Food Research International 44 (2011) 31953201

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Food Research International

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Relationship between the sensory quality of lentil (Lens culinaris) sprouts and their phenolic constituents
Agnieszka Troszyska a, Isabel Estrella b, Grzegorz Lamparski a,, Teresa Hernndez b, Ryszard Amarowicz a, Ronald B. Pegg c
a b c

Division of Food Science, Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, Tuwima 10, 10-474 Olsztyn, Poland Instituto de Ciencia y Tecnologa de Alimentos y Nutricin (ICTAN), Madrid, Spain Department of Food Science & Technology, The University of Georgia, 100 Cedar Street, Athens, GA, 30602-2610, USA

a r t i c l e

i n f o

a b s t r a c t
Green lentil seeds of the Aldona cultivar were germinated at 20 C for seven days in the dark, after which the resultant edible sprouts were subjected to sensory evaluation using quantitative descriptive analysis (QDA). A trained panel of nine members assessed differences in the sprouts based on their appearance, odour, taste, and texture. Changes in the prole of phenolic compounds from the sprouts were determined by HPLC-DAD and HPLC-MS. The results showed that the time period for germination played a key role on the sensory proles of the samples. Attributes that differentiated the sensory proles were as follows: colour, grassy odour, off-odour, green taste, bitter taste, sweet taste, beany taste, juiciness, brousness, and ourness. Principal component analysis (PCA) revealed that the rst and second principal component (PC1 and PC2) together explained 96.0% of the total variability for the sensory quality of the samples. HPLC determinations conrmed that during germination the phenolic composition of lentil seeds was modied both qualitatively and quantitatively with respect to ungerminated seeds. Based on partial least squares (PLS) regression analysis, a strong correlation was found between the negative sensory attributes (bitterness and astringency) and the chemical data set (catechin gallate and some types of avonols). 2011 Elsevier Ltd. All rights reserved.

Article history: Received 6 April 2011 Accepted 10 August 2011 Keywords: Lentils Germination Sensory quality Phenolic compounds Principal component analysis (PCA) HPLC-MS

1. Introduction In developed countries consumers are highly interested in wholesome food products; that is, so-called health-promoting foods (Boye, Zare, & Pletch, 2010; Campos-Vega, Loarca-Pia, & Oomah, 2010; Menrad, 2003; Urala & Lhteenmki, 2007; Verbeke, 2006). This category of foodstuffs includes, among others, grains of leguminous seeds and cereals as well as their co-products (Duranti, 2006; Messina, 1999; Saba et al., 2010). In Western European countries, there is a market for germinated seeds of legumes (Khandelwal, Udipi, & Ghugre, 2010; Urbano et al., 2005; Vidal-Valverde et al., 2002): they are consumed either fresh in the form of sprouts or, after drying, are used as additives in a variety of food products. The germination is technologically simple, inexpensive, and a widely employed processing technique for seeds. This approach has been known for centuries, especially in oriental cultures. According to the current state of knowledge, germinated seeds are characterized by higher contents of nutrients, notably amino acids, peptides, vitamins, and minerals, (Frias et al., 2002;

Corresponding author. E-mail address: greg@pan.olsztyn.pl (G. Lamparski). 0963-9969/$ see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2011.08.007

Kuo, Rozan, Lambein, Frias, & Vidal-Valverde, 2004) and lower levels of non-nutrients like trypsin inhibitors, galactosides, tannins, and lectins (Bau, Villaume, Nicolas, & Mjean, 1997; Chang & Harrold, 1988; Frias, Diaz-Pollan, Hedley, & Vidal-Valverde, 1995; Ibrahim, Habiba, Shatta, & Embaby, 2002; Savelkoul, Van der Poel, & Tamminga, 1992; Urbano et al., 2005) compared to their ungerminated analogues. Changes in the content of polyphenolic antioxidants for different legumes as a result of germination have also been reported (Bartolom, Estrella, & Hernndez, 1997; Lpez-Amors, Hernndez, & Estrella, 2006; Oomah, Caspar, Malcolmson, & Bellido, 2011). It is well known that polyphenolics, apart from possessing valuable biological properties, impart a high sensory activity to foods. Unfortunately, they typically introduce negative attributes such as bitterness and astringency, which are likely to reduce the intake of food products to a signicant extent (Drewnowski & Gomez-Carneros, 2000; Lesschaeve & Noble, 2005). Germination might be an important processing technique to change the composition of phenolic constituents and thereby modify/improve the sensory quality of the resultant sprouts. Details on such an approach, however, are sparse. The objective of this work was to report on changes in the phenolic compounds as a result of germination of green lentil seeds and their impact on the sensory quality of the edible sprouts.

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A. Troszyska et al. / Food Research International 44 (2011) 31953201 Table 1 Sensory attributes, their denitions, and anchors used in the descriptive analysis of green lentil sprout samples. Attribute Denition and anchors Visual impression of the sprouts (white to light creamy)

2. Materials and methods 2.1. Seeds and germination Raw green lentil (Lens culinaris L.) seeds of the Aldona cultivar (which were intended for germination, and then consumption as a vegetarian food product) were purchased from a health food shop in Olsztyn, Poland. Aldona is the major lentil cultivar grown in Southeastern Poland (i.e., the only region in Poland where lentil crops can be successfully grown) and comes from the LGR-2 variety pedigree. The green lentil seeds were rst soaked in deionized water for 3.5 h at room temperature. The imbibed seeds were distributed evenly in germination trays (Raszyn-Rybie Bionatura, Poland) which were then placed in a temperature- and humidity-controlled seed germinator (Economic Delux ECOO-065, Snijders, The Netherlands). The germination process was carried out at 20 C for seven days at 99% relative humidity in the dark. The seeds were wetted manually with deionized water from an atomizer spray bottle every 12 h. After germination, fresh sprouts were subjected to sensory evaluation. Seeds and sprouts designated for chemical analysis were frozen in liquid nitrogen and then lyophilized with a benchtop FreeZone 2.5 L freeze dry system (Labconco Corporation, Kansas City, MO, USA). 2.2. Sensory evaluation Sensory assessment of the samples was carried out by a panel consisting of nine members (i.e., six females and three males of ages between 28 and 54 years) selected, trained, and monitored according to ISO guidelines (ISO 8586-2, 1994). The panelists were already familiar with the sensory evaluation of legume sprouts based on their participation in a previous, but related, study (Troszyska, Szymkiewicz, & Woejszo, 2007). Quantitative descriptive analysis (QDA) was employed to determine differences in the sensory characteristics of the sprouts (Lawless & Heymann, 1999; Stone & Sidel, 1993). Prior to the study, a lexicon of sensory attributes/descriptors was developed by the panel in round-table sessions using a standardised procedure (ISO/DIS 13299, 1998). For attribute generation, all samples (n = 7) were presented to each assessor. Thirteen attributes related to the appearance, odour, taste, and texture of the sprouts were selected and thoroughly dened for proling (Table 1). The panelists evaluated the intensity perceived for each sensory descriptor on a continuous unstructured, graphical scale. The scales were ten cm in length and verbally anchored at each end. The left side of the scale corresponded to the lowest intensity of the attribute in question, while the right side was the highest. The metric measurements were automatically registered on a zero to ten continuous scale. Samples (i.e., ~10 g of sprouts) were presented to each assessor in three-digit random number coded plastic containers, covered with lids. Along with the samples, the panelist received a cup of spring water at room temperature for cleansing their palate. Two sessions were held per day and seven samples were presented in each session. Assessments were carried out at a sensory laboratory fullling the requirements of ISO 8589 (i.e., individual booths with appropriate ventilation, white lighting, and controlled temperature). The results were collected using a computerised ANALSENS system (IRZiBZ PAN, Olsztyn, Poland). 2.3. Analysis of phenolic compounds Phenolic compounds were extracted from lyophilised sprouts with 75% (v/v) aqueous ethanol at 70 C for 15 min at a solids-tosolvent ratio of 1:10 (w/v) (Amarowicz, Karama, & Shahidi, 2003). The extraction was repeated twice more, supernates combined, and ethanol evaporated under vacuum at 40 C in a Bchi Rotavapor R210 (Bchi Labortechnik AG, Flawil, Switzerland). The aqueous residue was lyophilised and then stored at 4 C until evaluated further.

Appearance Colour Odour Grassy Green Off-odour Taste Green Bitter Sweet Beany Astringent Pea-pod Aftertaste

The intensity of cut grass odour (none to very intensive) The intensity of fresh green pea odour (none to very intensive) Typical odour of aged oil (none to very intensive)

As for the corresponding odour (measured in the mouth) (none to very intensive) The intensity of a bitter taste (none to very intensive) The intensity of a sweet taste (none to very intensive) The intensity of a boiled legume seeds taste (none to very intensive) The intensity of dryness, roughness, and puckerness in the mouth (none to very intensive) The intensity of the taste typical of green pea-pod without pea seeds (none to very intensive) The sensation of pungency staying after the removal of sample from the mouth (low to high)

Texture Juiciness

Degree of juiciness perceived while chewing the sample 10 times (not juicy to juicy) Fibrousness Degree of brousness perceived while chewing the sample 10 times (not brous to brous) Flourness Degree of ourness perceived while chewing the sample 10 times (not oury to oury) The odour and taste reference standards employed for training of avour attributes were as follows: grassy, 1% cis-3-hexen-1-ol; green, commercial fresh green seeds of peas; off-odour, 0.01% iso-butyric acid solution; bitter, 0.2% caffeine solution; sweet, 3% sucrose solution; beany, dry lentil seeds mixed with hot water at a 1:3 (w/v) ratio; astringent; 0.1% tannin acid solution; and pea-pod, small pieces of pea-pods from green peas.

Lyophilised extracts (150 mg) were dissolved in 2 mL of methanol:water (4:1, v/v) and ltered through a 0.45 m cellulose acetate lter (Millipore) before HPLC analysis. The samples were prepared and extracted in duplicate. The HPLC-DAD analyses of polyphenolic compounds were performed using a Waters chromatographic system equipped with a Waters 996 photodiode-array detector and Millenium 32 chromatography manager software. The chromatographic system was equipped with an autoinjector, a quaternary pump, a photodiode-array detector 2001, (Waters Corporation, Milford, MA, USA) a Nova-Pak C18 column (300 3.9 mm; 4 m) and Millenium software. The analytical conditions for separation were those described by Dueas, Estrella, and Hernndez (2004). Two mobile phases were employed for elution: (A) water:acetic acid (98:2, v/v) and (B) water:acetonitrile:acetic acid (78:20:2, v/v/v). The gradient prole was 055 min, 100%20% A; 5570 min, 20%10% A; 7080 min, 10%5% A; and 80100 min, 100% B. The ow rate was 1 mL/min from the beginning to 55 min, and then 1.2 mL/min from this point onward. The column was re-equilibrated between sample injections with 10 mL of acetonitrile and 25 mL of the initial mobile phase. Detection was performed by scanning from 210 to 400 nm at an acquisition speed of 1 s. A volume of 100 L was injected. The samples were analysed in duplicate. For HPLC-MS analyses, mass spectra were obtained using a Hewlett Packard 1100 MSD (Palo Alto, CA, USA) equipped with an API source using an ESI interface. The solvent gradient and column employed were the same as those for HPLC-DAD analyses. ESI conditions were as follows: negative-ion mode; nitrogen was used as the nebulising gas, 40 psi; drying gas, 10 L/min at 340 C; voltage at capillary entrance, 4000 V; and variable fragmentation voltage, 100 V (m/z 200 to 1000), and 250 V (m/z 1000 to 2500). Mass spectra were recorded from m/z 100 to 2500.

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The standards (+)-catechin, trans p-coumaric acid, trans ferulic acid, quercetin-3-O-glucoside, kaempferol-3-O-glucoside, apigenin7-O-glucoside, tryptophan, tryptophol, and phloridzin (2,4,4,6tetrahydroxydihydrochalcone-2-O--glucoside) were purchased from Extrasynthse S.A.S. (Genay Cedex, France). Identication of compounds was carried out by comparison of the retention times and UV spectra with those of standards by HPLC-DAD and HPLC-MS. Other compounds for which no standards were available, such as trans p-coumaric acid derivatives, gallates, catechin glucoside, proanthocyanidins, avonols, avone and dihydrochalcone were identied by HPLC-DAD-MS according to Dueas, Sun, Hernndez, Estrella, and Spranger (2003) and Dueas et al. (2004). The acylated glycosides of quercetin and kaempferol were identied by HPLC-DAD-MS following the reports of Toms-Barbern, Gil, Ferreres, and Toms-Lorente (1992) and Dueas et al. (2003). Quantication was made using the external standard method with commercial standards. Calibration curves were developed by injecting different volumes of the stock solutions over the range of concentration observed for each of the compounds and using linear regression to dene the relationship of area sum versus concentration. The standards were subjected to the same conditions as those for the samples analysed. The trans p-coumaric acid derivative was quantied using the corresponding free acid calibration curve. Gallates, catechin glucoside, procyanidins, and prodelphinidin were quantied as (+)-catechin, and dihydrochalcone as phloridzin. In the case of avone and avonols, the quercetin glycosides were quantied as quercetin-3-O-glucoside, the kaempferol glycosides as kaempferol-3-O-glucoside, and the apigenin derivative as apigenin-7-O-glucoside. 2.4. Statistical analysis Sensory and chemical data were subjected to analysis of variance (ANOVA, StatSoft Inc., v. 8.0, Tulsa, OK, USA). Statistically signicant differences in the results were tested by Fisher's least signicant difference (LSD) test, at P 0.05. Principal component analysis (PCA) was applied to describe the variance among the whole sensory data obtained. PCA was also performed for general assessment of similarity/dissimilarity of the evaluated samples and in describing their sensory attributes. Partial least squares (PLS) regression was used to explore the relationship between the sensory attributes of taste (y-data) and phenolic compounds of sprouts (x-data). PCA and PLS were employed using the software packages StatSoft Inc., v. 8.0 and XL Stat v. 2010.5.03, Addinsoft, respectively. The sensory data presented are averages of duplicate determinations of two separate batches. 3. Results and discussion 3.1. Sensory evaluation Quantitative descriptive analysis (QDA) was used to determine attributes that inuenced the sensory quality of lentil sprouts. The QDA for this study was based on 14 attributes: one for appearance, three for odour, seven for taste, and three for texture (Table 1). The mean sensory ratings for the samples and the ANOVA are given in Table 2. The results show that there were highly signicant (P b 0.001) differences in the intensity of attributes such as colour (F= 35.9), grassy odour (F= 5.8), off-odour (F= 24.5), green taste (F= 8.0), bitter taste (F= 17.6), sweet taste (F= 6.9), beany taste (F= 23.7), juiciness (F= 6.5), brousnesses (F= 28.3), and ourness (F= 19.4) caused at the time of germination. The sensory proles of the one- and two dayold sprouts were very similar: they were perceived as mild in avour with the exceptions of the beany taste and ourness. It should be pointed out that these samples exhibited the least intensity in terms of bitter and astringent tastes, pea-pod taste, and brousness. The ndings indicated that prolonging the germination period over four days negatively

Table 2 Intensity of sensory attributes in the green lentil sprouts.a,b Sensory attribute Appearance Colour Odour Grassy Green Off-odour Taste Green Bitter Sweet Beany Astringent Pea-pod Aftertaste Texture Juiciness Fibrousness Flourness
a b

Days of germinations 1 0.3a 2 0.3a 3 0.8a 4 2.2b 5 4.1c 6 4.8c 7 4.3c

1.7a 0.3ab 0.0a

1.4a 0.3ab 0.0a

3.3b 0.6b 0.0a

4.5b 0.4ab 0.1a

3.9b 0.3ab 1.0a

4.5b 0.3ab 3.3b

3.3b 0.1a 5.0c

2.0cd 0.1a 2.3bc 6.5d 0.1a 2.0a 4.2a

2.6de 0.1a 3.3cd 5.4d 0.1a 2.6ab 4.5a

3.6e 0.4a 3.9d 3.7c 0.2ab 2.5ab 4.6a

1.6bcd 2.6b 2.1bc 2.8bc 0.3abc 3.1ab 4.6a

0.9abc 2.6b 1.9b 1.5ab 0.8bc 3.2ab 5.0a

0.5ab 4.5c 1.6b 0.6a 0.7c 3.4ab 4.9a

0.4a 5.9c 0.3a 0.2a 0.8c 4.2b 5.6a

0.6a 2.0a 7.6d

1.9abc 2.8ab 5.5cd

4.3d 3.6b 4.2bc

2.7c 5.8c 3.1b

2.5c 7.3d 1.6a

2.3bc 8.1de 0.6a

1.1ab 8.8e 0.2a

Mean descriptive analysis rating of green lentil sprouts on a 0 to 10 point scale. Values followed by the same letter (ae) in the same row are not signicantly different (P N 0.05).

affected the sensory quality of the sprouts. From the proles of six- and seven day-old sprouts, undesirable sensory attributes for the consumers such as bitter taste, off-odour, and brousness dominated. As indicated above, the sensory panel comprised six females and only three males: females are typically more sensitive to bitter avours then men, and this may explain why bitterness was associated so prevalently with phenolics (Bartoshuk, Duffy, & Miller, 1994). On the other hand, the samples were characterised by the least ourness and beany taste. It is well known that the major factor contributing to the reduction of consumer acceptability of legume products is associated with beany avour. Some studies have been designed to eliminate this negative attribute altogether in order to increase consumption of legumes (Braudo, Danilenko, Dianova, & Krokha, 2001; Uwaegbute, Iroegbu, & Eke, 2000). As our results showed, germination can be a relevant processing technique for modifying the beany taste note. Yet, further research is required: a separate approach for each legume in question should be considered, because beany avour is likely dependent on the type of legume. To visualize the differences between the sprouts, PCA was used; it was performed on the covariance matrix of the samples with no rotation. Two principal components (PC1PC2) were extracted, and they had eigenvalues greater than one (i.e., 34.9 and 3.6, respectively; Table 3). A third principal component (PC3) had an eigenvalue of just 1, but did not contribute much further toward explaining variations in the data. The rst (PC1) and second (PC2) component together explained ~ 96.0% of the variation in the sensory quality of the samples. PC1 explained the majority of the variations, comprising

Table 3 Eigenvalues of covariance matrix and related statistics. Principal component PC1 PC2 PC3 PC4 PC5 PC6 Eigenvalue 34.936 3.649 0.993 0.383 0.194 0.047 Total variance % 86.901 9.076 2.469 0.953 0.483 0.117 Cumulative eigenvalue 34.936 38.585 39.578 39.961 40.155 40.202 Cumulative % 86.901 95.978 98.447 99.400 99.883 100.000

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Table 4 Eigen analysis of the correlation matrix loadings of the principal components (PCs).a Sensory attribute Appearance Colour Odour Grassy Green Off-odour Taste Green Bitter Sweet Beany Astringent Pea-pod Aftertaste PC 1 PC 2 PC 3 PC 4 PC 5 PC 6

3
Day 1 Day 7

0.9722 0.0254

0.1666 0.1104 0.1186

0.0112

PC 2: 9,08%

Day 2

Day 6

0.7200 0.5403 0.2945 0.6099 0.6902 0.1636 0.8700 0.3808 0.2902

0.3119 0.0761 0.2408 0.2586 0.0439 0.1097

0.0032 0.0043 0.0027

-1

Day 5

Day 4

-2
Day 3

-3

0.8398 0.3986 0.3536 0.0750 0.0157 0.0702 0.9736 0.1775 0.0336 0.1318 0.0368 0.0265 0.8012 0.5367 0.1933 0.1151 0.0933 0.1032 0.9816 0.1848 0.0415 0.0064 0.0038 0.0222 0.9358 0.0313 0.1143 0.2794 0.0237 0.1780 0.9539 0.0526 0.1635 0.0164 0.2429 0373 0.8762 0.1791 0.3197 0.1461 0.2074 0.1835

-4 -10

-8

-6

-4

-2

10

PC 1: 86,90%
Fig. 1. Principal component analysis (PCA). Scores of sensory data for germinated green lentil sprouts.

Texture Juiciness 0.0567 0.9713 0.2198 0.0585 0.0323 Fibrousness 0.9963 0.0414 0.0595 0.0270 0.0357 Flourness 0.9747 0.1957 0.0702 0.0678 0.0377
a

0.0250 0.0131 0.0279

The most signicant loadings for each sensory attribute are highlighted in boldface.

86.9%, while 9.1% was attributable to PC2. PC1 and PC2 are plotted in Fig. 1. It can be seen that the distribution of the samples in the score plot (Fig. 1) was different according to the day of germination, which indicated their varied sensory characteristics. Two separate categories were distinguished along PC1: the samples marked as day 1, day 2, day 3, and day 4 were positively located along PC1 while the ones marked as day 5, day 6, and day 7 were negatively located along PC1. In the rst group, dominant attributes are ourness, juiciness, beany taste, green taste, and sweet taste while in the second group, they are off-odour, bitter taste, and brousness (Fig. 2). The PC factor loadings for the rst six PCs are given in Table 4. It can be seen that the attributes of colour, off-odour, green taste, bitter taste, sweet taste, beany taste, astringent taste, pea-pod taste, aftertaste, brousness, and ourness had a high loading (N 0.8) in PC1. It is common
2

knowledge that PC1 contains the most important information. Thus, these notes probably had a decisive effect on the variation in the sensory quality of the lentil sprouts. 3.2. Proling of the phenolic compounds The phenolic compounds identied by HPLC-DAD-MS in the green lentil crude extract are presented in Table 5 in their order of elution along with their wavelength maximum for detection, [M-H] ion, and dominant fragment ions. Compounds possessing a avanol structure (i.e., monomers, oligomers, and gallate derivatives) were the most abundant class of compounds detected in the lentil extracts, and a catechin glucoside was the predominant avanol (288.8 g/g). Flavonols, notably derivatives of quercetin and kaempferol with quercetin diglycoside being the most copious for this avonoid class, were also detected in the samples (Fig. 3). Non-avonoid compounds (e.g.,

Table 5 Phenolic compounds identied by HPLC-DAD-MS in the crude green lentil extract. Compoundsa max (nm) 276 276 279 278 274 279 279 279 280 310 309 256, 325 268, 266, 268, 265, 256, 268, 268, [MH] (m/z) 593 593 451 745 442 577 865 577 881 163 609 193 609 901 931 755 505 545 901 Fragment ions (m/z) 288, 289, 289 577, 169 289 577, 289 577, 163 301 285 755, 755, 285 301 285 755, 305 305 169

off-odour bitter tas te aftertas te pea-pod taste as tringent tas te c olour fibrous nes s green odour green tas te grass y odour s weet tas te juic ynes s flournes s beany tas te

-1

-2 -4

-3

-2

-1

PC 1 : 86,90%
Fig. 2. Principal component analysis (PCA). Loadings of sensory data for germinated green lentil sprouts.

Prodelphinidin dimer Prodelphinidin dimer Catechin glucoside Gallate procyanidin dimer Catechin gallate Procyanidin dimer Procyanidin trimer Procyanidin dimer Digallate procyanidin dimer Trans p-coumaric acid derivative Trans p-coumaric acid Quercetin diglycoside Trans ferulic acid Kaempferol glycoside, acylated Quercetin glycoside, with 2 sugar moieties Kaempferol glycoside, acylated Kaempferol rhamohexose-hexose Quercetin glycoside, acylated Kaempferol glycoside, acylated Kaempferol glycoside, acylated
a

PC 2 : 9,08%

289 289

354 317 348 318 348 325 317 318

285 609, 285

285

The compounds are presented in their order of elution from the Nova-Pak C18 column based on the chromatographic conditions described in Section 2.3.

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600 500 400 300 200 100 0 0 1 2 3 4 5 6 7

Germination time (days)

Fig. 3. Changes in the content of the classes of phenolic compounds (g/g) in extracts of the green lentil seeds and sprouts during germination. The symbols represent the following: hydroxycinnamates; catechins and proanthocyanidins; and avonols.

phenolic acids) were scarce in this type of lentil, and thus represented only a small percentage of the phenolic composition. The chromatographic approach of proling individual phenolic compounds in germinated lentil seeds is in line with that previously reported for leguminous seeds. (+)-Catechin and ()-epicatechin glucosides, quercetin glucoside, myricetin, as well as procyanidin B1 and B3 were the main phenolic compounds found in a crude extract of adzuki bean (Amarowicz, Estrella, Hernndez, & Troszyska, 2008; Ariga & Hamano, 1990; Ariga, Koshiyama, & Fukushima, 1988). Glucosides of avones and avonols were determined in the cotyledon of peas (Pisum sativum L.) (Dueas, Hernndez, & Estrella, 2006). High contents of quercetin-3-O-glucoside and myricetin-3-Oglucoside were also detected in raw cowpeas (Vigna sinensis L.) (Dueas, Fernndez, Hernndez, Estrella & Muoz, 2005). Caffeic, o-coumaric, ferulic, and sinapic acids were determined in the crude extract of red bean (Amarowicz & Troszyska, 2004), whereas vanillic, caffeic, p-coumaric, sinapic, and ferulic acids, as well as quercetin and kaempferol were found in a pea crude extract (Amarowicz & Troszyska, 2003). Flavonols were the dominant phenolic compounds

present in acetonic extracts of red and green lentils (Amarowicz et al., 2009, 2010). Germination has been identied as an effective technology for improving the quality of edible leguminous seeds by enhancing their digestibility, increasing their content of nutrients, and decreasing the levels of non-nutrients. With regard to important changes in these components, germination might be a valuable processing technique for modifying the sensory perception (Bau, Villaume, & Mjean, 2000; Uwaegbute et al., 2000). Details concerning the changes in levels of individual phenolic compounds that occur during germination of seeds and their inuence on sensory quality of the resultant sprouts is, however, still decient. From the onset of germination, there was a marked decrease in the contents of the proanthocyanidins and catechin glucoside: a majority of them were not detected in the sprouts! Furthermore, there was a signicant (P b 0.05) decrease in the concentration of hydroxycinnamates from the rst day of germination. Key changes in the phenolic proles were those of the avonol glycosides: the sprouts were found to be rich in acylated avonol glycosides, which were related to the decrease in the content of hydroxycinnamates. Based on the ndings, it was concluded that at two and three days of germination this corresponded to the lowest sum (170.9 g/g and 170.0 g/g, respectively) of identied polyphenolic compounds. The maximum content of polyphenols in the sprouts was determined after seven days of germination (i.e., 400.8 g/g, see Table 6). Bartolom et al. (1997) reported a decrease in (+)-catechin and procyanidin dimers and trimers in lentils after six days of germination. The results obtained by Dueas, Hernndez, Estrella, and Fernndez (2009) indicate that germination modies both the qualitative and quantitative polyphenolic composition of lupin (Lupinus angustifolius L.) seeds over time resulting in a signicant increase in avonoids contents. In the study of Lpez-Amors et al. (2006), various changes in the phenolic compounds occurred after germination that were not only dependent on the kind of leguminous seeds, but also on the process conditions (i.e., the presence of light and germination time). A general decrease in hydroxybenzoic acids was observed in lentils, beans, and peas. In the case of lentils, some hydroxycinnamic compounds were detected from the onset of germination. After four days

Table 6 Changes in the content of individual phenolic compounds (g/g) in extracts of green lentil seeds and sprouts during seven days of germination. Compounds Germination period (days) 0 Hydroxycinnamates Trans p-coumaric acid derivative Trans p-coumaric acid Trans ferulic acid Catechins and proanthocyanidins Prodelphinidin dimer Prodelphinidin dimer Catechin glucoside Gallate procyanidin dimer Catechin gallate Procyanidin dimer Procyanidin trimer Procyanidin dimer Digallate procyanidin dimer Flavonols Quercetin diglycoside Kaempferol glycoside acylated Kaempferol rutinoside-neohesperidoside Kaempferol glycoside acylated Kaempferol rhamohexose-hexose Quercetin glycoside acylated Kaempferol glycoside acylated Kaempferol glycoside acylated 6.4 0.3b 37.3 1.9c 10.1 0.9b 1 nda 8.6 0.8b nda 2 nda nda nda 3 nda nda nda 4 nda nda nda 5 nda nda nda 6 nda nda nda 7 nda nda nda

5.3 0.5b 14.1 0.9b 288.8 19.2c 31.9 1.9c 18.7 1.1a 59.5 2.4b 18.6 0.9a 100.2 12.1b 53.8 1.9b

nda nda 12.1 0.8b 9.3 0.9b 31.7 2.9b nda nda nda nda

nda nda nda 9.4 0.8b 31.8 1.1b nda nda nda nda

nda nda nda 5.1 0.8a 33.6 1.5b nda nda nda nda

nda nda nda 4.5 0.7a 43.3 3.7c nda nda nda nda

nda nda nda 6.3 0.7ab 53.4 3.7d nda nda nda nda

nda nda nda 3.7 0.5a 53.9 2.5d nda nda nda nda

nda nda nda 5.1 0.5a 71.9 4.0e nda nda nda nda

113.5 5.8e nda nda nda 19.4 0.8b 31.1 1.6b nda nda

158.4 5.8f nda 8.1 0.7b nda nda 20.7 2.5b 44.6 5.6b nda

74.1 2.6c nda nda nda nda nda 55.6 4.3bc nda

53.5 1.3c nda nda nda nda nda 68.8 7.1cd 9.0 0.8b

27.9 1.0ab 20.9 1.8b nda nda nda nda 82.5 6.3d 31.2 2.5c

28.3 2.1ab 63.5 7.7c nda nda nda nda 111.9 0.7e 33.4 1.8c

20.9 1.9a 82.6 5.3d nda nda nda nda 89.6 5.6d 33.8 1.2c

33.4 1.2 98.1 6.3e nda 18.6 1.8b nda nda 134.9 7.6f 38.8 2.0d

Values followed by the same letter (ae) in the same row are not signicantly different (P N 0.05); ndnot detected.

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of germination, avonol glycosides were noted in the beans; that is, compounds not detected in the raw samples.

3.3. Relationships between sensory and chemical results The relation between the taste attribute and phenolic compounds was studied by PLS regression (Fig. 4). PLS regression explained ~ 91.9% of the variation in the chemical data and 90.8% of the variation for the sensory result (i.e., taste) when the rst two PCs were employed. The ndings also indicated that catechin gallate and four forms of kaempferol glycosides (i.e., kaempferol glycosides 1, 2, 3, and 4, respectively) were highly positively related to the sensory attributes of peapod taste, bitter taste, aftertaste, and astringent sensation. The intensity of these sensory descriptors was greatest after seven days of germination. It should be stressed that the kaempferol derivatives were absent in the ungerminated seeds and that an increasing tendency in their accumulation was detected during seed germination (see Table 6). It is interesting that during germination acetylation of kaempferol and quercetin glycosides show the opposite trend. To our knowledge, this is the rst such observation to be reported and will require independent research for an explanation. The content of catechin gallate, on the other hand, was 18.7 g/g in the ungerminated seeds and increased continuously during sprouting to 71.9 g/g after the seventh day. According to literature, the sensory activity of phenolics is connected not only with their molecular structure but also to taste thresholds specic for each compound (Heini et al., 2008; van Gemert, 2003). Unfortunately, taste thresholds of polyphenols identied in the sprouts are not available. Documented taste threshold values for various polyphenols are scare, but those that do depend not only on the medium in which the polyphenols are presented, but also on the method of analysis employed. As an example, the taste threshold value of kaempferol in 5% aqueous ethanol is 20 mg/kg, whereas for beer it is almost
1

50 mg/kg (van Gemert, 2003). This suggests that the total concentration of kaempferol glycosides in the samples exceeded the taste threshold values. The question as to whether all forms of the kaempferol glycosides were taste-active compounds remains for future research. It also should be noted that non-phenolic compounds in legumes such as phytic acid can impart astringency, while peptides and saponins can contribute bitter notes; therefore, the PLS correlations between the chemical and sensory data do not represent a true cause and effect relationship. From the data it is evident that the germination time has a highly signicant impact on the polyphenolics content of lentil sprouts. According to the statistical multivariate methods employed in this work, namely PCA and PLS regression, it can be stated that the number of days in which germination progressed and the levels of phenolic compounds at various points in time modied the sensory quality of the lentil sprouts. Based on PLS regression, avonols and catechin gallate were highly positively related to bitterness and astringency of the spout samples; these compounds were the most dominant phenolics in seven day-old sprouts (see Table 6). From the sensory point of view, phenolic compounds can interact with other sprouts components as well as be converted to a number of volatile compounds (e.g., alcohols, aldehydes, and esters) thereby generating either desirable or non-desirable avour notes in the product. The impact of volatile compounds of sprouts on avour remains to be elucidated.

4. Conclusions The effect of individual phenolic compounds on the sensory prole of green lentil sprouts is reported here for rst time. It should be emphasised that the sprouts were only examined by a trained sensory panel. Thus, a consumer study is necessary to conrm these ndings.

0.75

SWEET TASTE GREEN TASTE

0.5

Day 3 Day 2

The second PLS axis

Day 4
0.25

BEANY TASTE
0

Day 5 Day 6 PEA POD TASTE

kaempferol glycoside 4 AFTERTASTE kaempferol glycoside 3 ASTRINGENT TASTE BITTER TASTE kaempferol glycoside 1 catechin gallate kaempferol glycoside 2

X procyanidin dimer gallate Y Obs

-0.25

quercetin diglycoside
coumaric acid quercetin glycoside kaempferol rutinoside

-0.5

Day 7
-0.75

Day 1

catechin glucoside

-1 -1

-0.75

-0.5

-0.25

0.25

0.5

0.75

The first PLS axis

Fig. 4. Partial least squares (PLS) regression biplot for scores and loadings of sensory and chemical data for the germinated sprouts.

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