Effects of nutrition strategy on the levels of nutrients ...

Effects of nutrition strategy on the levels of nutrients and bioactive compounds in blackberries Liaqat Ali, Beatrix W. Alsanius, Anna Karin Rosberg, Birgitta Svensson, Tim Nielsen & Marie E. Olsson European Food Research and Technology Zeitschrift für LebensmittelUntersuchung und -Forschung A ISSN 1438-2377 Volume 234 Number 1 Eur Food Res Technol (2012) 234:33-44 DOI 10.1007/s00217-011-1604-8

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Author's personal copy Eur Food Res Technol (2012) 234:33–44 DOI 10.1007/s00217-011-1604-8

ORIGINAL PAPER

Effects of nutrition strategy on the levels of nutrients and bioactive compounds in blackberries Liaqat Ali • Beatrix W. Alsanius • Anna Karin Rosberg Birgitta Svensson • Tim Nielsen • Marie E. Olsson

•

Received: 26 May 2011 / Revised: 26 September 2011 / Accepted: 8 October 2011 / Published online: 22 October 2011 Ó Springer-Verlag 2011

Abstract The effects of nutrition strategy on levels of nutrients and bioactive compounds in fruit and leaves of blackberries were studied in greenhouse-grown blackberry plants fertilised with combinations of two levels (low, high) of nitrogen (60 and 100 kg ha-1, respectively) and potassium (66.4 and 104 kg ha-1, respectively). Plant concentrations of organic phytochemicals were quantitatively analysed by high-performance liquid chromatography. High amounts of both fertilisers produced high amounts of all nutrients and bioactive compounds analysed in fruit except total acidity and ellagic acid. There were major differences in compounds affecting taste in fruit, e.g., sugars (fructose and glucose), total soluble solids and pH, and also in anthocyanin content. The concentrations of secondary metabolites, vitamin C and ellagic acid in fruit also varied significantly between treatments, although the differences were smaller. Storage of blackberries showed variable effects in the different levels of compounds, and the changes found were small. Nutrient regime did not affect blackberry leaves to the same extent, and only minor changes were found. The findings show that by optimising plant nutrition, phytonutrient levels can be maximised and maintained in fresh and stored berry crops, especially those grown in greenhouses, where conditions can easily be regulated.

L. Ali (&) B. W. Alsanius A. K. Rosberg B. Svensson M. E. Olsson Department of Horticulture, Swedish University of Agricultural Sciences, P.O. Box 103, 23053 Alnarp, Sweden e-mail: [email protected] T. Nielsen SIK, Ideon, 22370 Lund, Sweden

Keywords Nitrogen Potassium Fertilisation Nutrients HPLC Storage

Introduction Fresh soft fruits are highly perishable, and their quality and shelf-life can be greatly affected by different pre- and postharvest factors and by plant stress. Important quality parameters in berries are nutrient content and taste, which is determined by sugars, acidity and aroma compounds. In addition, there has recently been increasing interest in the content of other compounds with health-promoting properties in berries [1]. During the period 1995–2005, worldwide blackberry production area increased by 44% [2], and at least some of this increase is most likely due to consumer interest in various health-promoting effects. Numerous studies have shown that fruits and vegetables are a rich source of nutrients as well as non-nutrient molecules with antioxidant or other physiological effects, and it seems likely that given sufficient bioavailability, these compounds may be important constituents of a healthy diet. The health-promoting properties of plant-based foods have largely been attributed to their wide range of phytochemicals, many present at relatively high levels [3]. These bioactive compounds have a high antioxidant effect against reactive oxygen species (ROS), such as hydroxyl radicals 0 (OH), superoxide radicals (O2 ), singlet oxygen ( O2) and hydrogen peroxides (H2O2), produced as, e.g., by-products of normal metabolism [4–6]. Increased levels of these reactive oxygen species or free radicals create oxidative stress, which results in pathological conditions such as ischaemia–reperfusion damage, inflammatory and degenerative diseases, cancer, DNA damage and ageing [7–10]. In addition to their antioxidant properties, many components

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in fruit and vegetables have been suggested to help in sustaining memory, reducing blood pressure, strengthening the immune system and enhancing the response against pollutants and several skin diseases [11–13]. The level and composition of bioactive phenolics in berries vary according to genetics, climate factors, fertilisation and other cultural practices. Genotype has a major role in defining the level and composition of different compounds such as ellagitannins, flavonoids, total phenolics and sugars [14–17]. Climate factors such as seasonal variation, light intensity and fruit growing temperature (day/night) have also been found to influence quality factors [18–21]. Cultivation factors such as soil type, composts, mulching and fertilisation influence the water and nutrient supply to the plant and affect the nutritional composition and antioxidant activity of e.g. harvested berry fruit [22–24]. Among coloured fruits, berries, including blackberries, have been found to contain a wide range of bioactive compounds. Nutrients or otherwise health-promoting substances in berry fruits such as vitamin C, phenolic acids, ellagic acid, ellagitannins, flavonoids including anthocyanins, and carotenoids are widely available in blackberries, as are different sugars contributing to desirable taste [25, 26]. In addition, the leaves of Rubus species (blackberry and raspberry) are reported to be used in traditional medicine as anti-bacterial, anti-inflammatory, anti-diarrhoeic and antiviral agents, and also during pregnancy to shorten labour and make it easier [27, 28]. Rubus leaves have a great capacity as free radical scavengers and peroxide decomposers [26, 29–31]. Thus, blackberries could be considered a potent source of a number of health-promoting compounds related to protection against reactive oxygen species or free radicals, as well as other effects, and optimising the concentrations of these compounds would further contribute to the health effects. Plant nutrition is an important factor that can affect many aspects of berry fruits, such as crop yield, quality and, in particular, the levels and composition of bioactive compounds at harvest and during shelf-life [22, 24, 32]. Previous work on berry crops has shown that applying high amounts of compound fertiliser results in increased vegetative growth and yield, while it negatively affects some fruit quality parameters [24, 33]. Nitrogen (N) and potassium (K) in particular are reported to have pronounced effects on texture, aroma compounds and shelf-life after harvest [34]. Excessive supply of N has been shown to decrease quality in some berries and fruits such as chokeberries [33], strawberries [24] and grapes [35]. Potassium is the most mobile and abundant cation in berry fruits and is most likely associated with high acid levels, as a strong correlation between pH regulation and organic acid levels as affected by K supply has been reported [36]. Excess K

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Eur Food Res Technol (2012) 234:33–44

also induces antagonistic interactions with other nutrients, such as calcium and magnesium, in the leaves of many bramble cultivars [37–39]. Studies on grapes show interactive effects of N and K on the content of polyphenols [40]. Many decades of research have helped identify the ideal dose of N, P and K (and minor nutrients), but the results primarily relate to fruit production and yield. Knowledge about the effect of fertiliser application on nutrients and bioactive compounds in blackberries is very limited, especially concerning the antagonistic and synergistic interactions caused by fertilisation on phytochemical content at harvest and during shelf-life. With relevant data, adjusting plant nutrition and other cultivation factors could become future commercial practice in order to increase the content of phenols and other quality attributes during fruit shelf-life, especially in greenhouse cultivation where the conditions can be easily manipulated. The aim of this study was therefore to evaluate how plant nutrition strategy can be used to improve the content of nutrients and bioactive compounds in blackberries at harvest and during storage.

Materials and methods Experimental design Blackberries (Rubus fruticosus L. cv. Loch Ness) were grown in pots (5-L containers). Master coarse sifted sphagnum peat, H2-4, with clay, fibre length 10–35 mm; dry matter content 120 g L-1; organic matter content 80% of dry matter; dry bulk density 300 kg m-3; pH 6.0; EC 30 mS m-1; additives (kg m-3): limestone 4, dolomite 2, N–P–K 14–7–15 ? micro 1.2, micronutrients 0.05; product number 39965129 (Hasselfors, Orebro, Sweden) was used in a greenhouse experiment at the Department of Horticulture, SLU Alnarp, Sweden (55°390 3000 N, 13°50 000 E). A complete randomised block design (2 blocks; four nutrient treatments) was used and the experiment ran for one season, with greenhouse temperature set-point 20 °C, relative humidity 75% and light intensity 209 (PAR) mol m-2 s-1. One-year-old, outdoor vernalised (7 °C for 600 h) blackberry plants were trained to 80 cm height with three scions per plant and transferred to the greenhouse on February 5, 2008. Greenhouse temperature was increased gradually (night temperature: week 5: 0 °C, week 8: 5 °C, week 12: 10 °C; day temperature: week 5: 7 °C, week 8: 15 °C, week 12: 19 °C). Nutrient regime (T1, T2, T3, T4) comprised combinations of two levels (low, high) of N (60 and 100 kg ha-1, respectively) and K (66.4 and 104 kg ha-1, respectively) (Table 1). Plant nutrients (N, K and P, Ca, Mg, S, Mn, Fe, Zn, Cu, B, Mo, Cl, Na) were

Author's personal copy Eur Food Res Technol (2012) 234:33–44

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Table 1 Nitrogen (low: 60 kg ha-1, high: 100 kg ha-1) and potassium (low: 66.4 kg ha-1, high: 104 kg ha-1) regime used for blackberry plants grown under greenhouse conditions Treatment

N Low

T1

?

T2

?

K High

Low

High

?

12 after harvest, whereas green leaves were analysed without storage after freeze-drying (Labconco, Thermo Kyl AB Helsingborg, Sweden). 3–3.5 kg fruit was harvested for each treatment, and the fruit was divided into 15 boxes for analyses fresh at harvest day, or for storage (3 boxes for each day, and 200–250 g in each box). All analyses were performed in triplicate.

?

T3

?

T4

?

?

Chemicals

?

The nutrients were added by drip irrigation. The growing season was grouped into five phases with different nutrient levels to match the requirements and growth of the crop

distributed as nutrient solution by drip irrigation (two nozzles per container) with 4–5 irrigation events per day. Nitrogen was applied as magnesium nitrate (Mg (NO3)2*2H2O), calcium nitrate (Ca (NO3)2*4H2O) and potassium nitrate (KNO3). Potassium was applied as monopotassium phosphate (KH2PO4) and potassium nitrate (KNO3). The composition of the micronutrient solution was as follows (in lmol): Mn 23.8, Fe 49.3, Zn 0.96, B 18, Mo 0.14. Raspberry and blackberry are not yet common greenhouse crops under Scandinavian conditions. The choice of fertilization levels has been compared to common standard of Swedish growers and also re-evaluated by the growers. Under field conditions, 60 kg of N and 66.4 kg of K are used per ha [41]. Swedish growers supply 100 kg N/ha to blackberries. For the experiment, the N/K ratio was maintained for appointing the potassium level (104 kg ha-1). Nutrient levels of the macronutrients were managed with respect to plant requirement and growth in five distinct phases, where phase 1 and 2 comprised the vegetative phase and phases 3–5 the generative phase (phase 1: 5 weeks; phase 2: 6 weeks; phase 3: 5 weeks; phase 4: 6 weeks; phase 5: 4 weeks). Phase division of the nutrient composition is common practice in Swedish commercial production of Rubus-berries under greenhouse conditions and favours the crop’s vegetative and generative growth. During late season, no additional nutrients were supplied, and the plants were irrigated with tap water. Sampling and storage The effects of nutrition strategy were analysed in terms of the concentrations of nutrients and bioactive compounds in fresh green, fully mature leaves (4th from the top) and ripe berries, which were harvested on August 28th, 2008. The two blocks were not used for replication purpose, and therefore, the berries from the two blocks were pooled, and three samples were taken. The harvested fruit samples were stored in plastic containers at 2 °C and 75% relative humidity and analysed on days 1 (harvest day), 3, 6, 9 and

Ellagic acid, m-phosphoric acid, triethylamine and alltrans-b-carotene were purchased from Sigma-Aldrich Inc. (Laborchemikalien GmbH Germany). Ascorbic acid, dithiothreitol (DTT) and potassium di-hydrogen phosphate (KH2PO4) were purchased from VWR International (Geldenaaksebaan, Leuven, Belgium). Acetonitrile (Lichrosolv), acetone, ammonium acetate, dichloromethane (Lichrosolv), ethyl lactate (Lichrosolv), hydrochloric acid (HCl) and methanol (Lichrosolv) were obtained from Merck (Darmstadt, Germany). Ethanol was obtained from Solveco (Rosersberg, Sweden). All solvents were of HPLC-grade, and other chemicals were of reagent grade. Chlorophyll (chlorophyll a and b) and carotenoid (b-carotene and lutein) analysis For the extraction of carotenoids/chlorophylls, lyophilised leaf samples, 250 mg, were each extracted in 5 mL solvent consisting of 4/1 ethyl acetate/ethanol. BHT 0.1% was added as antioxidant [42]. Samples were then centrifuged, at 7,500g, and the supernatant analysed directly with HPLC. HPLC analysis of carotenoids/chlorophylls was performed as in [40] with some modifications. HPLC-system Merck-Hitachi La Chrome was used, with column Nucleosil EC-200 250*4,6(5 lm), and binary gradient with eluent A: 57/43 acetonitrile/methanol; eluent B: 52/38/10 acetonitrile/methanol/dichloromethane. Both eluents were modified with 0.02 M NH4C2H4O2 and 0.1% tri-ethylamine. Flow rate was 1.0 mL/min, and injection volume 15 lL. The gradient was 0–5 min 20% B, 5–20 min 20–80% B, 20–22 min 80–20% B, 22–25 min 20% B. Carotenoids and chlorophylls were quantified by external standards purchased from Sigma-Aldrich. Titrable acidity, pH and total soluble solids Analysis was performed according to standard method, as in AOAC. In short, pH and titrable acidity (TA) were determined using titration unit, Titroline easy (SCHOTT Instr. GmbH, Germany), 100 mM NaOH to endpoint pH 8.3. Values for TA are presented as percentage citric acid w/w (Table 1). Total soluble solids (TSS) were measured

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using termostated refractometer RFM 80 (Bellingham Stanley Ltd, UK). Vitamin C analysis


acid content was identified at 254 nm using a diode array detector and by comparing with retention times and quantified using an external standard ellagic acid (SigmaAldrich, USA). Anthocyanin analysis

Vitamin C was analysed in blackberry fruits and leaves. Fresh berry fruits were homogenized directly after harvest with a Waring Blender 8011G (Dynamics Corp., New Hartford, USA). 5 g homogenate and 25 mL 1.5% m-phosphoric acid were mixed and centrifuged at 12,500g for 12 min. For the leaf analysis, lyophilized and milled samples (equivalent to 5 g fresh weight) were used, and following the same extraction procedure as the fruits. The supernatants were treated with dithiothreitol (DTT) to reduce dehydroascorbic acid to ascorbic acid as in [42], and thereby measuring vitamin C. Samples were analysed by a 7,000 HPLC system (Merck-Hitachi Lachrome, Burladingen, Germany), and diode array detection was carried out at 248 nm. The column used was Phenomenex Synergy polar RP 250*4.6, (4 lm), flow rate 1 mL/min and injection volume 15 lL. The binary gradient contained eluent A: 20 mM K2HPO4/methanol 96/4 (pH 2.3) and eluent B: methanol. The gradient used was: 0–4 min 100% A, 4–4.5 min 100–20% A, 4.5–7.5 min 20% A, 7.5–8 min 20–100% A, 8–18 min 100% A. Quantification was made relative to external standard L(?) ascorbic acid (AnalaR Normapur, Leuven, Belgium).

Anthocyanins were analysed in the berries by extracting 1 g of lyophilised sample in 20 mL 50% ethanol (w/v) including 1% HCl at 2 °C for 20 h on a rotary shaker. The extract was centrifuged at 12,500g for 12 min and directly run on HPLC. The HPLC analyses were carried out using a 7,000 HPLC system (Merck-Hitachi Lachrome, Burladingen, Germany) equipped with diode array detector. The mobile phase consisted of A (5% formic acid/95% Millipore water) and B (50% acetonitrile/50% methanol). The flow rate was adjusted to 1.2 mL min-1 and the injection volume 5 lL. The binary gradient was: 0–5 min 5% eluent B, 5–20 min 5–40% B, 20–20.5 min 40–80% B, 20.5–22.5 min 80–5% B, 22.5–25 min 5% B. Separation was made with a Phenomenex Luna C8 column (250* 4.6, 5 lm) and fixed wave length 525 nm. The main anthocyanin compounds in blackberry fruit and leaves were identified by spectral characteristics with data given in the literature [44, 45]. Quantification was made by comparisons with a reference standard curve, cyanidin-3-glycoside (Polyphenols Laboratories, AS, Norway).

Ellagic acid analysis

Total phenolics and sugar analysis

The ellagic acid extraction protocol was based on a previous method described by Siriwoham et al. [43], with modifications to increase recovery. Three replicate samples of lyophilised fruit and leaves, each weighing 1 g, were placed in 20 mL of 70% acetone and homogenised using a rotary orbital shaker (Forma Scientific Inc., Ohio, USA) for 20 h at 2 °C. The extract was centrifuged at 12,500g for 12 min. For acid hydrolysis of the ellagic acid conjugate, aliquots of 1 mL of the supernatant were transferred to 1 mL of 4 M HCl and hydrolysed for 4 h at 95 °C. The hydrolysed extract (samples) was then cooled (in ice water, 10 min.) under green light to stop the reaction. The samples were centrifuged at 12,500g for 12 min and analysed on a 7,000 HPLC system (Merck-Hitachi Lachrome, Burladingen, Germany) using the method described in [43] with modifications to optimize reproducibility. The eluents consisted of: A (97% 20 mM KH2PO4 with pH adjusted to 2.3 (using conc. H3PO4) and 3% methanol) and B (acetonitrile, 99.7%). The flow rate was 1 mL min-1 and the injection volume 10 lL, with a Phenomenex synergy column (fusion RP 250 9 4.6 mm, 4 lm). The elution profile was as follows: 0–7 min 0% eluent B, 7–59 min 0–22% B, 59–65 min 22–55% B, 65–69 min 55–0% B. The ellagic

Fresh berries, 5 g of the homogenate (Waring Blender 8011G used), were mixed with 20 mL of ethanol (75% on a dry weight base). In the case of the leaves, lyophilised samples, equivalent to 5 g fresh sample, were mixed with 20 mL of 75% ethanol and were used for total phenolics and sugar extraction. The extracted samples were centrifuged at 12,500g for 12 min, and an aliquot of the supernatant was used for sugar analysis. Sugars were analysed by liquid chromatography apparatus consisting of a Kontron auto sampler 465, (Everett, USA) fitted with a pump (Waters 515). An isocratic gradient method with a mobile phase 70/30%, acetonitrile/water (v/v) was maintained for a run time of 14 min. Sugars were separated on an Asahipak NH2P-50 4E column (Shodex Inc. Shoko, America) using a LDC analytical refractive index detector (Refractometer 1V). Identification and quantification were performed using external standards D-fructose (Janssen Chimica, Geel, Belgium), glucose and sucrose (Sigma Chemical Co. USA). The total phenol content in blackberry fruit and leaves was determined by the Folin–Ciocalteau method as modified by Dewanto et al. [46]. Absorbance was measured at 765 nm using a Cary 50 Bio spectrophotometer (Varian, Australia). Quantification was made by comparing with

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external standard gallic acid, 3,4,5-trihydroxybenzoic acid (Sigma-Aldrich, Germany). Statistics Three replicates samples were used for each sampling occasion. Data for nutrient regime and storage effect on antioxidants were statistically evaluated by one-way ANOVA followed by Tukey’s studentised range test (HSD) using SAS (SAS Inst. Inc. Cary, North Carolina, USA). Pearson correlation coefficients (PROC CORR: SAS Inst. Inc. Cary, North Carolina, USA) were calculated to reveal the relationship between data. Differences were considered statistically significant at p \ 0.05. Principal component analysis [47] (Minitab 15: Minitab Inc.) was also made to test the load of discrimination for different bioactive compounds in relation to N and K strategies.

Results Blackberry fruit Principal component analysis of all organic phytochemicals analysed Principal component analysis is a method for identifying patterns in data, and to graphically express the data in such a way as to emphasize their similarities and differences. In this investigation, PCA was used in order to investigate how the different phytochemicals change over time and to analyze the overall correlation among the different variables simultaneously. When all organic phytochemicals analysed were considered using PCA, differences were found between the different nutrient regimes at harvest. Principal component (PC) 1 discriminated between the composition of blackberry fruit grown at low and high N supply, while PC 2 discriminated between the high N treatments subjected to low and high K supply and PC 3 discriminated between the high K treatments subjected to low and high N supply (Fig. 1a, b; values for PC 3 are not shown in the figure). In comparisons between day 1 (harvest day) and the third day of storage as well as between day 1 and the sixth day of storage, the first three principal components were decisive, with PC 1, PC 2 and PC 3 explaining 48.5, 24.5 and 12.5% (Fig. 1a) and 44.2, 25.9 and 13.9% (Fig. 1b) of the variation. Table 2 describes the load of the different bioactive phytochemicals on the three components. Comparison of phytochemicals between harvest and 3 days of storage showed that PC 1 was mostly dominated by different sugars (fructose, glucose, total sugars) as well anthocyanins and total soluble solids, whereas vitamin C, pH and total acidity dominated PC 2, while total phenolics and sucrose dominated PC 3. Beyond 3 days of storage, the

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differences between the treatments decreased; principal component 2 discriminated blackberry fruit provided with a combination of high N and low K supply from the three other treatments. After 6 days, no differences regarding the organic phytochemical composition were found between the two treatments with low N supply and the differences between the high N treatments decreased. When comparing phytochemicals between harvest and after 6 days of storage, the same compounds affected PC 1, but ellagic acid also had a substantial impact on PC 1. PC 2 and PC 3 displayed a different picture than for the first storage period. Vitamin C, pH, total acidity and sucrose dominated PC 2, whereas total phenolics, anthocyanins and ellagic acid were decisive for PC 3. Sugars (fructose, glucose, sucrose) and total soluble solids The high N treatments (T3 and T4) had the highest levels of total sugars at all storage times (Fig. 2, significance denotations not shown). Total soluble solids showed the same trend, with the highest and second highest found for the high N treatments, with the exception of day 12 when the low N, low K treatment (T1) was highest. There was no clear tendency for increased or decreased TSS during storage for any of the nutrient regimes tested. Titratable acidity and pH In blackberry fruit, significant differences were found for titratable acidity and pH both between treatments and during storage (Table 3; significance values shown only between treatments with the same storage time). At harvest, the highest pH was found in the high N, high K treatment (T3), whereas the highest titratable acidity was found in the low K treatments (T1 and T4). In treatments T1, T2 and T4, the pH increased during storage. The titratable acidity decreased during storage in T1 and T4, while no consistent changes could be found during storage in treatments T1 and T3. Vitamin C The treatment with high K levels (T2 and T3) showed the highest vitamin C content at all storage times, with the exception of day 9 (Table 3, significance values shown only between treatments with the same storage time). Treatment T4 (high N, low K) had the lowest vitamin C content at all storage times. In general, only small changes were found during storage in all treatments (Table 3). Total phenolics and anthocyanins The highest and second highest level of anthocyanins in the berries at harvest and during the first part of

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A

3

T3 S1

2

Second Component

Fig. 1 Principal component analysis on organic phytochemical composition of blackberry fruit grown under low and high nitrogen (60 and 100 kg ha-1) or potassium (66.4 and 104 kg ha-1) regime (T1 (filled circle): low N, low K; T2 (filled diamond): low N, high K; T3 (filled up triangle): high N, high K; T4 (filled down triangle): high N, low K). Filled symbols represent values at harvest (S1), open symbols values after 3 days (S3; a) and 6 days of storage. Loading of measured bioactive phytochemicals on principal components is shown in Table 2. PC 1, PC 2 and PC 3 were explaining 48.5, 24.5 and 12.5% (a) and 44.2, 25.9 and 13.9% (b) of the variation. The values for PC 3 are not shown in the figures


T2 S1 1

T2 S3

T1 S3 0

T3 S3 T1 S1

-1 -2

T4 S3 -3

T4 S1 -4 -5

-4

-3

-2

-1

0

1

2

3

4

5

First Component

B

3

T3 S1

Second Component

2

T2 S6 1

T2 S1

T1 S6

0

T3 S6

T1 S1

-1

T4 S6

-2

-3

T4 S1 -4 -5

-4

-3

-2

-1

0

1

2

3

4

5

First Component

storage (day 1 (harvest day) to 6) were found in treatment T4, whereas the lowest values for this storage time were found in T1 (Table 3, significance values shown only between treatments with the same storage time). No general pattern of change in the content of anthocyanins during storage could be found for any treatment. The total phenolics content decreased during storage in all treatments from day 1 to day 6. No consistent pattern of highest or lowest values in general during all storage times could be found for any treatment (Table 3). Ellagic acid The treatment with low N and high K (T2) showed in general a high level of ellagic acid at all storage times (Table 3, significance values shown only between

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treatments with the same storage time). With the exception of the harvest day, treatment T1 (low N, low K) showed low levels of ellagic acid during storage. For treatments T2, T3 and T4, the ellagic acid content decreased from the harvest day to the end of storage. Correlations The Pearson correlation was used as a measure of association for two variables, measuring both the strength and the direction of a linear relationship. The pH was positively correlated with vitamin C content (r = 0.53; p \ 0.0001), whereas titratable acidity was negatively correlated with pH (-0.64; p \ 0.0001) in the fruit. In addition, total sugar content was positively correlated with TSS (r = 0.58; p \ 0.001), fructose (r = 0.99; p \ 0.001), glucose (r = 0.99; p \ 0.001) and sucrose (r = 0.52; p \ 0.001).

Author's personal copy Eur Food Res Technol (2012) 234:33–44 Table 2 Loading of measured bioactive phytochemicals on principal components (PC) 1, 2 and 3 for the periods from harvest to day 3 and harvest to day 6

Source of data

Comparison between harvest day and day 3

Comparison between harvest day and day 6

PC 1

PC 1

PC 2

PC 3

PC 2

PC 3

Vitamin C

0.001

0.576

0.048

0.053

0.524

Tot. phenolics

0.192

0.102

0.666

0.016

0.193

Anthocyanins

0.375

-0.193

0.185

0.129

-0.04

0.584

TSS

0.371

0.216

-0.218

0.379

0.21

-0.287

0.502

-0.148

0.473

-0.102

pH Total acidity Fructose PC 1, PC 2 and PC 3 were explaining 48.5, 24.5 and 12.5% (Fig. 1a) and 44.2, 25.9 and 13.9% (Fig. 1b) of the variation

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0.222 -0.246 0.404

-0.43

-0.227

-0.138

-0.181

0.251 -0.12

-0.103 0.571

-0.491

-0.304

0.442

-0.101

-0.087 -0.1

Glucose

0.402

-0.128

-0.196

0.439

-0.096

Sucrose

0.226

-0.258

0.514

0.266

-0.321

0.119

Tot. sugars

0.404

-0.137

-0.175

0.44

-0.104

-0.085

0.126

0.174

0.201

-0.315

Ellagic acid

-0.19

Blackberry leaves Sugars The main soluble sugars found in blackberry leaves were fructose, sucrose and glucose. In blackberry leaves, the total sugar content was higher with the T1 and T3 treatments, whereas treatment T2 and T4 showed lower sugar content than T1. No significant differences could be found between treatments in fructose, glucose or sucrose in blackberry leaves (Table 4). Vitamin C, total phenolics and ellagic acid There was no significant treatment effect on vitamin C content in blackberry leaves (Table 4). The total phenolics in blackberry leaves showed no significant differences between treatments, nor did the ellagic acid content with the exception of treatment T3, which had higher levels of ellagic acid. Chlorophylls and carotenoids There was no significant effect of plant nutrition strategy on chlorophyll a and b content in blackberries (Table 5). Among the carotenoids, the major peaks identified were lutein and b-carotene. In blackberries, the treatment with low N and high K (T2) showed the significantly highest level of b-carotene, though no significant changes were found between treatments for the lutein content (Table 5).

Discussion Fertilisers are important in determining yield, nutrients and other quality parameters in horticultural crops [7]. Two of

-0.327

the most important nutrients for plant growth and function are N and K. Nitrogen availability is crucial for normal plant growth and development, as it is an essential component in proteins, and also for chloroplast structure and function [48]. The roles of K, on the other hand, have been suggested by Mpelasoka et al. [49] to fall into four different groups: (1) enzyme activation; (2) cellular transport processes and translocation of assimilates; (3) anion neutralisation, which is essential in maintenance of membrane potential; and (4) osmotic potential regulation and thereby an important mechanism in the control of turgour maintenance. The present study of different nutrient regimes, including higher or lower amounts of N and K, showed that the different levels of these elements significantly affected the content and chemical composition of sugars, pH and different bioactive compounds at harvest and during shelflife in both fruits and leaves of blackberries. The greatest differences between treatments for blackberry fruit were found in compounds which affect taste; sugar content and composition and acidity. Principal component analysis and analysis of total sugars during storage showed that the two treatments with high levels of N gave berries with the highest sugar content. In addition, PCA showed that the changes in anthocyanins, fructose and glucose content during the first days of storage showed similar patterns. It is difficult to conclude whether the similar changes in these compounds in fact have a common cause or are simply parallel processes. The highest titratable acid content at harvest was found in the treatments with low K content, as also reported in other studies [35, 50]. However, the opposite effect has been found in investigations of other berries, e.g., in highbush blueberries and strawberries, titratable acidity is reported to increase with the amount of K supplied [51, 52]. The levels of total sugars, total soluble solids, titratable acids and pH found in the present study are in the same range as found in another

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Fig. 2 Content (mg g-1 DW ± SD) of different sugars (fructose, glucose, sucrose) during storage of blackberry fruit grown with different nutrient regimes (T1, T2, T3 and T4). Storage temperature 2 °C and 75% relative humidity under low and high nitrogen (60 and 100 kg ha-1) or potassium (66.4 and 104 kg ha-1) regime (T1: low N, low K; T2: low N, high K; T3: high N, high K; T4: high N, low K)

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investigation with 18 cultivars and 21 selections of blackberries [53]. Previously, high N availability has been considered to over-stimulate vegetative growth, often resulting in weaker fruit development [54], and also poor fruit set and reduced sugars in tomatoes [55]. However, in the present study, there were no differences in berry yield between the different nutrition strategies (values not shown), and the high N supply did not reduce, but rather increased, the total sugar content. Another study reported no differences in yield of blackberry fruit with 56 or 112 kg N per ha [56], which are similar levels of N as were used in this investigation. In addition, K has been suggested to be involved in translocation of sugars into berries [57], and sugar unloading has been suggested to be linked to a K? channel [58]. The high N treatments used in the present study seem to have stimulated sugar translocation to the berries, particularly when the K level was high, which might indicate an involvement of K in sugar translocation, although only at sufficient N levels. However, even higher N levels might have produced different results. Cultivation and environmental factors, such as soil type, nutrient level and application strategy, have previously been shown to influence the nutrient supply to the plant and could thereby affect plant concentrations and composition of secondary metabolites, e.g., flavonoids, ellagitannins, vitamin C and phenolic compounds [23]. High N availability has been suggested to decrease the content of vitamin C and other antioxidants in the crop, because it results in rapid plant growth and thus resources are preferentially allocated to growth processes rather than to secondary metabolites [33, 59], though varying results have been shown in different investigations [60]. However, in this study, the highest levels of vitamin C at harvest, and on most storage days investigated, were found for treatments with high K, while high N only resulted in low content of vitamin C when the K level was also low. Previous studies on high N availability have more often reported lower vitamin C content, though this seems to be dependent on species and other factors [61]. The levels of vitamin C content found in the present study ranged from 11.8 to 22.1 mg 100 g-1 fresh weight, which was similar to or somewhat higher than the 12.3–17.5 mg 100 g-1 fresh weight reported previously [17, 62]. Ellagic acid content showed consistent results for the low N, high K treatment, which had the highest levels at harvest and during all storage days. The ellagic acid content was in the same range as found for blackberries in a previous investigation [63]. For total phenolics, the differences between treatments were small, although the highest content was found in the low N, high K treatment. The total phenolics content ranged from 182.3 to 215.6 mg GAE 100 g-1 FW, which is similar to or somewhat lower than that in previous


41

Table 3 Effect of different nutrition strategies on blackberry fruit subjected to different periods of storage Storage time (days)

Treatment

Vit. C (mg g-1 DW)

Tot. phenolics (mg g-1 DW)

Anthocyanins (mg g-1 DW)

EA (mg g-1 DW)

TSS (%)

pH

Titr. acidity (%)

1

T1

1.55 c

18.49 b

17.50 d

12.16 a

7.5 c

3.26 c

1.39 a

T2

1.72 b

20.66 a

19.38 c

11.78 a

7.5 c

3.31 b

1.24 b

T3

1.96 a

19.44 ab

20.46 b

9.92 b

7.6 b

3.62 a

1.15 c

T4

1.14 d

19.09 b

21.64 a

8.68 c

9.0 a

3.18 d

1.37 a

T1

1.52 c

17.83 ab

16.43 c

9.74 c

7.5 b

3.25 b

1.33 b

T2

1.77 a

18.27 a

17.58 b

13.28 a

7.5 b

3.31 a

1.33 b

T3 T4

1.61 b 1.14 d

16.81 b 18.54 a

17.92 b 19.54 a

11.97 b 11.69 b

7.5 b 8.16 a

3.31 a 3.20 c

1.42 a 1.35 b

T1

1.61 b

19.25 a

18.77 c

10.83 b

7.4 d

3.31 ab

1.28 c

T2

1.64 a

18.02 b

21.40 a

12.00 a

7.5 c

3.32 ab

1.28 c

T3

1.67 a

17.14 c

18.22 d

10.95 b

8.6 a

3.35 a

1.38 a

3

6

9

12

T4

1.04 c

17.61 bc

19.60 b

11.14 b

7.8 b

3.28 b

1.33 b

T1

1.83 a

18.19 b

17.80 c

10.04 c

7.5 b

3.35 a

1.15 c

T2

1.62 c

20.77 a

19.63 b

13.19 a

7.4 c

3.33 a

1.20 b

T3

1.70 b

17.27 c

22.04 a

10.71 b

7.5 b

3.35 a

1.28 a

T4

1.10 d

17.97 bc

17.20 d

10.79 b

8.06 a

3.31 a

1.26 a

T1

1.52 c

17.71 b

19.30 a

9.52 c

8.10 a

3.39 bc

1.08 d

T2

1.65 b

16.75 b

18.51 b

10.81 a

7.0 d

3.63 a

1.17 c

T3

1.76 a

20.18 a

19.96 a

10.89 a

7.8 b

3.43 b

1.19 b

T4

1.11 d

17.09 b

17.64 c

10.45 b

7.4 c

3.34 c

1.22 a

Mean values for all determinants, based on n = 3. Different letters within columns indicate significant differences between nutrition strategies T1, T2, T3 and T4 within each storage time (p \ 0.05). Values for significant differences between different storage times within each treatment are not shown in the table, but presented in ‘‘Results’’ section. Treatment (T)1: low N, low K; T2: low N, high K; T3: high N, high K; T4: high N, low K

Table 4 Mean values for the content of different bioactive compounds in blackberry leaves subjected to different nutrition strategies Vitamin C (mg g-1 DW)

Tot. phenolics (mg g-1 DW)

Ellagic acid (mg g-1 DW)

T1

4.44 a

18.83 a

T2

4.38 a

15.76 a

T3

4.44 a

T4

4.52 a

Treatment

Sugars (mg g-1 DW) Fructose

Glucose

Sucrose

Tot. sugars

76.85 b

3.68 a

5.56 a

18.05 a

27.30 a

76.61 b

3.30 a

5.14 a

16.31 a

24.75 b

16.46 a

89.55 a

3.74 a

5.59 a

16.80 a

26.15 ab

15.23 a

77.37 b

3.50 a

5.17 a

16.50 a

25.18 b

Mean values for all determinants, based on n = 3. Different letters within columns indicate significant differences between nutrition strategies T1, T2, T3 and T4 (p \ 0.05). Treatment (T)1: low N, low K; T2: low N, high K; T3: high N, high K; T4: high N, low K

investigations (204–326 mg GAE 100 g-1 FW) [14, 26, 64]. To conclude, a low level of N and a high level of K seem to have stimulated synthesis of the secondary metabolites vitamin C, ellagic acid and total phenolics in blackberries. For anthocyanin content, the differences between treatments were also small, but in this case, the highest or second highest content at harvest and after 2 days of storage was found in the high N treatments. A previous study observed that adequate N levels stimulated the activity of phenylalanine ammonia lipase (PAL), which is involved in anthocyanin synthesis [65]. Furthermore,

pigment biosynthesis might depend on the level of sugars [66]. Both these observations are supported by the results of the present study. The leaves of blackberry and other Rubus species (e.g. raspberry) have traditionally been used for their anti-bacterial, anti-viral and anti-inflammatory properties [27, 67]. They have also been shown to exert anti-microbial activity specifically against Heliobacter pylori, which is associated with pathologies such as peptic ulcer and gastric cancer [68]. As antioxidants and other secondary metabolites have been suggested as candidates for health-promoting properties [69], the possibility of modulating the concentrations

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Table 5 Mean values for the content of chlorophylls and carotenoids in blackberry leaves subjected to different nutrition strategies Chlorophylls (mg g-1 DW)

Carotenoids (mg g-1 DW)

Chlorophyll a

Chlorophyll b

Lutein

b-Carotene

T1

2.62 a

2.14 a

0.39 a

0.03 c

T2

2.86 a

2.18 a

0.40 a

0.05 a

T3

2.82 a

2.31 a

0.41 a

0.04 b

T4

2.73 a

2.19 a

0.40 a

0.04 b

Treatment

Mean values for all determinants, based on n = 3. Different letters within columns indicate significant differences between nutrition strategies T1, T2, T3 and T4 (p \ 0.05). Treatment (T)1: low N, low K; T2: low N, high K; T3: high N, high K; T4: high N, low K

of these compounds by adjusting nutrient supply to blackberry crops might be interesting. However, this study found only small differences between the treatments except for ellagic acid, which had a somewhat higher content in the high N, high K treatment. As the chlorophyll and carotenoid concentrations in the blackberry leaves were not affected by the levels of N or K supplied in the different treatments, it does not seem likely that the different sugar levels found in the blackberry fruit were the result of higher photosynthetic light absorption in the treatments having higher sugar levels. As the highest sugar level was found in the treatment with both high N and high K supply, it cannot only be due to that higher K might affect sugar translocation as been suggested previously [70], but in this case rather a synergetic and/or additative effect of both nutrients.

Conclusions Nitrogen and K levels significantly influenced the content of different quality attributes in blackberry fruit and leaves. Major changes were found in compounds affecting taste (48% of the total according to PCA), such as sugars (fructose and glucose), total soluble solids and pH, but also anthocyanins. The content of various secondary metabolites showed significant differences between the different nutrient regimes, though the differences were smaller. The treatment with high N and high K produced high levels of bioactive compounds and sugars in the fruit, but did not significantly affect ellagic acid or titritable acidity. The nutrient regimes did not affect blackberry leaves to the same extent. Cold-stored blackberries are a good source of nutrients and bioactive compounds. It can be concluded that by optimising plant nutrition, phytonutrient concentrations can be maximised and maintained in fresh and stored berry crops, especially those grown in the greenhouse, where the conditions can easily be regulated.

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Acknowledgments The authors are grateful to the Swedish Farmers’ Foundation for Agricultural Research (Stockholm) for financial support in this project. Our thanks to research engineer Karl-Erik Gustavsson for skilful technical assistance during HPLC analysis and to Assistant Professor Jan-Eric Englund for statistical advice. The Higher Education Commission (HEC) of Pakistan is gratefully acknowledged for a PhD scholarship.

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