Nutritional and Antioxidant Potential of Lentil Sprouts Affected by

Article pubs.acs.org/JAFC

Nutritional and Antioxidant Potential of Lentil Sprouts Affected by Elicitation with Temperature Stress ́ Michał Swieca* and Barbara Baraniak Department of Biochemistry and Food Chemistry, University of Life Sciences, Skromna Street 8, 20-704 Lublin, Poland ABSTRACT: The influences of temperature stress on antioxidant potential and nutritional quality of lentil sprouts were studied. Temperature treatments (TC, 1 h at 4 °C; TH, 1 h at 40 °C) significantly improved the nutraceutical potential without any negative effect on nutritional quality. In comparison to control, elicited sprouts were characterized by elevated content of condensed tannins, flavonoids, and total phenolics. The highest content of total phenolics and flavonoids was determined for 6-day-old TH sprouts −23.7 ± 0.87 and 2.50 ± 0.07 mg/(g of dry weight (DW)), respectively. The general trend of antiradical, lipid preventing, and reducing properties in elicited sprouts indicates a significantly improvement of these activities. The highest reducing power was determined for 6-day-old sprouts induced at TH (0.43 ± 0.02 mmol of Trolox/(g of DW)), while the lowest for 3-day-old sprouts elicited at TC (0.29 ± 0.02 mmol of Trolox/(g of DW)). Both modifications effectively elevated the ability to prevent lipids against oxidation (in 3-day-old sprouts a 3.3- and 4-fold increase for TC and TH, respectively). KEYWORDS: antioxidant activities, elicitation, nutritional value, phenolics, sprouting

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INTRODUCTION A properly composed diet may be of a high significance in the prevention of numerous diseases, and determination of individual nutrition is aimed at health-promoting, life quality improvement, and attenuation of symptoms of the process of organism aging. Interest in the possibilities of food modification at each stage of its obtaining, i.e., plant and animal breeding, technological processes, and conditions of product storage, has increased over recent years. These practices lead to obtaining food for special nutritional purpose, which, except for providing basic nutrients, is a source of biologically active substances regulating physiological processes in the human organism. 1 Since the major roles of plant secondary metabolites are to protect plants from biotic and abiotic stresses, some strategies for production of the metabolites based on this principle have been developed to improve the yield of such metabolites including phenolics. Temperature extremes, acting as abiotic stresses, induce local or systemic responseconcerning the whole plant organism. A key sign of such stresses at the molecular level is the accelerated production of reactive oxygen species (ROS) that activate enzymatic and nonenzymatic scavenging pathways or detoxification systems. One of the basic possible responses is overproduction of phenolics that play an important role in the regulation of plant metabolism (e.g., acting as signaling molecules and regulators of auxin transport), take a part in reduction of oxidative stress, and are a building material for cell barriers (e.g., lignin and suberin). On the other hand the nutraceutical potential of plant food is very often linked with phenolic content and composition. It is well-known that the content of phenolics in legume sprouts is generally affected by sprouting environmental and genetic factors such as cultivar, illumination, and temperature.2−5 Additionally, nutritional properties as well as the level and activity of bioactive compounds present in leguminous plants may be modified using a range of technological (e.g., thermal processing) and biotechnological practices (e.g., fermentation).6 Among them germination is one of the cheap and efficient technologies, but it © 2014 American Chemical Society

should be noted that during sprouting significant qualitative and quantitative changes in the content of polyphenolic compounds (at high degree responsible for antioxidative, anti-inflammatory, or anticancer activity) are observed.3,7 Usually, a significant elevation of polyphenol content, when the results are expressed in dry weight, is found.8,9 On the other hand when the results are presented in fresh weight, a significant decrease of their level is observed.3,10 It is possible to avoid unprofitable changes, from the point of view of lowered bioactivity, using elicitation (the natural defense mechanisms of plants), which constitutes one of the effective methods of secondary metabolite production improvement in numerous plant systems.11 Phenolics are primarily produced through the pentose phosphate, shikimate, and phenylpropanoid pathways.5,12 As a response on variable environmental conditions, plants modify their metabolism adjusting to existing conditions; thus stress factors may be a useful tool, which would allow one to modify the composition and activity of plant origin food.3,11,13,14 Attempts at modification of the composition and bioactivity of Fabaceae plants by cultivation condition changes are sparse. An increase in the level of polyphenols and increased digestibility and solubility of proteins were obtained by an application of variable lighting conditions (varicolored lightning conditions, γ-radiation).15 Elicitation of the phenylpropanoid pathway with UV radiation and oregano extract, fish proteins hydrolysate, and lactoferrin in order to increase the antioxidative potential of sprouts of mung beans was also described.13 So far, little has been reported about the effect of elicitation on the chemical and nutritional quality of legume sprouts. Thus, the objectives of this study were to determine the influence of elicitation with temperature stress on the polyphenolic content of lentils and their antioxidative abilities at different germination Received: Revised: Accepted: Published: 3306

September 5, 2013 March 11, 2014 March 14, 2014 March 14, 2014 dx.doi.org/10.1021/jf403923x | J. Agric. Food Chem. 2014, 62, 3306−3313

Journal of Agricultural and Food Chemistry

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Table 1. Data for Linearity, Limit of Detection (LOD), Limit of Quantification (LOQ), and Recovery for Phenolics Determination by HPLC Technique (n = 15)

a

compounds

detection

range (μg/mL)

r2a

LOD (μg/mL)

LOQ (μg/mL)

recovery (%)

gallic acid hydroxybenzoic acid caffeic acid benzoic acid salicylic acid syryngic acid chlorogenic acid sinapinic acid o-coumaric acid p-coumaric acid ferulic acid daidzein quercetin naringenin catechin

270 270 280 270 290 270 270 280 290 290 290 335 360 290 280

0.020−40 0.015−40 0.065−40 0.065−40 0.179−35 0.028−40 0.028−50 0.219−40 0.035−45 0.022−50 0.040−50 0.063−20 0.047−35 0.016−50 0.096−40

0.998 0.997 0.999 0.996 0.992 0.999 0.998 0.997 0.999 0.999 0.998 0.997 0.998 0.998 0.995

0.005 0.004 0.016 0.016 0.045 0.007 0.007 0.055 0.010 0.005 0.010 0.016 0.012 0.004 0.024

0.020 0.015 0.065 0.065 0.179 0.028 0.028 0.219 0.038 0.022 0.040 0.063 0.047 0.016 0.096

97.2 96.8 98.5 93.7 90.5 98.6 97.5 96.1 98.4 99.1 96.8 89.9 92.3 96.8 90.5

r2 = coefficient of determinination. Phenolic Analysis. Determination of Total Phenolic Compounds. The amount of total phenolics (TPC) was determined using Folin− Ciocalteau reagent. 16 The amount of total phenolics was calculated as a gallic acid equivalent (GAE) in milligrams per gram of dry mass (DW). Determination of Flavonoid Content. Total flavonoid content (TFC) was determined according to the method described by Lamaison and Carnat.17 Total flavonoid content was calculated as a quercetin equivalent (QE) in milligrams per gram of DW. Determination of Condensed Tannins Content. Condensed tannin content (CTC) was determined according to the method described by Sun et al.18 CTC was calculated as a (+)-catechin equivalent (CE) in milligrams per gram of DW. Quantitative−Qualitative Analysis of Phenolic. Samples were analyzed with a Varian ProStar high-performance liquid chromatrography (HPLC) system separation module (Varian, Palo Alto, CA, USA) equipped with Varian ChromSpher C18 reverse phase column (250 mm × 4.6 mm) and ProStar DAD detector. The column thermostat was set at 40 °C. The mobile phase consisted of 4.5% acetic acid (solvent A) and 50% acetonitrile (solvent B), and a flow rate of 0.8 mL min−1 was used. At the end of the gradient, the column was washed with 50% acetonitrile and equilibrated to the initial condition for 10 min. The gradient elution was used as follows: 0 min, 92% A; 30 min, 70% A; 45 min, 60% A; 80 min, 60% A; 82 min, 0% A; 85 min, 0% A; 86 min, 92% A; and 90 min, 92% A. Detection was carried out at 270 and 370 nm. Spectrum analysis and a comparison of their retention times with those of the standard compounds identified the phenolics in a sample. Quantitative determinations were carried out with the external standard calculation, using calibration curves of the standards. Detailed information concerning method validation is presented in Table 1. Phenolics were expressed in micrograms per gram of DM. 3 Antioxidant Aactivities. Antiradical Activity (ABTS). The experiments were carried out using an improved ABTS decolorization assay.19 Free radical scavenging ability was expressed as Trolox equivalent (TE) in millimoles per gram of DW. Reducing Power. Reducing power (RP) was determined by the method of Oyaizu.20 RP was expressed as Trolox equivalent in millimoles per gram of DW. Metal Chelating Activity. Chelating power (CHP) was determined by the method of Guo and co-workers.21 CHP was expressed as ethylenediaminetetraacetic acid (EDTA) equivalent in milligrams per gram of DW. Inhibition of Linoleic Acid Peroxidation. The inhibition of the hemoglobin-catalyzed peroxidation of linoleic acid (LPI) was determined.22 The activity was expressed as quercetin equivalent (Q) in milligrams per gram of DW. Total Antioxidant Capacity Index. Four complementary antioxidant methods were intergraded to obtain the total antioxidant

stages. Special emphasis was placed on the nutritional quality of sprouts, including protein and starch contents, and their potential bioavailability.

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MATERIAL AND METHODS

Chemicals. Ferrozine (3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine), ABTS (2,2-diphenyl-1-picrylhydrazyl), α-amylase, pancreatin, pepsin, bile extract, Folin−Ciocalteau reagent, linoleic acid, ammonium thiocyanate, pepsin, chymotrypsin, and phenolic standards linoleic acid were purchased from Sigma-Aldrich Co. (Poznan, Poland). All other chemicals were of analytical grade. Materials. Lentil (Lens culinaris Medik.) seeds var. Tina were purchased from the Horticulture and Seed Enterprises PNOS S.A. in Ozarów Mazowiecki, Poland. Germination. Seeds were sterilized in 1% (v/v) sodium hypochloride for 10 min, then drained, and washed with distilled water until they reached neutral pH. They were placed in distilled water and soaked for 6 h at 25 °C. Seeds were dark germinated 6 days, at 25 °C, in a growth chamber on Petri dishes (φ 125 mm) lined with absorbent paper (approximately 150 seeds per dish). Seedlings were watered with 5 mL of Milli-Q water daily. Elicitation. For the experiments, 2-day-old sprouts were incubated for 1 h at 4 and 40 °C (TC and TH, respectively). Temperatures for treatments were selected according to previous screening studies (sprouts vigor, phenolics content, and antiradical potential). Plates were transferred to the climatic chamber, discovered, and incubated for 1 h at appropriate temperature. After that, sprouts were cultivated under standard conditions (see the section Germination). Sprout samples were gently collected, weighed (fresh mass), rapidly frozen, and kept in polyethylene bags at −20 °C. For each treatment, three replicates were taken for analysis. Flour Preparation. Sprouts were dried in a forced-air oven at 50 °C for 12 h to moisture content < 10%.8 After that, sprouts were ground in a labor mill and sieved (60 mesh). Sprout flours were stored at 4 °C. Growth Analysis. In order to determine the influence of elicitation on the vigor of the sprouts, the growth factor was proposed. The growth factor was defined as an amount of fresh weight (in grams) obtained on the second, third, fourth, and sixth days of germination from 1 g of dormant seeds. Phenolic Content and Antioxidant Activities. Extract Preparation. 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, 6800 relative centrifugal force (rcf)) fractions were collected, combined, and used for further analysis. 3307

dx.doi.org/10.1021/jf403923x | J. Agric. Food Chem. 2014, 62, 3306−3313


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determined using α-N-benzoyl-DL-arginine-p-nitroanilidehydrochloride (BAPNA) as the substrate.29 TIA was expressed as trypsin inhibitor units per milligram of DW sample. One trypsin unit was defined as the increase by 0.01 absorbance units at 410 nm of the reaction mixture. Chymotrypsin Inhibitors Activity. A 100 mg sample was extracted with 4 mL of 0.05 M phosphate buffer pH 7.6 at 4 °C for 2 h. Chymotrypsin inhibitor activity (CIA) was determined using sodium beznzylo-DL-tyrosine-p-nitroanilide (BTpNA) as the substrate.30 CIA was expressed as chymotrypsin inhibitor units per milligram of DW sample. One chymotrypsin unit was defined as the increase by 0.01 absorbance units at 410 nm of the reaction mixture. α-Amylase Inhibitors Activity. For extraction, 100 mg (DW) of lentil sprouts was suspended in 4 mL of 50 mM phosphate buffer, pH 7.6, then stirred for 2 h at 4 °C, and centrifuged at 3000g for 60 min. α-Amylase inhibitor (αAI) activity was measured according to the method described by Alonso and co-workers. 6 One inhibitory unit was defined as the amount of αAI that completely inhibited one enzyme unit (the amount of α-amylase which realized 1 μmol of reducing groups/(1 min at 40 °C) in the reaction conditions). Statistic Analysis. All experimental results were mean ± standard deviation (SD) of three parallel experiments. Two-way analysis of variance (ANOVA and Tukeỳs posthoc test) was used to compare groups within different elicitors. α values < 0.05 were regarded as significant.

capacity index (AI). 10 The index may be useful for evaluation the total antioxidant potential of sprouts from different germination conditions with respect to control. The AI was calculated as the sum of relative activities (RA) for each antioxidant chemical method divided by the number of methods (n).

AI = ΣRA (n)/n RA was calculated as follows:

RA = Ax/Ac where Ax = activity of modified sprouts for the method and Ac = activity of control sprouts determined for the method. Soluble Protein Content. A 500 mg sample was twice extracted with 5 mL of 0.5 M NaCl solution in 0.05 M Tris-Hcl buffer, pH 7.0, at 4 °C for 2 h. After centrifugation (10 min, 6800 rcf) fractions were collected, combined, and used for further analysis. Total protein contents were determined with the Lowry method,23 using bovine serum albumin as the standard protein. Nonprotein Nitrogen Content. A 100 mg sample was twice extracted with 5 mL of 10% ethanol (v\v) at 4 °C for 2 h. After centrifugation (10 min, 6800 rcf) fractions were collected, combined, and used for further analysis. Nonprotein nitrogen was determined with 2,4,6-trinitrobenzene sulfonic acid (TNBS) according to the methods described by Habeeb,24 using L-leucine as the standard. Total Starch Content. The total starch (TS) content was determined after dispersion of the starch.25 The glucose content was determined by using the standard dinitrosalicylic acid (DNSA) method.26 Total starch was calculated as glucose × 0.9. The free reducing sugar content of the samples was determined in order to correct the obtained total starch values. The sucrose content of the samples was also determined in order to correct the obtained total starch values. The samples dispersed in sodium acetate buffer, pH 5.0, were treated with 200 μL 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−1) for 30 min at 37 °C. After centrifugation, reducing sugars were analyzed in the supernatants using the DNS reagent. Digestion in Vitro. Simulated mastication and gastrointestinal ́ digestion was performed according to Swieca and co-workers.27 In Vitro Protein digestibility. The in vitro protein digestibility was evaluated on the basis of the total soluble protein content and the content of protein determined after digestion in vitro.10

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RESULTS Diverse biological effects of legumes food are commonly considered in terms of phenolic content. In the light of this, increasing of the antioxidant potential by phenolics overproduction in lentil sprouts was performed using natural plant stress response pathways. Sprouts metabolism was induced by incubation of 2-day-old sprouts at low (4 °C, TC and high 40 °C, TH) temperature. Since the aim of the study was to determine the influence of temperature treatments, the results obtained for elicited sprouts were always compared to those obtained for control sprouts from the corresponding day of germination. One of the most important factors during trials aimed to overproduce secondary metabolites by elicitation is a negative influence of stressed factors on plant growth and yield. Generally, in our studies growth conditions did not influence negatively on fresh mass yield (Table 2). Extracts obtained from sprouts at different stages of germination (control and elicited) were preliminarily studied for their phenolics content (Table 2). Sprouts obtained by elicitation were characterized by elevated content of condensed tannins, flavonoids, and total phenolics. A small reduction of flavonoids content (about 6% in comparison to control) was only found for 6-day-old sprouts induced at 4 °C. Induction at 40 °C most effectively increased the flavonoids content in the sprouts; in comparison to the control condition, their amounts were higher by about 28%, 43%, and 49% for 3-, 4-, and 6-day-old sprouts, respectively. We have previously demonstrated that growth conditions affected significantly the qualitative and quantitative changes in polyphenols profiles of sprouts;3,4,7,14 thus, a further, precise analysis was performed. An exemplary profile of lentil sprouts phenolics is presented in Figure 1. In comparison to control, during growth of elicited sprouts an increase of salicylic and chlorogenic acids content was observed. Both treatments reduced also the content of syringic acid in sprouts; its level was lower by about 88%, 84%, and 52% in the third, fourth, and sixth 230 days of cultivation, respectively. Additionally, in 3-, 4-, and 6-day-old sprouts determined amounts of caffeic and p-coumaric acids (except p-coumaric acid content in 6-day-old TH sprouts) were significantly lower than those found for control sprouts. Both treatments caused significant changes of flavonoids fractions. Surprisingly, the

PD/% = 100 − [(Pr/Pt × 100] where PD = in vitro digestibility of the protein, Pt = total protein content, and Pr = content of proteins after in vitro digestion. In Vitro Starch Digestibility. The in vitro digestibility of starch was evaluated on the basis of total starch content (TS) and resistant starch (RS) determined after digestion in vitro. Resistant and Potentially Bioavailable Starch Content. The resistant (RS) and potentially bioavailable starch (AS) content was analyzed on the basis of results obtained after simulated gastrointestinal digestion. After centrifugation (3000g, 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 starch (TS, 2.8). RS was calculated as glucose × 0.9. AS content was calculated as the difference between TS and RS. In Vitro Starch Digestion Rate and Expected Glycemic Index. The digestion kinetics and expected glycemic index (eGI) of the lentil sprouts were calculated in accordance with the procedure established by Goni and co-workers.25 eGI was calculated using the equation proposed by Granfeldt and co-workers:28 eGI = 8.198 + 0.862HI. The hydrolysis index (HI) was calculated on the basis of the starch hydrolysis curve (0−180 min) as the percentage of total glucose released over 180 min in comparison to that released from white bread over the same duration. Hydrolase Inhibitors Activity. Trypsin Inhibitors Activity. A 100 mg sample was extracted with 4 mL of 0.05 M phosphate buffer, pH 7.6, at 4 °C for 2 h. The trypsin inhibitor activity (TIA) was 3308



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Table 2. Effect of Different Sprouting Conditions on Yield and Phenolics Composition of Lentil Sproutsa cultivation conditionsb total phenolics (mg/(g of DW))

total flavonoids (mg/(g of DW))

condensed tannins (mg/(g of DW))

growth factor (g)

time of germination (days)

C

TC

2 3 4 6 2 3 4 6 2 3 4 6 2 3 4 6

19.8 ± 0.12a 19.8 ± 0.30a 19.9 ± 0.19a 19.4 ± 0.17a 1.84 ± 0.04c 1.87 ± 0.03c 1.71 ± 0.10bc 1.68 ± 0.06b 6.06 ± 0.16f 5.63 ± 0.24f 4.94 ± 0.12bc 4.33 ± 0.01a 2.51 ± 0.07b 2.79 ± 0.01 cd 3.11 ± 0.15e 3.96 ± 0.10f

21.2 ± 0.66bc 21.2 ± 1.00bc 21.6 ± 0.11c

TH 20.1 ± 0.34ab 21.7 ± 0.49bc 23.7 ± 0.87d

2.10 ± 0.06d 1.77 ± 0.08bc 1.58 ± 0.02a

2.39 ± 0.05e 2.44 ± 0.02e 2.50 ± 0.07e

5.82 ± 0.08ef 5.26 ± 0.04d 4.80 ± 0.03b

5.73 ± 0.08ef 5.25 ± 0.22cde 4.57 ± 0.31a

2.70 ± 0.00c 3.02 ± 0.18de 4.23 ± 0.07g

2.29 ± 0.10a 3.19 ± 0.15 e 4.08 ± 0.11fg

Means in rows, for the respective components followed by different small letters are significantly different at α = 0.05. bC, control; TC, sprouts induced at 4 °C; TH, sprouts induced at 40 °C.

a

in the phenylpropanoid metabolism such as synthesis of different classes of flavonoids. In order to analyze the effect of elicitation and subsequent changes in the sprouts phenolics profile on the antioxidant capacities, four methods based on different modes of antioxidant action were used. The general trend of free radical scavenging, lipid preventing, and reducing properties in elicited lentil sprouts showed that induction significantly improved these activities (except the radical scavenging ability of 3-day-old sprouts). It is shown in Table 4 that temperature treatments caused a significant increase of radical scavenging ability of 4- and 6-day-old sprouts. Between elicited sprouts, the highest reducing power was determined on day 6 for sprouts induced at 40 °C (0.43 mmol of TE/(g of DW)), while the lowest, in those obtained on day 3 in those elicited at 4 °C (0.35 mmol of TE/(g of DW)). These parameters were higher than those determined for the respective control sprouts by 19% and 34%, respectively. It should be noted that, in comparison with samples cultivated under control conditions, sprouts obtained by induction at 40 °C (effect was not observed in the case of induction at 4 °C) were characterized by high chelating power; for 3-, 4-, and 6-day-old sprouts its activity was about 2-fold higher. Both studied modifications of sprouting (TH, TC) effectively elevated the ability to prevent lipids against oxidation. The best results were obtained on day 3, a 3.3- and 4-fold increase for TH and TC, respectively. Complementary antioxidant methods were intergraded to obtain the total antioxidant capacity index. All of the studied conditions significantly improved the nutraceutical value of sprouts. The highest antioxidant potentials were obtained for 6-day-old sprouts (2.25 and 3.11 for TC and TH, respectively) (Table 4). Nowadays, one of the main areas of research is the search for new food products that in spite of nutritional quality also possess natural ingredients with biological activity. 1 Here it is presented that the induction of sprouts at 4 and 40 °C has allowed us to obtain a low-processed food with significantly enhanced antioxidant potential. Since elicitation may in some cases negatively influence nutrients content, we also placed a special emphasis on the nutritional quality of the sprouts. On germination in control conditions, only a slight reduction in protein content was observed (Table 5). Elicitation by both

Figure 1. HPLC−DAD elution profile (270 nm) of standard and lentil samples ((A) 2-day-old sprouts; (B) standard): (1) hydroxybenzoic acid, (2) (+)-catechin, (3) vanillic acid, (4) chlorogenic acid, (5) syringic acid, (6) p-coumaric, (7) salicylic acid, (8) ferulic acid, (9) sinapinic acid, (10) o-coumaric acid, (11) daidzein, (12) quercetin, (13) naringenin, and (AV) internal standard. For details, see Materials and Methods.

levels of daidzein in elicited sprouts were lower than those determined for the control. It should be also noted that treated sprouts were characterized by significantly higher levels of (+)-catechin (Table 3). Phenolic compounds were determined by both spectrophotometric and HPLC techniques. Spectrophotometric methods are less precise (with respect to HPLC); however, they allow for better visualization of the effect of elicitation (changes are more visible). On the other hand, more accurate qualitative and quantitative data provided by HPLC technique coupled with diode array detection (DAD) technique allow to speculate about biological process taking place in the stressed plants, e.g., synthesis of the lignin precursors and changes 3309



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Table 3. Effect of Different Sprouting Conditions on Phenolics Composition of Lentil Sproutsa,b

Means, in columns, for the respective components followed by different small letters are significantly different at α = 0.05. bC, control; TC, sprouts induced at 4 °C; TH, sprouts induced at 40 °C.

a

Table 4. Effect of Different Sprouting Conditions on Antioxidant Activity of Lentil Sproutsa cultivation conditionsb time of germination (days) radical scavenging ability (mmol of TE/(g of DW))

reducing power (mmol of TE/(g of DW))

chelating power (mg of EDTA/(g of DW))

inhibition of lipids peroxidation (mg of Q/(g of DW))

relative antioxidant index

2 3 4 6 2 3 4 6 2 3 4 6 2 3 4 6 3 4 6

C 5.75 3.69 2.23 2.76 0.27 0.29 0.30 0.32 0.20 0.23 0.24 0.17 3.19 2.81 4.54 1.17 1 1 1

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.44e 0.59bcd 0.25ac 0.19ac 0.02a 0.02a 0.02ab 0.01b 0.03bc 0.05bcd 0.03 cd 0.01b 0.34b 0.27b 0.34c 0.17a

TC

TH

3.63 ± 0.00bc 4.56 ± 0.51de 3.88 ± 0.66bcd

4.11 ± 1.76cde 4.64 ± 0.69de 4.20 ± 0.71d

0.35 ± 0.04bcd 0.39 ± 0.03cde 0.38 ± 0.01d

0.36 ± 0.00c 0.38 ± 0.02de 0.43 ± 0.02e

0.15 ± 0.04b 0.27 ± 0.02d 0.07 ± 0.02a

0.45 ± 0.03f 0.46 ± 0.03f 0.38 ± 0.02e

11.3 ± 0.34g 7.55 ± 0.19d 6.90 ± 0.43d 1.80 1.47 2.25

9.42 ± 0.47f 7.13 ± 0.23d 8.21 ± 0.32e 1.93 1.63 3.11

Means, in rows, for the respective activities followed by different small letters are significantly different at α = 0.05. bC, control; TC, sprouts induced at 4 °C; TH, sprouts induced at 40 °C.

a

from corresponding days of germination), levels of total starch in 3-day-old sprouts elicited at 40 °C and 4-day-old sprouts elicited at 4 °C were significantly lower. Treatment at 4 °C caused a significant increase of potentially bioavailable starch and eGI value in 3-day-old sprouts. On the other hand treatment at 40 °C caused a significant decrease of resistant starch content in sprouts; with respect to control sprouts from corresponding days of germination its content was lower by about 15% and 18% for 3- and 4-day-old sprouts, respectively. Between elicited sprouts, the highest eGI was determined for 3-day-old sprouts induced at 4 °C (76.7; elevation by 13% in comparison to control 3-day-old sprouts), whereas the lowest was for 4-day-old sprouts induced at 40 °C (59.8; reduction by

temperature stresses of 2-day-old sprouts caused a significant decrease in protein content only in 4- and 6-day-old sprouts: however, it did not affect protein digestibility. The processes of elicitation were associated with significant changes in the nonprotein nitrogen content. The highest levels of free amino acids and peptides were determined for 6-day-old sprouts elicited at 40 °C95.1 mg/(g of DW) (increase by 25% in comparison to control sprouts). It should be noted that the used modification of sprouting caused a significant elevation of activity of trypsin and chymotrypsin inhibitors but, as was mentioned before, treatments had no significant effects on protein digestibility (Table 5). Elicitation of sprouts influenced also starch degradation during sprouting (Table 6). In comparison to control (sprouts 3310



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Table 5. Effect of Thermal Stress on Nonprotein Nitrogen and Protein Content, Protein Digestibility, and Protease Inhibitor Activities in Sproutsa time of germination (days)

cultivation conditionsb

2 3

protein (mg/(g of DW))

C C TC TH C TC TH C TC TH

4

6

253 253 260 254 246 227 227 243 213 221

± ± ± ± ± ± ± ± ± ±

nonprotein nitrogen (mg/(g of DW))

2.85d 1.45d 3.87e 2.22de 1.98c 4.32b 2.99b 1.87c 4.01a 0.99b

27.6 34.5 49.9 34.1 56.4 56.8 54.3 77.0 92.7 95.1

± ± ± ± ± ± ± ± ± ±

protein digestibility (%)

2.65a 1.76b 2.89c 1.04b 4.23d 1.65d 2.78d 2.99e 1.72f 4.78f

79.5 75.2 78.2 72.9 70.5 72.8 66.9 66.5 63.1 64.2

± ± ± ± ± ± ± ± ± ±

trypsin inhibitor activity (U/(mg of DW))

1.98f 4.56def 1.23e 0.99 cd 1.23bcd 1.98bcd 2.56ab 2.03a 3.56a 0.45a

4.34 4.30 6.35 7.94 4.02 6.48 5.26 4.50 6.09 4.84

± ± ± ± ± ± ± ± ± ±

0.36a 0.26a 0.40 cd 0.24e 0.25a 0.11d 0.55bc 0.23c 0.15b 0.86ab

chymotrypsin inhibitor activity (U/(mg of DW)) 0.49 0.36 0.78 0.88 0.49 0.67 0.93 0.72 0.83 0.79

± ± ± ± ± ± ± ± ± ±

0.04b 0.07a 0.00 cd 0.02de 0.08b 0.02c 0.13d 0.05c 0.09d 0.28cde

a Means, in columns, for the respective components or activities followed by different small letters are significantly different at α = 0.05. bC, control; TC, sprouts induced at 4 °C; TH, sprouts induced at 40 °C.

Table 6. Effect of Thermal Stress on Starch Content and Digestibility, Expected Glycemic Index (eGI), and α-Amylase Inhibitor Activitiesa time (day) 2 3

4

6

cultivation conditionsb C TC TH C TC TH C TC TH

total starch (mg/(g of DW) 273 282 302 259 270 222 264 301 306 299

± ± ± ± ± ± ± ± ± ±

0.55 cd 4.33ed 13.8f 7.43b 1.25bc 4.15a 19.9b 8.15f 3.60f 12.87fe

bioavailable starch (mg/(g of DW)) 159 177 201 162 164 113 177 201 184 201

± ± ± ± ± ± ± ± ± ±

4.98b 3.25c 1.33e 3.31b 7.11bc 4.44a 2.67c 1.32e 4.87d 9.11de

resistant starch (mg/(g of DW) 114 105 101 96.6 107 109 86.8 100 123 95.6

± ± ± ± ± ± ± ± ± ±

3.07f 0.25e 3.18c 0.13b 2.93e 2.42ef 2.80a 3.12 cd 5.33g 6.63abc

eGI 59.0a 67.6b 76.7d 66.9b 71.6c 59.8a 70.0c 64.5b 65.5b 65.8b

starch digestibility (%) 60.7 62.8 67.3 62.6 60.5 51.0 67.1 66.8 67.7 59.9

± ± ± ± ± ± ± ± ± ±

1.37b 0.15b 0.44c 1.12b 1.27b 2.69a 1.43c 1.94c 1.18c 2.21b

α-amylase inhibitor activity (AiU/(g of DW)) 61.5 59.8 33.9 77.8 33.7 51.9 74.9 35.4 279 227

± ± ± ± ± ± ± ± ± ±

8.15bc 5.50bc 2.00a 17.1c 20.0a 14.5abc 3.47c 10.6a 39.5d 29.9d

a Means, in columns, for the respective components or activities followed by different small letters are significantly different at α = 0.05. bC, control; TC, sprouts induced at 4 °C; TH, sprouts induced at 40 °C.

polyphenols with other organic substances such as carbohydrate or protein. 32 On germination, protein, carbohydrates, and minerals become more bioavailable and bioaccessible.3,9,10,27 In this study the gradual reduction of protein content and digestibility was also determined. On the third and fourth days of germination a significant decrease of total starch content and digestibility was observed. These data agree with the findings of Ghavidel and Prakash for the germinated green gram, cowpea, lentil, and chickpea.8 Similar results were also obtained by Urbano and coworkers during germination of lentil.33 Changes of nutritional and nutraceutical potential occurring during sprouting depend on process conditions.3,5,14 Additionally, sprouting conditions can have important effects on the composition of secondary metabolites with potential prohealth properties.5 Although we did not investigate the mechanism of phenolic compound stimulation by temperature stress, previous studies have shown that most phenolic compounds are generated by the phenylpropanoid pathway, which is stimulated by biotic and abiotic stress.5,12,34 In response to stress, plants induce endogenous plant hormones, which, in turn, induce enzymes involved in the phenylpropanoid pathway, including phenylalanine ammonia lyase (PAL), thereby resulting in the accumulation of phenolic compounds.11,34,36 In comparison to control, during growth of elicited sprouts an increase of chlorogenic acid and (+)-catechin content was observed. However, chlorogenic acid was probably synthesized de novo; in the case of (+)-catechin some additional factors, such as the

about 16% in comparison to control 4-day-old sprouts). It should be mentioned that both treatments (except 3-day-old TC sprouts) caused an increase of α-amylase inhibitors activity in sprouts; however only in cases of 4-day-old TC and 6-dayold TH sprouts may this fact be linked with reduction of starch digestibility. In the others cases there was no simple relationship between eGI values, α-amylase inhibitor activity, starch digestibility, and resistant starch content (Table 6).

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DISCUSSION Modern communities are strictly focused on consumption of functional and direct food with desired properties; thus, there is need for search new tools that may be useful in designing such products. In recent years, food legumes have attracted a great deal of attention due to their functional components and healthpromoting effects in relation to the prevention of chronic diseases including cardiovascular diseases, obesity, diabetes, inflammation, and cancer. 1 The role of germination as a method affecting the quality of food is well-documented.3,4,8,27,31 The gradual reduction in phenolic content and the antioxidant potential of lentil sprouts during germination confirmed by previous studies fully justifies the use of elicitation in order to improve the nutraceutical properties of sprouts.3,4,7,8,31 A significant statistical reduction was also observed in the total phenolic, tannin, and flavonoids contents of black soybeans;2 soybeans, lentil, and alfalfa;9 and cowpea, lentil, chickpea, and green gram8 due to the effect of germination. Losses may result from leaching into the soak water and binding of 3311



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rate of condensed tanins degradation and/or the activation of endogenous enzymes (e.g., hydroxylases and polyphenoloxydases), played an important role.3,8 Additionally, higher levels of p-coumaric acid in treated sprouts might be involved in overproduction of coumaryl-CoA, a key compound in flavonoids biosynthesis.5,37 This statement may be also supported by the fact that elicited sprouts contained higher amounts of quercetin. An increase of cell barrier precursors such as p-coumaric, ferulic, and syringic acids was also observed that may be linked with cross-talk response on stress conditions.36 Finally, an increased level of salicylic acid content in sprouts induced by heat and cold shocks may confirm the appearance of stress and the role of this compound in the signal transduction pathway.34,38 Induction of sprouts metabolism by low and high temperature and subsequent elevation of phenolics was translated into an increase of antioxidant potential. Up to now, there are some reports concerning improvement of the nutraceutical potential of sprouts by elicitation. An increase in the free radical scavenging properties of dark germinated mung bean sprouts using natural elicitors, such as fish protein hydrolysates, lactoferrin, and oregano extract, is reported.13 Also, Feng and co-workers 39 obtained a significant elevation in the antioxidant potential of black soybean sprouts by treatment with components of fungal cell walls. In studies of Oh and Rajashekar 40 chilling and high light intensity during growth of radish and alfalfa sprouts caused qualitative and quantitative changes in phenolic profiles and antioxidant activities. The effect of salinity stress on the nutritional quality of buckwheat sprouts was also studied.41 A cited researcher observed an increase in phenolic compounds, carotenoids, and antioxidant capacity in the sprouts compared with the control. In the light of our results and cited studies, it may be stated that elicitation has been demonstrated to be a useful method for improving the antioxidant potential of low-processed foods. Because modification of the growth (elicitation with abiotic and biotic factors) often has a negative impact on the sprouts growth and nutritional quality of food (content and bioaccessibility of nutrition and increased activity of hydrolases inhibitors), an evaluation of the influence of treatments was performed. In recent literature there are only a few reports concerning the influence of elicitation on the nutritional quality of sprouts.10,27 A decrease in the protein content and subsequent elevation of the nonprotein nitrogen fraction may be explained by induction of proteolytic systems in response to stress conditions.8,11,35 As had been reported by previous studies (cross-talk stress response theory), 36 a slight elevation of protease inhibitors activity was observed; however there was not any strong influence. Changes in total starch content in elicited sprouts may be due to modification of seedling metabolism in response to stress conditions, especially with the increased demand for energy needed to produce secondary metabolites. 11 Generally, growth conditions used to modify sprouts metabolism allow one to obtain product with a significantly enhanced phenolics content and antioxidant capacity. What is important is that modification did not affect negatively the growth and nutritional quality of the sprouts. In the light of epidemiological studies these kinds of sprouts (with enhanced antioxidant potential and lowered glycemic index) may be a predisposed product for specific consumers and consist of a useful tool for creating healthy eating habits.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: + 48-81-4623327. Fax: +48-81-4623324. E-mail: michal. [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

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Nutritional and Antioxidant Potential of Lentil Sprouts Affected by

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