Effect of Illumination on the Content of Melatonin, Phenolic

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Article

Effect of Illumination on the Content of Melatonin, Phenolic Compounds and Antioxidant Activity During Germination of Lentils (Lens culinaris L.) and Kidney Beans (Phaseolus vulgaris L.) Yolanda Aguilera, Rosa Liébana, Teresa Herrera, Miguel Rebollo, Carlos Sanchez-Puelles, Vanesa Benítez, and María Ángeles Martin-Cabrejas J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf503613w • Publication Date (Web): 13 Oct 2014 Downloaded from http://pubs.acs.org on October 20, 2014

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Journal of Agricultural and Food Chemistry

Effect of Illumination on the Content of Melatonin, Phenolic Compounds and Antioxidant Activity During Germination of Lentils (Lens culinaris L.) and Kidney Beans (Phaseolus vulgaris L.) YOLANDA AGUILERA, ROSA LIÉBANA, TERESA HERRERA, MIGUEL REBOLLO-HERNANZ,

CARLOS

SANCHEZ-PUELLES,

VANESA

BENÍTEZ,

MARÍA A. MARTÍN-CABREJAS*

Instituto de Investigación de Ciencias de la Alimentación (CIAL). Facultad de Ciencias, Universidad Autónoma de Madrid, C/ Nicolás Cabrera 9, 28049 Madrid, Spain.

* Corresponding author:

[email protected] Tel.: 0034 91 497 8678 Fax: 0034 91 497 3826

1 ACS Paragon Plus Environment


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ABSTRACT

2

This study reports the effects of two different illumination conditions during germination

3

(12 h light/12 h dark vs. 24 h dark) in lentils (Lens culinaris L.) and kidney bean

4

(Phaseolus vulgaris L.) on the content of melatonin and phenolic compounds, as well as

5

the antioxidant activity. Germination led to relative increase in melatonin content and

6

significant antioxidant activity whilst the content of phenolic compounds decreased. The

7

highest melatonin content was obtained after 6 days of germination under 24 h dark for

8

both legumes. These germinated legume seeds with improved levels of melatonin might

9

play a protective role against free radicals. Thus, considering the potent antioxidant

10

activity of melatonin, these sprouts can be consumed as direct foods and be offered as

11

preventive food strategies in combating chronic diseases through the diet.

12

13

14

KEYWORDS

15

Melatonin, phenolic compounds, antioxidant capacity, germination, legumes, sprouts.


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INTRODUCTION

17

Lentils and beans are the most widely consumed legume seeds by a large part of the

18

world’s human population. Many studies have been carried out to determine the role of

19

legumes as preventive agents in the diet of vulnerable populations (diabetes, obesity,

20

cardiovascular diseases, etc). However, the utilization of legumes is limited by the

21

presence of antinutrient compounds, such as protease inhibitors, non-protein amino acids,

22

lectins, saponins and flatulence compounds and, hence, they should be processed before

23

consumption. In this regard, germination has been identified as a low-cost technology to

24

improve the quality of legumes, by enhancing their digestibility and increasing their

25

content of bioactive compounds.1, 2−5 Phenolic compounds have been widely studied as

26

antioxidants due to its ability in querching free radicals contributing to total antioxidant

27

capacity and their protection role against highly prevalent diseases.6,7 Recently, the

28

attenuation of oxidative stress by germinated food consumption is reached through

29

increases in antioxidant levels in plasma and antioxidant enzyme activity in different

30

animal tissues.8 Increases of bioactive compounds during legume germination vary

31

greatly depending on the plant species, seed varieties and germination.9,10 So far, little

32

information has been reported about the effects of germination on melatonin content,

33

hormone that has been described as an effective free scavenger showing antioxidant

34

properties against lipid peroxidation due to its amphipathic nature.11

35

Melatonin (N-acetyl-5-methoxytryptamine), an indolamine, is a ubiquitous and highly

36

conserved molecule occurring in animals (vertebrates and invertebrates), bacteria, mono-

37

and multicellular algae, and plants. Melatonin plays a key role in plant physiology as

38

mechanism to increase the survival and perpetuation of species as circadian regulator,

39

cytoprotector and regulator of plant growth.12,13 Melatonin might be involved into

40

mechanisms of preservation of chlorophyll, promotion of photosynthesis and stimulation 3 ACS Paragon Plus Environment


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of root development. In mammals, besides acting as a regulator of circadian and seasonal

42

rhythms, melatonin is involved in antioxidative systems, vascular tone, and inhibition of

43

infections and tumors.14−16 Melatonin has been detected in plant foods, including cereals,

44

vegetables and fruits and roots.17,18 Indeed, plant tissues seem to contain much higher

45

melatonin levels than those observed in animals.15 Melatonin concentration has been

46

shown to vary from a few picograms to micrograms per gram of plant material.19

47

Nowadays, the beneficial effects of melatonin derived from the consumption of plant

48

foods are being considered on human health.20 In the last years, attention was also paid to

49

incorporate plant foods with high melatonin levels as a dietary supplement to increase its

50

blood plasma levels in humans and consequently, its scavenging and antioxidant action.21

51

Thus, we have focused this work on the impact of germination conditions in the

52

melatonin and phenolic compound content, as well as the antioxidant activity of legumes.

53

The objectives of the present study were to examine melatonin content under two

54

different illumination germination conditions (12 h light/12 h dark and 24 h dark) for 3, 6

55

and 8 days in two common legumes (Lens culinaris L. and Phaseolus vulgaris L.).

56

Moreover, the content of phenolic compounds was determined beside melatonin

57

to evaluate their influence on the antioxidant activity as a strategy to design novel foods

58

for improving consumer’s health.

59

MATERIALS AND METHODS

60

Plant materials

61

Two varieties of common legumes: lentil (Lens culinaris L., var. Salmantina) and kidney

62

bean (Phaseolus vulgaris L., var. Pinta) were provided by Institute of Food Science,

63

Technology and Nutrition (CSIC, Madrid). They were stored in dark and dry conditions at

64

refrigeration.

65

Processing conditions


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Firstly, three batches of seeds (20 g of lentils and 50 g of beans) were sterilized in 1%

67

NaClO solution for 30 min to reduce bacterial and fungal growth. Seeds were washed

68

three times with distilled water and then soaked in distilled water for 5 h for seed

69

hydration (ratio 1 g: 20 mL seed: water). They were germinated in an incubator

70

(Construcciones Frigoríficas, Confri, S.L.) at 20 ºC and 80 % RH, illumination conditions

71

of photoperiodic cycle (12 h light/12 h dark) and darkness (24 h dark) for 3, 6 and 8 days.

72

Lentil and kidney bean sprouts were withdrawn, frozen in N2, freeze-dried and milled.

73

Flours were passed through a 50 µm sieve and stored at 4-6 ºC for further analysis.

74

Melatonin extraction and quantification by HPLC-MS/MS

75

Extraction procedure was conducted according to Manchester et al.22 and Ansari et al.23

76

with some modifications. Briefly, lentil and kidney bean flours (2 g) were extracted with

77

10 mL of methanol (MeOH). Extracts were stored for 16 h at 4 ºC in darkness. After a

78

brief centrifugation at 2000 g for 15 min at room temperature; the supernatants were

79

filtered under vacuum by a filter (11µm, Whatman) and they were evaporated to dryness

80

under N2 (g). The residues were resuspended in 2 mL of water, after a mild ultrasonic

81

treatment for 2 min at room temperature; the reconstituted extracts were filtered using the

82

solid phase extraction (SPE, cartridge C-18, Waters). The extracts obtained were

83

evaporated to dryness by use of evaporator centrifuge (Speed Vac SC 200, Savant, USA).

84

The residues were dissolved in MeOH/ H20 (80: 20; v/v) + 0.1 % formic acid. Melatonin

85

was determined by HPLC-ESI-MS/MS triple quadrupole (Varian 1200L with API-ES

86

between 10 and 1500 Da range mass). Melatonin was recorded using multiple reaction

87

monitoring (MRM) mode by selecting ion 233 (M + H+) at the first quadrupole (Q1),

88

fragmented in Q2 using Ar and analyzing resulting ion m/z 130, 131, 159, and 174 at Q3,

89

measuring at 174 (Figure 1). The system consisting of two pumps Varian Prostar 210 and

90

Intensity Trio C18, (150mm x 2.0 mm ID, S-3µm, 12nm BRYTC18032150) (Brucker)



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column, at a flow rate of 0.2 mL/min. Acetonitrile containing 0.1 % formic acid (solvent

92

A) and 0.1 % formic acid in H20 (solvent B) as mobile phase were pumped with the

93

following gradient: 5 % solvent A (0-4 min), 5-100 % solvent A (4-15 min), 100 %

94

solvent A (15-18 min) and 100-5 % solvent A (18-25 min) to recover initial conditions.

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The injection volume was 50 µL. Quantification was performed using external standard

96

method, using pure melatonin (Sigma-Aldrich Química, Spain) dissolved in MeOH/ H20

97

(80:20; v/v) containing 0.1 % formic acid, showing good regression coefficient (R2 =

98

0.9989). Samples and standard solutions were analyzed by triplicate. Melatonin content

99

was expressed as ng/g dry weight (DW).

100

Phenolic compounds extraction and determination

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The extraction of phenolic compounds was carried out following Gou et al.24 method with

102

some modifications. Germinated flours (2 g) were blended using 30 mL of 80% chilled

103

acetone (1:2, w/v) for 5 min and vortex for 10 min. The homogenates were then

104

centrifuged at 2054 g for 5 min. Supernatants were collected, and the extraction was

105

repeated three times. All the supernatants were pooled and evaporated until 10% of the

106

supernatants. The soluble phytochemical extracts were brought to 10 mL in water and

107

were kept at -40 °C until analysis. For the extractions of bound phytochemicals, the

108

residues from above soluble free extraction were flushed with N2 and hydrolyzed directly

109

with 20 mL of 4 M NaOH at room temperature for 1 h with shaking. The mixture was

110

acidified to pH 2 with concentrated HCl, centrifuged at 2958 g for 5 min, and extracted

111

three times with ethyl acetate. The ethyl acetate fractions were evaporated to dryness, and

112

were reconstituted into 1.5 mL of methanol and stored at −40 °C until analysis.

113

The phenolic compounds were determined by Folin–Ciocalteu colorimetric method

114

according to Singleton, Orthofer and Lamuela-Raventos25 using gallic acid (GAE) as

115

standard. Phenolic compounds were expressed as mg GAE/ 100g DW.


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Oxygen Radical Absorbing Capacity (ORAC) assay

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The above methanol extracts were used for determining the radical absorbance activity by

118

ORAC method using fluorescein as a fluorescence probe.26 Briefly, the reaction was

119

carried out at 37 °C in 75 mM phosphate buffer (pH 7.4) and the final assay mixture (200

120

µL)

121

dihydrochloride (12 mM), and antioxidant standard (Trolox [10-80 µM] or sample

122

extracts). A Polarstar Galaxy plate reader (BMG Labtechnologies GmbH, Offenburg,

123

Germany) to read fluorescence at wavelengths of 485nm excitation and 520nm emission

124

was used. The equipment was controlled by the Fluostar Galaxy software version (4.31-0)

125

for fluorescence measurement. Black 96-well untreated microplates (PS Balck, Porvair,

126

Leatherhead, UK) were used. The plate was automatically shaken before the

127

preincubated, and the fluorescence was recorded every minute for 80 min. All reaction

128

mixtures were prepared in duplicate and at least 3 independent runs were performed for

129

each sample. Fluorescence measurements were normalized to the oxidation control

130

(phosphate buffer) and stability control (no antioxidant). From the normalized curves, the

131

area under the fluorescence decay curve (AUC) was calculated as:

contained

fluorescein

(70

nM),

2,2-azobis

(2-methyl-propionamidine)-

i=80

AUC = 1 + ෍ fi / f0 i=1

132

Where f0 is the initial fluorescence reading at 0 min and fi is the fluorescence reading at

133

time i. The net AUC corresponding to a sample was calculated as follows:

134

net AUC = AUC antioxidant−AUC blank

135

The net AUC was plotted against the antioxidant concentration and the regression

136

equation of the curve was calculated. The ORAC value was obtained by dividing the

137

slope of the latter curve between the slopes of the Trolox curve obtained in the same



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assay. Final ORAC values were expressed as µmol of Trolox equivalents/g of dry sample

139

(µmol TE/g DW).

140

Statistical analysis

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Each sample was analyzed in triplicate. Data were expressed as mean ± standard

142

deviation (SD). The data were analyzed by-one way analysis of variance (ANOVA) and

143

post-hoc Duncan test. Differences were considered to be significant at p < 0.05. The

144

statistical analysis was performed by SPSS 17.0.

145

RESULTS AND DISCUSSION

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Germination process

147

The changes in biomass and percents of germination under different germination

148

conditions are shown in Table 1. Legume biomass increased from 111% to 162% and

149

lentil sprouts showed higher levels than bean sprouts at the same germination conditions.

150

The highest rise was reached in lentil under 8th day 12 h light/12 h dark. The success of

151

this processing exhibited good viability for both legumes, reaching 100% in case of bean

152

sprouts at 12 h light/12 h dark on the 8th germination day. However, lentil exhibited

153

lower germination percentage on the 8th day due to fungal growth. The parameter of

154

development of radicle was different not only dependent on the kind of leguminous seeds,

155

but also on process conditions (i.e. the presence of light and germination time). The

156

length of bean radicle was higher than of lentils, irrespective of light conditions and

157

germination time. These differences could be attributed to changes on bioactive

158

compounds (melatonin) that promote vegetative plant growth.12

159

Melatonin

160

Different methods have been developed for melatonin identification in plant extracts.

161

Melatonin was detected in raw lentil and kidney beans varieties studied and

162

chromatograms are shown in Figure 2. From the results, kidney beans exhibited higher


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melatonin

content

than

lentils

(1.0

and

0.4

ng/g

DW,

respectively).

164

These data explain the different elongation showed by legume sprouts that could be due

165

to melatonin levels. This molecule exhibits auxinic activity in a similar way to indole-3-

166

acetic acid (IAA). 12 In comparison to other plant foods, the results obtained were higher

167

than those reported in other seeds like sweet corn and rice,27 being a good dietary

168

melatonin source. The available scientific data of melatonin in foods are scarce because a

169

few food items have been analyzed. Melatonin levels are greatly variable between plant

170

species, apart from seeds, fruits (12-36 pg/g WW), roots protected from light as carrots

171

and onions or processed foods as fermented drinks (0.1-35 ng/mL) and olive oil (108

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pg/mL) are also natural sources of melatonin.19

173

Germination brought about a noticeable increase in melatonin (p < 0.05) and this effect

174

was time-dependent in all seeds studied (Figure 3). It was reported higher melatonin

175

levels at complete darkness28 and relative increases in cucumber and red cabbage

176

germinated seeds.29,30 To our knowledge, the present work is the first report on the

177

melatonin content of lentil and bean sprouts to improve the content of this bioactive

178

compound. In this sense, melatonin content increased significantly in lentil sprouts during

179

both germination process, showing similar values under 12 h light/12 h dark and 24 h

180

dark conditions (Figure 3a). In both illumination conditions, the highest contents were

181

shown on the 6th germination day (2.3 and 2.5 ng/g DW under 12 h light/12 h dark and

182

24 h dark, respectively). After the 6th germination day, the levels of melatonin decreased

183

in lentil sprouts, being more pronounced under 24 h dark (72% of decrease respect to the

184

6th germination day). Similar behavior was found in bean sprouts (Figure 3b). On the 6th

185

processing day, germination significantly influenced melatonin accumulation in both

186

germination conditions, up to 2.4-fold at 12 h light/12 h dark and more accentuated, up to

187

9.4-fold at 24 h dark conditions. Finally, it is worth emphasizing that a sharp melatonin



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decrease (94%) from the 6th to 8th germination day was observed at 24 h darkness (0.6

189

ng/g DW) and less accentuated (38% of decrease) at 12 h light/12 h dark (1.5 ng/g DW).

190

Thus, germination in darkness for 8 days did not cause melatonin increase either in lentils

191

or beans. Our data were in agreement with those of Zielinski et al.,31 who found contents

192

of melatonin in lentil, vetch and soybean seeds as well as increases in melatonin during

193

seven days of germination. Hence, melatonin content in lentil showed maximum levels at

194

6th germination day either at 12 h or 24 h dark while melatonin content in bean reached

195

the highest content at 24 h dark. From our results, melatonin was mainly influenced by

196

photoperiods; darkness regimes led to more this bioactive compound in kidney bean and

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these results were in agreement with those of Murch et al.27

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The melatonin originated from these sprouts may play a role as a health promoting

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substance with clear antioxidant and anti-inflammatory properties; its genomic effects,

200

and its capacity to modulate mitochondrial homeostasis, are linked to the redox status of

201

cells and tissues.16 From our results, the sprout intake, especially germinated beans, could

202

be considered as a food source of dietary melatonin.

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Phenolic compounds

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The free, bound (cell wall-associated) and total phenolic compounds (TPC) of lentil and

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bean sprouts are shown in Figure 4. TPC in raw lentil and bean seeds were 488 and 379

206

mg GAE/100g DW, respectively. These legume seeds seem to be a good source of

207

phenolic compounds and our results are within the range of previously reported data.32−34

208

The predominant phenolic compounds in lentils and beans are catechins and

209

procyanidins;35,36 however, the phenolic composition of legume seeds may vary among

210

cultivars and genotypes. Differences between them may be attributed to differences in the

211

color of the seed coat as has been reported in the literature,32 lentil was darker than kidney

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bean. Lentil seeds showed 88% of free phenolic contents respect to TPC (Figure 4a);


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while kidney bean seeds exhibited a higher percentage of free phenols (92%) and

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consequently, a lower percentage of bound phenols (8%) (Figure 4b). These results are in

215

agreement to those found in other legume seeds.24,37

216

Our results exhibited that TPC decreased dramatically in lentil sprouts at both darkness

217

germination conditions (12 h and 24 h dark), while kidney bean sprouts exhibited relevant

218

decreases at 12 h light/12 h dark but no relevant changes at 24 h dark germination

219

conditions. These results are in agreement with the findings of Khandelwal et al.32 and

220

Troszyńska et al.38

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Respect to lentil, decreases of phenolic compounds (total, free and bound forms) were

222

initiated at 3rd germination day and maintained during germination conditions (12 and 24

223

h dark) (Figure 4a). The germination process of 12 h light/12 h dark reduces significantly

224

up to 72% respect to raw lentil on the 6th germination, but any further process not

225

exhibited any significant changes (p < 0.05). In this sense, the concentration of free

226

phenolic acids showed the maximum decrease on the 6th germination day (from 450 to

227

124 mg GAE/100g DW). Decreasing tendency was also observed in bound phenols, being

228

more accentuated on the 3rd germination day (from 38 to 5 mg GAE/100g DW).

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Regarding 24 h dark lentil germination, a similar trend was observed but lower reduction

230

in TPC was detected (50% of decrease at the 6th germination respect to control). This fact

231

may be due to the relative amounts of free phenolic compounds present in these sprouts

232

and also, the significant increases of bound phenolics (up to 1.6-fold respect to raw lentil

233

seed) exhibited under 24 h darkness (Figure 4a). Although this fraction is hardly

234

noticeable in both legumes, bound phenols are more likely glycoside flavonols, subjected

235

to the action of colonic microflora, releasing aglycones that might be absorbed in a lesser

236

extent and, probably, degraded to simpler phenolic derivatives and thus, they may play a

237

special role on health.39



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Concerning kidney beans, 12h light/12h dark led to a TPC reduction in a time-dependent

239

manner (Figure 4b). The contents of free phenolic under 12 h light/12 h dark exhibited

240

decreases (70% reduction at the 3rd germination day as compared to raw bean) and

241

contributed up to 93% of TPC of bean sprouts. Bean sprouts at 24 h dark exhibited an

242

increase of TPC due to the increases either of free or bound phenol forms but there were

243

not significant (p < 0.05) in a time-dependent manner. A similar trend was also observed

244

in germinated black beans (Phaseolus vulgaris L.) with no significant difference of

245

phenolic contents among them.34 However, decreases in phenolic contents of legumes

246

following germination have been reported by several authors.32,40 These decreases may be

247

attributed to the increases in the activity of enzymes responsible for the oxidation of

248

endogenous phenolic compounds and other catabolic enzymes in raw and processed

249

legumes. During germination, enzymes are activated, resulting in the hydrolysis of

250

various components, including carbohydrate, protein, fiber and lipid, as well as phenolic

251

compounds.32 Thus, the phytochemical quality of sprouts depends on many factors such

252

as legume cultivar and germination conditions. This means that optimum germination

253

conditions needs to be defined for legume seeds to improve the functional quality of the

254

sprout.

255

Antioxidant activity

256

The antioxidant capacity of lentil and bean seeds (Figure 5) was similar between them

257

(20.2 and 24.0 µmol TE/g DW, respectively). The results obtained were lower than those

258

reported by Xu et al.41 and Aguilera et al.35 but similar to Xu and Chang37 and Guajardo-

259

Flores et al.34 in common beans. In general, germination brought about an enhancement

260

of the antioxidant potential of legumes, in agreement with other studies.7,10,34,42

261

Germination time directly affected the antioxidant activity of lentil and bean sprouts. The

262

antioxidant activity of both sprouts increased with longer germination time (p < 0.05),


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except at day 6. Among legume seeds, antioxidant activity also varied following different

264

germination conditions. ORAC values were up to 2.4-fold and 2.2-fold higher after

265

sprouting on the 8th germination day at 12 and 24 h dark for lentil sprouts, respectively.

266

In general, lower increases were detected in bean sprouts (1.6 and 1.9-fold on the 6th

267

germination day at 12 and 24 h dark, respectively). In addition, it should be pointed out

268

that different light conditions during germination significantly influenced the antioxidant

269

capacity of sprouts.43 Among all of the sprouted legumes tested, it was noted that both

270

samples germinated on the 6th germination day under 12 and 24 h dark showed the lowest

271

antioxidant activities, with values ranged from 14.8 to 18.9 µmol TE/g DW. Interestingly,

272

these samples corresponded to those exhibited the lowest phenolic compound levels but

273

the maximum contents of melatonin. In general, the antioxidant properties of melatonin

274

and its ability to scavenge several radical species as part of its bioactivity have been

275

described in the literature.44,45 Melatonin has been found to neutralize the most toxic

276

oxidizing agents generated within cells, such hydroxyl radical (•OH) and peroxynitrite

277

anion (ONOO-). In addition, melatonin reportedly scavenges singlet oxygen (1O2),

278

superoxide anion radical (O2•–), hydrogen peroxide (H2O2), nitric oxide (NO•) and

279

hypochlorous acid (HClO).46

280

Finally, after the 8th germination day, a significant increase of antioxidant activity was

281

also detected for both legume sprouts as mentioned before. Thus, this effect could be

282

attributed to higher accumulation of compounds as melatonin degradation with radical

283

scavenging activity. Longer application of germination resulted in further increment of

284

antioxidant capacity, showing major ability of sprouts to prevent oxidative stress on the

285

8th germination. The pronounced values of antioxidant capacity on eight day have an

286

inverse correlation with values of melatonin in sprouts (r = 0.61-0.75, p < 0.01). This

287

behavior may result from the indirect antioxidant properties of melatonin, as the



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288

activation of several antioxidant enzymatic cascades, including glutathione peroxidase,

289

superoxide dismutase and glutathione reductase and the accumulation of degradation

290

products, highly effective scavengers (AFMK, AMK and cyclic 3-hydroxymelatonin),46,47

291

bringing about the increased antioxidant capacity levels of bean sprouts on the 8th

292

germination day.48 Thus, the legume sprouts obtained in this study are valuable sources of

293

natural antioxidants that most likely could positively influence the overall antioxidant

294

status in humans.

295

Germination led to decreases on phenolic compounds and relative improvements in

296

melatonin levels, bringing about significant increases of antioxidant activity in lentil and

297

bean sprouts. Optimal germination conditions for melatonin levels seemed to be under 24

298

h dark at the 6th germination day for both sprouts, even though antioxidant activity

299

increased on the 8th day. These germinated legume seeds with improved levels of

300

melatonin might play a role because of the potential enhancement of the nutraceutical

301

value of these seeds. Thus, considering the potent antioxidant activity of melatonin, these

302

sprouts can be consumed as direct foods or adopted in the future consumer’s food

303

practices and be offered as preventive food strategies in combating chronic diseases

304

through the diet.

305

ABBREVIATIONS USED

306

AFMK

N(1)-acetyl-N(2)-formyl-5-methoxykynuramine

307

AMK

N(1)-acetyl-5-methoxykynuramine

308

DW

Dry Weight

309

GAE

Gallic Acid Equivalents

310

HPLC-MS/MS

High-performance

311

spectrometry

312

MRM

liquid

chromatography

tandem

mass

Multiple Reaction Monitoring 14 ACS Paragon Plus Environment

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313

TE

Trolox Equivalents

314

TPC

Total Phenolic Compounds

315

WW

Wet Weight

316 317

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318

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319

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2. Martín-Cabrejas, M.A.; Díaz, M.F.; Aguilera, Y.; Benítez, V.; Mollá, E.; Esteban, R.M.

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Influence of germination on the soluble carbohydrates and dietary fibre fractions in

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non-conventional legumes. Food Chem. 2008, 107, 1045-1052.

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3. Vadivel, V.; Stuetz, W.; Scherbaum, V.; Biesalski, H.K. Total free phenolic content

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and health relevant functionality of Indian wild legume grains: Effect of indigenous

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FINANCIAL SUPPORT

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This research was financially supported by Second Call for Interuniversity Cooperation

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Projects University Autónoma de Madrid and Santander Bank with United States (2013-

470

2014).



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FIGURE CAPTIONS Figure 1. Melatonin spectrum and mass accuracy Figure 2. HPLC-MS/MS chromatograms of raw lentil (a), and raw kidney bean (b) Figure 3. Melatonin content in raw and germinated lentils (a) and kidney beans (b) Figure 4. Bound, free and total phenolic compounds in raw and germinated lentils (a) and kidney beans (b) Figure 5. Antioxidant activity in raw and germinated lentils (a) and kidney beans (b)


Page 23 of 30


Table 1. Changes in Seed/seedlings Biomass and Percent of Germination at Different Illumination Conditions of Germination

Illumination

% Increase in Germination

Legume

conditions of

Development of fresh weight of

% Germination

time (day) germination Lentil

radicle (cm) seeds/seedlings

3

137 ± 8a

92 ± 8b

1.4 ± 0.1 a

6

141 ± 9a

58 ± 5a

2.6 ± 0.1 b

8

162 ± 5b

59 ± 5a

4.5 ± 0.2 c

3

138 ± 5a

83 ± 7b

1.4 ± 0.1 a

6

150 ± 6b

83 ± 7b

4.1 ± 0.2 c

8

156 ± 10b

65 ± 6a

7.3 ± 0.2 d

3

104 ± 6a

47 ± 3a

1.0 ± 0.1 a

6

126 ± 9b

84 ± 5b

3.7 ± 0.2 b

8

133 ± 9c

100 ± 4c

6.1 ± 0.4 d

3

90 ± 7a

46 ± 4a

1.9 ± 0.1 a

6

104 ± 8a

89 ± 5b

5.6 ± 0.2 c

8

111 ± 9a

96 ± 5b

10.6 ± 0.6 e

12 h light/12 h dark

24 h dark

Bean 12 h light/12 h dark

24 h dark

Mean ± SD (n = 3) Mean values of each row of different illumination conditions followed by different superscript letter significantly differ when subjected to Duncan’s multiple range test (p

Effect of Illumination on the Content of Melatonin, Phenolic

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