Food Chemistry 180 (2015) 219–226

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Production of ready-to-eat lentil sprouts with improved antioxidant capacity: Optimization of elicitation conditions with hydrogen peroxide Michał S´wieca ⇑ Department of Biochemistry and Food Chemistry, University of Life Sciences, Skromna Str. 8, 20-704 Lublin, Poland

a r t i c l e

i n f o

Article history: Received 14 August 2014 Received in revised form 30 January 2015 Accepted 6 February 2015 Available online 17 February 2015 Chemical compounds studied in this article: ABTS (PubChem CID: 9570474) Hydrogen peroxide (PubChem CID: 784) Malondialdehyde (PubChem CID: 10964) trans-Cinnamic acid (PubChem CID: 444539) p-Coumaric acid (PubChem CID: 637542)

a b s t r a c t This study evaluates the optimal conditions for elicitation with H2O2 for improving the antioxidant capacity of lentil sprouts. Generally, except for 3-day-old sprouts, elicitation increased phenolic content (in respect to the control). The highest phenolic content was determined for 2-day-old sprouts treated with 15 mM H2O2 (0.71 mg/g f.m.). All the studied modifications increased the antioxidant potential of sprouts. The highest elevation (3.2-fold) was found for 5-day-old sprouts (single 15 mM H2O2 treatment). A significant increase was also found on the 2nd and 4th days (2.13- and 2.14-fold, respectively). Elicitation induced tyrosine and phenylalanine ammonia-lyases activities. H2O2 treatments induced the activity of catalase – especially for 2-day-old sprouts treated with 150 mM H2O2 (597 U/g f.m.). Elicitation with H2O2 is a useful tool for designing some features of sprouts. Phenolic content and antioxidant capacity are strongly affected by concentration of the elicitor, and time and intervals of its application. Ó 2015 Elsevier Ltd. All rights reserved.

Keywords: Antioxidant capacity Elicitation Hydrogen peroxide Phenylpropanoids Sprouting

1. Introduction A rapid increase in demand for preparations improving functioning and the quality of life has been observed in recent years in highly-developed countries. This demand has focused mainly on functional foods of natural origin and nutraceuticals. A properly composed diet may have significant impact in the prevention of numerous diseases, the improvement of the quality of life and the attenuation of symptoms accompanying aging process (Zhao, 2007). Interest in the possibility of food modification at each stage of production (plant and animal breeding, technological processes, and conditions of product storage) has increased over recent years. The pro-health properties of food of plant origin are strongly determined by secondary metabolite content, including polyphenols (Zhao, 2007). Polyphenols belong to a group of compounds with well-documented antioxidant, antitumor, and anti-inflammatory properties. Sprouting seems to be an effective process for improv⇑ Tel.: +48 81 4623327; fax: +48 81 4623324. E-mail address: [email protected] http://dx.doi.org/10.1016/j.foodchem.2015.02.031 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.

ing the nutritional and nutraceutical quality of legume food (López-Amorós, Hernández, & Estrella, 2006; Silva et al., 2013). Unfortunately, during the germination of a seed, a decrease in phenolic antioxidant content is observed, which results in a subsequent decrease in the antioxidant potential of food (Cevallos-Casals & Cisneros-Zevallos, 2010; S´wieca & Baraniak, 2013; S´wieca, Gawlik-Dziki, Kowalczyk, & Złotek, 2012). Elicitation leads to oxidative stress through an increase in reactive oxygen (ROS) and nitrogen species (NOS) levels. On the one hand, ROS and NOS damage attacks the most sensitive biological macromolecules. These species also act as signaling compounds. An elicitor is a factor stimulating any type of plant defense and causing the induction of phenolics biosynthesis. Elicitors might be of either biotic or abiotic origin. In plants, polyphenols act as defense (against herbivores, microbes, viruses or competing plants) and signal compounds (to attract pollinating or seed dispersing animals), as well as protect plants from oxidation. Usually, oxidative damage and increased resistance under environmental stresses can be correlated with the efficacy of the antioxidative defense system and increased stress tolerance (Zhao, Lawrence, & Verpoorte, 2005).

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M. S´wieca / Food Chemistry 180 (2015) 219–226

Hydrogen peroxide (H2O2) is a strong oxidizing agent that is commonly used in medicine, agriculture, and the food industry where it is used as a bleaching agent in wheat flour, edible oil, egg white, etc. It may also be used as an antimicrobial agent in food, e.g. milk, and as a sterilizing agent for food packaging materials. In plants, ROS, including hydrogen peroxide, also contribute in the stress signaling cascade, and thus may be used for the induction (elicitation) of plant resistance (Vasconsuelo & Boland, 2007; Zhao et al., 2005). Elicitation is an effective technique used in bioreactor systems for the overproduction of metabolites with potential biological activity e.g. phenolics (Matkowski, 2008). Phenolics are primarily produced through the pentose phosphate (PPP), shikimate and phenylpropanoid pathways. The oxidative PPP provides precursor erythrose-4-phosphate for the shikimate pathway. The shikimate pathway converts these sugar phosphates into aromatic amino acids such as phenylalanine and tyrosine, which become the precursors for the phenylpropanoid pathway that synthesizes phenolics (Shetty, 2004). In plants, these amino acids are transformed into trans-cinnamic acid and p-coumaric acid via phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase, respectively. The synthesis of phenolic compounds is accompanied by the stimulation of these enzymes. Plants modify their metabolism to adjust to variable environmental conditions. This enables the modification of their composition and consequent changes of the activity of plant-origin food (Feng, Song, Lee, & Huang, 2010; Gawlik-Dziki, S´wieca, Dziki, & Sugier, 2013; Gawlik-Dziki, S´wieca, & Sugier, 2012; McCue, Zheng, Pinkham, & Shetty, 2000; Pérez-Balibrea, Moreno, & García-Viguera, 2011; Randhir, Lin, & Shetty, 2004; Złotek, S´wieca, & Jakubczyk, 2014; S´wieca & Baraniak, 2013; S´wieca, Baraniak, & Gawlik-Dziki, 2013; S´wieca et al., 2012). Despite the analysis of final effects (changes in bioactive component levels and bioactivity itself), the cited papers lack data concerning the mechanisms for acquiring new features. Currently, there is no study reporting the selection of the optimal concentration, time of exposure and intervals of elicitor treatments considering the effectiveness of these biotech treatments in the creation of some features of low-processed food. The purpose of this study was to evaluate the optimal conditions of elicitation using hydrogen peroxide for improving the antioxidant capacity of ready-to-eat lentil sprouts. We focused on the activities of enzymes involved in plant defense and phenolic synthesis and metabolism. 2. Materials and methods 2.1. Plant material and growth conditions Lentil seeds var. Tina were purchased from PNOS S.A. in Ozarów Mazowiecki, Poland. Seeds were sterilized in 1% (v/v) sodium hypochloride for 10 min, then drained and washed with distilled water until they reached neutral pH (6.8). After that, they were placed in distilled water and soaked for 6 h at 25 °C. Seeds were dark germinated (25 °C, 85% relative humidity) for 5 days in a growth chamber on Petri dishes (/ 125 mm) lined with absorbent paper (approximately 150 seeds per dish). Seedlings were watered with 5 ml of Milli-Q water daily. For the experiments, 15 mM and 150 mM H2O2 were selected as abiotic elicitors. All solutions were freshly prepared before each application. For Ox1 treatment, 1-day-old seedlings were watered only once with 5 ml of 15 or 150 mM H2O2 (single treatment; Ox1– 15 and Ox1–150, respectively) and then cultivated under standard conditions (watered with distilled water). For Ox2 treatments, 1-day-old seedlings (since the first day of cultivation) were watered daily (to the end of sprouting) with 5 ml of 15 mM and 150 mM

H2O2 (continuous treatment; Ox2–15 and Ox2–150, respectively). Sprout samples were gently collected, weighed and rapidly frozen and kept in polyethylene bags at 20 °C. Three independent experiments were carried out. 2.2. Growth analysis In order to determine the influence of elicitation on sprout growth the morphological characteristic (length of roots and stalk) and biomass accumulation (10 sprouts mass) were determined. 2.3. Phenolics content Lentil flours (0.2 g) were extracted three times with 4 ml of acetone/water/ hydrochloric acid (70:29:1, v/v/v). After centrifugation (10 min., 6800g) fractions were collected, combined and used for further analysis. The amount of total phenolics was determined using Folin–Ciocalteau reagent (Singleton, Orthofer, & Lamuela-Raventos, 1974). To 0.5 ml of the sample, 0.5 ml H2O, 2 ml Folin–Ciocalteau reagent (1:5 H2O) were added, and after 3 min, 10 ml of 10% Na2CO3 and the contents were mixed and allowed to stand for 30 min. Absorbation at 725 nm was measured in a UV–vis spectrophotometer. The amount of total phenolics was calculated as a gallic acid equivalent (GAE) in mg per g of fresh mass (f.m.). 2.4. Antioxidant activities Antiradical activity was carried out using an improved ABTS decolorization assay (Re et al., 1999). Free radical scavenging ability was expresses as Trolox equivalent in mg per g of fresh mass (f.m.). Reducing power was determined by the method of Oyaizu (1986). Reducing power was expressed as Trolox equivalent in mg per g of fresh mass (f.m.). Chelating power was determined by the method of Decker and Welch (1990). Chelating power was expressed as EDTA equivalent (EDTA) in mg per g of fresh mass (f.m.). The inhibition of the hemoglobin-catalyzed peroxidation of linoleic acid was determined according to Goupy, Vulcain, CarisVeyrat, and Dangles (2007). The activity was expressed as quercetin equivalent (Q) in mg per g of fresh mass (f.m.). Four complementary antioxidant methods were intergraded to obtain the total antioxidant activity index (IA) (1). The index may be useful for evaluation total antioxidant potential of sprouts from different germination conditions in respect to control. The IA was calculated as the sum of relative activities (RA) (2) for each antioxidant chemical methods divided by a number of methods (S´wieca & Baraniak, 2013).

IA ¼

RRAðnÞ 4

ð1Þ

RA was calculated as follows:

RA ¼

Ax ; Ac

ð2Þ

where: Ax-activity of modified sprouts for the method, Ac-activity of control sprouts determined for the method 2.5. Oxidative damage The degree of lipid peroxidation was measured in terms of malondialdehyde (MDA) content, as described by Dhindsa, Plumb-dhindsa, and Thorpe (1981). Samples (0.2 g) were homogenized in 2 ml of 5% trichloroacetic acid (TCA) solution and centrifuged at 13,500g for 15 min at room temperature. The

M. S´wieca / Food Chemistry 180 (2015) 219–226

supernatant of the tissue extract was mixed with an equal volume of 20% (v/v) TCA, containing 0.5% (v/v) thiobarbituric acid (TBA). The mixture was heated at 96 °C for 30 min, cooled in ice and centrifuged at 9500g for 10 min. The content of MDA was expressed as nmol MDA per g of fresh mass (f.m.).

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stop the reaction, samples were centrifuged (15,000g, 10 min) and absorbance at 290 nm was measured in an ultraviolet spectrophotometer. One unit was defined as the amount of enzyme that produced 1.0 lg trans-cinnamic acid per min under the conditions of the assay. The results were presented as U per g of fresh mass (Assis et al., 2001).

2.6. Enzymatic activities 2.7. Statistic analysis 2.6.1. Extract preparation All enzyme extract procedures were conducted at 4 °C. For phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL), 200 mg of lyophilized sample were ground with 2 ml extracting buffer (0.2 M boric acid buffer containing, 1 mM EDTA, and 50 mM b-mercaptoethanol, pH 8.8). The extracts were then homogenized and centrifuged at 12,000g at 4 °C for 30 min, and the supernatant was collected. For polyphenol oxidase (PPO), catalase (CAT) and guaiacol peroxidase (POD), 200 mg of the sample were ground with 4 ml of 100 mM sodium phosphate buffer (pH 6.4) containing 0.2 g of polyvinylpolypyrrolidone. The extracts were then homogenized and centrifuged at 12,000g at 4 °C for 30 min, and the supernatant was collected. 2.6.2. Enzyme assay 2.6.2.1. Catalase (CAT) assay. CAT [EC 1.11.1.6] activity was measured following Aebi (1984). The decomposition of H2O2 was followed by an absorbance decrease at 240 nm for 1 min. One unit was defined as the amount of enzyme that decomposed 1.0 lmol H2O2 per min under the conditions of the assay. The results were presented as U per g of fresh mass (f.m.). 2.6.2.2. Peroxidase (POD) assay. POD activity was determined using guaiacol as the substrate (Ippolito, El Ghaouth, Wilson, & Wisniewski, 2000). The reaction mixture consisted of 0.1 ml of crude extract and 2 ml of guaiacol (8 mM, in 100 mM sodium phosphate buffer, pH 6.4) incubated for 1 min at 30 °C. The increase in absorbance at 460 nm was measured after 1 ml H2O2 (24 mM) was added. The activity of POD was expressed as U, where U = 0.001 OD460/min under the conditions of the assay. The results were presented as U per g of fresh mass (f.m.). 2.6.2.3. Polyphenol oxidase (PPO) assay. For the PPO assay, 100 ll of extract were incubated with 2 ml 0.05 M phosphate buffer (pH 7.0) and 0.5 ml 0.5 M catechol at 24 °C for 5 min, and absorbance at 398 nm was measured with an ultraviolet spectrophotometer. The PPO activity was expressed as U, where U = 0.001 OD398/min under the conditions of the assay (Galeazzi, Sgarbieri, & Costantinides, 1981). The results were presented as U per g of fresh mass (f.m.). 2.6.2.4. Tyrosine ammonia-lyase (TAL) assay. For the TAL assay, 100 ll of the extract were incubated with 0.9 ml 0.02 M L-tyrosine at 30 °C for 60 min. After incubations, 0.5 ml 10% trichloroacetic acid (TCA) was added to stop the reaction, samples were centrifuged (15,000g, 10 min) and absorbance at 310 nm was measured in an ultraviolet spectrophotometer. One unit was defined as the amount of enzyme that produced 1.0 lg p-coumaric acid per min under the conditions of the assay. The results were presented as U per g of fresh mass (Assis, Maldonado, Munoz, Escribano, & Merodio, 2001). 2.6.2.5. Phenylalanine ammonia-lyase (PAL) assay. For the PAL assay, 300 ll of the extract were incubated with 1.2 ml 0.02 M L-phenylalanine and 2 ml of the PAL extracting buffer at 30 °C for 60 min. After incubations, 0.5 ml 10% TCA was added to

All experimental results were mean ± S.D. of three independent experiments (n = 9). The one-way analysis of variance (ANOVA) and Tukey’s post hoc test were used to compare groups within different elicitors. a values < 0.05 were regarded as a significant. 3. Results One of the most important factors in the production of food is the safety and effectiveness of the technologies used. The usefulness of elicitation for designing some desirable features of sprouts is documented; however, there has been no study of the kinetic changes of chemical composition and nutraceutical potential due to the frequency and time of elicitor application and its concentration. Additionally, previous studies have not included the influence of the biotechnologies used on sprout yield and/or the biochemical mechanism of the acquisition of these features. The induction of sprout metabolism by elicitation, besides its positive effects, may cause a simultaneous reduction in both growth rate and seedling vigor. Thus, the determination of biomass production and stress marker levels during studies involving the use of elicitation is very important. Among the studied growth conditions only one step elicitation with 15 mM H2O2 did not affect sprout yield. Generally, the studied concentrations of H2O2 under continuous elicitation strongly and negatively influenced biomass production (Table 1). This tendency was clearly visible for 3-dayold sprouts where, in comparison to the control, the mass of 10 sprouts was 20% and 30% lower for sprouts elicited with 15 and 150 mM H2O2, respectively. However, it should be noted that in sprouts normally consumed (except for 5-day-old Ox2–15 and Ox2–150 sprouts) reduction did not exceed 10% (Table 1). Despite significant impact on biomass accumulation and sprout morphology, the studied growth conditions did not cause oxidative damage. In the subsequent days of culture, the measured contents of malondialdehyde in the elicited sprouts did not differ significantly compared to the respective controls. Additionally, in the case of 5-day-old sprouts, single treated with 15 and 150 mM H2O2 the determined levels of malondialdehyde were about 14% lower compared with the control sprouts (Fig. 1). H2O2 treatments induced an enzymatic defense responsible for the detoxification of hydrogen peroxide (Table 2). In comparison to the corresponding control, catalase activity was significantly elevated in all treated sprouts (regardless of the day of cultivation). The highest activity was determined for 2-day-old sprouts treated with 150 mM H2O2 (597 U/g FM). On the 5th day of cultivation, except for Ox2–150, catalase activity did not differ significantly among the studied sprouts. The activity of peroxidase, involved in the synthesis of the physical barriers such as suberin or lignin, was mainly induced in sprouts continuously treated with 150 mM H2O2 – the highest activity was found on the 3rd day (121.02 kU/g FM); however, the highest percentage increase was found on the 4th day (an increase of about 100% compared to the control sprouts). During germination, except for the 1st day, the activity of polyphenol oxidase did not significantly change in the control sprouts; however, treatments increased its activity especially in 3- and 5-day old sprouts. On the 4th day of cultivation, a significant elevation of PPO activity was found for sprouts treated with 150 mM H2O2 (an increase of about 27% and 70% for single and continuous

M. S´wieca / Food Chemistry 180 (2015) 219–226

222

Table 1 Influence of elicitation conditions (elicitor concentration, time and intervals of its application) on the lentil sprouts growth. 10 sprouts mass (g)

Root length (mm)

Stalk length (mm)

1-day-old

C

0.58 ± 0.02a

5.90 ± 0.25a

2-day-old

C Ox1–15 Ox1–150 Ox2–15 Ox2–150

0.75 ± 0.03c 0.72 ± 0.02bc 0.66 ± 0.02b 0.72 ± 0.03bc 0.66 ± 0.02b

18.80 ± 0.75b 19.80 ± 0.60b 19.90 ± 0.50b 19.80 ± 0.80b 19.90 ± 0.60b

7.50 ± 0.30a 8.50 ± 0.20b 8.30 ± 0.20b 8.50 ± 0.35b 8.30 ± 0.25b

0.00

3-day-old

C Ox1–15 Ox1–150 Ox2–15 Ox2–150

0.94 ± 0.02e 0.96 ± 0.04ef 0.84 ± 0.03d 0.76 ± 0.02dc 0.66 ± 0.03b

30.60 ± 0.80c 28.50 ± 1.15c 31.30 ± 1.00cd 29.70 ± 0.75c 28.10 ± 1.15c

20.90 ± 0.50e 18.90 ± 0.75de 24.00 ± 0.70f 18.30 ± 0.45d 13.00 ± 0.50c

4-day-old

C Ox1–15 Ox1–150 Ox2–15 Ox2–150

1.05 ± 0.03f 0.94 ± 0.02e 0.97 ± 0.04ef 0.94 ± 0.03e 0.89 ± 0.02de

36.10 ± 1.10e 39.40 ± 1.00f 40.30 ± 1.60f 36.70 ± 1.10e 33.10 ± 0.85cd

31.60 ± 0.95gh 31.10 ± 0.80gh 29.90 ± 1.20g 30.70 ± 0.90gh 22.10 ± 0.55e

5-day-old

C Ox1–15 Ox1–150 Ox2–15 Ox2–150

1.11 ± 0.04f 1.10 ± 0.03f 1.06 ± 0.03f 0.96 ± 0.04e 0.92 ± 0.04de

38.10 ± 1.50ef 52.30 ± 1.60g 41.30 ± 1.00f 38.00 ± 1.50ef 34.30 ± 1.40cde

38.20 ± 1.55i 37.80 ± 1.15i 40.60 ± 1.05i 33.20 ± 1.35gh 41.70 ± 1.70i

Means, in columns, followed by different small letters are significantly different at p < 0.05. C, control sprouts; Ox-15, one-step elicitation with 15 mM H2O2; Ox1–150, onestep elicitation with 150 mM H2O2; Ox2–15, continuous elicitation with 15 mM H2O2; Ox2–150, continuous elicitation with 150 mM H2O2.

700 600

MDA content [nmol/ g f.m.]

500

b b

b

b

b

b

c

bc

bc

b

abc b b

bc

bc

b

b

abc

a a

a

400 300 200 100

1-day-old

Ox2-15

Ox2-150

Ox1-150

C

Ox1-15

Ox2-15

4-day-old

Ox2-150

Ox1-150

C

Ox1-15

Ox2-15

3-day-old

Ox2-150

Ox1-150

C

Ox1-15

Ox2-15

2-day-old

Ox2-150

Ox1-150

C

Ox1-15

C

0

5-day-old

Fig. 1. Influence of elicitor concentration, time and frequency of its application on oxidative damage in lentil sprouts. Means in columns followed by different small letters are significantly different at p < 0.05. C, control sprouts; Ox-15, one-step elicitation with 15 mM H2O2; Ox1–150, one-step elicitation with 150 mM H2O2; Ox2–15, continuous elicitation with 15 mM H2O2; Ox2–150, continuous elicitation with 150 mM H2O2.

treatment, respectively, was observed compared to the control) (Table 2). In response to oxidative stress by H2O2, plants overproduce phenolics that are involved in signaling, antioxidant defense and/ or synthesis of mechanical barriers. The highest phenolic content was determined for 2-day-old sprouts treated with 15 mM H2O2 (0.71 mg/g f.m.): an increase of about 44% regarding the control. Generally, except for 3-day-old Ox1–150 and Ox2–15 sprouts, elicitation caused a significant elevation of phenolic content compared to the appropriate control sprouts (Fig. 2). One step elicitation is more effective than continuous treatments based on the results presented in Fig. 2. Additionally, phenolics were synthesized de novo. Elicitation induced the activity of the two main enzymes responsible for phenolic synthesis: tyrosine ammonialyase (TAL) and phenylalanine ammonia-lyase (PAL). About a

twofold increase in PAL activity was found in 3-day-old Ox2–150 sprouts. Moreover, elicitation with 150 mM H2O2 significantly decreased PAL activity in 2-day-old sprouts. A negative effect of treatment was observed in cultures treated at 24-h intervals with 15 mM H2O2, where PAL activities were significantly lower than those determined for the appropriate controls. TAL activity was most effectively elevated in Ox1–150 sprouts – compared to the corresponding control, an increase of about 70% and 270% for 2- and 3-day-old sprouts, respectively (Table 3). Phenolic overproduction is linked with a subsequent elevation of antioxidant capacity. With respect to control conditions, elicitation caused an increase in antioxidant capacity in the all studied sprouts. The antioxidant potential of sprouts was evaluated based on four complementary methods. On the 2nd day, 24 h after treatment, there were no significant changes in the reducing potential, the ability to protect lipids against oxidation and to quench free radicals; however, the chelating power was significantly increased. The highest increase in the ability to quench free radicals was observed in 4- and 5-day-old sprouts continuously treated with 15 and 150 mM H2O2 (50% increase compared to the control). Generally, a significant increase in the studied activity was observed in 4-day-old sprouts compared to the control. A significant decrease in reducing ability was observed in all treated sprouts, except for 3-, 4- and 5-day-old sprouts Ox2–150, where increases of 20%, 15% and 15% were determined, respectively. In comparison to the control, all treatments effectively increased the ability to chelate the metal ions of 2-day-old sprouts (5-fold and 3.5-fold for Ox1 and Ox2, respectively) (Table 4). The ability to protect lipids against oxidation was significantly increased in all the studied sprouts, except for 2-day-old ones, compared to the controls. For Ox2–15 and Ox1–150 sprouts, increases of 18%, 61% and 44%, 38%, respectively, were determined on the 4th and 5th days. Because multiple reaction mechanisms are usually involved in measuring the antioxidant capacity of a complex food system, there is no simple universal method by which ‘‘total antioxidant capacity’’ can be measured accurately and quantitatively. Thus, for a better evaluation of the total antioxidant potential and the effectiveness of the treatments used, we

M. S´wieca / Food Chemistry 180 (2015) 219–226

223

Table 2 Changes of activity of enzymes involved in the antioxidant response and phenolics metabolism affected by elicitation with hydrogen peroxide-effect of concentration, time and intervals of its application. Catalase activity (U/g f.m.)

Peroxidase activity (U/g f.m.)

Polyphenol oxidase activity (kU/g f.m.)

1-day-old

C

174.60 ± 17.51a

159.67 ± 5.72b

3.73 ± 0.04a

2-day-old

C Ox1–15 Ox1–150 Ox2–15 Ox2–150

486.00 ± 82.46bcd 310.45 ± 57.77b 430.00 ± 87.64bcd 310.45 ± 57.77b 430.00 ± 87.64bcd

293.50 ± 38.84ef 443.20 ± 17.73g 596.67 ± 17.64h 443.20 ± 17.73g 596.67 ± 17.64h

9.01 ± 0.44fg 8.09 ± 0.32e 8.58 ± 0.13f 8.09 ± 0.32e 8.58 ± 0.13f

3-day-old

C Ox1–15 Ox1–150 Ox2–15 Ox2–150

448.00 ± 79.46bcd 605.47 ± 107.40cde 603.33 ± 107.02cde 510.00 ± 63.79bcd 530.80 ± 61.32cd

126.67 ± 27.22ab 146.40 ± 5.86bc 190.40 ± 7.62c 181.67 ± 35.85cb 331.67 ± 37.86ef

9.09 ± 0.17g 8.32 ± 0.35fe 4.50 ± 0.14b 7.29 ± 0.09d 12.02 ± 0.73h

4-day-old

C Ox1–15 Ox1–150 Ox2–15 Ox2–150

420.00 ± 40.53b 431.60 ± 41.65bc 533.49 ± 51.48cd 474.00 ± 49.04bc 716.00 ± 41.57e

101.67 ± 30.97a 196.80 ± 7.87c 292.00 ± 11.68e 156.67 ± 15.87bc 248.33 ± 30.97de

4.37 ± 0.16b 6.12 ± 0.24c 4.59 ± 0.29b 6.85 ± 0.80cd 9.13 ± 0.29fg

5-day-old

C Ox1–15 Ox1–150 Ox2–15 Ox2–150

444.00 ± 71.11bc 683.41 ± 109.45de 597.22 ± 95.64cde 516.00 ± 75.19cd 544.00 ± 70.65cd

139.17 ± 12.87b 150.40 ± 6.02b 152.80 ± 6.11b 144.78 ± 17.13b 213.33 ± 31.74cd

6.73 ± 0.30cd 3.36 ± 0.17a 4.63 ± 0.20b 6.71 ± 0.43cd 6.69 ± 0.14d

Means, in columns, followed by different small letters are significantly different at p < 0.05. C, control sprouts; Ox-15, one-step elicitation with 15 mM H2O2; Ox1–150, onestep elicitation with 150 mM H2O2; Ox2–15, continuous elicitation with 15 mM H2O2; Ox2–150, continuous elicitation with 150 mM H2O2.

0,9

ef

0,8 0,7

f e

0,6 Total phenolics [mg/ g f.m.]

ef

f

de

e

e e

cd

0,5

c

0,4

e d

cd bc bc bc bc

b

a

a

0,3 0,2 0,1

1-day-old

Ox2-15

Ox2-150

Ox1-150

C

Ox1-15

Ox2-15

4-day-old

Ox2-150

Ox1-150

C

Ox1-15

Ox2-15

3-day-old

Ox2-150

Ox1-150

C

Ox1-15

Ox2-15

2-day-old

Ox2-150

Ox1-150

C

Ox1-15

C

0

5-day-old

Fig. 2. Influence of elicitor concentration, time and frequency of its application on total phenolic content in lentil sprouts. Means in columns followed by different small letters are significantly different at p < 0.05. C, control sprouts; Ox-15, onestep elicitation with 15 mM H2O2; Ox1–150, one-step elicitation with 150 mM H2O2; Ox2–15, continuous elicitation with 15 mM H2O2; Ox2–150, continuous elicitation with 150 mM H2O2.

introduced the total antioxidant activity index (IA). It should be noted that all the studied modifications of germination increased the antioxidant potential of sprouts. The highest IA value (3.2) was calculated for 5-day-old Ox1–15 sprouts; however, a significant elevation was also found for 2-day-old, Ox-15 and 5-dayold Ox1–15 sprouts (2.13 and 2.14, respectively) (Table 4).

4. Discussion Treatment of plants with elicitors causes an array of defense reactions, including the accumulation of a range of plant defensive secondary metabolites, such as antioxidants, in intact plants. The sequentially occurring events in elicitor-induced defense responses are multi-stage and briefly include: the perception of an elicitor

by a receptor, the transfer of the elicitor signals to secondary messengers (e.g. G proteins, protein kinases), NADPH oxidase activation and reactive oxygen species (ROS) production, early defense gene expression, plant hormones synthesis (ethylene, salicylic acid and jasmonate), late defense response gene expression, and secondary metabolite accumulation. The elicitors induce production of desirable metabolites, but the concentration, time and intervals of treatment required for maximum secondary metabolite accumulation are a characteristic of each plant species and normally are preceded by an increase in the activity of the metabolic enzymes involved (Vasconsuelo & Boland, 2007). These facts indicate the importance of determining empirically the optimum conditions of elicitation time and elicitor concentration for each system in particular. Consequently, for a better understanding of the effect of elicitation on antioxidant synthesis, kinetic studies are needed (Zhao et al., 2005). The effectiveness of elicitation as a tool to enhance the production of secondary metabolites depends on a complex interaction between the elicitor and the plant cell (Vasconsuelo & Boland, 2007). Between the main factors that can affect this interaction and thereby the elicitation response are elicitor specificity, concentration and treatment intervals and culture conditions such as growth stage, medium composition, light should be mentioned. The key role of elicitor concentration and treatment intervals observed in this study was also confirmed in many other studies, e.g. induction of benzophenanthridine alkaloid accumulation by sequential treatment with methyl jasmonate, salicylic acid and yeast extract in the Eschscholtzia californica suspension cultures (Cho et al., 2008). When designing some features of low-processed food (sprouts), the factors mentioned above are especially considered as an important, due to the clearly defined shelf life of the final product: for lentil sprouts, depending on the subsequent processing (raw-eaten, stir-fried or steamed), this period is between the 2nd and 5th day of cultivation. In this study, sprouts represent a biotechnological module – a ‘‘biofermentor’’, and elicitation is a tool of metabolic engineering aimed at metabolism manipulation to obtain the determined features of a given food – therapeutic benefits such

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Table 3 Influence of elicitor concentration, time and intervals of its application on the activity of enzymes involved in phenolics synthesis. Tyrosine ammonia-lyase activity (lg/min/g f.m.)

Phenylalanine ammonia-lyase activity (lg/min/g f.m.)

1-day-old

C

96.02 ± 31.27b

173.42 ± 4.51a

2-day-old

C Ox1–15 Ox1–150 Ox2–15 Ox2–150

159.17 ± 20.98cd 170.25 ± 21.36cd 278.50 ± 26.35e 170.73 ± 21.36cd 278.50 ± 26.35e

572.17 ± 14.88f 605.97 ± 11.78f 383.11 ± 7.36b 605.97 ± 11.78f 383.11 ± 7.36b

3-day-old

C Ox1–15 Ox1–150 Ox2–15 Ox2–150

139.11 ± 10.15c 162.48 ± 5.90c 524.71 ± 41.30f 115.72 ± 19.07c 233.90 ± 64.89d

423.88 ± 11.02d 436.77 ± 11.36d 475.42 ± 12.36e 323.99 ± 8.42b 832.50 ± 21.65h

4-day-old

C Ox1–15 Ox1–150 Ox2–15 Ox2–150

73.86 ± 4.02b 129.03 ± 5.70c 127.94 ± 8.10c 48.01 ± 17.23ab 103.41 ± 54.09abcd

383.18 ± 9.96cd 341.15 ± 8.87bc 358.46 ± 9.32c 276.22 ± 7.18b 412.25 ± 10.72d

5-day-old

C Ox1–15 Ox1–150 Ox2–15 Ox2–150

70.17 ± 58.87abc 86.22 ± 2.89b 59.99 ± 5.30b 44.32 ± 6.96a 94.79 ± 21.04b

344.76 ± 8.96bc 557.28 ± 14.49f 366.01 ± 9.52c 322.95 ± 8.40b 416.41 ± 10.83d

Means, in columns, followed by different small letters are significantly different at p < 0.05. C, control sprouts; Ox-15, one-step elicitation with 15 mM H2O2; Ox1–150, onestep elicitation with 150 mM H2O2; Ox2–15, continuous elicitation with 15 mM H2O2; Ox2–150, continuous elicitation with 150 mM H2O2.

Table 4 Influence of elicitation conditions (elicitor concentration, time and intervals of its application) on the antioxidant capacity of lentil sprouts. Radical scavenging ability (mg TE/g f.m.)

Inhibition of lipids peroxidation (mg Q/g f.m.)

Chelating power (mg EDTA/g f.m.)

Reducing power (mg TE/g f.m.)

Antioxidant index

1-day-old

C

2.32 ± 0.07g

1.22 ± 0.01f

0.63 ± 0.07c

3.03 ± 0.1g

–

2-day-old

C Ox1–15 Ox1–150 Ox2–15 Ox2–150

1.82 ± 0.01e 2.10 ± 0.26efg 1.91 ± 0.11ef 2.10 ± 0.26efg 1.91 ± 0.11ef

1.31 ± 0.03g 1.21 ± 0.07fg 1.29 ± 0.13fg 1.21 ± 0.07fg 1.29 ± 0.13fg

0.42 ± 0.09bc 2.26 ± 0.22g 1.26 ± 0.07e 2.26 ± 0.22g 1.26 ± 0.07e

2.41 ± 0.06f 2.48 ± 0.02f 2.46 ± 0.12ef 2.48 ± 0.02f 2.46 ± 0.12ef

1.00 2.13 1.52 2.13 1.52

3-day-old

C Ox1–15 Ox1–150 Ox2–15 Ox2–150

1.56 ± 0.03cd 1.28 ± 0.11b 1.71 ± 0.31cde 1.73 ± 0.12de 1.86 ± 0.02f

0.80 ± 0.03d 0.85 ± 0.08de 0.88 ± 0.01e 0.94 ± 0.07de 1.29 ± 0.11fg

0.36 ± 0.02b 0.72 ± 0.01d 0.32 ± 0.05b 1.15 ± 0.04e 0.32 ± 0.03b

1.87 ± 0.09d 1.57 ± 0.08bc 1.79 ± 0.04d 1.65 ± 0.02c 2.24 ± 0.08e

1.00 1.18 1.01 1.58 1.22

4-day-old

C Ox1–15 Ox1–150 Ox2–15 Ox2–150

1.10 ± 0.17ab 1.48 ± 0.06cd 1.25 ± 0.16ab 1.65 ± 0.16de 1.39 ± 0.12bc

0.56 ± 0.05a 0.68 ± 0.06bc 0.60 ± 0.02a 0.81 ± 0.01d 0.77 ± 0.08dc

0.22 ± 0.06ab 1.09 ± 0.20e 0.54 ± 0.09c 0.31 ± 0.04b 0.31 ± 0.04b

1.59 ± 0.06bc 1.58 ± 0.12bc 1.47 ± 0.20ab 1.60 ± 0.11bc 1.84 ± 0.06d

1.00 2.14 1.41 1.34 1.31

5-day-old

C Ox1–15 Ox1–150 Ox2–15 Ox2–150

1.17 ± 0.05a 1.10 ± 0.02a 1.13 ± 0.05a 1.44 ± 0.04c 1.75 ± 0.02e

0.60 ± 0.02a 0.67 ± 0.03b 0.55 ± 0.00a 0.70 ± 0.01c 0.63 ± 0.02ab

0.16 ± 0.03a 1.57 ± 0.00f 0.19 ± 0.06a 0.59 ± 0.00c 0.09 ± 0.03a

1.46 ± 0.08ab 1.34 ± 0.02a 1.41 ± 0.01b 1.52 ± 0.10b 1.67 ± 0.05bc

1.00 3.20 1.01 1.78 1.06

Means, in columns, followed by different small letters are significantly different at p < 0.05. C, control sprouts; Ox-15, one-step elicitation with 15 mM H2O2; Ox1–150, onestep elicitation with 150 mM H2O2; Ox2–15, continuous elicitation with 15 mM H2O2; Ox2–150, continuous elicitation with 150 mM H2O2.

as hypoglycemic, anticancer, antioxidant, anti-inflammatory, antimicrobial and anticholesterol effects. During induction of sprout metabolism, the most important factors seem to be elicitor concentration, the time of its application and intervals of treatment. In most previous studies, treatments have been applied on dry seeds soaked in the tested solution (Feng et al., 2010; McCue et al., 2000). In contrast, in these studies stress factors were applied not on seeds but on already growing sprouts. This procedure was designed to avoid any disadvantageous influence of stress on seed germination and any further lowering of sprout vigor (biomass production). In these studies an adverse effect, observed after ‘‘overloading’’ of an elicitor, was

observed in sprouts continuously treated with 150 mM hydrogen peroxide (Table 1). The overproduction of phenolics in response to treatments is diverse – higher concentrations of stress factors do not always cause a liner increase in desirable features. It should be noted that in this study the maximal effect of elicitation, an increase of phenolics level by about 65% in respect to appropriate control sprouts, was observed after treatment with 15 mM H2O2 (Fig. 2). A similar relationship was observed by Pérez-Balibrea et al. (2011) in a study of the effect of exogenous elicitors (methionine, tryptophan, chitosan, salicylic acid and methyl jasmonate) on bioactive component content in broccoli sprouts. Although the residual hydrogen peroxide content in treated

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sprouts was not studied, however according to results concerning lipids peroxidation and increased activities of catalase, it may be indirectly stated that the elicitor was effectively degraded (Fig. 1, Table 2). Activation of phenolic synthesis usually means an increase in the antioxidant capacity of low-processed food. In this study, the effect of elicitation on the antioxidant potential of sprouts was determined based on four antioxidant tests that can capture the different modes of action of antioxidants – reducing abilities (reducing power), antiradical ability (ABTS test), preventive effect against lipid oxidation (inhibition of the hemoglobin-catalyzed peroxidation of linoleic acid) and the ability to chelate transition metals ions – potential substrates of the Fenton reaction (chelating power) (Prior, Wu, & Schaich, 2005). The conditions of elicitation in this study caused significant changes in the phenolics profile and antioxidant capacity of lentil sprouts (Fig. 2, Table 4) and most importantly the results are comparable or much better than those shown in other studies. Additionally, in the study by S´wieca and Baraniak (2013) continuous and single treatment of 2-day-old lentil sprouts with 20 mM H2O2 and 200 mM H2O2 caused 1.6- and 1.9-fold increase in total antioxidant capacity of 4-day-old sprouts. The effect of temperature stress (1 h induction at 4 and 40 °C) also improved the phenolics content and antioxidant capacity of lentil sprouts; however, after 4 days of sprouting an increase did not exceed 20% compared to the control (S´wieca, Surdyka, Gawlik-Dziki, Złotek, & Baraniak, 2014). Also, the study considered the efficacy of UV-B treatment and precursor feeding with phenolic synthesis precursors in relation to the production of polyphenolrich lentil sprouts and the highest obtained increases in phenolics content and antioxidant capacity were not higher than 19% and 27%, respectively (S´wieca, Se˛czyk, & Gawlik-Dziki, 2014). In comparison with the cited studies, in this research a 1.7- and 1.6-fold increase in the antioxidant capacity of Ox1–15 and Ox1–150 4-day-old sprouts was observed. In this study, all variants of treatments increased the antioxidant capacity of sprouts; however, the final effect was strictly determined by the hydrogen peroxide concentrations, time of application and intervals of treatments. Differentiation of plant responses to the intervals of treatments had also previously been observed by Gawlik-Dziki et al. (2012). Single or continuous treatments of broccoli sprouts with yeast extracts and willow bark infusion were able to differentiate plant response linked with an elevation of the antioxidant capacity of food. In contrast to these studies, where generally the single treatment increased nutraceutical potential in higher degree, better results have been obtained after continuous elicitation (Gawlik-Dziki et al., 2012). Treatments also stimulated the activity of plant defense-related enzymes. In a study performed by McCue et al. (2000), low pH and salicylic acid treatments increased pea sprout vigor and phenolic content. Based on concomitant stimulation of glucose-6-phosphate dehydrogenase, the key enzyme of the pentose-phosphate pathway, they suggested that this pathway produces the critical precursors for the synthesis of phenolic secondary metabolites (important for plant growth, stress response and lignifications). The pentose-phosphate pathway delivers the reducing power (NADPH2) and intermediate metabolites that are necessary for synthesis of aromatic amino acids such as phenylalanine and tyrosine. These amino acids are converted by the action of PAL and TAL into p-coumaric and t-cinnamic acids. Induction of PAL and TAL activity, linked with a subsequent elevation of phenolic levels and antioxidant capacity, seems to confirm the role of elicitation as a tool able to elevate the de novo synthesis of antioxidants. So far, similar to these studies, there have only been a few studies into the elicitor-mediated changes of PAL and TAL activities in the production low-processed food; however, the key role of these enzymes in phenolic synthesis is well-documented (Shetty, 2004; Zhao et al.,

225

2005; Matkowski, 2008). The induction of PAL and TAL activity by exogenous salicylic acid application has been previously observed in cassava cell suspensions (Dogbo et al., 2012). Also, Guo, Yuan, and Wang (2011) determined an increase in PAL activity and antioxidant activity after induction of broccoli sprouts with 176 mM sucrose. Most importantly, in the lentil sprouts an increase in TAL activity observed after elicitation seems to be largely responsible for the overproduction of phenolics (Table 3). This observation is confirmed by previous studies by S´wieca, Se˛czyk et al. (2014) concerning improvement of the phenolic content and antioxidant activity of lentil sprouts affected by UV-B treatment and feeding with the phenylpropanoid pathway precursors. As mentioned above, plant stress response is strongly linked with the overproduction of phenolics acting as signaling compounds, antioxidants and/or cell wall precursors. The phenolic metabolism is complicated; however, from a nutraceutical standpoint the action of guaiacol peroxidases and polyphenol oxidases should be mentioned. Guaiacol peroxidase participates in lignin and suberin synthesis that are involved in enhancing cell structure (Fukushima, 2001). Polyphenol oxidase is a copper-containing enzyme which is responsible for browning in plants (it catalyzes two distinct reactions: the o-hydroxylation of monophenols to o-diphenols and the oxidation of o-diphenols to o-quinones) (Udayasekhara Rao & Deosthale, 1987). These two enzymes consume phenolic compounds; hence, they may significantly decrease the levels of the latter in plant food. An increase in these activities has previously been determined in elicited plant e.g. pea sprouts (McCue et al., 2000), or lettuce (Złotek & Wójcik, 2014). Most importantly, the increase in the above activities was mainly observed in lentil sprouts continuously treated H2O2 (Ox2–150). It may be speculated that this is the reason why the increased activity of PAL and TAL in these sprouts was not translated into an elevated content of phenolics and antioxidant capacity (Tables 1–3, Fig. 2).

5. Conclusion In the light of this and previous studies, elicitation seems to be an effective biotech technique for use in the overproduction of metabolites with potential biological activity. Additionally, the application of minimal processing methods, especially treatments with H2O2, may preserve the shelf-life of sprouts and reduce food microorganisms without affecting their sensory and nutritional quality (S´wieca & Baraniak, 2013; S´wieca et al., 2013). During the design of some features of low-processed food (sprouts) factors such as the concentration of elicitors and the time and intervals of its application should be considered. These studies represent the first attempt to determine the effects of time and the method of elicitation on plant (sprouts) stress response linked with the overproduction of phenolic compounds and the improvement of antioxidant capacity. Additionally, our results are related to biochemical changes, such as the level of oxidative damage, the activities of enzymes involved in sprouts stress response and phenolic metabolism. As the use of abiotic elicitors is cheap and relatively easy to employ in the sprout industry, this biotechnology seems to be an alternative to conventional techniques which are applied to improve the levels of health promoting phytochemicals and the bioactivity of low-processed food.

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