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Oral Presentation

Conference Paper · November 2018

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Oral Presentation

Effects of Irrigation Practices and Lateral Distance on Color and Carotenoids Composition of Carrot roots (Daucus carota L.)

İlknur Kutlar Yaylalıa*, Derya Arslanb, Duran Yavuza, Nurcan Yavuza

a Department of Agricultural Structures and Irrigation, Faculty of Agriculture, Selçuk University,

42031, Konya, Turkiye

b Division of Food Sciences, Department of Food Engineering, Faculty of Engineering and Architecture,

Necmettin Erbakan University, Konya, Turkiye *Corresponding Author E-mail: nur@selcuk.edu.tr

Abstract Field experiments were carried out during winter season of 2014 in the conditions of clay loamy soil to study the effect of lateral distance, irrigation interval and irrigation levels on the concentrations of carotenoid compounds (β-carotene, α-carotene, lutein), total lipids and also CIE Lab color parameters of carrot roots. The plants were subjected to two deficit irrigation (DI) (75% and 50%), two lateral distance (40 and 80 cm) and two irrigation intervals (10 and 15 days). Significant effects of lateral distance and deficit irrigation practices were observed

• n the carrot roots, implying that lower lateral distance (40 cm) and 75% deficit irrigation

provides carrots with higher concentrations of carotenoid compounds and total lipids than the

• ther practices. Irrigation interval didnot influence all the characteristics analysed.

The agricultural treatments applied in the field study didnot show significant influences on total carotenoids content, but apparent differences were detected for individual carotenoid

• compounds. This indicated that spectrophotometric analyses of the total carotenoids were not

sensitive enough to represent the effects of field treatments applied in the assay. Keywords: Daucus carota L., irrigation, lateral distance, carotenoids, color, total lipids. Introduction The optimum production of field crops can be reached through application of adequate amount of irrigation. Improving growth and yield have been reported in carrot with increasing frequency of irrigation (Nortje and Henrico, 1986) and drought leads to significant reductions in the quality of this crop (Lada et al., 2004). The management of irrigation water is important, since it governs evapotranspiration, water use efficiency, moisture extraction pattern, and nutrient uptake. Carrot plants are cultivated widely in semi-arid climates, where the scarcity of water resources is the major factor limiting irrigated agriculture. To optimise water resources deficit irrigation (DI) strategies are employed, in which moderate water stress is applied during part

• f the seasonal cycle of plant development (Kriedemann and Goodwin, 2003).

374 Vegetable quality greatly varies under the influence of different soil and climatic conditions. The breeder must determine which quality attributes are important to consumers and develop methods to rapidly and accurately assess these attributes. The consumer quality can include such diverse attributes as vitamin content, absence of antimetabolic compounds, flavour, texture, colour, appearance and convenience. Carrot (Daucus carota L.) is one of the most important horticultural root crop that has gained popularity in recent decades due to increased awareness of its nutritional value (Arscott and Tanumihardjo, 2010) and increased consumption of this crop has been recommended to help prevent the onset of free radical-mediated diseases. Carrot roots are rich in carotenoids (α-, β-, γ -, and ζ -carotenes, β-zeacarotene, and lycopene) predominantly β-carotene (45–80%) accompanied by α-carotene that together constitute up to 95% of total carotenoids (Simon and Wolff, 1987; Gross, 1991). Carotenoid molecules consist of a long central chain with a conjugated double-bond system, which is a light absorbing chromophore which are responsible for the natural yellow to red fat-soluble pigments in many fruits and vegetables (Simon and Wolf, 1987). Carotenoids determine root colour and they also affect the perception of carrot taste and flavour that influence the consumer preference (Alasalvar et al., 2001; Habegger and Schnitzler, 2005). They are directly related to the perception of their quality, as colour does influence the consumers’ preferences (Mele´ndez-Martı´nez et al., 2004; Mele´ndez-Martı´nez et al., 2005). In addition these piments show vitamin A activity (Mele´ndez-Martı´nez et al., 2005), potential antioxidant capacity and involvement in the prevention or protection against serious human health disorders, such as heart disease, cancer and macular degeneration, among others (Fraser and Bramley, 2004; Giovanucci, 1999; Krinsky, 2001). In order to apply the optimum DI strategy, a good knowledge of the effects of DI on fruit quality is necessary. Our objective was to determine the influence of DI and lateral distance in a semi-arid environment, individual carotenoid components and colour parameters were evaluated in field-grown carrots. Material and Method Plant Material, Growth Conditions, and Experiment Design The study was carried out during 2014 in Konya city (center Turkey), around Kaşınhanı province where intensive carrot farming exists, under controlled field conditions. First of all, the soil and irrigation water samples were taken and were subjected to some physical and chemical analysis. Maestro (vilmorin) cultivar of carrot was was planted in a 1.5 da experimental field which is one of the most common cultivar grown in the province and. The treatments comprised of two lateral distances (40 and 80 cm), two irrigation intervals (5 and 10 days), and three irrigation levels (%100, %75 and %50 of plant water requirement). These 12 treatment combinations were replicated four times in a factorial randomized block design consisting of 42 parcels. The carrots were sown by a hand-operated seeding machine

• n a profiled surface. Each experimental unit consisted of five rows of 2.5 m long and 40 cm

(or 80 cm) apart. Each plot size covered an area of 10.0 × 2.4 m (24 m2). Carrot seeds (cv. Vilmorin) at a rate of 3 kg/fed were sown on both sides of each row. After homogenous growing of plants was achieved, drip irrigation system was launched on the plots. A surface

375 drip irrigation system was used with a flow rate of 4 l h−1 and a spacing of 0.50 m, the wettable area was 0.50 m2 by linear meter of drip line. All plots received a constant level of N, P and K at ratios of 60 kg/da. Super phosphateand K were added at 15 kg P2O5 /fed doses, after 4 and 8 week from the plantation, respectively. The carrot plants were sprayed twice against pests with a biological preparation NeemAzal-T/S (05%) and against diseases the plants were sprayed once with Champion (0.2%). Konya is located in the middle of Anatolia facing the interior drought regions and is subject to a continental climate characterized by cold weather throughout the winter and also to a subtropical climate distinguished by dry summers. The average annual temperature varies between 11.4oC, relative humidity 58% and annual rainfall is approximately 323 mm to. According to the long-term average values, the rainfall in plant growing season corresponds to 30% of total rainfall. Colour measurement A colorimeter [Minolta Chroma meter CR 400 (Minolta Co., Osaka, Japan)] was used to assess carrot colour and the CIELAB colorimetric system was applied. The colour meter was calibrated against a standard calibration plate of a white surface and set to CIE Standard Illuminant C. Each time 20 ml of samples put into a petri dish, and the liquid probe of the instrument was immersed into the dish sitting on the white tile, and readings of the CIE lab coordinates are recorded. The L*, a*, b* values are average of ten readings (Criado et al., 2004). Determination of carotenoids

• Reagents. n-Hexane, and tert-butyl hydroxytoluene (BHT) (special grade), potassium

hydroxide were purchased from Sigma (Steinheim, Germany ); and chloroform, methyl tert- butyl ether (MTBE) (HPLC grade), ethanol, diethyl ether, magnesium hydroxide carbonate, methanol (HPLC grade), sodium chloride and potassium hydroxide was obtained from Merck (Darmstadt, Germany). The standards lutein, β-carotene, α-carotene, all-trans-retinol palmitate at 99% purity was obtained from Sigma-Aldrich (St. Louis,MO, USA) Samples and solvents were filtered, using 0,45-μmMillipore™membranes (Billerica,MA,USA) before being injected to HPLC. Instrumentation The LC system consisted of a series 1050 chromatograph with a quaternary pump system, a diode array detector (Hewlett-Packard, 1100 series), a column thermostat (Agilent 1100 series), an on-line degassing system, and a ChemStation data system (Hewlett-Packard, Waldbronn, Germany). Carotenoids were separated with a Vydac 201TP54 reverse phase C18 column (5 μm, 250×4.6 mm i.d.). Separation was carried out with an injection volume of 20 μL, a flow rate of 0.9 mL.min−1 and the column temperature set at 24 °C. The UV–visible spectrum was obtained for a wavelength ranging from 200 to 600 nm and the chromatograms analyzed at 450 nm. The HPLC system's software tentatively identified each carotenoid according to its order of elution in the column, chromatographic behaviour (e.g., retention time and spectral characteristics), UV– visible absorption spectrum (absorption maximum wavelength, λmax) and the fine-structure spectrum (%III/II) obtained for each injection. The carotenoids were

376 quantified by HPLC, using the external calibration curve of β-carotene, α-carotene and lutein at a minimum of six levels of concentration. Each concentrations were injected into the HPLC and the linear regression equations were acquired by plotting the quantity of standard injected against the peak area. Good correlations were obtained (r2=0.994), reporting the concentration

• f each carotenoid analysed. The percent recoveries for the standard compounds ranged from

90% to 94% (Cortés et al., 2004). Table 1. Mobile phase gradient programme for determination of carotenoids by HPLC

time (min) MeOH+AAa (%) H2O (%) TBME+ethyl acetate (50:50) (%)

95 5 3 100 5 95 5 10 86 14 15 75 25 22 95 5 23 100

aAA, 0.1 M ammonium acetate

Sample extraction The extraction procedure was carried out with slight modifications (Huck et al., 2000). Triplicate 25-g aliquots of the ground sample were placed in a conical flask together with 50 mg solid magnesium hydroxide carbonate to neutralize any organic acids together with 25 mg

• BHT. Thirtyfive milliliters ethanol and hexane (4:3, v/v) were added with the internal

standard (100 µL). For the extraction of the carotenoids, the samples were homogenized for 2 min using an Ultra-Turrax T25 homogenizer (IKA, Staufen, Germany) at darkness. The resulting suspension was filtered through a Glas Fibre filter pad (GF/A Whatman, Maidstone, U.K.) in a Buchner funnel under vacuum. The residue was shaken again with 35 mL ethanol and hexane (4:3, v/v), 2x12.5 mL ethanol and 12.5 mL n-hexane (until it was colorless) and the residue was discarded. The homogenizer was washed with 20 mL ethanol and hexane (4:3, v/v). The filter pad was washed with two further aliquots of hexane–EtOH. The combined hexane–EtOH filtrates were transferred into a separating funnel. 2x50 mL 10% sodium chloride solution and 3x50 mL H2O were added and mixed. The aqueous phase was drowned off. The residue was shaken with 10 mL diethyl ether+10 mL methanolic KOH+0.1% (w/v) BHT at room temperature. The liquid extracts were combined. 20 mL of diethyl ether was added to the extract and extracted twice with 50 mL of 10% NaCl (w/v). The ether phase was washed three times with 50 mL of H2O until a neutral pH was obtained. It was filtered in the presence of anhydrous Na2SO4. To ensure that the water was totally eliminated, we added 10 mL of absolute ethanol, evaporating at 45°C until dryness. The residue was dissolved with 4 mL of diethyl ether and placed in an amber glass flask, the solvent was evaporated under N2. These extracts were then placed in amber-coloured bottles containing nitrogen atmosphere and stored at −20 °C until time for chromatographic

• determination. The carotenoid extract was reconstituted with 1 mL of MeOH/TBME (70:30,

v/v) at the moment of injection. Before injection, the extracts were filtered through a 0.45-μm membrane.

377 Determination of total carotenoids The preweighed samples of ground carrot were put in ethyl acetate (20mL per each mg). The extract obtained was centrifuged at 5000 rpm for about 10 min. The supernatant was separated and absorbance was read at 400–700 nm on UV spectrophotometer. Maximum absorbance of total carotenes is at 470 nm. The amounts of total carotenoid pigments present in the samples were calculated according to the formula of Lichtentaler and Wellburn (1985). Formula used in the calculation is: Ca = 10.05 A662 – 0.766 A644; Cb = 16.37 A644 – 3.140 A662; Cx+c = 1000 A470 – 1.280 Ca – 56.7 Cb/230. Statistical analysis The results are reported as mean values of the three replicates and standard deviations. Analysis of variance was used to evaluate irrigation interval, lateral distance and deficit irrigation depended differences regarding the parameters analyzed, with or no transformed data using SPSS 10.0 for Windows. Significant difference was defined as P<0.05. In case of significance, differences between mean values were evaluated using the Duncan's multiple range test. Research Findings and Discussion Tables 2 and 3 represents the values of total and individual carotenoids, total lipids and color parameters in carrot roots after different field treatments. β-carotene was the main carotenoid

• f carrot roots having concentrations between 144.13-231.29 mg kg-1 f.w., α-carotene and

lutein followed β-carotene with concentrations slightly over 40 and over 3 mg kg-1 f.w. (mean

• f all treatments), respectively.

The compounds belonging to carotenoid group (β-carotene, α-carotene, lutein) as well as total lipids reached higher amounts with the decrease in lateral distance. Regarding the effects of the irrigation interval on carrots, no statistically significant differences were found for all the tested parameters including total carotenoids, individual carotenoid compounds, total lipids and color indices. Unlikely, a previous report suggests that frequent irrigations discourage good root colour formation (Nuńez et al., 1997) and another study reports that long intervals between irrigations can cause the development of thinner roots (Nortjè and Henrico, 1986). Table 3 summarizes color data for changes due to agricultural practice. The statistical study showed that significant differences were not found for all the factors under study (P<0.001). When the deficit irrigation applied the carrot roots contained higher amounts of carotenoids, where 5 days irrigation intervals exhibited this effect more notably than 10 days intervals. Nevertheless, the results regarding the influence of deficit irrigation, irrigation intervals and lateral distance on total carotenoid values were not statistically significant with P<0.001 probability level. Deficit irrigation resulted in lower levels of total lipids which was more evident for high deficit ratio (50% water requirement met). Just like the effect of lateral distance, deficit irrigation influenced the carotenoid compounds. Deficit irrigated samples had higher values of carotenoid compounds, than those of totally irrigated samples, where 75% deficit led to the highest amounts with mean values of 231.3, 46.6 and 4.0, for β-carotene, α-

378 carotene, lutein; respectively. 100% irrigation level resulted in the lowest carotenoid compounds. The agricultural treatments applied in the field study didnot show significant influences on total carotenoids content. The higher total carotenoid levels in roots from lower lateral distance was not high enough to be considered statistically significant. The same trend was

• bserved for the deficit irrigation effect. This may be a result of analysis methods. On the
• ther hand, it was questionable that the values obtained by spectrophotometric analyses of the

total carotenoids were not statistically significant however the HPLC determination of individual carotenoid compounds resulted in significant differences against field experiments. This might be attributed to the less sensitivity of spectrophotometric method, as this method has some disadvantages about quantification such as lack of selectivity, low sensitivity and/or higher limit of detection (Shishehbore and Aghamiri, 2014) when compared to chromatographic techniques. Table 2. Total and individual carotenoids and lipid content in carrots after different field treatments.

irrigation interval lateral distance deficit irrigation total lipids (%) total carotenoids (mg/kg) β-carotene α-carotene lutein

5 days 40 cm 100% 2.24±0.21 243.06±28.74 145.34±5.26 35.3±1.55 2.80±0.32 75% 1.84±0.26 266.01±16.87 261.17±18.31 51.6±2.65 4.70±0.20 50% 1.13±0.09 254.31±19.11 248.48±27.51 51.5±2.02 4.13±0.15 80 cm 100% 1.23±0.08 247.11±22.47 139.89±24.80 35.0±2.21 2.71±0.20 75% 1.09±0.02 271.86±20.22 183.16±18.75 42.6±2.30 3.62±0.23 50% 1.46±0.21 245.76±14.23 159.78±13.41 39.7±1.20 3.12±0.13 10 days 40 cm 100% 1.81±0.12 239.90±17.98 174.56±22.28 43.1±2.11 3.50±0.24 75% 1.58±0.16 248.01±19.49 258.61±31.96 54.4±1.45 4.22±0.21 50% 1.37±0.25 268.71±31.41 184.20±5.52 42.8±1.34 3.49±0.30 80 cm 100% 1.23±0.16 251.16±3.57 116.73±5.67 34.4±1.35 2.48±0.27 75% 1.16±0.03 253.92±5.82 222.22±8.33 37.9±1.07 3.29±0.19 50% 1.15±0.22 244.41±11.05 166.19±20.98 38.2±1.20 3.19±0.28 *mean value±standard deviation

379 Table 3. Colour indices of carrots after different field treatments.

irrigation interval lateral distance deficit irrigation L* a* b*

5 days 40 cm 100% 53.33±3.70 25.80±2.73 42.14±3.85 75% 52.65±3.37 23.82±2.76 40.82±2.31 50% 52.87±2.53 25.38±0.95 39.42±0.44 80 cm 100% 54.50±0.86 24.74±1.29 40.68±2.20 75% 54.74±0.54 25.05±3.24 40.85±4.73 50% 54.25±3.11 24.53±1.91 41.16±3.05 10 days 40 cm 100% 51.30±1.95 23.95±0.57 37.53±0.29 75% 53.82±0.27 26.00±0.56 41.40±2.46 50% 52.57±1.44 26.97±1.62 42.73±2.43 80 cm 100% 52.79±3.07 24.07±1.61 38.85±3.14 75% 51.30±1.64 25.38±1.30 40.32±4.38 50% 50.76±3.06 24.77±2.74 38.60±4.71 Total carotenes for different carrot cultivars were reported between 133-196 mg/kg (Karkleliene et al., 2012). Mech-Nowak (2012) reported total carotenes for seventeen carrot cultivars varying between 10-280 mg/kg (spectrophotometric method) and β-carotene between 10-171 mg/kg (HPLC method). Arscott and Tanumihardjo (2010) reviewed the α- carotene, β-carotene and lutein concentrations of orange coloured carrots ranging between 10- 70, 18-128 and 0-2.6 ppm, respectively. The total carotenoids and β-carotene contents of carrots in this study were slightly higher than those reported by Karkleliene et al. (2012) and Mech-Nowak (2012), while the remaining were similar to the values obtained in the present study. Growing conditions such as temperature, soil moisture, rainfall, light intensity and day length have a significant effect on the quality of carrot roots (Bloksma et al., 2003). Carrots are more tolerant to drought than other vegetable crops due to their extensive root system (Lada et al., 2004). However, the availability of soil moisture throughout the growing season is one of the most vital production requirements for carrots (Suojala, 2000). Excessive soil moisture results in pale coloured roots (Joubert et al., 1994; Rubatzky et al., 1999). Carrots require a steady supply of moisture and available soil moisture needs to be maintained above 50% of plant available water throughout the growth season (Manosa, 2011). Correlations between tested parameters showed that the carotenoid compounds were strongly correlated to L* (lightness) with very high correlation coefficient percentages of 0.80, 1.00 and 0.90, respectively (Table 5). Unlike the individual carotenoid compounds, total carotenoids did not show significant correlation with colour indices. Total carotenoid values were not found suitable to make correlations to CIELAB color coordinates. Total carotenoids exhibited only a slight correlation with lutein (R2=0.33, P<0.05). The color indices also correlated with each other, where the correlation was higher between a* and b* 0.77, while this percentage was 0.43 between L* and b*.

380 Table 4. Duncan multiple comparison test results from statistical analysis of carotenoids and lipids in carrot samples after different field treatments.

Treatments n total carotenoids β-carotene α-carotene lutein total lipids (%) L* a* b*

Irrigation interval Lateral distance Deficit irrigation

5 days 10 days

18 254.68 189.64 42.59 3.51 1.50 53.72 24.89 40.85 18 251.02 187.08 41.77 3.36 1.38 52.09 25.19 39.91

40 cm 80 cm

18 253.33 212.06 a 46.37 a 3.81 a 1.66 a 52.76 25.32 40.66 18 252.37 164.66 b 37.99 b 3.07 b 1.22 b 53.06 24.76 40.10 100 % 75 % 50 % 12 243.31 144.13 c 37.03 c 2.87 c 1.63 a 52.98 24.64 39.80 12 259.95 231.29 a 46.58 a 3.96 a 1.42 ab 53.13 25.06 40.85 12 253.29 189.66 b 42.94 b 3.48 b 1.28 b 52.61 25.41 40.48

a, b, c: Mean values with a different superscript differ significantly (P≤0.05)

Table 5. Correlations between carotenoid compounds and L* values of carrot samples after different field treatments.

Treatments β-carotene α-carotene lutein L* a*

total carotenoids β-carotene α-carotene

• 0.33*
• 0.80**

0.80**

• 0.84**

0.90** 0.80** 1.00**

• Lutein

b*

• 0.90**

0.43**

• 0.77**

*: Correlation is significant at the 0.05 level. **: Correlation is significant at the 0.01 level. The maximum correlations was found between the α-carotene and L* (R2= 1.00), and α- carotene and lutein (R2= 0.90). In terms of correlations between carotenoid compounds and colour parameters, β-carotene followed α-carotene (R2=0.80). Carotenoid compounds was also positively correlated to each other, such as β-carotene showed correlation between lutein (R2= 0.84), and α-carotene (R2= 0.80). Several studies have correlated the color indices with the pigment content of different crops (Ameny & Wilson 1997; Arias et al. 2000; Ruiz et al. 2005; Cardarelli et al. 2008). Correlation between carotenoid content and the color measurements of apricots were also reported and an estimation of the carotenoid content in apricots by using a portable colorimeter were suggested by the same authors (Ruiz et al. 2005). Results and Suggestions It can be concluded that lower lateral distance led to increase in carotenoid compounds and total lipids contents. Deficit irrigated samples had higher values of carotenoid compounds (β- carotene, α-carotene, lutein), than those of totally irrigated samples, as the treatments where 75% of water requirement met led to the highest amounts. The colour L*, b* and a* values were not significantly affected by the irrigation and lateral distance treatments.

381 Unlike the individual carotenoid compounds, total carotenoids did not show significant correlation with colour indices. Total carotenoid values were not found suitable to make correlations to CIELAB color coordinates. HPLC determination of individual carotenoid compounds resulted in significant differences against field experiments, however the spectrophotometric determination of total carotenoids did not reflect the differences between treatments well enough to comment, probably due to the less sensitivity of this method. Acknowledgment: A part of this study was financially supported by the office of Selçuk University

Scientific Research Projects (Project No:14401017).

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