Saudi Journal of Biological Sciences (2015) 22, 409–416

King Saud University

Saudi Journal of Biological Sciences

www.ksu.edu.sa
www.sciencedirect.com

ORIGINAL ARTICLE

Elicitation with abiotic stresses improves pro-health

constituents, antioxidant potential and nutritional
quality of lentil sprouts
Micha Świeca *

Department of Biochemistry and Food Chemistry, University of Life Sciences, Skromna Str. 8, 20-704 Lublin, Poland

Received 20 October 2014; revised 14 December 2014; accepted 15 December 2014

Available online 22 December 2014

KEYWORDS Abstract Phenolic content and antioxidant potential of lentil sprouts may be enhanced by treat-
Antioxidant activity; ment of seedlings in abiotic stress conditions without any negative influence on nutritional quality.
Elicitation; The health-relevant and nutritional quality of sprouts was improved by elicitation of 2-day-old
Lentil; sprouts with oxidative, osmotic, ion-osmotic and temperature stresses. Among the sprouts studied,
Nutrients; those obtained by elicitation with osmotic (600 mM mannitol) and ion-osmotic (300 mM NaCl)
Phenolics; shocks had the highest total phenolic content levels: 6.52 and 6.56 mg/g flour, respectively. Oxida-
Sprouting tive stress significantly enhanced the levels of (+)-catechin and p-coumaric acid. A marked eleva-
tion of the chlorogenic and gallic acid contents was also determined for sprouts induced at 4 C and
40 C. The elevated phenolic content was translated into the antioxidant potential of sprouts, espe-
cially the ability to reduce lipid oxidation. A marked elevation of this ability was determined for
seedlings treated with 20 mM, 200 mM H2O2 (oxidative stress) and 600 mM mannitol (osmotic
stress); about a 12-fold, 8-fold and 9.5-fold increase in respect to control sprouts. The highest ability
to quench free radicals was observed in sprouts induced by osmotic stress (IC50- 4.91 and 5.12 mg/
ml for 200 mM and 600 mM mannitol, respectively). The highest total antioxidant activity indexes
were determined for sprouts elicited with 20 mM H2O2 and 600 mM mannitol: 4.0 and 3.4, respec-
tively. All studied growth conditions, except induction at 40 C, caused a significant elevation of
resistant starch levels which was also affected in a subsequent reduction of starch digestibility.
Improvement of sprout quality by elicitation with abiotic stresses is a cheap and easy biotechnol-
ogy and it seems to be an alternative to conventional techniques applied to improve the health pro-
moting phytochemical levels and bioactivity of low-processed food.
ª 2014 The Author. Production and hosting by Elsevier B.V. on behalf of King Saud University. This is an
open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

* Tel.: +48 81 4623327; fax: +48 81 4623324.

E-mail address: [email protected]
Peer review under responsibility of King Saud University. 1. Introduction

Food quality and functionality may be modified at each stage of

its production including plant and animal breeding, technolog-
Production and hosting by Elsevier
ical processing, endogenous ingredients and/or by modification
http://dx.doi.org/10.1016/j.sjbs.2014.12.007
1319-562X ª 2014 The Author. Production and hosting by Elsevier B.V. on behalf of King Saud University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
410 M. Świeca

of storage conditions (Francis et al., 2012). In the case of low sured to characterize the effect of these elicitors were
processed food, such as sprouts, the quality and potential bioac- biomass yield, phenolic content, and antioxidant activities
tivity are mainly determined by seed quality and conditions of (the abilities to prevent lipids and scavenge free radicals, che-
germination (Świeca et al., 2014a; Świeca and Baraniak, lating and reducing power).
2014b; Swieca et al., 2013a; Pérez-Balibrea et al., 2011;
Tsurunaga et al., 2013). 2. Material and methods
Plants subjected to environmental stresses produce reactive
oxygen species (ROS), thus antioxidant activity is of funda- 2.1. Materials and preparation of sprouts
mental importance to their life. ROS can damage vital cellular
macromolecules, e.g. proteins, DNA, and lipids; however, on 2.1.1. Sprouting
the other hand, they play an important role in the regulation
Lentil seeds var. Tina were purchased from PNOS S.A. in
of plant metabolism, acting as regulators of auxin transport
Ozarów Mazowiecki, Poland. Seeds were sterilized in 1% (v/
and/or signaling compounds during response to stress condi-
v) sodium hypochloride (Sigma-Aldrich, USA) for 10 min,
tions (Fujita et al., 2006; Shetty, 2004). To reduce excess
then drained and washed with distilled water until they
ROS, plants have developed an antioxidant defense system,
reached neutral pH. They were placed in distilled water and
which comprises enzymatic and non-enzymatic components.
soaked for 6 h at 25 C. Seeds were dark germinated for 8 days
Non-enzymatic response is mainly linked with overproduction
in a growth chamber (SANYO MLR-350H) on Petri dishes (/
of antioxidants e.g. phenolics. Phenolics are primarily pro-
125 mm) lined with absorbent paper. Seedlings were watered
duced through the pentose phosphate, the shikimate and the
with 5 ml of Milli-Q water daily. Sprout (8-day-old) samples
phenylpropanoid pathways (Shetty, 2004). The antioxidant
were gently collected, weighed (fresh mass), rapidly frozen
potential of phenolics is bound with: (a) radical-scavenging
and kept in polyethylene bags at 20 C. For each treatment,
abilities; (b) the ability to chelate metal transition ions; (c)
three replicates were performed.
reducing power; (d) prevention of lipids and other biomole-
cules against oxidation; (e) inhibition of prooxidant enzymes;
2.1.2. Elicitation
(f) activation of enzymatic defense system (Fernandez-
Panchon et al., 2008; Gawlik-Dziki et al., 2012). Elicitation conditions were selected in previous screening stud-
Numerous studies have indicated the positive correlation ies. For the experiments, temperature (4 C and 40 C – TC
between consumption of phenolic-rich food and health. and TH, respectively), H2O2 (20 mM and 200 mM – Ox1
Legume phenolics exhibit therapeutic benefits such as hypogly- and Ox2, respectively), mannitol (200 mM and 600 mM –
cemic, anticancer, antioxidant, anti-inflammatory, antimicro- Os1 and Os2, respectively) and NaCl (100 mM and 300 mM
bial and anticholesterol effects (Zhao, 2007). Germination is – S-Os1 and S-Os2, respectively) were selected as abiotic elici-
one of the most common and effective processes for improving tors. All solutions were freshly prepared before each applica-
the quality of legumes (Świeca et al., 2012; Paja˛k et al., 2013; tion. Mannitol (Os1, Os2), NaCl (S-O1, S-O2) and H2O2
Ghavidel and Prakash, 2007; Cevallos-Casals and Cisneros- (Ox1) treatments were applied by watering daily (not soaking)
Zevallos, 2010). There are several reports about the effect of 2-day-old sprouts with 5 ml of test solution. For Ox2 (200 mM
germination methods on the nutraceutical value of legumes H2O2) treatment 2-day-old seedlings were only once watered
including, soybeans, mung beans or lentils. Among these strat- with 5 ml of 200 mM H2O2 and then cultivated under standard
egies those employing biotic and abiotic elicitors have been conditions. For temperature conditioning treatment, 2-day-old
widely studied (Świeca et al., 2014a,b, 2013a; Pérez-Balibrea sprouts were incubated at 4 C and 40 C (TC and TH, respec-
et al., 2011; Gawlik-Dziki et al., 2013; Oh and Rajeshekar, tively) for 1 h and then cultivated under standard conditions.
2009; Baenas et al., 2014). These techniques effectively increase Sprout (8-day-old) samples were gently collected, weighed
production of phenolics and other compounds involved in (fresh mass), rapidly frozen and kept in polyethylene bags at
plant response to stress and, although not yet applied to large 20 C.
scale production, have proved to be a very efficient procedure
on a laboratory scale (Ahmed and Baig, 2014). Thus, elicita- 2.1.3. Growth analysis
tion of sprouts seems to be a promising alternative to other In order to determine the influence of elicitation on sprout
conventional biotechnological techniques used for improving vigor the growth ratio was proposed. The growth ratio was
the yield of plant secondary metabolites and the nutraceutical defined as an amount of fresh weight obtained from 1 g of dor-
potential of low-processed food. Additionally, the application mant seeds after germination.
of minimal processing methods may preserve the shelf-life of
sprouts and reduce food microorganisms without affecting 2.1.4. Flour preparation
the sensory and nutritional quality (Peñas et al., 2009). Sprouts were dried in a forced-air oven at 50 C for 12 h
The hypothesis proposed by these studies is that the pheno- (Ghavidel et al., 2007). After that sprouts were grounded in
lic content and antioxidant potential of ready-to-eat lentil a labor mill, and sieved (60 mesh). Sprout flours were stored
sprouts may be enhanced by elicitation of seedlings in abiotic at 4 C.
stress conditions. Cross-talk between different signaling path-
ways is very common in plant defense responses; thus we sup- 2.2. Phenolic content and antioxidant activities
pose that all the studied factors will be effective inductors of
phenolic synthesis (Fujita et al., 2006). On the other hand,
2.2.1. Extract preparation
on the basis of studies concerning plant responses to abiotic
stresses we assumed that some qualitative and quantitative dif- Lentil flours (0.2 g) were extracted three times with 4 ml of
ferences between them would be found. The parameters mea- acetone/water/hydrochloric acid (70:29:1, v/v/v). After
Elicitation with abiotic stresses improves nutritional quality of lentil sprouts 411

centrifugation (10 min, 8000g) fractions were collected, combined 2.2.3.5. Total antioxidant activity index (AI). Four comple-
and used for further analysis. mentary antioxidant methods were intergraded to obtain the
total antioxidant activity index (AI) (Świeca et al., 2014b).
2.2.2. Phenolic analysis The index may be useful for evaluation of the total antioxidant
The amount of total phenolics was determined using potential of sprouts from different germination conditions
Folin–Ciocalteu phenol reagent (Singleton et al., 1974). The with respect to the control.
amount of total phenolics was calculated as a gallic acid
equivalent (GAE) in mg/g of flour. 2.3. Nutritional quality
Total flavonoid content was determined according to the
method described by Lamaison and Carnet, 1990. Total flavo- 2.3.1. Digestion in vitro
noid content was calculated as a quercetin equivalent (QE) in Simulated mastication and gastrointestinal digestion were per-
mg/g of flour. formed according to Swieca et al. (2013a). Lentil sprouts
Condensed tannin content was determined according to the (150 mg of flour) were homogenized in 3.5 ml of simulated sal-
method described by Sun et al. (1998). Condensed tannin con- ivary fluid (2.38 g Na2HPO4, 0.19 g KH2PO4 and 8 g NaCl,
tent was calculated as a (+)-catechin equivalent (CE) in mg/g 200 U a-amylase (E.C. 3.2.1.1) in 1 l H2O, pH 6.75) and sha-
of flour. ken for 10 min at 37 C. Next, the samples were adjusted to
Qualitative–quantitative analysis of phenolics was per- pH 1.2 with HCl (5 mM), suspended in 1.25 ml of simulated
formed using a Varian ProStar HPLC System separation gastric fluid (300 U ml 1 of pepsin A, EC 3.4.23.1 in 0.03 M
module (Varian, Palo Alto, CA) equipped with Varian HCl, pH 1.2) and shaken for 120 min at 37 C. After simulated
ChromSpher C18 reverse phase column (250 mm · 4.6 mm) gastric digestion, samples were adjusted to pH 6 with 0.1 M
and ProStar DAD detector (Swieca and Baraniak, 2014c). NaHCO3 and suspended in simulated intestinal juice (0.05 g
of pancreatin (activity equivalent 4·USP) and 0.3 g of bile
2.2.3. Antioxidant activities extract in 2.0 ml of 0.1 M NaHCO3; adjusted to pH 7 with
2.2.3.1. Antiradical activity (ABTS). The experiments were 1 M NaOH and finally 1.25 ml of 120 mM NaCl and 5 mM
carried out using an improved ABTS decolorization assay KCl was added to the sample. The samples thus prepared
(Re et al., 1999). Free radical scavenging ability was expressed underwent in vitro intestinal digestion for 120 min.
as IC50 in mg flour per ml.
2.3.2. Protein content and digestibility
2.2.3.2. Reducing power (RP). Reducing power was deter- A 500 mg of sample was twice extracted with 5 ml of 0.5 M
mined by the method of Oyaizu, 1986. Reducing power was NaCl solution in 0.05 M Tris–HCl buffer pH 7.0 at 4 C for
expressed as quercetin equivalent (Q) in lg/g of flour. 2 h. After centrifugation (10 min, 8000g) fractions were col-
lected, combined and used for further analysis. The total pro-
2.2.3.3. Metal chelating activity (CHP). Chelating power was tein contents were determined with the Lowry method (1951),
determined by the method of Decker and Welch (1990). Che- using bovine serum albumin as the standard protein.
lating power was expressed as EDTA equivalent (EDTA) in The in vitro protein digestibility was evaluated on the
lg/100 mg of flour. basis of total soluble protein content and the content of
protein determined after digestion in vitro (Świeca et al.,
2.2.3.4. Inhibition of linoleic acid peroxidation (LPI). The anti- 2013b).
oxidant activity was determined as the degree of inhibition
of the hemoglobin-catalyzed peroxidation of linoleic acid 2.3.3. Non-protein nitrogen
(Groupy et al., 2007). Inhibition of linoleic acid peroxida-
A 100 mg of sample was twice extracted with 5 ml of 10%
tion was expressed as quercetin equivalent (Q) in lg/mg
ethanol (v/v) at 4 C for 2 h. After centrifugation (10 min,
of flour.

Table 1 Phenolic composition of lentil sprouts elicited by different abiotic stresses.

Cultivation conditions Total phenolics [mg/g flour] Total flavonoids [mg/g flour] Condensed tannins [mg/g flour]
C 4.40 ± 0.03b 0.38 ± 0.02b 0.91 ± 0.03d
Ox1 4.74 ± 0.14bc 0.59 ± 0.01d 0.86 ± 0.03cd
Ox2 3.96 ± 0.13a 0.50 ± 0.00c 0.63 ± 0.01a
Os1 5.13 ± 0.22c 0.50 ± 0.02c 1.19 ± 0.04e
Os2 6.52 ± 0.13d 0.70 ± 0.11e 1.73 ± 0.05f
S-O1 4.19 ± 0.15a 0.43 ± 0.04b 0.76 ± 0.05b
S-O2 6.56 ± 0.33d 0.79 ± 0.02e 1.63 ± 0.11f
TC 4.02 ± 0.14a 0.28 ± 0.01a 0.88 ± 0.02c
TH 5.10 ± 0.34b 0.50 ± 0.04c 0.88 ± 0.12bc
Means in columns followed by different letters are significantly different at p = 0.05.
Each value represents the mean of 3 independent experiments (±SD).
C, control; Ox1, induction with 20 mM H2O2; Ox2, induction with 20 mM H2O2; Os1, induction with 200 mM mannitol; Os2, induction with
600 mM mannitol; S-O1, induction with 100 mM NaCl; S-O2, induction with 300 mM NaCl; TC, induction at 4 C; TH, induction at 40 C.
412 M. Świeca

8000g) fractions were collected, combined and used for fur-

0.27 ± 0.01b
0.72 ± 0.12d

C, control; Ox1, induction with 20 mM H2O2; Ox2, induction with 200 mM H2O2; Os1, induction with 200 mM mannitol; Os2, induction with 600 mM mannitol; S-O1, induction with 100 mM
0.17 ± 0.01a

0.14 ± 0.00a
0.10 ± 0.01a
0.12 ± 0.01a
1.06 ± 0.12e

0.39 ± 0.02c
1.56 ± 0.14f
ther analysis. Non-protein nitrogen was determined with

Daidzein
2,4,6-trinitrobenzene sulfonic acid (TNBS) according to the
methods described by Habeeb (1966), using L-leucine as the
standard.

2.69 ± 0.11d

0.93 ± 0.04b
0.09 ± 0.04a
0.02 ± 0.00a
0.01 ± 0.00a

0.02 ± 0.00a
4.32 ± 0.20e

1.30 ± 0.05c
6.80 ± 0.27f
Kemferol
2.3.4. Starch content and digestibility
The total starch (TS) content was determined after dispersion
of the starch granules in 2 M KOH (50 mg sample, 6 ml KOH)

0.60 ± 0.22bc 3.54 ± 0.6bc

0.05 ± 0.10a 4.16 ± 0.2d
2.67 ± 0.62e 3.87 ± 0.1d

0.53 ± 0.06c 9.65 ± 1.5d

0.23 ± 0.14ab 4.33 ± 0.4d

0.74 ± 0.06c 2.80 ± 0.2b
1.74 ± 0.64de 1.40 ± 0.2a

1.49 ± 0.03d 1.24 ± 0.0a

1.71 ± 0.17d 11.17 ± 0.1e
at room temperature (30 min, constant shaking) and hydroly-

Quercetin
sis of the solubilized starch with 80 ll (1 mg/ml) amyloglucosi-
dase (14 U mg 1; EC 3.2.1.3) at 60 C for 45 min (Goni et al.,

Means in columns followed by different letters are significantly different at p = 0.05. Each value represents the mean of 3 independent experiments (±SD).
19967). The glucose content was determined by using the stan-
dard dinitrosalicylic acid (DNSA) method (Miller, 1959).

Naringenin
Total starch was calculated as glucose ·0.9. The free reducing
sugar content of the samples was determined in order to cor-
rect the obtained total starch values. The sucrose content of
the samples was also determined in order to correct the

2.09 ± 0.9bcd
obtained total starch values. The samples dispersed in sodium

2.94 ± 0.9cd

2.30 ± 0.2bc
Ferulic acid

1.86 ± 0.1b

2.86 ± 0.1d

1.41 ± 0.2b
1.08 ± 0.1a

0.98 ± 0.1a

1.16 ± 0.0a
acetate buffer, pH 5.0, were treated with 200 ll of (10 mg in
1 ml of 0.4 M sodium acetate buffer, pH 5.0) invertase (EC
3.2.1.26; 300 U/mg) for 30 min at 37 C. After centrifugation,
reducing sugars were analyzed in the supernatants using the

7.22 ± 0.7bc

8.38 ± 0.6bc
7.13 ± 0.3b
7.83 ± 0.2b
11.48 ± 0.8d

6.65 ± 0.8b
4.06 ± 0.2a

4.49 ± 0.3a
8.64 ± 0.4c
p-coumaric acid Benzoic acid Salicylic acid Chlorogenic acid Caffeic acid
DNS reagent. The resistant (RS) and potentially bioavailable
(AS) starch content was analyzed on the basis of results
obtained after digestion in vitro. After centrifugation (8000g,
15 min) and removal of supernatant, the pellet was dispersed
with 2 M KOH, hydrolyzed with amyloglucosidase, and then
liberated glucose was quantified, as described above, for total

43.47 ± 2.5c 11.42 ± 0.5cd

37.25 ± 4.6b 5.89 ± 0.3b

42.88 ± 1.7c 6.91 ± 0.0b

33.86 ± 2.6b 11.94 ± 0.1d

28.30 ± 1.2a 3.05 ± 0.8a

27.50 ± 0.6a 3.27 ± 0.2a

30.45 ± 2.3ab 8.65 ± 0.2c

39.54 ± 2.2bc 14.20 ± 1.0e

42.50 ± 2.5c 9.96 ± 0.5c

starch (TS). Resistant starch (RS) was calculated as glucose
·0.9. The potentially bioavailable starch (AS) content was cal-
Phenolic composition of lentil sprouts cultivated under abiotic stress conditions.

culated as the difference between TS and RS.

The in vitro digestibility of starch was evaluated on the basis

NaCl; S-O2, induction with 300 mM NaCl; TC, induction at 4 C; TH, induction at 40 C.
of total starch content (TS) and resistant starch (RS) deter-
mined after digestion in vitro according to Świeca et al.
(2013a).

2.4. Statistical analysis

18.68 ± 2.9de

17.50 ± 2.3cd
6.07 ± 1.0b

6.22 ± 1.6b
1.57 ± 1.4a
22.82 ± 2.6e
13.50 ± 1.9c

15.68 ± 0.2c
28.34 ± 1.0f

All experimental results were mean ± S.D. of three parallel

experiments (n = 9). One-way analysis of variance (ANOVA
and Tukey test) was used to compare groups within different
elicitors. P values <0.05 were regarded significant.
35.02 ± 6.3abd
33.08 ± 1.0bc

42.12 ± 2.9d
37.52 ± 1.0b

30.95 ± 1.5b
29.21 ± 2.6b
24.85 ± 0.9a
51.43 ± 2.7e

34.11 ± 2.4c

3. Results and discussion

Currently, one of the main areas of research is the search for

0.78 ± 0.02b 39.86 ± 6.8de

1.59 ± 0.00c 39.24 ± 0.8d

0.96 ± 0.16b 21.10 ± 0.9b
0.66 ± 0.42ab 121.28 ± 2.1g

6.56 ± 0.1a
0.48 ± 0.08a 48.56 ± 1.9e

0.58 ± 0.03a 26.77 ± 2.1c

1.42 ± 0.02c 28.95 ± 1.0c

1.05 ± 0.61bc 110.14 ± 3.0f
(+) catechin

new food products that, in addition to nutritional quality, also

Germination Compounds [lg/g flour]

possessed natural ingredients with biological activity (e.g.,

antioxidant, antiviral, antihypertensive, etc). There are some
reports describing the application of elicitation in order to
improve the nutraceutical potential of sprouts (Świeca et al.,
3.79 ± 0.14d
Gallic acid

2014a,b, 2013a; Pérez-Balibrea et al., 2011; Gawlik-Dziki

et al., 2013; Oh et al., 2009).
In this study, elicitation of sprout metabolism by abiotic
elicitors significantly changed the qualitative and quantitative
profile of phenolics. The levels of total phenolics and flavo-
conditions

noids determined in sprouts treated with 200 mM and

Table 2

600 mM mannitol and at 40 C were significantly higher in

S-O1
S-O2
Ox1
Ox2
Os1
Os2

TH
TC

comparison with those from the control (Table 1). Among

C

*
Elicitation with abiotic stresses improves nutritional quality of lentil sprouts 413

the sprouts studied, those obtained by elicitation by osmotic biotic and abiotic signaling (Fujita et al., 2006). There is much
(Os2) and ion-osmotic (S-O2) shocks had the highest total phe- evidence that generation of ROS during stress in plant induces
nolic content levels: 6.52 and 6.56 mg/g flour, respectively, overproduction of phenolics (Shulaev et al., 2008). These com-
(Table 1). In comparison with control total flavonoid and con- pounds are mainly produced to protect plants from biotic/abi-
densed tannin contents were also elevated; an increase of about otic stresses such as photooxidation stress, reactive oxygen
90% for osmotic stress and 79% for salt-osmotic stress species, wounds, disease and herbivores (Shetty, 2004). Appli-
(Table 1). In response to shock treatments lentil sprouts accu- cation of different cultivation conditions (biotic and abiotic
mulated a number of phenolic compounds. The qualitative– stresses) can result in an enhanced production of these antiox-
quantitative analysis of phenolics indicated the presence of ele- idants. These findings were attributed to the biochemical
ven hydroxycinnamic and hydroxybenzoic acids and six flavo- changes in sprout metabolism, which might influence the pro-
noids. It was found that in 8-day-old sprouts p-coumaric, duction of compounds, such as anthocyanins and flavonoids,
benzoic acids and (+)-catechin were the dominant phenolic or release aglycones from conjugated glycosides due to enzy-
components (Table 2). Among the studied elicitors, the highest matic activation (Shetty, 2004). Although, all the studied mod-
amounts of polyphenolics were determined for sprouts treated ifications caused the overproduction of polyphenolic the
with H2O2 and mannitol (Table 2). The stimulated influence of qualitative analysis suggests that response differs significantly.
these factors was clearly visible in the case of chlorogenic, feru- An elevation of plant polyphenolics in response to elicitors
lic, o-coumaric and salicylic acids. It should be noted that was also observed by Swieca et al. (2014a) and Randhir et al.
among the studied modifications of sprouting a direct oxida- (2004). The cited investigators reported an induction of the
tive stress (Ox1, Ox2) produced with H2O2 significantly pentose phosphate pathway, the shikimate and the phenylprop-
enhanced the levels of (+)-catechin and p-coumaric acid. A anoid pathways during plant growth under stress condition.
marked elevation of chlorogenic and gallic acid contents was Plants increase the level of salicylic acid (signaling compound)
determined for sprouts incubated at 4 C and 40 C, about and other phenolics acting as antioxidants and/or precursors
the threefold and twofold increase in respect to control, respec- for the synthesis of lignin, suberin and phenolic barriers. High
tively. The dominant flavonoid in the lentil sprouts was (+)- levels of p-coumaric acids might be involved in the overproduc-
catechin. Daidzein and quercetin levels in sprouts were tion of coumarylCoA, a key compound in flavonoid biosynthe-
increased about twofold in sprouts obtained by elicitation with sis (Shetty, 2004). High amounts of p-coumaric, ferulic and
mannitol. An extreme reduction in benzoic acid levels, linked caffeic acids (potential precursors of barriers) in lentil sprouts
with elevation of salicylic acid content, was observed in sprouts treated with mannitol may suggest that plant response in this
treated with 200 mM mannitol, 100 mM NaCl and incubated case is mainly bound with sealing up the cells. These observa-
at 4 C (Table 2). tions seem to confirm results obtained by Bandeoğlu et al.
Under normal conditions, during germination lentil pro- (2004). It should be noted that, similar to studies performed
duces various phenolics (Świeca et al., 2012). Data concerning by Dixon (2001), the stress conditions applied caused a signifi-
polyphenolic profiles of lentil sprouts are in agreement with cant increase in protecting compounds e.g. chlorogenic acid.
those obtained by Cevallos-Casals et al. (2010) and Ghavidel Also, Feng et al. (2010) reported the accumulation of flavo-
et al. (2007). Studies on the stress signaling pathway suggest noids in soybean seedlings under stress. Similar results were
that ROS signaling plays a key role in the cross-talk between also reported in this work for osmotic stress conditions.

16
C Ox1 Ox2 Os1 Os2 S-O1 S-O2 TC TH
14 e

12
cd cd
10 c
c bc cd
8 d
b d

6
a a c
c bc
bc
4 ab f
e
e de
d abcd a d
2 b c bc a
cd cd
b b
a
0

Antiradical activity Reducing power Chelating power Lipids peroxidation Total antioxidant
IC50 [mg flour/ ml] [µg Q/ mg flour] [µg EDTA/100 mg flour] inhibition activity index
[µgQ/ mg flour]

Figure 1 Antioxidant activities of sprouts cultivated under abiotic stress conditions C, control; Ox1, induction with 20 mM H2O2; Ox2,
induction with 20 mM H2O2; Os1, induction with 200 mM mannitol; Os2, induction with 600 mM mannitol; S-O1, induction with
100 mM NaCl; S-O2, induction with 300 mM NaCl; TC, induction at 4 C; TH, induction at 40 C. Means followed by different letters are
significantly different at p < 0.05. Each value represents the mean of 3 experiments (±SE).
414 M. Świeca

Changes in phenolic content were generally associated with

225.08 ± 11.25cd

C, control; Ox1, induction with 20 mM H2O2; Ox2, induction with 20 mM H2O2; Os1, induction with 200 mM mannitol; Os2, induction with 600 mM; mannitol; S-O1, induction with 100 mM
4.75 ± 0.31cd

102.86 ± 2.01de
56.22 ± 3.37cd
the changes in antioxidant capacity of the studied sprouts. The

258.52 ± 5.25a
33.44 ± 3.63a
226.21 ± 4.84c

87.07 ± 4.35c
antioxidant potential of sprouts was evaluated on the basis the
four complementary methods. Radical scavengers acted in a
dosage-dependent manner (data not shown), thus calculation
TH

of the 50% inhibitory concentration (IC50) was possible. The

highest abilities to quench free radicals were observed in

240.34 ± 12.02d
54.87 ± 3.29cb
5.07 ± 0.08d

46.26 ± 1.40b
226.96 ± 3.80c

286.60 ± 2.80c

83.86 ± 4.19c
108.19 ± 1.13f
sprouts induced with osmotic stress – Os1 and Os2 (IC50-
4.91 and 5.12 mg flour per ml, respectively), whereas the lowest
was in salt-ionic case (100 mM NaCl- IC50- 12.88 mg flour per
ml) (Fig. 1). A significant decrease of reducing abilities was
TC

observed in sprouts treated with 100 mM NaCl and incubated

175.22 ± 8.76b 172.57 ± 8.63ab 197.50 ± 9.87c 198.26 ± 9.91c 244.29 ± 10.01d
181.49 ± 6.37a 207.83 ± 2.97b 251.80 ± 3.68d 250.49 ± 8.56d 280.39 ± 10.05e

47.19 ± 2.83ab
245.92 ± 11.96a 264.82 ± 10.18a 288.40 ± 3.82c 276.70 ± 4.58b 296.86 ± 2.51d

at 4 C (1.07 and 1.22 lg Q/g flour, respectively), while the

2.96 ± 0.15a

57.99 ± 1.37a

90.90 ± 4.58ef 78.44 ± 10.88de 56.57 ± 5.79c

80.95 ± 4.05c

highest abilities were recorded for seedlings treated with

600 mM mannitol and 300 mM NaCl (2.21 and 1.92 lg Q/g
flour, respectively), (Fig. 1). The highest chelating power was
S-O2

determined for sprouts from Ox1, S-O2 and TH treatments

(7.36, 5.21 and 5.40 lg EDTA/g flour, respectively), (Fig. 1).
4.83 ± 0.09d

70.23 ± 2.68b 74.89 ± 2.06b

71.65 ± 3.58b
a
42.65 ± 2.56

Among the studied activities the ability to protect lipids

against peroxidation was affected by elicitation at the highest
degree. A marked elevation of this ability was determined
S-O1

for sprouts treated with 20 mM, 200 mM H2O2 and 600 mM

mannitol, about a 12-fold, 8-fold and 9.5-fold increase above
68.48 ± 3.42ab

that for the sprouts cultivated at control conditions, respec-

b
2.91 ± 0.00a

52.06 ± 3.12

tively (Fig. 1). Due to the fact that antioxidant capacity of

complex food systems is created by the activities that differ
in the mode of action, there is no simple and universal method
Os2

by which ‘‘total antioxidant capacity’’ can be measured accu-

rately and quantitatively. Thus, for better evaluation of the
65.16 ± 3.26ab
de
3.74 ± 0.09b

96.72 ± 2.22c

70.70 ± 11.39cd 92.25 ± 7.00e

60.56 ± 3.63

total antioxidant potential the total antioxidant activity index

(AI) was proposed. It should be noted that all studied modifi-
cations of sprouting caused an elevation of the antioxidant
Os1

potential of sprouts. The highest values of AI were determined

NaCl; S-O2, induction with 300 mM NaCl; TC, induction at 4 C; TH, induction at 40 C.

for sprouts elicited with 20 mM H2O2 and 600 mM mannitol:

Influence of elicitation on growth rate and nutritional quality of sprouts.

4.0 and 3.4, respectively (Fig. 1).

4.68 ± 0.14d

71.25 ± 3.56b
104.05 ± 1.22e
f
70.94 ± 4.26

Means in rows followed by different letters are significantly different at P < 0.05.

Sprouting improves nutritional value of lentil, however neg-

atively affects phenolics content and antioxidant activity, thus
looking forward for an effective, safe and acceptable method
Ox2

of elevation of antioxidant activities is very important. Gener-

ally, in these studies the elevated concentrations of phenolics
Each value represents the mean of 3 independent experiments (±SD).
255.28 ± 7.89a 244.30 ± 17.82a
99.12 ± 0.82cd
67.94 ± 4.08ef

93.96 ± 24.18
227.99 ± 5.83c 189.83 ± 1.51a

221.69 ± 11.08cd 150.34 ± 7.52a

61.54 ± 3.08a
4.14 ± 0.01c

(especially flavonoids) were linked with a subsequent increase

of antioxidant potential of sprouts, which may suggest that
these compounds act as effective antioxidants (Table 4). The
level of phenolics was most effectively increased by application
Cultivation conditions
Ox1

of H2O2 and mannitol as elicitors. Data concerning antioxi-

dant activity are in agreement with the previous reports by
cb
4.56 ± 0.01d

33.59 ± 6.01a
Non-protein nitrogen [mg/g flour] 96.00 ± 1.48c

86.84 ± 4.34c
54.25 ± 3.25

Shetty (2004) and Randhir et al. (2004). The cited investigators

reported that elicitation of bean seeds with fish protein hydrol-
ysates, lactoferrin and oregano extract enhanced production of
phenolic antioxidants and ability to quench free radicals. The
C

nutraceutical value of edible sprouts was also effectively

enhanced by Gawlik-Dziki et al. (2013). Elicitations of sprouts
with salix bark extract and yeast extract significantly increased
Resistant starch [mg/g flour]

their reductive potential and their abilities to prevent lipids

Total starch[mg/g flour]
Protein digestibility [%]

Potentially bioavailable

Starch digestibility [%]

against oxidation and to scavenge free radicals. It should be

Proteins[mg/g flour]

noted that sprouts obtained in this studies had a very strong

starch [mg/g flour]

ability to prevent lipids. It may be speculated that similarly

Growth ratio

to the studies performed by Gawlik-Dziki et al. (2013), an

increase of this ability was linked with increased concentra-
Table 3

tions of flavonoids. Additionally, the high reducing potential

of seedlings may be due to the high content of hydroxycin-
*

namic acids, which are able to reduce iron ions (Table 4).
Elicitation with abiotic stresses improves nutritional quality of lentil sprouts 415

Table 4 Relationships between phenolics and antioxidant activities in lentil sprout (Pearson correlation coefficient).
Total phenolics Total flavonoids Condensed tannins Hydroxycinnamic acids Hydroxybenzoic acids
Antiradical activity 0.71 0.56 0.73 0.79 0.59
Reducing power 0.91 0.81 0.81 0.89 0.66
Chelating power 0.01 0.25 0.16 0.37 0.36
Lipid peroxidation inhibition 0.21 0.55 0.17 0.34 0.46

As it was presented elicitation is an effective method for (Feng et al., 2010). It should be noted that in our studies 2-
improving nutraceutical quality of sprout but sometimes it day–old sprouts were elicited and undesirable effects of abiotic
may negatively influence growth and nutritional quality of elicitors (environmental shock) were reduced. Additionally,
sprouts. Thus, the influence of studied factors on biomass elicitation is based on the natural mechanisms involving in
accumulation, protein and starch content and digestibility plant response to pathogen, thus microbial contaminations
was studied. Water deficient conditions, caused by mannitol are probably reduced (Peñas et al., 2009). According to the lit-
and NaCl treatments, significantly reduced the growth of lentil erature data it is also known that in genus Lens there is no
sprouts. Cultivations watered with high osmotic potential solu- overproduction of ‘‘dangerous’’ metabolites such as alkaloids,
tions (about – 1.4 MPa; 600 mM mannitol (Os2) and 300 mM toxins (Lin and Lai, 2006).
NaCl (S-O2)) were characterized by the largest biomass reduc-
tion – about 35% when compared to control sprouts. In con- 4. Conclusion
trast, apart from Os1 and Ox1, the treatments applied did not
cause any significant biomass reduction in 8-day-old sprouts The results of this study show that application of abiotic elic-
(Table 3). In spite of the fact that lentils are known to be salt itors (environmental shocks) was an effective method for
sensitive in this work only higher concentration of NaCl neg- improvement of sprout pro-health potential via an increase
atively influenced plant growth. This finding is partially in of phenolic contents and subsequent elevation of antioxidant
opposition to results obtained by Bandeoglu et al. (2004). potential. Innovative application of elicitors on 2-day-old
The citied investigators determined a statistically significant sprouts (not seed) allowed the elimination of the unfavorable
reduction of biomass in 9-day-old lentil seedlings after treat- influence of the factors employed on germination yield and
ments with 100 mM and 200 mM NaCl (62% and 43%, biomass production. Assuming that the optimal germination
respectively). conditions are those which most effectively increase the antiox-
Total protein content was significantly reduced in sprouts idant potential without any negative influence on biomass
obtained at Ox1 and Ox2 conditions; in comparison with con- accumulation and nutritional quality the elicitation with
trol conditions their levels were lower about 16.7 and 20.4%, 20 mM H2O2 for the future applications is recommended. As
respectively. In respect to control seedlings sprouts from these the use of abiotic elicitors is cheap and relatively easy to adopt
cultures were also characterized by elevated protein digestibil- in the edible sprout production industry, this biotechnology
ity. Protein digestibility and non-nitrogen contents determined seems to be an alternative to conventional techniques applied
for sprouts elicited with 100 mM and 300 mM NaCl and to improve the health promoting phytochemical levels and bio-
600 mM mannitol were significantly lower than those deter- activity of low-processed food.
mined for control sprouts. Changes in total starch content
caused by elicitation did not exceed 15%. The highest total
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