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Accepted Manuscript Melatonin, phenolics content and antioxidant activity of germinated selected legumes and their fractions Hend M. Saleh, Amal A. Hassan, Esam H. Mansour, Hany A. Fahmy, Abo ElFath A. El-Bedawey PII: DOI: Reference:

S1658-077X(17)30185-6 http://dx.doi.org/10.1016/j.jssas.2017.09.001 JSSAS 285

To appear in:

Journal of the Saudi Society of Agricultural Sciences

Received Date: Revised Date: Accepted Date:

15 June 2017 17 August 2017 8 September 2017

Please cite this article as: Saleh, H.M., Hassan, A.A., Mansour, E.H., Fahmy, H.A., El-Fath A. El-Bedawey, A., Melatonin, phenolics content and antioxidant activity of germinated selected legumes and their fractions, Journal of the Saudi Society of Agricultural Sciences (2017), doi: http://dx.doi.org/10.1016/j.jssas.2017.09.001

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Melatonin, phenolics content and antioxidant activity of germinated selected legumes and their fractions

Hend M. Saleh1, Amal A. Hassan2, Esam H. Mansour2*, Hany A. Fahmy1, and Abo El-Fath A. El-Bedawey2

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Special Food and Nutrition Department, Food Technology Research Institute, Agricultural Research Center, Giza, Egypt

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Department of Food Science and Technology, Faculty of Agriculture, Menoufia University, 3251 Shibin El-Kom, Egypt

*Corresponding author E-mail:[email protected] Tel: (+02) 01222328188 Fax: (+02) 048-2228187

Melatonin, phenolics content and antioxidant activity of germinated selected legumes and their fractions

Abstract This study was aimed to evaluate the effects of germination process on melatonin, total phenols, total flavonoids and antioxidant activities of broad beans (Vicia faba L.), lupine seeds (Lupinus albus), chickpea seeds (Cicer arietinum L.), lentil seeds (Lens culimaris), fenugreek seeds (Trigonella foenum-graecum L.) and common beans (Phaseolus vulgaris) fractions (cotyledons, radicles and seed hulls). Radicle length of germinated legumes and sensory properties of legumes after 6 days of germination were also evaluated. Fenugreek and chickpea seeds had higher melatonin (54.22 and 24.42 ng/g), total phenols (5.79 and 5.68 mg gallic acid /g) and total flavonoids (8.86 and 8.43 mg quercetin /g), respectively than other legumes however, common beans showed the lowest values. Broad beans and lentil seeds had the highest antioxidant activities while lupine seeds showed the lowest value among all other legumes. The mean values of melatonin for cotyledons, radicles and seed hulls were increased by 386.26, 261.98 and 183.22%, respectively compared with the third day of germination. The increases in melatonin content of legume fractions were parallel to the increases in radicle lengths, total phenols, total flavonoids and antioxidant activities throughout the germination time. Overall acceptability of legumes had rating scores ranged between like moderately and like very much except for chickpea which had rating score described as like slightly. Key words: Melatonin, legumes, germination, radicle length, total phenols

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1. Introduction Melatonin (N-acetyl-5-methoxytryptamine) is synthesized from tryptophan in mammalian pineal gland during the night (Huether et al., 1992). Melatonin is able to catalyze endogenous antioxidant enzymes such as catalase and superoxide dismutase. A positive correlation was found between plasma melatonin level and oxidative stress (Rodriguez et al., 2004, Retier et al., 2005). Melatonin has antioxidant properties as catabolites or/and a direct free radical scavenger (Reiter et al., 2009, Galano et al., 2013, Tan et al., 2014). Melatonin has several biological effects such as immune defense, anti-inflammation, metabolic syndrome and antitumor activities (Kitagawa et al., 2012, Calvo et al., 2013, Xin et al., 2015, Farez et al., 2016). Melatonin is detected in seeds, roots, fruits, and leaves in plants (Hattori et al., 1995). The role of melatonin in plants is similar to that in animals (Posmyk and Janas, 2009). Its content in plant tissues was much higher than in animal tissues (Tan et al., 2012). Legumes are good and economical sources of protein, minerals, vitamins B and bioactive compounds thus newly received growing interest. Germination enhances the nutritive value of legumes by forming enzymes which reduce or remove the anti-nutritional and indigestible factors in legumes (Mansour and EL-Adawy, 1994, Khalil and Mansour, 1995). Bioactive compounds such as phenolic compounds and melatonin are increased during germination of legumes and their levels vary depending on the plant species, seed varieties, and germination conditions (Paucar-Menacho et al., 2010, Aguilera et al., 2015). Consumption of germinated seeds reduced oxidative stress by increasing the antioxidant levels in blood plasma and antioxidant enzyme activities in different animal tissues (Mohd Esa et al., 2013). Little information is available on the effect of germination process on melatonin content in edible seeds (kidney beans, lentil seeds, alfalfa, chicory, rape, red kale and sunflower seeds) (Kim and Cho, 2011, Aguilera et al., 2014 and 2015). To the best of our knowledge there are no reports concerning the effect of germination process on the melatonin

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content in broad beans, lupine seeds chickpea seeds and fenugreek seeds. Therefore this work was conducted to evaluate the effects of germination time in the dark at room temperature (~25°C) for 6 days on melatonin content, total phenols, total flavonoids and antioxidant activities of broad beans, lupine seeds, chickpea seeds, lentil seeds, fenugreek seeds and common beans fractions (cotyledons, radicles and seed hulls). Radicle length of selected legumes and sensory properties of legumes after 6 days of germination were also evaluated.

2. Materials and methods 2.1 Legumes Three batches (5 kg of each batch) of Broad beans (Vicia faba L.), lupine seeds (Lupinus albus), chickpea seeds (Cicer arietinum L.), lentil seeds (Lens culimaris), fenugreek seeds (Trigonella foenum-graecum L.) and common beans (Phaseolus vulgaris) were grown during the season of 2015, and obtained from Field Crops Research Institute, Agricultural Research Center, Giza, Egypt. Legumes were cleaned by hand to remove foreign materials and stored in polyethylene containers at 4°C until use. 2.1.1 Germination process Legumes were germinated according to Khalil and Mansour (1995). Three batches of each legume (200 g/batch) were sterilized by soaking in ethanol for 1 min. The seeds were washed by distilled water then soaked in distilled water (1:10 w/v) for 12 h at room temperature (~25°C). The soaked seeds were kept between thick layers of cotton cloth and allowed to germinate in the dark at room temperature (~25°C) for 6 days. The germinated seeds were rinsed with distilled water. Germinated seeds were separated by hand into cotyledons, radicles and hulls and dried separately at 50°C overnight in an electric draught oven. Raw legumes and dried germinated legume fractions were ground to pass through a 60 mesh sieve and stored in screw cap plastic containers at 4°C.

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During germination process, sample from each legume was taken daily from the third day up to the sixth day of germination for determining radicle length as well as melatonin, total phenols, total flavonoids and antioxidant activity of legume fractions. 2.2 Preparation of extracts Raw legumes and germinated legume fractions extracts were prepared as described by Cao et al. (2006). Two gram of raw legumes and germinated legume fractions were extracted with 10 ml methanol for 45 min in darkness using sonication in an ultrasonic bath (Bandelin electronic, RK 514 H, Berlin, Germany). The mixture was centrifuged at 4500×g for 10 min (Harrier18/80 refrigerated MSE centrifuge, UK). The supernatant was filtered through a 0.45 µm filter (Hydrophilic PTFE, Millipore, Mississauga, Canada). The residue was washed twice with methanol, thus the filtrates combined and adjusted to a final volume of 10 ml. The resultant extract was used for determining melatonin, total phenol and total flavonoid contents. 2.3 Determination of melatonin Melatonin was determined by HPLC Agilent (Series 1200) equipped with auto-sampling injector, solvent degasser, ultraviolet (UV) detector set at 250 nm and quaternary HP pump (series 1050). The column temperature was maintained at 30°C. Chromatographic condition was applied as described by Lin et al., (2012) with some modification. An isocratic mobile phase consisted of methanol: acetonitrile: 0.5% acetic acid solution (4:1:5, v/v/v) and its flow rate was 0.5 ml/min. UV detection wave length was 250 nm. Injection volume was 10 µL. The column oven temperature was set at 30°C. Melatonin quantification was performed using melatonin standard (Sigma-Aldrich, St. Louis, MO, USA) dissolved in methanol 80% containing 0.1% formic acid. Melatonin content was expressed as ng/g dry weight sample. 2.4 Determination of total phenols

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Total phenols content was measured by Folin-Ciocalteu reagent (LOBA chemie, India) according to the method of Singleton and Rossi (1965) with some modification. Two hundred microliters of extract was mixed with 1 mL of diluted Folin-Ciocalteu reagent, after standing for 8 min at 24°C, 0.8 mL of sodium bicarbonate (7.5%) solution was added to the mixture. After standing in the dark for 30 min at 24°C, absorbance was measured at 765 nm using T60 UV-visible Spectrophotometer (Leicestershire LE17 5BH, UK). Total phenols content was expressed as mg gallic acid/g dry weight sample. 2.5 Determination of total flavonoids Aluminum chloride colorimetric method was used for the determination of total flavonoids as described by Zhishen et al. (1999). The absorbance of the reaction mixture was measured at 510 nm using T60 UV-visible Spectrophotometer (Leicestershire LE17 5BH, UK). Total flavonoids content was expressed as mg quercetin/g dry weight sample. 2.6 Determination of antioxidant activity The antioxidant activity of legume extracts was measured using 1, 1-diphenyl-2picrylhydrazyl, DPPH, (LOBA chemie, India) as free radical substrate according to the procedure described by Braca et al., (2002). The absorbance of the reaction mixture was measured at 517 nm using T60 UV-visible Spectrophotometer (Leicestershire LE17 5BH, UK). Control was prepared using 50 μL of 80% methanol instead of the extract. The percentage of inhibition of the DPPH radical by the extracts (antioxidant activity) was calculated according to the following equation: % inhibition = (A – B) ÷ A × 100 A = the absorbance of the control and B = the absorbance of the extract sample. 2.7 Sensory evaluation of germinated legumes Sensory properties of germinated legumes were carried out by ten-trained panellists who represented graduate students and staff members in the Department of Food science and

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Technology, Faculty of Agriculture, Menoufia University, Shibin El-Kom, Egypt. Randomly coded samples were served to panelists individually. Five sensory attributes were evaluated (appearance, taste, odor, color, texture and overall acceptability) using nine-points hedonic scale for each trait where 9 = like extremely, 8 = like very much, 7 = like moderately, 6 = like slightly, 5 = neither like nor dislike, 4 = dislike slightly, 3 = dislike moderately, 2 = dislike very much and 1 = dislike extremely. The observations were converted to equivalent numerical scores. 2.8 Statistical analysis Data were presented as mean of three replicates and two determinations for each replicate ± standard deviations. Melatonin, total phenols, total flavonoids and antioxidant activity of selected legumes were analyzed using one-way analysis of variance. Two way randomized blocks design was used for germinated legumes data. Comparisons among means were performed using LSD test. The differences were considered significant at the 5% level (p≤0.05) using a Statistical Analysis System (SAS, 2008).

3. Results and discussion 3.1 Melatonin, total phenols, total flavonoids and antioxidant activity of selected raw legumes Melatonin, total phenols, total flavonoids and antioxidant activity of selected raw legumes are presented in Table 1. Raw legumes showed considerable variations in their melatonin, total phenols, total flavonoids and antioxidant activity. Fenugreek seeds followed by chickpea seeds had the highest (p≤0.05) melatonin, total phenols and total flavonoids as compared with other legumes. However, common beans had the lowest (p≤0.05) corresponding compounds. Although fenugreek seeds contained the highest melatonin (54.22 ng/g), total phenols (5.79 mg gallic acid /g) and total flavonoids (8.86 mg quercetin /g), the broad beans had the highest

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(p≤0.05) antioxidant activity (92.67%) followed by lentil seeds (90.32%). On the other hand, lupine seeds showed the lowest antioxidant activity (78.29%) followed by common beans (84.52%). Comparable melatonin value (43 ng/g) was reported by Manchester et al. (2000) for fenugreek seeds. Melatonin values of common beans (5.85 ng/g) and lentil seeds (4.79 ng/g) were much higher than the values reported by Aguilera et al. (2015) for kidney beans (1.0 ng/g) and lentil seeds (0.5 ng/g). Also they found that total phenolic contents in lentil seeds and kidney beans were 487.5 and 379.1 mg gallic acid/100g, respectively. Salem et al. (2014) reported that faba beans, chickpea, lentil and fenugreek seeds contained 33.65, 57.94, 60.39 and 56.14 mg gallic acid /g total phenols, 6.39, 5.54, 5.54 and 6.58 mg quercetin /g total flavonoids and 95.16, 72.80, 81.65 and 90.69% antioxidant activity, respectively. The total phenolic compounds of broad beans (5.04 mg gallic acid /g) and lupine seeds (5.38 mg gallic acid /g) were lower than the value (7.11 mg gallic acid /g) reported by Boudjou et al. (2013) for faba beans and the value (8.56 mg gallic acid /g) reported by Dueñas, et al. (2009) for lupine seeds. These differences might be due to interspecies variation, growing conditions, storage conditions and extraction procedures.

3.2 Radicle length of legumes Radicle length of legumes was affected (p≤0.05) by germination time and legume types (Table 2). Radicle length of legumes was significantly (p≤0.05) increased by increasing germination time. Similar results were reported by Xue et al. (2016) for mung beans, black beans and soybeans and Aguilera et al. (2014, 2015) for lentil seeds and kidney beans. At the sixth day of germination, the mean of radicle length of legumes was increased by 85.06% as compared with the third day of germination. There were significant (p≤0.05) differences in radicle length among legumes. Lupine seeds had the highest (p≤0.05) mean radicle length

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(6.5 cm) followed by fenugreek seeds (5.46 cm). However, common beans showed the lowest mean radicle length (3.24 cm) followed by lentil seeds (3.74 cm). Aguilera et al. (2014) reported that kidney beans had higher radicle length (1.9-10.6 cm) than lentil seeds (1.4-7.3 cm) throughout germination period. Xue et al. (2016) reported much higher radicle length values for mung beans (24.77 cm), soybeans (22.93 cm) and black beans (18.50 cm) after six day of germination.

3.3 Melatonin of germinated legumes Melatonin contents of legumes were affected (p≤0.05) by germination time and legume fractions (Table 3). Melatonin contents of legume cotyledons, radicles and hulls were significantly (p≤0.05) increased as germination time progressed. This increase could be attributed to the synthesis of melatonin during germination. Similar increases in melatonin content during germination period were reported by Aguilera et al. (2014, 2015) for kidney beans and lentil seeds and Kim and Cho (2011) for alfalfa, chicory, rape, red kale and sunflower seeds. The increases in melatonin content in legume fractions were parallel to the increase in radicle length throughout germination time (Table 2). Aguilera et al. (2015) reported that positive correlations were found between length of the sprouts and melatonin content (r = 0.877−0.879) in endosperms and radicles of lentil seeds. Mean melatonin content of legumes was increased in the following order 386.26% for cotyledons, 261.98% for radicles and 183.22% for seed hulls as compared with the third day of germination. Mean melatonin contents of legume cotyledons were ranged in the following order chickpea seeds (250.06 ng/g), fenugreek seeds (122.07 ng/g), common beans (96.54 ng/g), lupine seeds (92.83 ng/g), lentil seeds (53.56 ng/g) and broad beans (7.79 ng/g). There was no significantly (p>0.05) difference in mean melatonin content in legume cotyledons between common beans and lupine seeds. Mean melatonin contents of legumes radicles of

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common beans (221.94 ng/g) followed by fenugreek seeds (92.59 ng/g) were much higher (p≤0.05) than those of other legumes which ranged from 19.28 to 34.49 ng/g. Non-significant (p>0.05) difference in mean melatonin content was observed in radicles between chickpea seeds and lentil seeds.

On the other hand, fenugreek seed hulls revealed much higher

(p≤0.05) mean melatonin content (196.71 ng/g) than those of other legumes which ranged from 0.60 to 18.45 ng/g. Melatonin contents of chickpea seeds were much higher in the cotyledons (54.46-471.49 ng/g), than in the radicles (28.86-100.56 ng/g) and seed hulls (10.44-21.05 ng/g) throughout germination time. Lupine and lentil seed fractions had similar trend to chickpea seeds, although their melatonin contents were much lower. Different results were reported by Aguilera et al. (2015) who found that melatonin level in lentil hulls was much higher than endosperm and radicle during germination period from 3 to 10 days. Melatonin contents of common beans were much higher in the radicles (144.27-432.31 ng/g), than in the cotyledons (63.72-208.96 ng/g) and seed hulls (10.18-12.07 ng/g) throughout germination time. Similar trend was observed for broad beans. However, melatonin contents of fenugreek seeds were higher in the seed hulls (96.41-290.08 ng/g) than in the cotyledons (73.20-161.95 ng/g) and radicles (18.54-154.4 ng/g).

3.4 Total phenols of germinated legumes Total phenol contents of legumes were affected (p≤0.05) by germination time and legume fractions (Table 4). Total phenol contents of legume cotyledons, radicles and seed hulls were significantly (p≤0.05) increased by increasing germination time. This increase could be attributed to the synthesis of phenol compounds during germination process. Similar increases in total phenol contents during germination period were reported by the following researchers Xue et al. (2016) and Khang et al. (2016) for mung beans, black beans and

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soybeans, Fouad and Rehab (2015) for lentil seeds, Salem et al. (2014) for faba beans, chickpea seeds, lentil seeds and fenugreek seeds and Dueñas et al. (2009) for lupine seeds. On the other hand, decreases in the total phenolic compounds during germination process were reported by Aguilera et al. (2015)

e as et al. (2015) and ros

s a (2011) for

kidney beans and lentil seeds. Aguilera et al. (2014) reported that lentil seeds and kidney beans showed highly decreased and no change, respectively of total phenol content during germination periods. The increases in total phenol contents in legume fractions were parallel to the increases in radicle length and melatonin throughout germination time (Tables 2 and 3). Mean total phenol content was increased in the following order 61.68% for radicles, 41.65% for seed hulls and 39.72% for cotyledons as compared with the third day of germination. These values were much lower than the values reported by Boudjou et al. (2013) who found that total phenolic content (35.92 mg gallic acid /g) in raw faba bean hulls were higher than in cotyledons (2.20 mg gallic acid /g) by sixteen-fold. Sreerama et al. (2010) reported that total phenolic content in raw chickpea seed coats and cotyledons were 75.9 and 15.2 mg gallic acid /g, respectively. There was no significant (p>0.05) difference in mean total phenol content in cotyledons between chickpea seeds (9.03 mg gallic acid /g) and fenugreek seeds (9.06 mg gallic acid /g), however significant (p≤0.05) differences in mean total phenol contents were observed among other legumes. Non-significant (p>0.05) difference in mean total phenol contents were observed in radicles among lentil seeds, fenugreek seeds and common beans as well as between broad beans and chickpea seeds. Non-significant (p>0.05) difference in mean total phenol content was observed in hulls between fenugreek seeds and common beans however, their mean total phenol contents were higher (p≤0.05) than other legumes. The results also

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showed that there were significant (p≤0.05) differences in mean total phenol contents in hulls among broad beans, lupine seeds, chickpea seeds and lentil seeds.

3.5 Total flavonoids of germinated legumes Total flavonoid contents of legumes were affected (p≤0.05) by germination time and legume fractions (Table 5). Total flavonoid contents of legume cotyledons, radicles and seed hulls were significantly (p≤0.05) increased by increasing germination time. This increase could be attributed to the synthesis of flavonoid compounds during germination. Fouad and Rehab (2015) reported that total flavonoid content in lentil seeds was gradually increased and reached the maximum (4.96 mg catechin /g) after six days of germination. Similar increases in total flavonoid contents during germination period were also reported by Pajak et al. (2014) and Kim et al. (2012) for mung beans. However, decreases in the total flavonoid content during germination process were reported by Salem et al. (2014) for faba beans, chickpea seeds, lentil seeds and fenugreek seeds. The increases in total flavonoid contents in legume fractions were parallel to the increases in radicle length, melatonin and total phenols throughout germination time (Tables 2, 3 and 4). Mean total flavonoid content was increased in the following order 134.88% for seed hulls, 85.43% for radicles, and 63.64% for cotyledons as compared with the third day of germination. Boudjou et al. (2013) reported that total flavonoid was concentrated in the raw faba bean hulls (0.67 mg quercetin /g) with over five-fold the content in the cotyledons (0.12 mg quercetin /g). Sreerama et al. (2010) reported that total flavonoid contents in raw chickpea seed coats and cotyledons were 12.6 and 7.5 mg/g catechin, respectively. There were significant (p≤0.05) differences in mean total flavonoid contents among legume cotyledons and radicles. Fenugreek seed hulls had higher (p≤0.05) mean total flavonoid content than other legumes. Non-significant (p>0.05) difference in mean total flavonoid

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content was observed in hulls between lupine seeds and chickpea seeds and between lentil seeds and common beans.

3.6 Antioxidant activity of germinated legumes Antioxidant activities of legumes were affected (p≤0.05) by germination time and legume fractions (Table 6). Antioxidant activities of legume cotyledons, radicles and seed hulls were significantly (p≤0.05) increased by increasing germination time. Similar increases in antioxidant activities during germination period were reported by the following researchers Xue et al. (2016) and Khang et al. (2016) for mung beans, black beans and soybeans, Aguilera et al. (2014, 2015) for kidney beans and lentil seeds, Fouad and Rehab (2015) for lentil seeds, Salem et al. (2014) for chickpea seeds, lentil seeds and fenugreek seeds and Dueñas et al. (2009) for lupine seeds. However, Guajardo-Flores et al. (2013) reported that antioxidant activity of black beans did not change by germination process. The increases in antioxidant activity of legume fractions were parallel to the increases in radicle length, melatonin, total phenols and total flavonoids throughout germination time (Tables 2, 3, 4 and 5). Mean total antioxidant activity was increased in the following order 24.49% for seed hulls, 16.76% for radicles, and 7.73% for cotyledons as compared with the third day of germination. Boudjou et al. (2013) reported that antioxidant activity of raw faba bean hulls (91.72%) was much higher than cotyledons (3.67%). Sreerama et al. (2010) reported that the radical scavenging activity of chickpea seed coats was much higher than cotyledon fractions. Lentil seeds also exhibited more antioxidant activity in seed coats than cotyledons (Dueñas et al., 2006). Mean antioxidant activity of broad bean cotyledon (96.82%) followed by lupine seed cotyledon (95.22%) was higher (p≤0.05) than other legumes. Also non-significant (p>0.05) difference in mean antioxidant activity was observed between lentil seed and fenugreek seed

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cotyledons. However mean antioxidant activities in other legume cotyledons were significantly (p≤0.05) different. Lentil seed (96.10%) followed by chickpea seed (94.81%) radicles had higher (p≤0.05) mean antioxidant activity than other legumes. Non-significant (p>0.05) difference in mean antioxidant activity was observed between broad bean and fenugreek seed radicles, however mean antioxidant activities in other legume radicles were significantly (p≤0.05) different. A significant (p≤0.05) difference in mean antioxidant activity was observed among legume seed hulls. The mean antioxidant activity of broad bean hulls (96.38%) followed by fenugreek seed hulls (95.51%) was higher (p≤0.05) than other legumes.

3.7 Sensory properties of legumes Sensory properties of legumes after six day of germination are presented in Table (7). Nonsignificant (p>0.05) difference in appearance scores was observed among broad beans, lupine seeds, lentil seeds and fenugreek seeds and between chickpea seeds and common beans. Nonsignificant (p>0.05) differences in taste, odor, color and overall acceptability scores were observed among all legumes except for common beans and chickpea seeds. Non-significant (p>0.05) difference in texture scores was observed among lupine seeds, lentil seeds and fenugreek seeds and among broad beans, lupine seeds and common beans. Chickpea seeds had the lowest (p≤0.05) odor text re and overall acceptabilit scores; however common beans had the lowest (p≤0.05) taste score as compared with other leg mes. Although overall acceptability rating score of chickpea seeds were lower (p≤0.05) than other leg mes it had rating score described as like slightly. However, other legumes had rating scores ranged between like moderately and like very much. Wołejs o et al. (2007) reported that the effects of germination on the sensory profile of lintel and mung bean sprouts were found to depend

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on the type of legumes. Also, quantitative descriptive analysis demonstrated significant differences between the lintel and mung bean sprouts for bitterness, taste, and juiciness.

4. Conclusion From the above results, it could be concluded that legumes showed considerable variations in their melatonin, total phenols, total flavonoids and antioxidant activities. Melatonin, total phenols, total flavonoids and antioxidant activities of legume cotyledons, radicles and seed hulls were increased as germination time progressed. The increases in melatonin content of legume fractions were parallel to the increases in radicle length, total phenols, total flavonoids and antioxidant activities throughout germination time. Overall acceptability had rating scores ranged between like moderately and like very much except for chickpea which had rating score described as like slightly.

Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper.

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Table 1 Melatonin, total phenols, total flavonoids and antioxidant activities of selected raw legumes Legumes

Melatonin (ng/g)

Total phenols (mg/g gallic acid)

Total flavonoids (mg/g quercetin)

Antioxidant activity (%)

4.43±0.10e Broad bean 5.04±0.001e 92.67±0.20a 4.31±0.020d 16.47±0.30c Lupine 5.38±0.001c 78.29±0.18f 2.62±0.020e 24.42±0.30b Chickpea 5.68±0.001b 86.70±0.24d 8.43±0.020b e d c 4.79±0.10 Lentil 5.21±0.001 90.32±0.17b 5.56±0.030 54.22±0.50a Fenugreek 5.79±0.003a 87.68±0.11c 8.86±0.020a d 5.85±0.10 Common bean 4.50±0.002f 84.52±0.08e 4.33±0.006d Means in the same column with different letters are significantly different (p≤0.05)

19

Table 2 Radicle length (cm) of selected legumes as affected by germination time and legume types 1 Germination time (days) Means 3 4 5 6 Broad bean 3.40±0.36 4.13±0.32 5.20±0.26 5.50±0.50 4.56d Lupine 5.23±0.25 5.37±0.32 6.50±0.50 8.50±0.50 6.50a Chick pea 2.90±0.36 4.90±0.36 5.77±0.25 6.63±0.35 5.05c Lentil 2.37±0.21 4.10±0.10 4.23±0.25 4.27±0.25 3.74e Fenugreek 3.50±0.50 5.07±0.51 5.97±0.55 7.30±0.26 5.46b Common bean 2.27±0.25 2.90±0.10 3.60±0.36 4.20±0.30 3.24f 2 d c b a Means 3.28 4.41 5.21 6.07 1 Means in the same column with different letters are significantly different (p≤0.05)

Legumes

2

Means in the same row with different letters are significantly different (p≤0.05)

Table 3 Melatonin (ng/g) of selected legumes as affected by germination time and legume fractions Legumes

Germination time (days) 4 5

3 6 Cotyledon Broad bean 4.10±0.02 5.51±0.10 7.81±0.10 13.75±0.20 Lupine 17.83±0.3 84.37±0.7 93.61±0.60 175.50±1.7 Chickpea 54.46±0.5 165.06±1.1 308.83±1.8 471.49±2.2 Lentil 17.33±0.3 17.42±0.30 89.60±0.80 89.89±0.70 Fenugreek 73.20±0.8 116.20±0.9 136.93±1.1 161.95±1.1 Common bean 63.72±0.7 71.96±0.60 101.51±0.9 208.96±1.3 2 d c b Means 38.44 76.75 123.05 186.92a Radicle Broad bean 13.80±0.20 16.20±0.40 18.40±0.20 28.70±0.30 Lupine 16.27±0.30 29.72±0.20 30.82±0.30 33.65±0.80 Chickpea 28.86±0.40 36.62±0.40 98.86±0.90 100.56±1.1 Lentil 5.64±0.10 17.33±0.30 41.50±0.60 73.49±0.80 Fenugreek 18.54±0.30 96.42±0.80 100.98±1.0 154.40±1.2 Common bean 144.27±1.1 150.14±1.10 161.06±1.2 432.31±1.9 2 Means 37.90d 57.75c 75.27b 137.19a Hull Broad bean 0.52±0.02 0.61±0.02 0.63±0.02 0.65±0.02 Lupine 1.12±0.01 1.79±0.05 7.93±0.10 9.36±0.30 Chickpea 10.44±0.2 16.51±0.30 18.74±0.30 21.05±0.70 Lentil 4.41±0.10 8.08±0.20 28.98±0.40 32.32±0.20 Fenugreek 96.41±0.8 125.08±0.7 275.27±1.8 290.08±1.6 Common bean 10.18±0.10 10.85±0.10 11.92±0.20 12.07±0.20 2 d c b Means 21.51 27.15 57.25 60.92a 1 Means in the same column with different letters are significantly different (p≤0.05) 2

Means in the same row with different letters are significantly different (p≤0.05) 20

1

Means

7.79e 92.83c 250.06a 53.56d 122.07b 96.54c 19.28e 27.62d 33.23c 34.49c 92.59b 221.94a 0.60f 5.02e 16.69c 18.45b 196.71a 11.69d

Table 4 Total phenols (mg gallic acid /g) of selected legumes as affected by germination time and legume fractions Legumes 3

Germination) time (day 4 5

1

6

Cotyledon Broad bean 5.20±0.002 5.32±0.002 5.88±0.002 5.88±0.003 Lupine 5.74±0.001 5.94±0.002 6.41±0.002 6.72±0.008 Chickpea 6.30±0.002 7.97±0.003 9.89±0.001 11.94±0.001 Lentil 5.66±0.003 6.53±0.001 7.13±0.001 7.97±0.006 Fenugreek 7.25±0.001 8.28±0.001 10.02±0.001 10.74±0.001 Common bean 7.81±0.004 8.23±0.003 9.32±0.002 9.71±0.002 2 d c b Means 6.32 7.04 8.11 8.83a Radicle Broad bean 1.81±0.002 3.88±0.002 4.12±0.002 4.97±0.004 Lupine 3.34±0.001 4.33±0.002 5.05±0.001 5.48±0.001 Chickpea 1.61±0.002 3.34±0.001 4.77±0.001 5.19±0.002 Lentil 4.92±0.001 5.17±0.002 5.30±0.003 5.36±0.001 Fenugreek 4.35±0.002 4.89±0.003 5.42±0.001 5.54±0.001 Common bean 4.00±0.003 4.86±0.002 5.76±0.002 5.87±0.007 2 Means 3.34d 4.41c 5.07b 5.40a Hull Broad bean 3.51±0.001 3.87±0.002 4.46±0.001 5.10±0.001 Lupine 2.82±0.002 3.59±0.002 4.43±0.001 5.66±0.002 Chickpea 2.91±0.002 3.58±0.004 4.03±0.004 5.33±0.001 Lentil 3.58±0.002 4.97±0.002 5.04±0.002 5.62±0.001 Fenugreek 5.25±0.007 5.29±0.006 5.50±0.001 5.61±0.004 Common bean 5.27±0.002 5.39±0.002 5.43±0.003 5.76±0.005 2 d c b Means 3.89 4.45 4.82 5.51a 1 Means in the same column with different letters are significantly different (p≤0.05) 2

Means in the same row with different letters are significantly different (p≤0.05)

21

Means

5.57e 6.19d 9.03a 6.81c 9.06a 8.76b 3.69c 4.46b 3.72c 5.18a 5.04a 5.03a 4.32c 4.12d 3.96e 4.63b 5.41a 5.46a

Table 5 Total flavonoids (mg quercetin /g) of selected legumes as affected by germination time and legume fractions Legumes 3

Germination time (day) 4 5

1

6

Cotyledon Broad bean 4.30±0.02 4.36±0.02 4.48±0.01 4.93±0.01 Lupine 3.28±0.01 3.45±0.03 3.66±0.03 3.85±0.03 Chickpea 8.08±0.01 8.48±0.02 8.53±0.02 8.86±0.02 Lentil 6.73±0.02 7.23±0.02 7.35±3.97 8.78±0.02 Fenugreek 9.23±0.09 10.48±0.11 15.36±0.04 24.52±0.27 Common bean 5.32±0.02 6.53±0.02 7.67±0.03 9.52±0.02 2 d c b Means 6.16 6.76 7.84 10.08a Radicle Broad bean 1.59±0.01 1.93±0.03 1.93±0.02 2.39±0.07 Lupine 1.49±0.02 1.53±0.05 2.73±0.10 3.67±0.12 Chickpea 1.53±0.05 2.20±0.02 3.16±0.76 4.53±0.02 Lentil 3.18±0.01 3.35±0.03 3.42±0.06 3.45±0.07 Fenugreek 3.24±0.01 3.92±0.18 4.56±0.08 5.04±0.01 Common bean 0.92±0.07 1.06±0.04 1.43±0.02 3.05±0.06 2 Means 1.99d 2.33c 2.87b 3.69a Hull Broad bean 0.25±0.01 0.29±0.01 0.77±0.01 0.92±0.01 Lupine 1.38±0.04 1.37±0.09 1.46±0.03 1.56±0.03 Chickpea 0.61±0.05 0.56±0.04 2.09±0.14 2.94±0.01 Lentil 1.32±0.08 2.42±0.03 2.98±0.10 3.60±0.10 Fenugreek 3.01±0.12 3.57±0.01 4.53±0.03 5.63±0.03 Common bean 1.16±0.06 1.94±0.02 2.75±0.06 3.54±0.02 2 d c b Means 1.29 1.69 2.43 3.03a 1 Means in the same column with different letters are significantly different (p≤0.05) 2

Means in the same row with different letters are significantly different (p≤0.05)

22

Means

4.52e 3.56f 8.49b 7.52c 14.90a 7.26d 1.96e 2.36d 2.86c 3.35b 4.19a 1.61f 0.56d 1.44c 1.55c 2.58b 4.19a 2.35b

Table 6 Antioxidant activity (%) of selected legumes as affected by germination time and legume fractions Legumes 3

Germination time (day) 4 5

1

6

Cotyledon Broad bean 94.81±0.39 96.26±0.14 97.88±0.31 98.33±0.28 Lupine 91.24±0.28 95.95±0.31 96.05±0.43 97.68±0.31 Chickpea 88.02±0.36 92.74±0.39 94.67±0.39 99.16±0.34 Lentil 91.23±0.98 93.82±0.17 95.90±0.23 96.44±0.15 Fenugreek 90.59±0.30 92.71±0.30 95.84±0.13 98.79±0.29 Common bean 86.61±0.14 88.33±0.14 90.41±0.30 94.01±0.20 2 d c b Means 90.41 93.30 95.12 97.40a Radicle Broad bean 89.43±0.76 91.56±0.39 94.32±0.17 96.34±0.36 Lupine 58.57±0.23 95.03±0.08 95.90±0.09 96.40±0.23 Chickpea 93.18±0.28 94.57±0.23 95.46±0.24 96.05±0.45 Lentil 92.55±0.14 96.28±0.06 97.36±0.35 98.22±0.15 Fenugreek 83.00±0.22 93.95±0.34 96.69±0.31 98.02±0.23 Common bean 77.49±0.12 91.64±0.22 94.92±0.08 95.92±0.22 2 Means 82.92d 93.83c 95.72b 96.82a Hull Broad bean 92.46±0.21 96.30±0.15 98.28±0.28 98.52±0.15 Lupine 60.03±0.49 81.89±0.48 90.11±0.23 98.02±0.09 Chickpea 46.25±0.44 47.29±0.14 62.17±0.23 64.32±0.44 Lentil 70.87±0.36 96.00±0.14 96.00±0.14 99.11±0.31 Fenugreek 93.92±0.22 95.12±0.23 95.44±0.28 97.55±0.24 Common bean 66.43±0.22 68.38±0.28 75.07±0.12 77.49±0.15 2 d c b Means 71.59 80.76 86.13 89.12a 1 Means in the same column with different letters are significantly different (p≤0.05) 2

Means in the same row with different letters are significantly different (p≤0.05)

23

Means

96.82a 95.22b 93.64d 94.35c 94.48c 89.84e 92.91c 86.47e 94.81b 96.10a 93.66c 89.99d 96.38a 82.48d 54.98f 90.23c 95.51b 71.84e

Table 7 Sensory properties of selected legumes after six day of germination Overall Legumes Appearance Taste Odor Color Texture acceptability a a a a bc 8.25 7.63 8.00 8.38 7.63 7.95a Broad bean ±1.03 ±0.93 ±0.53 ±0.74 ±0.74 ±0.65 7.63ab 7.00a 7.75a 8.13a 7.75abc 7.65a Lupine ±1.41 ±1.69 ±0.71 ±0.99 ±0.71 ±0.51 7.13b 6.75a 6.25b 7.38a 6.00d 6.30b Chickpea ±0.83 ±0.71 ±0.71 ±0.74 ± 0.76 ±0.41 8.50a 7.00a 7.88a 8.25a 8.63a 8.05a Lentil ±0.53 ±1.07 ±0.83 ±1.03 ±0.52 ±0.50 8.13a 7.00a 8.25a 8.25a 7.88ab 7.85a Fenugreek ±0.35 ±1.07 ±0.46 ±1.04 ±0.83 ±0.60 6.75b 6.38b 7.38a 8.00a 6.88c 7.35a Common bean ±1.16 ±0.74 ±0.52 ±1.07 ±0.83 ±0.74 LSD 0.97 1.07 0.65 0.96 0.75 0.58 Means in the same column with different letters are significantly different (p≤0.05)

24

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