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JOURNAL OF FOOD COMPOSITION AND ANALYSIS

Journal of Food Composition and Analysis 19 (2006) 277283 www.elsevier.com/locate/jfca

Original Article

Effect of germination on legume phenolic compounds and their antioxidant activity

pez-Amoro s, T. Herna ndez, I. Estrella M.L. Lo
Instituto de Fermentaciones Industriales. CSIC, Juan de la Cierva 3, 28006-Madrid. Spain Received 23 September 2003; received in revised form 16 April 2004; accepted 16 June 2004

Abstract This work has studied the effects of varying germination conditions for beans, lentils and peas, at semi-pilot scale, on bioactive compounds such as avonoid and non-avonoid phenolic compounds. It has also evaluated the free radical scavenging activity of these samples. The legumes studied contain different hydroxybenzoic acids and aldehydes, hydroxycinnamic acids and derivatives, avonol glycosides, and avan-3-ols and procyanidins. The results obtained indicate that germination modies the quantitative and qualitative phenolic composition of legumes, and the changes depend on the type of legume and the germination conditions. These changes inuence the functional properties of the legumes as consequence of the variation in antioxidant activity. Peas and beans undergo a signicant increase in antioxidant activity after germination, whereas lentils show a decrease. r 2004 Elsevier Inc. All rights reserved.
Keywords: Lentils; Peas; Beans; Germination; Phenolic compounds; Antioxidant activity

1. Introduction Legumes contain a high concentration of proteins, carbohydrates and dietary ber and make an important contribution to human diet in many countries. Legumes have to be processed prior to consumption due to their content of antinutritional compounds, such as trypsin inhibitors, phytic acid, a-galactosides (Agustin et al., 1989; Vidal-Valverde et al., 2002). Germination is one of the most common and effective processes for improving the quality of legumes, and germinated legumes are widely consumed all around the word. The process is inuenced by external factors such as germination time and presence or absence of light both of which aid or inhibit the germination process in relation to the reserve materials of the seed (Ridge, 1991). Soaking legume seeds in water must precede germination.
Corresponding author. Tel.: +34-91-562-2900; fax: +34-91-5644853. ndez). E-mail address: thernandez@i.csic.es (T. Herna

During germination, some of the seed reserve materials are degraded and used for respiration and synthesis of new cell constituents of the developing embryo, therefore causing signicant changes in the biochemical, nutritional and sensory characteristics of these legumes. It is known that the germination process generally improves the nutritional quality of legumes, not only by the reduction of antinutritive compounds, but by augmenting the levels of free amino acids, available carbohydrates, dietary ber, and other components (Danisova et al., 1995; Urbano et al., 1995; Ayet et al., 1997; Vidal-Valverde et al., 2002, 2003), and increasing the functionality of the seeds due to the pezsubsequent increase in the bioactive compounds (Lo s et al., 2001; Fr as et al., 2002; Vidal-Valverde Amoro et al., 2002). Legumes contain several phenolic compounds (Bar et al., 1997; Lo pez-Amoro s et al., 1998a, b; tolome pez-Amoro s, 2000; Duen Lo as et al., 2002; Troszynska et al., 2002), which are considered to be natural antioxidants, representing an important group of bioactive compounds in foods, and may prevent the development

0889-1575/$ - see front matter r 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.jfca.2004.06.012

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of many diseases such as atherosclerosis, cancer (Formica and Regelson, 1995; Kupulainen and Salonen, 1996). As a consequence of this activity, the presence of phenolic compounds in food has in recent years come to be viewed in a positive light by both scientists and consumers and has resulted in a push to procure food with specic benecial effects such as functional foods. Phenolic compounds not only effectively prevent oxidation in foods; they also act as protective factors against oxidative damage in the human body (Jovanovic et al., 1996; Haslam, 1998; Aviram, 2000; Castillo et al., 2000; Kikuzaki et al., 2002). The antioxidant activity of phenolics is related to their chemical structure. In general the avonoid compounds present a stronger antioxidant activity than nonavonoids, and the combined forms, such as glycosides, have lower activity than free forms (Rice-Evans et al., 1996). Among the non-avonoid compounds are the hydroxycinnamics which contribute most to antioxidant activity (Meyer and Andreasen, 1999). Little is known about the changes in the individual phenolic compounds during the germination of legumes et al., 1997; Lo pez-Amoro s, 2000; Lo pez(Bartolome s et al., 2001); however, much information exists Amoro in the literature about the changes observed in total condensed tannins. The germination of soybean, lentil and vicia, evaluated by several methods, modify the antioxidant activity indicating differences which exist among the types of legumes as well as in germination conditions, which are not always related to the content of some of the antioxidant compounds (Kojima et al., 1997; Zielinski, 2002). In this work we have carried out a study of the germination of three different legumes, beans, peas and lentils, on a semi-pilot scale, under varying germination conditions, in order to know the variations in the individual seed phenolic compounds. The inuence of the process on the antioxidant capacity of the legumes has also been evaluated.

drained off, watered to neutral pH, and soaked in distilled water for 5 h and 30 min. The hydrated seeds were located in six trays, and germinated by capillary in a seed germinator (G-120 Snijders, Holland) for 2, 4 and 6 days with light (named L2d, L4d and L6d, respectively) and without light (named NL2d, LN4d and LN6d, respectively) at 20 1C and 99% relative humidity. The germinated seeds were freeze-dried and ground to pass 0.18-mm sieve for the analysis. 2.3. Extraction Two portions (2 10 g) of legume ours from raw and from each of the germination conditions were macerated with 3 80 mL of a solution of methanolHCl (10/000)/water (80:20 v/v), following the method of Duen as et al. (2002). An aliquot of this methanol solution (200 mL) was extracted four times with diethyl ether and four times with ethyl acetate, and the organic fractions were combined, dried with anhydrous Na2SO4 and evaporated to dryness under vacuum. The residue was dissolved in methanol/water (1:1, v/v), to analyze by HPLC-photodiode array detector (PAD). All samples were ltered through a 0.45 mm cellulose acetate lter (Millipore) before injection. 2.4. HPLC-PAD analysis The chromatographic system is provided by a 996 photodiode-array detector (Waters, Milford, MA, USA) and a column Spherisorb ODS2 C18 (300 3.9 mm). Two mobile phases were employed for elution, solvent A: water/acetic acid (98:2) and solvent B: water/ acetonitrile/acetic acid (78:20:2). The gradient prole was from 0 to 55 min, 10020% A; from 55 to 57 min, 2010% A; from 57 to 80 min, 105% A; from 80 to 90 min, 50% A; the ow rates used were 1.0 mL/min (055 min) and 1.2 mL/min (5590 min). Scanning from 210 to 360 nm performed detection. A volume of 25 mL was injected. The samples were analyzed in duplicate. 2.5. Identication and quantication of the compounds

2. Materials and methods 2.1. Samples Beans (Phaseolus vulgaris L. variety La Granja) lentils (Lens culinaris L., var. Castellana) and peas (Pisum sativum L., variety Elsa) were purchased and used for the germination process. 2.2. Germination process Prior to germination, the seeds (500 g) were soaked in 2500 mL of water containing 0.7% sodium hypochlorite solution for 30 min at room temperature. The seeds were Chromatographic peaks were identied by comparing retention times, UV spectra and data of UV spectral et al., 1993, 1996), with those of parameters (Bartolome standards, or those of previously puried and identied rez-Ilzarbe et al., 1992), recorded in the procyanidins (Pe same chromatographic conditions with the PAD. The standards of hydroxybenzoic compounds, protocatechuic, p-hydroxybenzoic, p-hydroxyphenylacetic and vanillic acids, p-hydroxybenzoic and p-vanillic aldehydes, and the hydroxycinnamic acids, trans pcoumaric and trans ferulic, and the (+) catechin, are from Aldrich Chimie (Germany); the procyanidin B2, and the avonols (quercetin-3-rhamnoside, quercetin-3-

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rutinoside, kaempferol-3-glucoside, kaempferol-3-ruti` se (France). The cis-pnoside) are from Extrasynthe coumaric and cis ferulic acids were obtained from the corresponding trans isomers after exposition to the UV light. Compounds with the same shape and wavelengths maxima of UV spectrum of hydroxycinnamic acids, but with different retention time were identied as derivatives of these free acids (Bengoechea et al., 1995). Compounds with UV spectra corresponding to that of procyanidins, the standards of which were not available, were identied as procyanidins B3 and C1 agree with the rez-Ilzarbe et al. compounds previously identied by Pe (1992), and as a tetramer procyanidin, based on the et al., 1996). study of data of UV spectral (Bartolome Quantication was made using the external standard method at 280 and 340 nm according to the maximum absorption of each compound. The calibration curves were made by injection of different volumes from the stock solutions over the range of concentration observed for each of the compounds, using a lineal regression for the relationship of area sum versus concentration, under the same conditions as for the samples analyzed. Hydroxycinnamic acid derivatives were quantied using the calibration curves of the corresponding free acid, and procyanidins with the calibration curve of (+) catechin.

hydrazil (DPPH). The percentage of remaining DPPH against the sample concentration was plotted to obtain the amount of antioxidant (mg of legume ours) necessary to decrease absorbance by 50%. A smaller IC50 value corresponds to a higher antioxidant activity. The samples were prepared and measured separately in duplicate.

3. Results and discussion 3.1. Raw legumes Beans, peas and lentils contain different concentrations (Table 1) of the hydroxybenzoic phenolic compounds, protocatechuic, p-hydroxybenzoic and vanillic acids; p-hydroxyphenylacetic acid was only found in beans and the aldehyde p-hydroxybenzoic only in lentils. The hydroxycinnamic compounds show a more changeable behavior, trans-ferulic acid is present in the three seeds, but cis-ferulic acid is found solely in beans. Trans p-coumaric acid is present in lentils and peas, and the cis p-coumaric acid in peas. Only in lentils have we identied a derivative of trans-p-coumaric acid, and in very low concentration, the (+)-catechin together with the procyanidin dimers B2, B3, the trimer C1, and a tetramer procyanidin. 3.2. Germination During the soaking period before germination, all of the phenolic compounds identied in beans, peas and lentils undergo a drastic decrease (Table 1) which was

2.6. Antioxidant activity In the methanol solution the antioxidant activity (IC50) was determined by the BrandWilliams method (Brand-William et al., 1995), with 2,20 diphenyl-1-picryl-

Table 1 Concentration (mg/100 g d.w.) of phenolic compounds in raw and soaked legumes Compounds Beans Raw Protocatechuic acid p-Hydroxybenzoic acid p-Hydroxybenzoic aldehyde Vanillic acid p-Hydroxyphenylacetic acid trans p-Coumaric acid cis p-Coumaric acid trans p-Coumaric acid derivative cis p-Coumaric acid derivative trans Ferulic acid cis Ferulic acid cis Ferulic acid derivative (+)-Catechin Procyanidin B2 Procyanidin B3 Procyanidin C1 Procyanidin tetramer 32.841.4 32.336.1 nd 90.997.9 45.851.6 nd nd nd nd 342366 74.179.1 nd Soaked 16.517.5 11.913.7 nd 31.234.4 nd nd nd nd nd nd nd 206224 Peas Raw 206221 46.549.9 nd 19.422.2 nd 37.741.5 65.570.1 nd 31.933.7 9.110.9 nd nd Soaked 62.766.9 0.81.0 nd 2.02.6 nd nd nd 21.124.1 nd nd nd nd Lentils Raw 49.952.3 93.6100 13.315.3 73.679.6 nd 322342 nd 82.889.6 nd 20.925.7 nd nd 0.1 0.3 0.30.5 0.30.5 t 0.20.3 Soaked 5.97.5 21.325.1 nd 2.63.8 nd nd nd 19.023.0 nd nd nd nd nd nd t nd nd

Note: Values are lower and higher of two samples analyzed in duplicate; nd: not detected; and t: trace.

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also observed in various varieties of peas by Bishnoi and Khetarpaul (1998). A decrease in other components such as carbohydrates and phytates was also observed (Vidal-Valverde and Fr as, 1992; Bressani and Elias, 1980). In the soaked beans, a derivative of cis ferulic acid was detected, possibly originating from the two isomers of ferulic acid present in the raw beans. A trans pcoumaric acid derivative from the corresponding free acid was also identied in soaked peas. Procyanidins were not detected in lentils; only traces of B3 were found. After germination, various changes in the phenolic compounds occurred which are not only dependent on the type of seeds, but also on the process conditions, the presence of light and germination time. A general decrease in hydroxybenzoic acids, compared with raw seeds, was observed in beans, peas and lentils after soaking (Table 1), along with an overall increase during germination, with the exception of protocatechuic acid (Tables 2, 3 and 4) where no increase took place. This last acid, completely absent in the germinated legumes, presents two hydroxyl
Table 2 Concentration (mg/100 g d.w.) of phenolic compounds in germinated beans Compounds Protocatechuic acid p-Hydroxybenzoic acid p-Hydroxybenzoic aldehyde Vanillic acid Vanillic aldehyde trans p-Coumaric acid trans p-Coumaric acid derivative trans Ferulic acid cis Ferulic acid cis Ferulic acid derivative Quercetin-3-rutinoside Quercetin-3-rhamnoside Kaempferol-3-rutinoside Kaempferol-3-gluicoside NL2d nd 4.56.1 26.328.1 nd nd nd 20.422.6 2.22.8 nd 9.712.9 nd nd nd nd NL4d nd 7.39.3 42.647.0 nd nd 54.1 57.9 44.646.4 28.030.4 nd nd nd nd nd nd

groups in the ortho position, which allows it to form quinones due to enzymes action; this compound can at the same time interact with proteins bringing about stable complexes which are removable with great difculty under analysis conditions. The p-hydroxybenzoic aldehyde has been detected only after germination, in beans, peas and lentils, and the concentration is larger in the samples where germination has taken place with light, with the sole exception being peas (Tables 2, 3 and 4). The vanillic aldehyde is detected in germinated beans and lentils, at the end of the process, with a higher concentration being observed for those samples with light (Tables 2 and 4). The p-hydroxybenzoic and p-vanillic aldehydes are present only in the germinated seeds, and they could originate from the degradation of the seed lignin as a consequence of enzymatic oxidation (Dagley, 1971). The hydroxycinnamic compounds show different behaviors in the three seeds. Thus, in beans, the pcoumaric compounds neither detected in raw nor soaked beans (Table 1), increases during the germination period, even though it does not appear to be very

NL6d nd 11.515.7 14.416.8 74.577.9 6.47.6 102113 nd 49.854.0 24.326.5 Nd ndn 297311 305323 72.778.3

L2d nd 12.715.1 33.640.0 nd nd nd 4.95.9 17.7 19.5 nd nd nd nd nd nd

L4d nd 16.320.9 59.566.1 nd nd 27.532.1 nd 40.146.3 4.05.8 nd 253260 nd nd nd

L6d nd 11.115.1 43.948.1 12.817.2 19.121.3 90.5101.3 nd 113117 nd nd 22952341 310326 916933 70.677.6

Note: Values are lower and higher of two samples analyzed in duplicate; nd: not detected; NL: without light; L: with light; and d: days of germination.

Table 3 Concentration (mg/100 g d.w.) of phenolic compounds in germinated peas Compounds Protocatechuic acid p-Hydroxybenzoic acid p-Hydroxybenzoic aldehyde Vanillic acid trans p-Coumaric acid cis p-Coumaric acid trans p-Coumaric acid derivative trans Ferulic acid NL2d nd 7.89.4 21.625.8 2.53.5 10.2 12.0 nd 10.212.0 nd NL4d nd 6.77.3 15.818.2 nd 14.817.4 nd nd 28.231.8 NL6d nd 12.515.5 111119 nd t nd nd nd L2d nd 8.210.0 11.113.9 nd 26.631.2 nd nd 17.120.2 L4d nd nd 3.94.9 nd t 51.457.0 nd nd L6d nd 18.320.7 19.222.2 nd 17.920.3 100108 nd nd

Note: Values are lower and higher of two samples analyzed in duplicate; nd: not detected; t: trace; NL: without light; L: with light; and d: days of germination.

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pez-Amoro s et al. / Journal of Food Composition and Analysis 19 (2006) 277283 M.L. Lo Table 4 Concentration (mg/100 g d.w.) of phenolic compounds in germinated lentils Compounds Protocatechuic acid p-Hydroxybenzoic acid p-Hydroxybenzoic aldehyde Vanillic acid Vanillic aldehyde trans p-Coumaric acid trans Ferulic acid trans p-Coumaric acid derivative Procyanidin B3 NL2d nd 7.89.4 20.222.0 nd nd 43.247.6 47.853.8 12.915.3 nd NL4d nd nd 22.224.6 nd nd nd 46.150.7 19.421.4 nd NL6d nd 9.211.6 39.644.4 5.27.0 nd 23.427.4 26.130.3 97.9105 t L2d nd nd 30.732.7 nd nd 25.127.5 18.320.5 8.19.9 nd L4d nd 15.318.1 41.445.4 nd 16.718.7 139148 4.76.1 22.833.6 nd L6d nd 18.320.3 47.454.2 nd 31.636.2 110116 157164 nd nd 281

Note: Values are lower and higher of two samples analyzed in duplicate; nd: not detected; t: trace; NL: without light; L: with light; and d: days of germination.

inuenced by the presence or absence of light (Table 2). The two isomers of ferulic acid appear during germination, which may have been formed from the cis ferulic acid derivative detected after soaking. More remarkable are the variations in peas (Table 3), in which, after the general decrease in the free acids, as a consequence of soaking (Table 1), the germination brings out a slight increase in the hydroxycinnamic compounds, which is higher with the presence of light. In lentils (Table 4), some of these compounds, which had decreased or disappeared with soaking, are now detected from the beginning of the germination period, showing different behavior depending on the compound. In general, in lentils and in the presence of light, the trans p-coumaric acid increases, while the corresponding derivative decreases; the trans ferulic acid also increases during germination with light. The hydroxycinnamic compounds are constituents of the cell wall, in various bonds and esteried forms, linked to arabinoxylans and lignin (Ishii, 1994). The changes observed in these compounds during germination, principally in the last stages of the process, could be explained by the action of the endogenous esterases, the presence of which is also greatly inuenced by the sample, as well as both the presence, or absence, of light. It is important to point out the presence of avonol glycosides (quercetin-3-rutinoside, quercetin-3-ramnoside, kaempferol-3-rutinoside and kaempferol-3-glucoside) in beans, after 4 days of germination, compounds that are not detected in the raw samples (Table 2). The highest concentration of these compounds was observed at 6 days and in the samples germinated with light. Various types of endogenous and exogenous factors, including hormones, nutrients, light and temperature exert regulatory inuences on the enzymes of avonoids biosynthesis, including avonols (Wiermann, 1981; Beggs et al., 1986). The presence of avonols glycosides in beans after germination could be related to the changes, which occur during the process, inuenced by the light, temperature and activation of the endogenous

enzymes of the seeds. The light accelerates and increases the formation of quercetin and kaempferol glycosides in the bean seeds. The procyanidins, present in the raw lentils, although in very low concentrations, have not been detected after the process, except for trace of the procyanidin B3 and only after 6 days of the germination without light (Table 4). These modications of procyanidins in lentils, as a consequence of germination, have also been observed by et al. (1997). Bartolome The endogenous enzymes of the legumes are activated during germination bringing about differences in the variations in the chemical composition of peas, beans and lentils. The enzymes which are most directly related with the phenolics are hydrolases and polyphenoloxydases whose activity increases during germination, albeit in a different manner, depending on the type of legume (Rao and Deosthale, 1987). This occurrence could also explain the differences in the variation of each of the phenolic compounds in the three legumes analyzed, as a consequence of the differing matrix of said seeds.

3.3. Antioxidant activity The germination process modies the antioxidant activity, measured by its free radical scavenging capacity, of peas, beans and lentils studied, but in a different manner in each of them (Table 5). After a germination period of 4 and 6 days, beans show higher antioxidant activity than raw seeds. Time and light are factors that seem to inuence this capacity, as they reach the highest values of antioxidant activity after 6 days of germination and in the absence of light. In the case of peas, germination increases antioxidant activity, from early stages of germination, this increase being most remarkable after 4 days and in the presence of light. The germination modies the antioxidant activity of lentils in a negative sense as raw lentils

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282 pez-Amoro s et al. / Journal of Food Composition and Analysis 19 (2006) 277283 M.L. Lo Table 5 Antioxidant activity (IC50) of germinated legumes Samples Raw L2d L4d L6d NL2d NL4d NL6d Peas 14.214.5 13.013.2 6.16.2 7.47.6 13.213.3 8.38.5 6.76.8 Beans 19.920.8 21.422.6 15.115.4 9.69.8 21.622.6 11.912.2 7.57.7 Lentils 4.95.1 5.96.1 9.39.5 7.47.6 9.110.9 7.47.6 7.27.4

Note: Values are the lower and higher of two samples analyzed in duplicate. L: with light; NL: without light; d: days of germination.

present greater antioxidant activity than the germinated ones. The results suggest that additional antioxidants other than polyphenols are present. Legumes contain, together with phenolics, other bioactive compounds such as vitamins and carotenoids in different concentrations (Sies et al., 1992; Prodanov et al., 1998; Atienza et al., 1999) that can also affect the antioxidant activity of the samples. These compounds may exert a synergetic effect between them and with phenolic compounds, which could be the origin of the observed differences in the antioxidant activity of seeds. Considering the optimum germination conditions to be those where the highest free radical scavenging activity is reached, in the case of peas it is after 4 days of germination under light. For beans, it is after 6 days of germination with no light present. Germination affected lentils in a negative way as it decreased antioxidant activity.

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4. Conclusions The germination process of beans, peas and lentils modied their phenolic composition. The metabolic changes observed seem to be affected by light and days of germination. From the results of the antioxidant activity evaluation, germination does not always improve the biological quality of legumes and therefore their functionality. Germination enhanced the functionality of peas and beans ours, but was negative in the case of lentils.

Acknowledgements This work was supported by the nanced projects ALI96-0480 and AGL2002-02905-ALI, through Span n Interministerial de Ciencia y Tecnoloish Comisio g a. The authors are grateful to Luis Pin al for technical assistance.

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