The effects of organic and conventional farm management and harvest time on the polyphenol content in different raspberry cultivars

Food Chemistry 301 (2019) 125295

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The effects of organic and conventional farm management and harvest time on the polyphenol content in different raspberry cultivars

T

Alicja Ponder, Ewelina Hallmann

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Warsaw University of Life Sciences, Faculty of Human Nutrition and Consumer Sciences, Department of Functional, Organic Food and Commodities, Nowoursynowska 159c, 02-776 Warsaw, Poland

ARTICLE INFO

ABSTRACT

Keywords: Organic raspberry Conventional raspberry Polyphenols Anthocyanins Flavonols

Raspberry fruits are a perfect source of polyphenols, including flavonols, anthocyanins. Some experiments have indicated that organic fruits contain more bioactive compounds than conventional fruits. The aim of this study was therefore to analyse and compare the concentration of bioactive compounds in organic vs. conventional raspberries and to determine the effects of harvest time and cultivar. Three cultivars of raspberry (‘Laszka’, ‘Glen Ample’ and ‘Glen Fine’) that were harvested in summer and one ‘Polka’ cv. that was harvested in autumn time from organic and conventional cultivation methods were used in the experiment. The contents of dry matter and polyphenols in the fruits were determined. The organic samples contained significantly more dry matter, phenolic acid and flavonoids, including myrycetin, quercetin, luteolin and quercetin-3-O-rutinoside. Harvest time was an important factor in raspberry fruit quality.

1. Introduction Raspberries are among the most popular berry fruits in Europe. Poland is the largest European producer of raspberries in both organic and conventional systems. Other major producers of raspberries are Serbia and Spain. In 2017, conventionally grown raspberries were grown on 29.3 thousand ha in Poland, and the production rate was 109.7 thousand tons. In the organic growing system, the production area is much smaller, amounting to 14,800 ha, and the production rate is 30.8 thousand tons (Database, 2019). Raspberry fruits are one of the best sources of polyphenolic compounds in the human diet. At present, over 6000 compounds belonging to the polyphenol group are known. Many studies indicate that biologically active compounds, such as flavonoids (including anthocyanins) and other compounds belonging to the phenolic acids, have strong antioxidant and anti-inflammatory properties (Baby, Antony, & Vijayan, 2017; Bobinaitė et al., 2016; Teng & Chen, 2019; Teng et al., 2017; Veljkovic et al., 2019; Wang et al., 2019). In organic farming systems, the use of synthetic plant protection chemicals (pesticides) and mineral fertilizers is forbidden. Organic fertilizers (manure, compost and green manures) are widely used, and there is no plant protection system that is understood and maintained as

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in conventional agriculture. Instead, prevention of diseases and pests is carried out, which enriches species biodiversity (Regulation (EC) No 834/2007). This also contributes to the increased synthesis of polyphenolic compounds in organic plants (Kazimierczak, Hallmann, & Rembiałkowska, 2014; Kazimierczak, Hallmann, Sokołowska, & Rembiałkowska, 2011). Polyphenolic compounds are produced by plants for defensive purposes when in states of increased biotic or abiotic stress. They can also be called “natural pesticides”. The content of biologically active compounds in raspberries depends mainly on the cultivation method as well as cultivar and harvest time. Many studies indicate that organic farming systems have a significant impact on the quality of berries produced (Asami, Hong, Barrett, & Mitchell, 2003; Hargreaves, Adl, Warman, & Rupasinghe, 2008; Tassoni, Tango, & Ferri, 2013; Stavanga et al., 2015; Papaioanou et al., 2018). Information about the content of individual polyphenolic compounds in raspberries from organic and conventional cultivation is limited. Most of the presented studies has shown the content of polyphenols only in a quantitative system and typically after only one year of experimentation. Therefore, carrying out research in both quantitative and qualitative systems is appropriate. In this study, the phenolic compounds were determined in a quantitative analysis and some polyphenol compounds were identified.

Corresponding author. E-mail address: [email protected] (E. Hallmann).

https://doi.org/10.1016/j.foodchem.2019.125295 Received 30 April 2019; Received in revised form 25 July 2019; Accepted 29 July 2019 Available online 30 July 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.

Food Chemistry 301 (2019) 125295

A. Ponder and E. Hallmann

2. Materials and methods

sulphate in autumn a year before raspberry planting; 3 doses in time of cultivation; plants protection system: Calypso 480 SC, Miros 20 SP, Zato 50 WG; Conventional farm no. 3: Czerwińsk nad Wisłą (52°25″N 20°23″E); sandy-clay middle soil II and III category (20% floatable particles), pH 6.0; fertilizers: Rosafert 5-12-24-3 250 kg ha−1 in autumn a year before raspberry planting; 4 doses in time of cultivation, plants protection system: Calypso 480 SC, Miros 20 SP, Zato 50 WG. Detailed information on the climate conditions (minimum and maximum temperature, number of hours of sunshine per day and rainfall) is presented in Fig. 1.

2.1. Origin of fruits The experiment was carried out in 2013–2014. Fruits of three raspberry cultivars (‘Laszka’, ‘Glen Ample’ and ‘Glen Fine’) were collected in the summer, and fruits of the 'Polka' cv. were collected in the autumn. The experiment was carried out on three organic farms and three conventional farms. Organic farm no. 1: Zakroczym (52°26″N 20°36″E); sandy middle soil IVa and IVb category (15% floatable particles) pH 5.5; fertilizer: cow manure in dose 35 t ha−1 one year before raspberry planting; plant protection system: Grevit 200 SL; Organic farm no. 2: Załuski (52°37″N 20°22″E); sandy middle soil, sandy-clay IV category (20% floatable particles), pH 5.5; fertilizer: cow manure in dose 30 t ha−1 one year before raspberry planting, no plant protection; Organic farm no. 3: Radzanów (51°33″N 20°51″E); sandy middle soil IVa and III category (10% floatable particles), pH 6.0; fertilizer: sheep manure, green manure 10 t ha−1 and 15 t ha−1 one year before raspberry planting, plant protection system: Bioczos 33 SL, Grevit 200 SL; Conventional farm no. 1: Czerwińsk nad Wisłą (52°23″N 20°20″E); sandy-loamy middle soil IV and III category (20% floatable particles), pH 5.5, fertilizers: Hydrocomplex 12-11-18; Superba 8-11-36 (200 kg ha−1, 150 kg ha−1) in autumn a year before raspberry planting; 3 doses in time of cultivation, plants protection system: Signum 33 WG, Miros 20 SP; Conventional farm no. 2: Czerwińsk nad Wisłą (52°23″N 20°20″E); sandy-loamy middle soil IV and III category (25% floatable particles), pH 5.5; fertilizers: ammonium nitrate, polyphosphate, magnesium

2.2. Plant material preparation The fruits for chemical analysis were harvested early in the morning from every production farm and immediately transported to the laboratory. Five hundred grams of fruits per sample were used in analyses. Each sample was divided into two parts. The first part was used for dry matter evaluation, and the second part was freeze-dried using a Labconco (2.5) freeze-dryer (Warsaw, Poland, −40 °C, pressure 0.100 mBa). After freeze-drying, the plant material was ground in a laboratory mill (A-11). The ground samples were then stored at −80 °C. 2.3. Dry matter content The dry matter content of the raspberries was measured before freeze-drying. The dry matter content was determined using the weight method (Polish Norm PN-R-04013:1988, 1988). Empty glass beakers were weighed, filled with fresh raspberries and weighed again. The samples were placed in a FP-25 W Farma Play (Poland) dryer set to 105 °C for 72 h. After 3 days, the samples were cooled to 21 °C and

Organic farms (2013)

(oC) 30.0

30.0

25.0

25.0

20.0

20.0 (B)

15.0

(A)

10.0 5.0

15.0

(B)

10.0

(A)

5.0

0.0

V

VI

VII

month

VIII

IX

0.0

X

maximum temperature

minimum temperature

Sun hours per day (A)

rainfall mm/month (B)

Conventional farms (2013)

(oC)

30.0

25.0

25.0

20.0

20.0

10.0

0.0

VII

month

VIII

IX

X

maximum temperature

minimum temperature

Sun hours per day (A)

rainfall mm/month (B)

Conventional farms (2014)

(B)

10.0

(A)

V

VI

15.0

(B)

5.0

V

(oC)

30.0

15.0

Organic farms (2014)

(oC)

(A)

5.0 VI

VII

month

maximum temperature Sun hours per day (A)

VIII

IX

0.0

X

minimum temperature rainfall mm/month (B)

V

VI

VII

month

VIII

IX

X

maximum temperature

minimum temperature

Sun hours per day (A)

rainfall mm/month (B)

Fig. 1. Weather conditions in experimental farms (organic and conventional) 2013–2014 in time of raspberry fruits development. 2

Food Chemistry 301 (2019) 125295

A. Ponder and E. Hallmann

weighed. The dry matter content was calculated for the raspberry samples based on their mass differences and given in units of 100 g−1 FW.

(A1) 6

2.4. Phenolic acid and flavonol separation and identification Polyphenols were measured by an HPLC method that was described previously in detail by Hallmann, Rozpara, Słowianek, and Leszczyńska (2019). One hundred milligrams of freeze-dried raspberry material was mixed with 5 mL of 80% methanol and shaken on a Micro-Shaker 326 M (Poland). Next, all samples were extracted in an ultrasonic bath (10 min, 30 °C, 5500 Hz). After 10 min of extraction, the raspberry samples were moved to a centrifuge (10 min, 3780g, 5 °C). The supernatant was collected in a clean plastic tube and re-centrifuged again (5 min, 31,180g, 0 °C). A total of 900 µL of supernatant was transferred to HPLC vials and analysed. For polyphenol compound separation and identification, a Synergi Fusion-RP 80i Phenomenex column (250 × 4.60 mm) was used. The analysis was carried out with the use of Shimadzu equipment (USA Manufacturing Inc, USA: two pumps LC20AD, controller CBM-20A, column oven SIL-20AC, spectrometer UV/ Vis SPD-20 AV). The phenolic compounds were separated under gradient conditions with a flow rate of 1 mL min−1. Two gradient phases were used, 10% (V:V) acetonitrile and ultra-pure water (phase A) and 55% (V:V) acetonitrile and ultrapure water (phase B). The phases were acidified by ortho-phosphoric acid (pH 3.0). The total time of the analysis was 38 min. The phase-time programme was as follows: 1.00–22.99 min 95% phase A and 5% phase B, 23.00–27.99 min 50% phase A and 50% phase B, 28.00–28.99 min 80% phase A and 20% phase B, and 29.00–38.00 min 95% phase A and 5% phase B. The wavelengths were 250 nm for flavonols and 370 nm for phenolic acids. The phenolic compounds were identified by using 99.9% pure standards (Sigma-Aldrich, Poland) and the analysis times for the standards (Fig. 2).

1 2

5

4 3

(A2)

5 1

2

4 3

2.5. Anthocyanin separation and identification

(A3)

The first step of sample purification for the anthocyanin analysis was combined with the analysis of the phenolic acids and flavonols. The samples were extracted with 80% methanol. After the first centrifugation (see previous section), 2.5 mL of supernatant was collected into a new plastic tube, then 2.5 mL of 10 mol hydrochloric acid and 5 mL of 100% methanol were added. The samples were gently shaken and put in a refrigerator (5 °C, 10 min). Next, 1 mL of extract was transferred into HPLC vials and analysed. The anthocyanins were separated under isocratic conditions with a flow rate of 1.5 mL min−1. One mobile phase, 5% acetic acid, methanol and acetonitrile (70:10:20), was used. The analysis time was 10 min at a. wavelength of 570 nm. The anthocyanins were identified by using 99.9% pure standards (Sigma-Aldrich, Poland) and the analysis times for the standards (Fig. 2).

1

2

3

Fig. 2. Chromatogram showing retention times for different groups of bioactive compounds in raspberry. (A1) phenolic (1) chlorogenic acid, (2) gallic acid, (3) caffeic acid, (4) ferulic, (5) p-coumaric acid, (6) ellagic acid (A2) flavonoids: (1) quercetin-3-O-rutinoside, (2) myrycetin, (3) quercetin, (4) luteolin, (5) kaempferol. (A3) anthocyanins: (1) cyanidin-3.5-O-di-glucoside, (2) delphinidin-3.5-di-O-glucoside, (3) pelargonidin-3.5-di-O-glucoside.

2.6. Statistical analysis The results obtained from the chemical analyses were statistically elaborated using Statgraphics Centurion 15.2.11.0 software (StatPoint Technologies, Inc., Warranton, VA, USA). The values presented in the tables are expressed as the mean values for the organic and conventional cultivation systems for the four raspberry cultivars ‘Laszka’, ‘Glen Ample’, ‘Glen Fine’ and ‘Polka’ and are separated for each year of the experiment (2013 and 2014) and the season (summer and autumn). The mean value for the organic raspberries was obtained from 30 individual measurements (n = 30) and 20 for conventional raspberries (n = 20). Individual raspberry cultivars were represented by (n = 24) for ‘Laszka’, (n = 18) for ‘Glen Ample’, (n = 8) for ‘Glen Fine’, (n = 12) for organic Polka and (n = 21) for conventional Polka. The statistical calculations were based on a two-way analysis of variance with the use of Tukey’s test (p = 0.05). A lack of statistically significant differences

between the examined groups is indicated by labelling with the same letters. A standard deviation (SD) was given with each mean value reported in the tables. 3. Results and discussion Raspberry fruits are one of the most popular fruits among consumers. In Poland, due to their seasonality, raspberries often appear in markets in the early summer (VI-VII) and again in late autumn (IX-X). The dry matter content in summer raspberry fruits is presented in Table 1. In both years of the experiment, organic raspberries contained 3

3.97 2.55 0.46 1.82 82.53 ± 12.08A 42.19 ± 6.04A 8.75 ± 1.58A 31.59 ± 4.65A

8.63 ± 4.26A 1.22 ± 0.2A

4.09 2.23 0.58 0.51

103.98 ± 11.22A

22.56 ± 5.16A

33.74 ± 4.5A

Total flavonols Quercetin-3-Orutinoside Myrycetin luteolin Quercetin kaempferol

Total anthocyanins

4

2.27A 1.73A 0.4A 0.35A

42.3 0.12 4.77 0.20 0.63 0.20 36.4

79.19 ± 0.79c

8.64 ± 0.79c 1.78 ± 0.77a

Total phenolic acids Gallic Chlorogenic Caffeic p-coumaric Ferulic Ellagic

Total flavonoids

Total flavonols Quercetin-3-Orutinoside

2.6c 0.05b 2.0a 0.12a 0.17b 0.12b 1.8c

121.54 ± 9.49c

± ± ± ± ± ± ±

13.45 ± 1.61b

Total polyphenols ± ± ± ± ± ± ±

3.9a 0.08b 3.09a 0.27b 1.99a 0.52a 8.4a

106.10 ± 14.02a 6.35 ± 3.38a 1.16 ± 0.26a

47.4 0.23 4.11 0.41 2.81 0.95 38.8

153.4 ± 27.7a

13.80 ± 0.35b

2013

2014

0.08A 0.18A 0.16A 0.49A

‘Glen Fine’ cv. (n = 8)

± ± ± ±

93.53 ± 1.24A

‘Glen Ample’ cv. (n = 18)

47.68 ± 8.63A

± ± ± ±

Dry matter

Cyanidin-3.5-O-diglucoside Pelargonidin-3.5-di-Oglucoside Delphinidin-3.5-di-Oglucoside

11.00 ± 1.24A 2.21 ± 0.74A

111.14 ± 12.26A

9.3A 0.12B 2.54B 0.39A 1.54A 0.37A 6.2A

Total flavonoids

± ± ± ± ± ± ±

47.9 0.31 3.55 0.65 2.34 0.87 40.2

Total phenolic acids Gallic Chlorogenic Caffeic p-coumaric Ferulic Ellagic

156.39 ± 14.94A 62.9 ± 3.4A 0.30 ± 0.56A 4.09 ± 3.19A 0.24 ± 0.11A 0.93 ± 0.37 0.20 ± 0.09B 57.1 ± 3.5A

15.26 ± 1.98 A

160 ± 21.8A

Dry matter

Total polyphenols

15.80 ± 2.37A

2014 13.80 ± 1.52B

± ± ± ± ± ± ±

6.5A 0.11B 1.69B 0.42B 0.46B 0.12B 5.7B

± ± ± ±

1.01B 0.34B 0.24B 0.46A

11.41 ± 1.91a 2.28 ± 0.82a

100.94 ± 1.91a

177.28 ± 15.15a 76.3 ± 1.5a 0.60 ± 0.07a 4.87 ± 2.25a 0.24 ± 0.06a 1.0 ± 0.04b 0.35 ± 0.20a 69.3 ± 3.1a

16.88 ± 2.86a

2014

32.76 ± 3.57A

20.72 ± 6.22A

44.58 ± 5.98B

98.06 ± 9.56B

2.36 0.67 0.32 0.47

103.06 ± 10.83A 4.99 ± 1.67B 1.17 ± 0.17B

44.8 0.31 3.23 0.36 1.23 0.61 39.1

147.9 ± 16.7 A

± ± ± ±

0.05B 0.09B 0.16A 0.17B

± ± ± ± ± ± ±

± ± ± ±

0.002 0.0177

N.S. N.S.

N.S.

0.032 0.0144 N.S. 0.0029 N.S. N.S. N.S.

0.0002

2013

Cultivars

34.13 ± 4.59a

21.00 ± 6.29a

N.S.

N.S.

2.44a 2.08a 0.48a 0.46a 102.93 ± 12.04a 47.79 ± 8.27a

3.71 1.87 0.51 0.53

N.S.3 N.S. N.S. N.S. 0.0312 < 0.0001 < 0.0001 N.S.

7.2b 0.13a 1.86a 0.35b 1.22a 0.28a 5.9a

110.76 ± 16.39a 7.85 ± 4.69a 1.23 ± 0.18a

46.7 0.36 2.78 0.40 1.66 0.73 40.8

157.5 ± 21.8a

15.71 ± 1.86a

2013

‘Laszka’ cv. (n = 24)

0.019

2013

Cultivation system

28.18 ± 2.94B

8.30 ± 1.28A

37.35 ± 3.35B

73.83 ± 7.41B

3.48 1.69 0.32 1.35

8.82 ± 1.13B 1.98 ± 1.1A

82.65 ± 1.13B

126.98 ± 10.63B 44.3 ± 3.1B 0.18 ± 0.11A 4.57 ± 1.8A 0.21 ± 0.11A 0.87 ± 0.28A 0.34 ± 0.18A 38.2 ± 2.4B

13.83 ± 1.39B

2014

2013

2

2013 1

Conventional raspberry (n = 20)

Organic raspberry (n = 30)

Table 1 The content of dry matter in (g/100 g FW) and polyphenols (mg/100 g FW) in examined raspberry fruits in 2013 and 2014 at summer time harvest.

± ± ± ± ± ± ±

2.4b 0.11b 2.73a 0.11a 0.32a 0.15b 2.7b

± ± ± ±

0.07b 0.13a 0.19ab 0.29ab

0.0018 N.S.

0.0031

0.035 N.S. N.S. N.S. N.S. 0.0262 0.035

0.011

< 0.0001

2014

Cultivation system

29.98 ± 1.97b

8.68 ± 0.76a

40.10 ± 2.31b

78.77 ± 4.6b

3.71 2.22 0.41 1.61

10.21 ± 1.15b 2.27 ± 1.09a

88.98 ± 1.15b

51.3 0.16 3.67 0.23 1.08 0.29 45.9

140.31 ± 6.67b

14.90 ± 1.19ab

2014

± ± ± ±

1.56a 0.61a 0.20a 0.30a

(continued on next page)

0.023 N.S.

< 0.0001

0.0025 0.0039 N.S. N.S. < 0.0001 < 0.0001 0.0057

0.0001

< 0.0001

2014

Cultivars

32.84 ± 3.14a

22.25 ± 4.21a

45.80 ± 6.35a

100.89 ± 8.88a

3.25 1.45 0.48 0.44

6.79 ± 1.90a 1.17 ± 0.16a

107.68 ± 9.88a

46.6 ± 7.6b 0.27 ± 0.07b 4.05 ± 2.18a 0.77 ± 0.46a 1.98 ± 1.14a 0.78 ± 0.28a 38.87 ± 5.1a

154.3 ± 16.4a

13.67 ± 1.68b

2013

‘Glen Ample’ cv. (n = 18)

A. Ponder and E. Hallmann

Food Chemistry 301 (2019) 125295

Food Chemistry 301 (2019) 125295

0.0002

< 0.0001

significantly more dry matter (p = 0.019 and p < 0.0001) than conventional raspberries. In 2013, the ‘Laszka’ cultivar was characterized by a significantly (p = 0.0002) higher level of dry matter content compared to the other investigated cultivars, but in 2014, this pattern was observed in ‘Glen Fine’ cv (p < 0.0001) instead. Raspberries are a perfect source of biologically active compounds in the human diet. Polyphenols have strong antioxidant and anticancer activities. Consumption of the fruits in sufficient quantities reduces the risk for major chronic diseases such as cancers, type 2 diabetes, obesity and cardiovascular disorders (Baby, Antony & Vijayan, 2018; Schell, Betts, Lyons, & Basu, 2019). We observed that only organic raspberries grown in 2014 contained a significantly greater total polyphenol content (p = 0.0122) when compared to conventional raspberries. In 2013, there were no significant differences in the total polyphenol content among the examined raspberry cultivars. In 2014, the situation was changed, and ‘Glen Fine’ cv. was characterized by a significant level of total polyphenol content (p = 0.0001). The anticancer properties of raspberries are the result of ellagitannins and ellagic acid in the fruits (Ismail et al., 2016). The raspberry fruit is a rich source of ellagitannins. Ellagic acid is released from ellagitannins by hydrolysis at the end of a biosynthetic pathway. As reported by Bobinaitė, Viškelis, and Venskutonis (2012), the range of ellagic acid (free and hydrolysed) is 119.8–323.5 mg/100 g FW for 19 raspberry cultivars. ‘Laszka’ cv. contained 255.8 mg/100 g FW and ‘Polka’ cv. 177.8 mg/100 g FW. Raspberries are a rich source of ellagic acid. In our experiment, we observed that the farming system affected the ellagic acid content only in 2014. Organic raspberries contained 57.1 mg/100 mg FW and conventional 38.2 mg/100 g FW (Table 1). In the present experiment, the total ellagitannin levels were much lower in ‘Laszka’ cv. (43.5 mg/100 g FW) and ‘Polka’ cv. (54.8 mg/100 g FW). Tannins (pre-cursors) of ellagitannins are part of the plant defence mechanism and are released from plant cells in response to biotic and abiotic environmental stressors (Zhang et al., 2011). In the present study, organic raspberries were characterized by a higher content of ellagic acid in comparison to conventional raspberries, but only for the summer harvest time. In organic farming systems, synthetic pesticides are prohibited. Therefore, plants need to create their own defence systems against pests and diseases (Young et al., 2005). ‘Polka’ cv. grown in the autumn time is a cultivar that produces fruits on young shoots. Plant shoots are removed in accordance with after-harvest requirements. Therefore, the raspberry plants produced much fewer phenolic acids in their fruits during autumn (Table 2). On the other hand, ellagitannins are produced as a response to UV radiation from the sun. We observed a similar effect on the ellagic acid content in autumn harvested raspberry fruits, but only in one year of the experiment (2014). Summer-harvested raspberries contained much more ellagic acid compared to those harvested in the autumn. As noted by Zafrilla, Ferreres, and Tomás-Barberán (2001), ellagic acid, among phenolic compounds, is characterized by a high antioxidant power (in Trolox equivalents): quercetin > kaempferol > ellagic acid > gallic acid > caffeic acid. It is worth noting that all the above mentioned polyphenolic compounds are found in raspberry fruits (Fig. 2). The total flavonoid content was significant in organic raspberries only in 2014. In the second year of the experiment, the organic fruits contained 93.53 mg/100 g FW of total flavonoids and the conventional fruits 82.65 mg/100 g FW. In 2014, the cultivar ‘Glen Fine’ was characterized by the highest level of total flavonoids, which including total flavonols. We noticed that farm management had an effect on most flavonoid compounds, except for kaempferol in 2013 and quercetin-3-O-rutinoside and quercetin in 2014. Organic raspberries contained significantly more myricetin (p = 0.0054) and luteolin (p = 0.0223) than conventional raspberries in both years of the experiment. We observed an effect of the cultivar on the individual flavonol content in raspberry fruits only in 2014. ‘Glen Fine’ cv. fruits contained significantly more quercetin and kaempferol compared to the other experimental cultivars (Table 1). However, the content of quercetin was significantly higher in

3

2

1

Data are presented as the mean ± SD with ANOVA p-value. Means in rows followed by the same letter are not significantly different at the 5% level of probability (p < 0.05). N.S. not significant statistically.

N.S. N.S. 31.95 ± 4.72a 27.06 ± 3.17

34.32 ± 4.7a

< 0.0001 N.S. N.S. N.S. 24.20 ± 5.92a 7.53 ± 1.15

9.86 ± 0.75a

< 0.0001 < 0.0001 0.0002 < 0.0001 N.S. N.S. 0.0374 0.0207 99.71 ± 11.88a 43.56 ± 9.31a 70.56 ± 8.31 35.97 ± 4.08

Total anthocyanins Cyanidin-3.5-O-diglucoside Pelargonidin-3.5-di-Oglucoside Delphinidin-3.5-di-Oglucoside

89.53 ± 11.91a 45.35 ± 6.46a

0.0129 0.040 0.0043 0.0411 0.0014 0.0137 N.S. < 0.0001 N.S. N.S. N.S. N.S. 4.16 2.68 0.47 1.81 ± ± ± ±

0.05b 0.11b 0.14b 0.20b

2.95 1.35 0.36 0.54

± ± ± ±

1.67a 1.05a 0.26a 0.37a 2013 2014

3.47 1.66 0.31 1.42

‘Glen Fine’ cv. (n = 8) ‘Glen Ample’ cv. (n = 18)

Myrycetin luteolin Quercetin kaempferol

Table 1 (continued)

2014

± ± ± ±

0.09a 0.18a 0.10a 0.84a

0.0082 0.0023 0.0446 N.S.

2014 2014 2013 2013

Cultivation system

Cultivars

Cultivation system

Cultivars

A. Ponder and E. Hallmann

5

Food Chemistry 301 (2019) 125295

A. Ponder and E. Hallmann

Table 2 The content of dry matter in (g/100 g FW) and polyphenols (mg/100 g FW) in examined raspberry fruits in 2013 and 2014 at autumn time harvest. Organic raspberry 'Polka' cv. (n = 12)

Conventional raspberry 'Polka' cv. (n = 21)

p-value /cultivation/

2013

2014

2013

2014

2013

2014

1

2

Dry matter

14.77 ± 1.46 A

12.83 ± 1.38 A

14.26 ± 1.08 A

13.25 ± 1.90 A

N.S.

N.S.

Total polyphenols Total phenolic acids Gallic Chlorogenic Caffeic p-coumaric Ferulic Ellagic

152.3 ± 5.5 A 43.9 ± 2.5 A 0.25 ± 0.11 A 2.64 ± 1.60 A 0.43 ± 0.29 A 2.28 ± 0.73 A 0.84 ± 0.16 A 37.4 ± 1.5 A

152.09 ± 0.9 A 81.1 ± 3.2 A 0.13 ± 0.04 A 4.12 ± 3.71 A 0.50 ± 0.37 A 1.21 ± 0.38 A 0.09 ± 0.01 A 71.4 ± 0.45 A

149.4 ± 15.9 A 40.8 ± 4.2B 0.24 ± 0.14 A 1.72 ± 0.97 A 0.38 ± 0.30 A 1.82 ± 0.75 A 0.72 ± 0.18 A 35.9 ± 3.1 A

142.7 ± 10.9 A 67.8 ± 1.90B 0.16 ± 0.006 A 3.40 ± 2.25 A 0.14 ± 0.10B 1.20 ± 0.34 A 0.18 ± 0.18 A 74.8 ± 1.98 A

N.S. 0.0353 N.S. N.S. N.S. N.S. N.S. N.S.

N.S. 0.045 N.S. N.S. 0.001 N.S. N.S. N.S.

Total flavonoids Total flavonols Quercetin-3-O-rutinoside Myrycetin Luteolin Quercetin Kaempferol

108.48 ± 0.02 A 11.88 ± 2.24 A 1.15 ± 0.04 A 2.95 ± 1.03 A 6.81 ± 2.55 A 0.38 ± 0.21 A 0.59 ± 0.47 A

71.38 ± 2.43 A 3.63 ± 0.34B 0.81 ± 0.37B 0.54 ± 0.16 A 0.22 ± 0.05 A 0.29 ± 0.12 A 1.76 ± 0.15 A

108.46 ± 0.02 A 13.57 ± 2.93 A 1.11 ± 0.09 A 3.11 ± 1.43 A 8.34 ± 3.27 A 0.45 ± 0.22 A 0.56 ± 0.52 A

74.84 ± 9.96 A 5.37 ± 1.29 A 2.46 ± 1.08 A 0.42 ± 0.12 A 0.26 ± 0.07 A 0.45 ± 0.18 A 1.79 ± 0.56 A

N.S. N.S. N.S. N.S. N.S. N.S. N.S.

N.S. 0.0039 0.0014 N.S. N.S. N.S. N.S.

Total anthocyanins Cyanidin-3.5-O-di-glucoside Pelargonidin-3.5-di-O-glucoside Delphinidin-3.5-di-O-glucoside

96.58 44.22 18.98 33.38

67.75 ± 2.52 A 34.23 ± 1.04 A 7.31 ± 0.56 A 26.20 ± 0.92 A

94.91 43.03 18.74 33.14

69.47 ± 9.12 A 35.18 ± 4.73 A 7.41 ± 0.87 A 26.88 ± 3.55 A

N.S. N.S. N.S. N.S.

N.S. N.S. N.S. N.S.

1 2

± ± ± ±

7.24 1.81 4.54 3.47

A A A A

± ± ± ±

13.06 A 3.54 A 7.26 A 4.62 A

Data are presented as the mean ± SD with ANOVA p-value. Means in rows followed by the same letter are not significantly different at the 5% level of probability (p < 0.05).

cultivars. In 2013, the highest amount of quercetin was found in ‘Laszka’ cv. fruits (0.51 mg/100 g FW), but in 2014 it was in ‘Glen Fine’ cv. (0.47 mg/100 g FW). Plants from organic systems are much more similar to plants from natural stages than those from conventional systems. This similarity is determined by the chemical structure (quantity and quality) of flavonoids. As noted by Mikulic-Petkovsek, Slatnar, Stampar, and Veberic (2012), wild forms of plants contained more (in quantity and quality) quercetin and quercetin derivatives compared to cultivated forms: cultivated raspberries (1.44 mg/100 g FW) and wild-grown raspberries (2.36 mg/100 g FW); garden blackberries (5.49 mg/100 g FW); and wild-grown blackberries (18.0 mg/ 100 g FW). In the case of kaempferol content, the wild forms of strawberry contained four times more compared to the cultivated form (0.89 mg/100 g FW and 0.19 mg/100 g FW, respectively). In the present experiment, organic raspberries were characterized by a higher level of kaempferol in both years of the investigation (Table 1). Among edible plants, berries with red, blue or purple colours constitute one of the most important sources of anthocyanins. In berry fruits, anthocyanins are abundant mostly in glucoside forms. Cyanidin derivatives appear to be the main anthocyanin in raspberries, as already reported by Mullen, Lean, and Crozier (2002). However, other cyanidin glycosides, as well as pelargonidin and delphinidin glycosides, have also been identified in raspberry fruits (Fig. 2). The content of anthocyanins in raspberry fruits is connected to the plants’ exposure to the sun. The results of this study are in accordance with those shown by Zorenc, Veberic, Koron, and Mikulic-Petkovsek (2017), who observed that in times of lower sun exposure (autumn time), raspberry fruits accumulated fewer anthocyanins (42.63 mg/100 g FW) compared to the summer time (47.53 mg/100 g FW). In both years of the experiment, we noticed that in the autumn, when the sun exposure of the fruits was lower, the raspberries contained fewer total anthocyanins compared to in the summer (Tables 1 and 2). The differences between the cultivars are an effect of genetic diversity. Bobinaitė et al. (2012) showed that raspberries with a light-pink colour contained fewer total anthocyanins (‘Begalinka’ cv, 2.1 mg/100 g FW) compared to deep-purple raspberries

organic fruits only in one year (2013). It is worth noting that organic raspberries contained a higher concentration of this flavonol in the summer harvest time (Table 1). In the autumn harvest conventional raspberries contained significantly greater total flavonols (5.37 mg/ 100 g FW) and quercetin-3-O-rutinoside (2.46 mg/100 g FW) than organic raspberries (3.63 mg/100 g FW) and (0.81 mg/100 g FW) (Table 2). Quercetin is produced by plants as a response to UV radiation (Xu et al., 2017). According to this theory, raspberry plants in organic farms had greater sun exposure (Fig. 1) in both years of the experiment compared to conventional plants, especially in the summer harvest time. Therefore, the fruits of organic raspberries harvested in the summer contained more quercetin compared to conventional ones. Due to the lower light intensity present in autumn, the raspberry plants were exposed to much less UV radiation stress. Therefore, the fruits contained less quercetin in comparison with the those harvested in the summer period. In the autumn harvest time, the plants grown in conventional farms received much more light, which contributed to increasing the content of quercetin in the raspberry fruits (Fig. 1). Organic fruits contained 0.38 mg/100 FW and 0.29 mg/100 g FW (2013 and 2014, respectively) and conventional 0.45 mg/100 g FW and 0.45 mg/100 g FW (2013 and 2014, respectively). Among the three investigated raspberry cultivars, ‘Glen Fine’ deserves attention, especially in the second experimental year (2014). This cultivar was characterized by the highest and most significant amounts of almost all the phenolic compounds identified in raspberries (Table 1, Fig. 2). The differences in the polyphenol compound contents among raspberry cultivars is a genetic factor. As reported by Anttonena and Karjalainen (2005), three raspberry cultivars were characterized by a significantly different content of quercetin in their fruits, ‘Balder’ cv. (1.80 mg/100 g FW), ‘Heisa’ (0.56 mg/100 g FW) and ‘Wild’ (0.34 mg/100 g FW) Furthermore, in different years of the experiment, a single cultivar (‘Algonquine’ cv.) contained different levels of quercetin in its fruits. In the first year, it was 1.12 mg/100 g FW, and in the second year, it was only 0.73 mg/100 g FW. In the present experiment, we found significant variation in the quercetin content among the examined raspberry

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A. Ponder and E. Hallmann

(‘Bristol’ cv., 325.5 mg/100 g FW). In the present experiment, among the three raspberry cultivars tested, the greatest total anthocyanin content was in ‘Laszka’ cv in the first year of the experiment (102.9 mg/ 100 g FW), but in the second year, ‘Glen Fine’ cv. (89.5 mg/100 g FW) had the greatest total anthocyanin content (Table 1). In the case of anthocyanins, we noticed that both the farm managementmethod and the cultivar strongly affected the level of bioactive compounds in the raspberry fruits, but only in the second experimental year. Organic fruits contained significantly greater total anthocyanins (103.9 mg/ 100 g FW and 82.5 mg/100 g FW) and cyanidin-3,5-di-O-glucoside (47.7 mg/100 g FW and 42 mg/100 g FW) in both years compared to conventional fruits. The 2014 ‘Glen Fine’ cultivar was characterized by the highest level of all anthocyanin compounds (Table 1). Similar results were presented by Crecente-Campo, Nunes-Damaceno, RomeroRodríguez, and Vázquez-Odériz (2012) for organic and conventional strawberries. They suggested that organic fruits contained greater total anthocyanins because their concentration was greater in the fruits’ peels and less in flesh. Kazimierczak, Hallmann, Kowalska, and Rembiałkowska (2015) showed that organic raspberries ‘Polka’ cv. contained 173.2 mg/100 g FW total anthocyanins and conventional only 137.8 mg/100 g FW, while work presented by Skupień, Ochniam, Grajkowski, and Krzywy-Gawrońska (2011), obtained a much lower content: for organic ‘Polka’ cv., 38.1 mg/100 g FW, and for conventional, 33.8 mg/100 g FW. Chaovanalikit and Wrolstad (2004) showed that the distribution of anthocyanins in cherries of some cultivars was 70% in the skin and 30% in the flesh (‘Royal Ann’ cv.) or 100% in the skin and 0% in the flesh (‘Montmorency’ cv.). Raspberry fruits, as well as strawberries, blackberries and wild strawberries, have even skin and flesh colouring. Based on the obtained results for the organic farming system, we suggest that the organic farming system could be more comparable to plants that are harvested from a natural state. As reported by Veberic, Slatnar, Bizjak, Stampar, and Mikulic-Petkovsek (2015), wild raspberry fruits are characterized by a higher level of total anthocyanins compared to those same species from farming. Cultivated blackberries had an anthocyanin level of 191.5 mg/100 g FW, whereas the wild form of blackberries had 281.2 mg/100 g FW; garden strawberries 15.8 mg/100 g FW and wild strawberries 47.9 mg/100 g FW; and cultivated raspberries 62.1 mg/100 g FW and wild grown raspberries 70.2 mg/100 g FW. This is related to the increased content of protective chemicals of fruits in the harder conditions posed by organic farming.

References Anttonena, M. J., & Karjalainen, R. O. (2005). Environmental and geneticvariation of phenolicc ompounds in red raspberry. Journal of Food Composition and Analysis, 18, 759–769. Asami, D. K., Hong, Y. J., Barrett, D. M., & Mitchell, A. E. (2003). Comparison of the total phenolic and ascorbic acid content of freeze-dried and air-dried marionberry, strawberry, and corn grown using conventional, organic, and sustainable agricultural practices. Journal of Agricultural and Food Chemistry, 51, 1237–1241. Baby, B., Antony, P., & Vijayan, R. (2017). Antioxidant and anticancer properties of berries. Critical Reviews in Food Science and Nutrition, 1–17. Bobinaitė, R., Viškelis, P., & Venskutonis, P. R. (2012). Variation of total phenolics, anthocyanins, ellagic acid and radical scavenging capacity in various raspberry (Rubus spp.) cultivars. Food Chemistry, 132, 1495–1501. Bobinaitė, R., Viskelis, P., Bobinas, C., Mieželienė, A., Alenčikienė, G., & Venskutonis, P. R. (2016). Raspberry marc extracts increase antioxidative potential, ellagic acid, ellagitannin and anthocyanin concentrations in fruit purees. LWT - Food Science and Technology, 66, 460–467. Chaovanalikit, A., & Wrolstad, A. E. (2004). Total anthocyanins and total phenolics of fresh and processed cherries and their antioxidant properties. Journal of Food Science, 69, 67–72. Council Regulation (EC) No 834/2007 of 28 June 2007 on organic production and labelling of organic products and repealing Regulation (EEC) No 2092/91. Crecente-Campo, J., Nunes-Damaceno, M., Romero-Rodríguez, M. A., & Vázquez-Odériz, M. L. (2012). Color, anthocyanin pigment, ascorbic acid and total phenolic compound determination in organic versus conventional strawberries (Fragaria x ananassa Duch, cv Selva). Journal of Food Composition and Analysis, 28, 23–30. Eurostat Database (2019). https://ec.europa.eu/eurostat. Hallmann, E., Rozpara, E., Słowianek, M., & Leszczyńska, J. (2019). The effect of organic and conventional farm management on the allergenic potency and bioactive compounds status of apricots (Prunus armeniaca L.). Food Chemistry, 279, 171–178. Hargreaves, J. C., Adl, M. S., Warman, P. R., & Rupasinghe, H. P. V. (2008). The effects of organic and conventional nutrient amendments on strawberry cultivation: Fruit yield and quality. Journal of the Science of Food and Agriculture, 88, 2669–2675. Ismail, T., Calcabrini, C., Diaz, A. R., Fimognari, C., Turrini, E., Catanzaro, E., ... Sestili, P. (2016). Ellagitannins in cancer chemoprevention and therapy. Toxins, 8, 151–172. Kazimierczak, R., Hallmann, E., Sokołowska, O., & Rembiałkowska, E. (2011). Bioactive substances content in selected species of medical plants from organic and conventional production. Journal of Research and Applications in Agricultural Engineering, 56(3), 200–205. Kazimierczak, R., Hallmann, E., & Rembiałkowska, E. (2014). Effects of organic and conventional production systems on the content of bioactive substances in four species of medicinal plants. Biological Agriculture and Horticulture, 34(4), 1–11. Kazimierczak, R., Hallmann, E., Kowalska, K., & Rembiałkowska, E. (2015). Biocompounds content in organic and conventional raspberry fruits. Acta Fytotechnica et Zootechnica, 18, 40–42. Mikulic-Petkovsek, M., Slatnar, A., Stampar, F., & Veberic, R. (2012). HPLC–MSn identification and quantification of flavonol glycosides in 28 wild and cultivated berry species. Food Chemistry, 135, 2138–2146. Mullen, W., Lean, M. E. J., & Crozier, A. (2002). Rapid characterization of anthocyanins in red raspberry fruit by high-performance liquid chromatography coupled to single quadrupole mass spectrometry. Journal of Chromatography A, 966, 63–70. Papaioanou, M., Chronopoulou, E. G., Ciobotari, G., Efrose, R. C., Sfichi-Duke, L., Chatzikonstantinou, M., ... Labrou, N. E. (2018). Cosmeceutical properties of two cultivars of red raspberry grown under different conditions. Cosmetics, 5(1), 1–20. Polish Norm PN-R-04013:1988 (1988). The estimation of dry matter in fruits and vegetables, published by Polish Standard Committee. 1–5. Schell, J., Betts, N. M., Lyons, T. J., & Basu, A. (2019). Raspberries improve postprandial glucose and acute and chronic inflammation in adults with type 2 diabetes. Annals of Nutrition and Metabolism, 74, 165–174. Skupień, K., Ochniam, I., Grajkowski, J., & Krzywy-Gawrońska, E. (2011). Nutrients, antioxidants, and antioxidant activity of organically and conventionally grown raspberries. Journal of Applied Botany and Food Quality, 84, 85–89. Stavanga, J. A., Freitag, S., Foito, A., Verrall, S., Heide, O. M., Stewart, D., & Sønsteby, A. (2015). Raspberry fruit quality changes during ripening and storage as assessed by colour, sensory evaluation and chemical analyses. Scientia Horticulturae, 195, 216–225. Tassoni, A., Tango, N., & Ferri, M. (2013). Comparison of biogenic amine and polyphenol profiles of grape berries and wines obtained following conventional, organic and biodynamic agricultural and oenological practices. Food Chemistry, 139, 405–413. Teng, H., Fang, T., Lin, Q., Song, H., Liu, B., & Chen, L. (2017). Red raspberry and its anthocyanins: Bioactivity beyond antioxidant capacity. Trends in Food Science & Technology, 66, 153–165. Teng, H., & Chen, L. (2019). Polyphenols and bioavailability: An update. Critical Reviews in Food Science and Nutrition. https://doi.org/10.1080/10408398.2018.1437023, 1-12. Veberic, R., Slatnar, A., Bizjak, J., Stampar, F., & Mikulic-Petkovsek, M. (2015). Athocyanin composition of different wild and cultivated berry species. LWT - Food Science and Technology, 60, 509–517. Veljkovic, B., Djordjevic, N., Dolicanin, Z., Licina, B., Topuzovic, M., Stankovic, M., ... Dajic-Stevanovic, Z. (2019). Antioxidant and anticancer properties of leaf and fruit extracts of the wild raspberry (Rubus idaeus L.). Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 47, 2, 359–367. Wang, P., Cheng, Y. Ch., Hung, y. Ch., Lee, Ch. H., Fang, J. Y., Li, W. T., ... Pan, T. L. (2019). Red raspberry extract protects the skin against UVb-induced damage with

4. Conclusions The present study confirms that farm management method may have a significant impact on the phenolic composition of raspberry fruits. However, in addition to the agronomic factors arising from the farming system, the plant cultivars appeared to have an important effect on the quality and quantity of bioactive compounds. It is worth pointing out that the vast-majority of the research on the quality of organic fruits is focused only on one-year experimentation. There are major changes in the chemical composition of raspberry fruits with the passage of time and production in organic systems. The choice of a proper cultivar and harvest time also has a significant impact on the polyphenol content of raspberry fruits. Declaration of Competing Interest Authors declare NO Conflict of Interest. Acknowledgements This paper has been published under the support of: Polish Ministry of Higher Education within founds of Faculty of Human Nutrition and Consumer Sciences, Warsaw University of Life Sciences (WULS), for scientific research. 7

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A. Ponder and E. Hallmann antioxidative and anti-inflammatory properties. Oxidative Medicine and Cellular Longevity. https://doi.org/10.1155/2019/9529676. Zafrilla, P., Ferreres, F., & Tomás-Barberán, F. A. (2001). Effect of processing and storage on the antioxidant ellagic acid derivatives and flavonoids of red raspberry (Rubus idaeus) jams. Journal of Agriculture and Food Chemistry, 49, 3651–3655. Zhang, J., Wang, X., Yu, O., Tang, J., Gu, X., Wan, X., & Fang, C. (2011). Metabolic profiling of strawberry (Fragaria×ananassa Duch.) during fruit development and maturation. Journal of Experimental Botany, 62, 1103–1118. Zorenc, Z., Veberic, R., Koron, D., & Mikulic-Petkovsek, M. (2017). Impact of raspberry

(Rubus idaeus L.) primocane tipping on fruit yield and quality. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 45, 417–424. Young, J. E., Zhao, X., Carey, E. E., Welti, R., Yang, Sh.-Sh., & Wang, W. (2005). Phytochemical phenolics in organically grown vegetables. Molecular Nutrition & Food Research, 49, 1136–1142. Xu, Y., Charles, M. T., Luo, Z., Mimee, B., Veronneau, P.-Y., Rolland, D., & Roussel, D. (2017). Preharvest ultraviolet c irradiation increased the level of polyphenol accumulation and flavonoid pathway gene expression in strawberry fruit. Journal of Agriculture and Food Chemistry, 65, 9970–9979.

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