.002

34.86 ± 0.270 10.36 ± 0.258 6.77 ± 0.480 5.13 ± 0.231 2.69 ± 0.080 2.09 ± 0.042

n, number of fruits under study.

hydrochloric acid (1% v/v) (1 g of sample in 10 mL of 1% HCl methanol). The samples were held at ambient temperature for 1 h. After 1 h, the mixture was filtered through pleated Whatman no. 2 filter paper (Whatman International Limited, Kent, UK), and permeates were used for analysis. Before the identification of polyphenols, fresh and frozen samples were freeze-dried as described earlier. Phenolics were extracted from freeze-dried samples using 80% aqueous methanol. The mixture was sonicated for 15 min and centrifuged at room temperature for 15 min. Extracted samples were filtered through a 0.45 Rm poly(tetrafluoroethylene) syringe-tip filter (Chromafil Xtra, Macherey-Nagel GmbH & Co. KG), and extracts were used for HPLC analysis. The amount of phenolics was expressed as milligrams per 100 g of fresh weight (FW). Determination of Total Polyphenol Content. The total phenols content was determined according to the modified colorimetric Folin− Ciocalteu method.29 A 0.2 mL of apple extract and 1.8 mL of deionizer water were added to a 23 mL test tube. Ten milliliters of Folin− Ciocalteu reagent (1:10) was added to the solution, and finally 8 mL of 7.5% sodium carbonate (Na2CO3) solution was transferred into the test tubes. The color was developed in 120 min, and the absorbance was read at 765 nm by a spectrophotometer (Jenway 6300, Bibby Scientific, UK). The measurements were performed in triplicates for each sample, and the average value was interpolated on a gallic acid calibration curve and expressed as grams of gallic acid per kilogram of sample equivalents (g GAE/kg). Antioxidant Activity Determination. The ABTS assay followed the method of Arnao et al. with some modifications.30 The results were expressed as millimoles of trolox equivalents (TE) per 100 mL of sample. Additional dilution was needed if the measured ABTS value was over the linear range of the standard curve. For the DPPH assay 0.2 mL of the apple extract was diluted with methanol (2 mL), and 1 mL of DPPH solution (0.5 mM) was added. After 15 min, the absorbance was measured at 517 nm.31 The results were expressed as 1675

dx.doi.org/10.1021/jf405003u | J. Agric. Food Chem. 2014, 62, 1674−1682


Article

Table 2. Polyphenol Compounds in ‘Idared’ and ‘Fuji’ Apple Varieties (Milligrams per 100 g FW)a part of fruit

polyphenol

flesh

peel + flesh

quercetin glycosidec

peel

quercetin glycosidec

‘Idared’

procyanidin B2 phloridzin (−)-epicatchin phloretin xyloglucoside chlorogenic acid

3.13 0.72 0.45 nd 8.05

± 0.027a ± 0.021a ± 0.012a

procyanidin B2 phloridzin (−)-epicatchin phloretin xyloglucoside chlorogenic acid 24.416 24.997 25.243 rutin

12.41 1.54 1.12 nd 9.16 0.30 nd 0.68 2.93

± 0.433a ± 0.093a ± 0.021b

procyanidin B2 phloridzin (−)-epicatchin phloretin xyloglucoside chlorogenic acid caffeic acid 24.181 24.440 24.997 25.213 rutin quercetin

23.47 4.32 3.33 nd 2.54 nd 0.56 0.11 1.16 0.51 3.26 0.08

± 0.013b ± 0.127a ± 0.036b

± 0.083a

± 0.133a ± 0.016a ± 0.025aa ± 0.050a

± 0.064a ± ± ± ± ± ±

0.048b 0.001b 0.078b 0.007b 0.058b 0.001a

‘Fuji’ ndb 0.57 ± 0.012b 1.16 ± 0.078b nd 3.82 ± 0.024b 9.48 0.87 1.70 nd 4.69 0.13 0.17 0.09 0.95

± 0.348b ± 0.026b ± 0.049a

31.41 nd 6.39 nd nd nd 3.88 0.95 5.53 2.66 8.33 nd

± 0.131a

± ± ± ± ±

0.029b 0.005b 0.005a 0.005b 0.020b

± 0.094a

± ± ± ± ±

0.004a 0.002a 0.012a 0.009a 0.136a

Each value is expressed as the mean ± standard deviation (n = 3). Within the same row, means followed by different letters are significantly different at p ≤ 0.05 (ANOVA, Fisher’s LSD). bnd, not detectable. cQuercetin glycosides retention times (min): peaks of quercetin-3-glucoside, galactoside, arabinoside, xyloside, and rhamnoside. a

millimoles of trolox equivalents (TE) per 100 mL of sample. Additional dilution was needed if the measured DPPH value was over the linear range of the standard curve. Identification of Phenolics. The analytical HPLC system employed consisted of a Varian LC system (Agilent, Avondale, PA, USA) equipped with a ProStar 230 solvent delivery module and a ProStar 330 PDA detector. Phenolic compound separation was done with an OmniSpher C18 column (250 × 4.6 mm inner diameter, 5 μm, Agilent, USA) protected with a guard column (ChromSep 1 cm × 3 mm, Varian, USA). The data were collected and analyzed on an IBM computing system equipped with Star Chromatography Workstation software (version 5.52). The same solvents and gradient elution program were used in the determination of phenolic acids and flavonols. Solvent A was 0.1% phosphoric acid, and solvent B was 100% HPLC grade methanol. The elution conditions were as follows: 0−30 min from 5 to 80% B; 30−33 min, 80% B; 33−35 min, from 80 to 5% B; with a flow rate = 0.8 mL/min.32 Phenolic standards were used to generate characteristic UV−vis spectra and calibration curves. Individual phenolics in the sample were tentatively identified by comparison of their UV−vis spectra and retention times with spiked input of polyphenolic standard. Three replicated HPLC analyses were performed for each sample. Color Measurement. The color of the apple purée was determined by using a colorimeter (Minolta CR-300) prior to the chemical analysis. Color values, for each fruit, were calculated using the averaging mode with five replications. The color measurements were made using the L*a*b* system. L* measures lightness and varies from 100 for perfect white to zero for black, approximately as the eye would evaluate it. The a* parameter measures redness when positive and greenness when negative, and b* measures yellowness when positive and blueness when negative. Statistical Analysis. All measurements were done in triplicate, and data were expressed as the mean ± standard deviation. The experimental

data were subjected to a one-way analysis of variance (ANOVA), and Fisher’s LSD values were calculated to detect significant differences (p ≤ 0.05) between mean values. Statistical analyses were performed with the statistical program MS Excel (Microsoft Office 2007 Professional).

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RESULTS AND DISCUSSION The physicochemical parameters of both apple varieties are shown in Table 1. The water content of the total fresh weight of both apple varieties was ca. 84%, and the content of soluble solids ca. 13 °Brix. pH value differed from 3.4 ± 0.006 (‘Idared’) to 3.8 ± 0.017 (‘Fuji’). ‘Idared’ apples had a higher acidity (0.3 ± 0.020 g 100 g−1 of malic acid) than ‘Fuji’ apples (0.2 ± 0.001 g 100 g−1 of malic acid), whereas the amount of ascorbic acid was higher in ‘Fuji’ apples (8.4 mg 100 g−1). Mean values of ascorbic acid and soluble solids content were at a range of those reported for other commercial apple cultivars.33 The total sugar content ranged from 11.6 mg (‘Idared’) to 12.4 mg (‘Fuji’) (Table 1). The total sugar contents were slightly lower than those obtained in studies on apple cultivars grown in, for example, Slovenia and Brazil.34,35 Color parameters of ‘Fuji’ apples indicated that the flesh was lighter green (a* parameter was negative) and the coordinate b* indicates a slight yellow color, whereas ‘Idared’ apples had a light red flesh (a* parameter was positive). In both apple varieties, TPC was highest in the peel, followed by the flesh + peel and the flesh, which is in accordance with the results of other researchers.36,37 However, ‘Idared’ apple peel had a higher TPC than ‘Fuji’ (5.7 ± 0.182 and 3.3 ± 0.082 g GAE/kg of peel, respectively). 1676



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Table 3. Total Polyphenol Content and Antioxidant Activity in Processed Apple Purée during Storagea frozen 0 days

30 days

TPC (g GAE/L) DPPH (mmol trolox/100 mL) ABTS (mmol trolox/100 mL)

0.27 ± 0.005b 0.81 ± 0.003a 3.42 ± 0.349a

0.30 ± 0.012a 0.70 ± 0.021b 3.48 ± 0.073a

TPC (g GAE/L) DPPH (mmol trolox/100 mL) ABTS (mmol trolox/100 mL)

0.25 ± 0.005c 0.92 ± 0.001a 0.77 ± 0.031c

freeze-dried 180 days

‘Idared’ 0.27 0.57 3.78 ‘Fuji’ 0.37 ± 0.005a 0.33 0.68 ± 0.081b 0.24 2.34 ± 0.081b 2.85

0 days

30 days

180 days

± 0.004b ± 0.019c ± 0.134a

0.30 ± 0.009c 1.67 ± 0.007a 1.99 ± 0.385c

0.34 ± 0.008a 0.70 ± 0.047b 3.29 ± 0.031b

0.32 ± 0.011b 0.65 ± 0.010b 3.73 ± 0.233a

± 0.009b ± 0.039c ± 0.131a

0.32 ± 0.010a 0.69 ± 0.005a 2.45 ± 0.406b

0.32 ± 0.004a 0.71 ± 0.014a 3.24 ± 0.146a

0.34 ± 0.022a 0.52 ± 0.062b 3.65 ± 0.042a

a Each value is expressed as the mean ± standard deviation (n = 3). Within the same row, means followed by different letters are significantly different at p ≤ 0.05 (ANOVA, Fisher’s LSD).

Figure 1. Loss of total polyphenols (%) in frozen (A, ‘Idared’; C, ‘Fuji’) and freeze-dried (B, ‘Idared’; D, ‘Fuji’) apple purée, prepared with an addition of sugars (S, sucrose; G, glucose; F, fructose; T, trehalose; 5, 5%; 1, 1%): black bars, immediately after preparation; light gray bars, after 3 months of storage; dark gray bars, after 6 months of storage. Error bars represent standard deviations of each data (n = 3).

purée and 25.9, 29.3, and 27.6 for ‘Idared’ apple purée on days 0, 30, and 180, respectively. In general, the highest levels of polyphenols were lost in the apple flesh (without peel) followed by flesh + peel, and peel (data not shown). Significant losses during processing can be explained by the oxidation of phenolic compounds during the slow freezing process achieved in a laboratory freezer (−18 °C). These losses were detected also in freeze-dried samples. In some samples an increase of polyphenols during storage occurred. The higher amount of polyphenols during storage in frozen apple purée (peel + flesh) may be attributed to better extraction efficiency of polyphenols from frozen apple purée than from fresh apple fruit (purée) because of cellular disruption during freezing and thawing.41 It is known that AOA is influenced not only by the content of the polyphenols but also by phenolic compositions. The results of antioxidant activity (Table 3) obtained with the use of the ABTS·+ radical were higher compared to the results of assays performed using the DPPH radical (Table 3), which is in accordance with ref 1. The results obtained by the DPPH method (Table 3) indicate a decrease of AOA during storage of the samples of both apple varieties. This was not the case with AOA measured with the ABTS method. The AOA determined with the ABTS method increased during storage. This can be explained by the nature of the two radicals applied. Like ABTS•+, DPPH reacts with both electron and hydrogen donors, although more slowly, and as result of that, the DPPH radical can

Antioxidant activity in both ABTS and DPPH assays was positively correlated with TPC in the peel, flesh + peel, and flesh. Ki Won et al. found that quercetin, epicatechin, and procyanidin B2 significantly contribute to the total antioxidant activity of apples.37 As shown in Table 2 the phenolics in apple flesh consisted of four main compounds, procyanidin B2, phloridzin, (−)-epicatchin, and chlorogenic acid. The apple peels contained additional quercetin glycoside such as glucoside, galactoside, xyloside, arabinoside, rhamnoside, and rutinoside. The main quercetin glycoside in the peel of both apple varieties was quercetin rutinoside (rutin). Mean values of phenolic compounds presented in Table 2 were at the range of those reported by Carbone et al. and Ceymann et al. for other commercial apple cultivars.38,39 Differences in extraction procedures or quantification methods could explain the different results for the same cultivar as could be influenced by pre- and postharvest factors.40 A difference in the levels of TPC between fresh and processed apple purées was detected, as shown in Table 3. The content of TPC in unprocessed apple fruit ranged from 0.9 g GAE/kg (‘Fuji’ apples) to 1.2 g GAE/kg (‘Idared’ apples). The highest retention of TPC in processed frozen samples was observed in (unprocessed 100% TPC) ‘Fuji’ apple purée: 29.4, 43.5, and 38.3% on days 0, 30, and 180 respectively. Retention of TPC in (unprocessed 100% TPC) ‘Idared’ apples purée was slightly lower at 23.3, 25.9, and 23.9% on days 0, 30, and 180, respectively. In freeze-dried samples, retention of TPC was 37.7, 37.7, and 40.0% for ‘Fuji’ apple 1677


quercetin

23.867 24.664 24.912

23.867 24.664 24.912

nd 0.44 0.60 nd 3.90 0.26 0.35 0.19 nd ± ± ± ± 0.016i 0.004g 0.003h 0.001i

± 0.004h ± 0.001g

1.90 0.47 0.63 nd 6.87 0.35 0.08 0.47 nd

4.63 0.58 0.67 0.03 6.66 0.41 0.48 0.29 ndc

0.021d 0.004e 0.001i 0.001a

nd 0.38 0.55 nd 5.63 0.33 0.44 0.21 nd

± 0.027c ± 0.011g ± 0.012f ± ± ± ±

3.85 0.81 1.28 0.10 7.18 0.54 0.67 0.34 nd

0.038a 0.003h 0.010g 0.022e 0.013f 0.002c 0.006c 0.020d

± ± ± ± ± ± ± ±

0.018bc 0.026b 0.005h 0.001bc 0.004i 0.007c 0.049c 0.009bc 0.001a

± ± ± ± ± ± ± ± ±

2.84 0.91 0.54 0.09 4.59 0.41 0.50 0.33 0.14

0.021a 0.001d 0.014b 0.001ab 0.012c 0.003b 0.004b 0.001b

± ± ± ± 0.022h 0.002f 0.003g 0.001h

± 0.006i ± 0.007i

± ± ± ± ± ± ± ±

1S

2.72 1.33 0.89 nd 6.82 0.52 0.73 0.34 nd

3.19 0.69 1.03 0.08 6.44 0.30 0.40 0.23 nd

± ± ± ± 0.019e 0.005a 0.007a 0.001b

‘Fuji’ ± 0.016a ± 0.014a ± 0.007b nd 0.56 0.68 nd 6.16 0.42 0.51 0.22 nd ± ± ± ± 0.016g 0.001b 0.006d 0.004g

± 0.001d ± 0.004e

0.014bc 0.007e 0.012c 0.003d 0.021d 0.001d 0.004c 0.001e

± ± ± ± ± ± ± ±

‘Idared’ ± 0.017ab ± 0.006f ± 0.009e ± 0.011c ± 0.031g ± 0.001e ± 0.004d ± 0.001e 2.67 0.75 1.18 0.06 7.02 0.40 0.49 0.23 nd

1G

5G

2.58 0.70 0.73 nd 6.73 0.35 0.48 0.24 nd

2.56 0.64 1.09 0.08 6.82 0.25 0.36 0.21 nd

0.018c 0.002g 0.002d 0.001c 0.004e 0.001f 0.001e 0.001f

± ± ± ± 0.027f 0.001e 0.001f 0.004f

± 0.017b ± 0.006c ± 0.004d

± ± ± ± ± ± ± ±

5F

nd 0.50 0.79 0.08 7.03 0.38 0.54 0.26 nd

2.58 0.86 1.03 0.08 7.90 0.54 0.65 0.32 nd

± ± ± ± ± ± ±

± ± ± ± ± ± ± ±

0.004f 0.001c 0.001b 0.024c 0.006c 0.009c 0.004e

0.024c 0.007c 0.015e 0.016c 0.019b 0.006b 0.001b 0.001c

1F

nd 1.09 0.97 0.10 7.44 0.36 0.50 0.27 nd

3.11 0.55 0.91 0.02 5.17 0.22 0.31 0.24 nd

± ± ± ± ± ± ±

± ± ± ± ± ± ± ±

0.016b 0.001a 0.001a 0.026b 0.011d 0.010e 0.007d

0.018bc 0.005i 0.001f 0.001e 0.024h 0.007g 0.002f 0.002e

5T

nd 0.53 0.58 0.05 11.10 0.52 0.62 0.35 nd

3.27 1.06 1.64 0.11 8.02 1.01 0.73 0.41 nd

± ± ± ± ± ± ±

± ± ± ± ± ± ± ±

0.002e 0.006h 0.001c 0.024a 0.008a 0.004b 0.004c

0.026bc 0.018a 0.014a 0.001a 0.016a 0.011a 0.002a 0.004a

1T

Each value is expressed as mean ± standard deviation (n = 3). Within the same row, means followed by different letters are significantly different at p ≤ 0.05 (ANOVA, Fisher’s LSD). bQuercetin glycoside retention times peaks of quercetin-3-glucoside, galactoside, arabinoside, xyloside, and rhamnoside. cnd, not detectable.

a

(+)-catechin (−)-epicatchin chlorogenic acid caffeic acid rutin quercetin glycosidesb

quercetin

(+)-catechin (−)-epicatchin chlorogenic acid caffeic acid rutin quercetin glycosidesb

5S

control

Table 4. Polyphenolic Profile of Freeze-Dried Apple Purée with Addition of Sugars after 6 Months of Storage (Milligrrams per 100 g FW)a

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± 0.001f ± 0.001d ± 0.007b ± 0.005b ± 0.001g ± 0.009c ± 0.006i ± 0.004e

± 0.015f ± 0.005g

± 0.001d ± 0.001c

7.25 0.33 nd 0.43 0.20 ± 0.027b ± 0.009c

a

quercetin glycosidesb

5.01 0.48 nd 0.46 0.35

± 0.002h ± 0.001d

10.10 0.55 nd 0.70 0.45 ± 0.021h ± 0.005de

rutin 23.867 24.664 24.912 25.213

± 0.009bc

Each value is expressed as the mean ± standard deviation (n = 3). Within the same row, means followed by different letters are significantly different at p ≤ 0.05 (ANOVA, Fisher’s LSD). bQuercetin glycoside retention time peaks of quercetin-3-glucoside, galactoside, arabinoside, xyloside, and rhamnoside. cnd, not detectable.

0.025c 0.003a 0.004a 0.016a 0.008a ± ± ± ± ± 9.10 0.79 0.20 1.36 0.61 ± 0.021a ± 0.017f

15.36 0.42 nd 0.57 0.36 ± 0.018d ± 0.011b

8.25 0.60 nd 0.87 0.51 ± 0.015g ± 0.014g

5.46 0.33 nd 0.52 0.44 0.018e 0.005e 0.001b 0.003c 0.003d ± ± ± ± ±

± 0.001a ± 0.005ab ± 0.009d ± 0.001c

7.68 0.71 nd 1.24 0.76 ± 0.014h ± 0.004g ‘Idared’

4.07 0.43 nd 0.90 0.41 quercetin glycosidesb

9.23 0.74 0.67 nd 0.58 7.28 ± 0.008e 0.53 ± 0.001e ndc 0.96 ± 0.009b nd rutin 23.867 24.664 24.912 25.213

± 0.021a ± 0.005c ± 0.010b

5.33 ± 0.011g 0.31 ± 0.004i nd 0.81 ± 0.003e nd

8.17 ± 0.021c 0.38 ± 0.006h nd 0.73 ± 0.001g nd ‘Fuji’ 8.23 ± 0.012d 0.49 ± 0.004d nd 0.60 ± 0.004e 0.36 ± 0.003d

7.98 0.47 0.18 0.81 0.35

± 0.001c ± 0.001a ± 0.006f ± 0.008ab

± 0.011f ± 0.004f ± 0.016d ± 0.004d

5.85 0.46 nd 0.74 0.75

1F 5F 1G 5G 1S 5S control

Table 5. Polyphenolic Profile of Frozen Apple Purée with Addition of Sugars after 6 Months of Storage (Milligrams per 100 g FW)a

9.21 1.06 nd 0.92 0.99

5T

± 0.021a ± 0.009a

8.44 0.81 1.40 0.73 nd

± ± ± ±

1T

underestimate fast reactors. DPPH is also more selective than ABTS•+ in the reaction with H-donors. For instance, the inhibition coefficient f for a series of tea catechins was found to be almost equal to the number of active OH-groups inherent in catechol and pyrogallol fragments.42 In contrast to ABTS•+, DPPH does not react with flavonoids, which contain no OHgroups in the B-ring as well as with aromatic acids containing only one OH-group.43,44 With regard to ABTS•+, nonspecific side reactions are common and could cause an overestimation of antioxidant activity. ABTS•+ reacts with any hydroxylated aromatics independently of their real antioxidative potential.44 Some examples may verify this statement. The AOA of resorcinol determined with microsomal model was found to be less than that of catechol and p-hydroquinone by 150- and 10-fold, respectively. At the same time, AOA measured with the ABTS method for resorcinol (2.49) was significantly superior to those for catechol (1.43) and p-hydroquinone (1.33).45 In more recent years research on the influence of sugars upon polyphenols has focused on anthocyanins and color stability of fruit and vegetable products. Numerous studies have been performed to elucidate such effects, however, revealing contradictory results. Increased loss of anthocyanin color or uninfluenced anthocyanin color in the presence of saccharides was reported by Malien-Aubert et al., Dyrby et al., and Hubbermann et al.46−48 In contrast, improved pigment stability ́ upon sugar addition was reported by Kopjar et al., Scibisz and Mitek, and Kopjar et al.26,49,50 In our research, the loss of TPC in all apple samples, determined immediately after sugar addition and after processing, was smaller compared to the findings in the samples without sugar addition (Figure 1). The apple polyphenol profile was mostly preserved in the freeze-dried samples with sugar addition during 6 months of storage (Table 4). In the ‘Idared’ apple samples we observed a reduction of quercetin aglycon and an increase of quercetin glycosides, which is observed in all ‘Idared’ samples with added sugars. In the freeze-dried apple samples the highest impact on (+)-catechin preservation had S 5% (‘Idared’) and G 5% (‘Fuji’) 4.6 and 2.7 mg/100 g FW, respectively. The highest impact on (−)-epicatechin preservation had T 1% (‘Idared’) and G 5% (‘Fuji’) 1.1 and 1.3 mg/100 g FW, respectively, whereas that on the phenolic acids (chlorogenic acid and caffeic acid) had T 1% (‘Idared’) and T 5% (‘Fuji’). Addition of T 1% had the highest impact on flavonol preservation in both apple cultivars. Flavonols were present in the ranges from 6.0 to 10.2 mg/100 g FW and from 1.2 to 12.5 mg/100 g FW in ‘Idared’ and ‘Fuji’ apple samples, respectively. Rutin accounted for most of the flavonols, 4.6−8.0 and 0.4−11.0 mg/100 g FW in ‘Idared’ and ‘Fuji’ apple samples, respectively. Results obtained by an HPLC method showed that the samples in which T 1% was added had the highest levels of polyphenols in both apple cultivars ‘Idared’ and ‘Fuji’ (16.3 and 13.7 mg/100 g FW, respectively). In the frozen samples after 6 months of storage, a different polyphenolic profile was identified (Table 5). It consists only of quercetin glycosides, of which rutin had the largest share. It can be seen that samples of both apple varieties, ‘Idared’ and ‘Fuji’, had the highest proportion of quercetin glycosides with addition of T 5% (12.2 and 16.7 mg/100 g FW, respectively) detected by HPLC. As in the case of apple samples without sugars, there was an increase of AOA measured with the ABTS method during storage in all ‘Idared’ apple samples, whereas in ‘Fuji’ apple samples the increase of AOA during storage was noted in

0.017b 0.003b 0.010a 0.004g

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Figure 2. Antioxidative activity measured with ABTS method (mmol trolox/100 mL) for ‘Idared’ frozen (A) and freeze-dried (B) and ‘Fuji’ frozen (C) and freeze-dried (D) (S, sucrose; G, glucose; F, fructose; T, trehalose; 5, 5%; 1, 1%): black bars, immediately after preparation; light gray bars, after 3 months of storage; dark gray bars, after 6 months of storage. Each value is expressed as the mean ± standard deviation (n = 3). Among the different sugars within the same period of storage, different letters are significantly different at p ≤ 0.05 (ANOVA, Fisher’s LSD).

Figure 3. Antioxidative activity measured with DPPH method (mmol trolox/100 mL) for ‘Idared’ frozen (A) and freeze-dried (B) and ‘Fuji’ frozen (C) and freeze-dried (D) (S, sucrose; G, glucose; F, fructose; T, trehalose; 5, 5%; 1, 1%): black bars, immediately after preparation; light gray bars, after 3 months of storage; dark gray bars, after 6 months of storage. Each value is expressed as the mean ± standard deviation (n = 3). Among the different sugars within the same period of storage, different letters are significantly different at p ≤ 0.05 (ANOVA, Fisher’s LSD).

In this study, the highest effect on polyphenol retention in freeze-dried apple purées was observed in the samples with addition of T 1%, whereas in frozen samples higher preservation of polyphenols was in samples with addition of T 5%. The difference in the extent of the effect of sugars on TPC and AOA preservation in the particular apple variety could also depend on the complex apple matrix. In such complex natural systems, in which the presence of other compounds can lead to numerous reactions, the stability of the system will be limited by the extent to which physicochemical reactions can occur. Because the degradation mechanisms of polyphenols in the presence of sugars are still unknown, further investigations on processing (in this case freezing and freeze-drying) that induce degradation of polyphenols and the effect of different sugars on its preservation are needed.

samples with addition of S 5%, S 1%, and G 5%. In frozen ‘Idared’ apple purée, antioxidative activity was the highest in the samples with an addition of T 5% and in freeze-dried samples with an addition of T 1% (3.71 and 5.33 mmol trolox/100 mL, respectively). Those results are equivalent to polyphenol content measured by HPLC.51 The highest antioxidative activities measured in ‘Fuji’ apple purée with the addition of T 5%, in both frozen and freeze-dried samples, were 6.7 and 5.1 mmol trolox/100 mL, respectively. With addition of sucrose the AOA in freeze-dried ‘Fuji’ apple samples decreased (Figure 2). During the first 30 days of frozen and freeze-dried ‘Idared’ apple purée storage, high AOA preservation after addition of S 5%, G 5%, F 5%, and T 5% was noted. In frozen ‘Fuji’ apple purée during the first 30 days of storage the highest preservation of AOA was noted in samples with added T 5%. However, in freeze-dried ‘Fuji’ apple purée during the first 30 days of storage the highest preservation ability of AOA was noted in samples with added S 5%. After the storage, the highest AOA in both frozen and freeze-dried ‘Fuji’ and ‘Idared’ apple purées was with addition of S 5% and G 5%, respectively (Figure 3).

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*(A.L.) Phone: (385) 91 576 6066. Fax: (385) 31 207 115. E-mail: [email protected]. 1680



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(16) Golding, J. B.; Mcglasson, W. B.; Wyllie, S. G.; Leach, D. N. Fate of apple peel phenolics during cool storage. J. Agric. Food Chem. 2001, 49, 2283−2289. (17) Mareczek, A.; Leja, M.; Ben, J. Total phenolics, anthocyanins and antioxidant activity in the peel of the stored apples. J. Fruit Ornamental Plant Res. 2000, 8, 59−64. (18) Awad, M. A.; De Jager, A. Flavonoid and chlorogenic acid concentrations in skin of ‘Jonagold’ and ‘Elstar’ apples during and after regular and ultra low oxygen storage. Postharvest Biol. Technol. 2000, 20, 15−24. (19) Lattanzio, V.; Di Venere, D.; Linsalata, V.; Bertolini, P.; Ippolito, A.; Salerno, M. Low temperature metabolism of apple phenolics and quiescence of Phlyctaena vagabunda. J. Agric. Food Chem. 2001, 49, 5817−5821. (20) Kozłowicz, K.; Kluza, F. Experimental characteristics of freezing of apple-pear purée with sweetening substances addition. Acta Agrophys. 2006, 7, 105−112. (21) Genin, N.; René, F. Analyse du rôle de la transition vitreuse dans les procédés de conservation agroalimentaires. J. Food Eng 1995, 26, 391−408. (22) Desai, K. G. H.; Park, H. J. Recent developments in microencapsulation of food ingredients. Dry Technol. 2005, 23, 1361−1394. (23) Denis, M. C.; Furtos, A.; Dudonne, S.; Montoudis, A.; Garofalo, C.; Desjardins, Y.; Delvin, E.; Levy, E. Apple peel polyphenols and their beneficial actions on oxidative stress and inflammation. PLoS One 2013, 8, e53725. (24) Balasundram, N.; Sundram, K.; Samman, S. Phenolic compounds in plants and agri-industrial by-products, antioxidant activity, occurrence, and potential uses. Food Chem. 2006, 99, 191− 203. (25) Komes, D.; Lovric, T.; Kovacevic Ganic, K. Aroma of dehydrated pear products. LWT−Food Sci. Technol. 2007, 40, 1578− 1586. (26) Kopjar, M.; Pilizota, V.; Hribar, J.; Simcic, M.; Zlatic, E.; Nedic Tiban, N. Influence of trehalose addition and storage conditions on the quality of strawberry cream filling. J. Food Eng. 2008, 87, 341−350. (27) Egan, H.; Kirk, R.; Sawyer, R. The Luff Schoorl method. Sugars and preserves. In Pearson’s Chemical Analysis of Foods, 8th ed.; Longman Scientific and Technical: Harlow, UK, 1981; pp 152−153. (28) Tee, E. S.; Siti Mizura, S.; Kuladevan, R.; Young, S. I.; Khor, S. C.; Chin, S. K. Laboratory Procedures in Nutrient Analysis of Foods; Tee, E. S., Siti Mizura, S., Kuladevan, R., Young, S. I., Khor, S. C., Chin, S. K., Eds.; Division of Human Nutrition, Institute for Medical Research: Kuala Lumpur, Malaysia, 1987; pp 47−54. (29) Ough, C. S.; Amerine, M. A. Phenolic compounds. In Methods for Analysis of Musts and Wines; Ough, C. S., Amarine, M. A., Eds.; Wiley: New York, 1998; pp 196−221. (30) Arnao, M. B.; Cano, A.; Acosta, M. The hydrophilic and lipophilic contribution to total antioxidant activity. Food Chem. 2001, 73, 239−244. (31) Brand-Williams, W.; Cuvelier, M. E.; Berset, C. Use of a free radical method to evaluate antioxidant activity. Lebensm. Wiss. Technol. 1995, 28, 25−30. (32) Jakobek, L.; Šeruga, M.; Novak, L.; Medvidović-Kosanović, M. Flavonols, phenolic acids and antioxidant activity of some red fruit. Dtsch. Lebensm.-Rundsch. 2007, 103, 369−378. (33) Planchon, V.; Lateur, M.; Dupont, P.; Lognay, G. Ascorbic acid level of Belgian apple genetic resources. Sci. Hortic. 2004, 100, 51−61. (34) Mikulic, P. M.; Stampar, F.; Veberic, R. Parameters of inner quality of the apple scab resistant and susceptible apple cultivars Malus domestica Borkh. Sci. Hortic. 2007, 114, 37−44. (35) Vieira, F. G. K.; Borges, G. D. S. C.; Copetti, C.; Amboni, R. D. D. M. C.; Denardi, F.; Fett, R. Physico-chemical and antioxidant properties of six apple cultivars Malus domestica Borkh, grown in southern Brazil. Sci. Hortic. 2009, 122, 421−425. (36) Ayala-Zavala, J. F.; Rosas-Dominguez, C.; Vega-Vega, V.; Gonzalez-Aguilar, G. A. Antioxidant enrichment and antimicrobial

This research was supported by the Croatian Ministry of Science, Education and Sport. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Agricultural Institute (Osijek, Croatia) for providing the raw materials required for experiments. We also thank Hayashibara Co. for providing a trehalose.

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ABBREVIATION USED TPC, total polyphenol content; AOA, antioxidant activity; ABTS, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate); DPPH, 2,2-diphenyl-1-picrylhydrazyl; DCPIP, 2,6-dichlorophenol indophenols

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Effects of Sugar Addition on Total Polyphenol Content and Antioxidant

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