Z Lebensm Unters Forsch A (1997) 205: 290 294
O R I G I N A L PA P E R
Begona Bartolome ? Isabel Estrella ? Teresa Hernandez
Changes in phenolic compounds in lentils (Lens culinaris)
during germination and fermentation
Received: 16 December 1996 / Revised version: 6 March 1997
AbstractmGermination and fermentation have been pro-
posed to improve the nutritive value of legumes. This paper
reports the changes that occur in phenolic compounds in
lentils (Lens culinaris var. vulgaris) during germination and
fermentation. Levels of low-molecular-weight phenolic
compounds (benzoic acids and aldehydes, hydroxycin-
namic acids and derivatives, flavan-3-ols and procyanidins)
were determined. Germination did not appreciably vary the
content of phenolic compounds, although important struc-
tural changes in procyanidin-type compounds were ob-
served. Fermentation led to a general increase in the content
of phenolic compounds. Gentisic acid, p-hydroxyphenyl-
propionic acid, tryptophol and three other unknown com-
pounds were detected in fermented lentils, but not in raw
lentils. The influence of these changes on the potential
antinutritional effects associated with phenolic compounds
in legumes, as well as on the antioxidant properties attrib-
uted to these compounds is discussed.
Key wordsmLens culinaris ? Phenolic compounds
Germination ? Fermentation ? Antinutritional effects
Introduction
Lentils (Lens culinaris) constitute an important source of
foods for humans in Mediterranean countries. They have a
high protein content of good biological value, with a
limited amount of sulphur-containing amino acids. Lentils
are also rich in complex carbohydrates, starch and dietary
fibre; however, they contain some antinutritional factors
(trypsin and chymotrypsin inhibitors, phytic acid, con-
densed tannins, lectins, saponins etc.) which could limit
their consumption [1, 2].
B. Bartolome ? I. Estrella ? T. Hernandez ( )
Instituto de Fermentaciones Industriales (CSIC), Juan de la Cierva 3,
E-28006 Madrid, Spain
Springer-Verlag 1997
Condensed tannins are polymers of flavan-3-ols. Studies
of animals have provided evidence that tannins reduce
growth rates and lead to poor efficiencies of feed utilization
[3]. In vitro studies have clearly shown that these polymers
bind to food proteins [4] and have an inhibitory action
against digestive enzymes [5]. Tannins may also reduce the
bioavailability of iron-containing compounds in lentils [6].
However, over the last few years, the presence of tannins
(and phenolic compounds in general) in food has been
viewed in a much more positive light. Phenolic compounds
not only effectively prevent oxidation in food systems [7],
but they also may act as protective factors against oxidative
damage in the human body [8].
Germination and fermentation have been proposed to
improve the nutritive value of lentils and lentil-based
products for human consumption. It has been reported
that germination of lentils brings about an increase in the
levels of free amino acids and vitamins (ascorbic acid,
riboflavin, thiamin and niacin) [9, 10]. In addition, the
amounts of antinutrients (trypsin inhibitors, phytates, a-
galactosides and saponins) decrease with germination
[9 12]. The condensed tannin content is observed to
increase with germination, but this is thought to be related
more to the accessibility in the assay rather than to
metabolic changes [11].
Fermentation modifies the organoleptic characteristics
of lentils, which could make them more attractive than the
raw seeds to the consumer [13]. It has been shown that
fermentation increases the in vitro digestibility of protein
and starch, as well as the riboflavin content [14, 15],
whereas levels of antinutrients in lentils (trypsin inhibitors,
phytic acid, a-galactosides) are reduced by fermentation
[16 18]. In a time course study a significant increase in the
condensed tannin content as fermentation proceeds has
been observed, although the preparation step was the source
of the main variation [17].
Determination of the levels of condensed tannins in
lentils before and after processing has generally been
achieved using colorimetric methods (HCl/butanol and
vanillin assays) [11, 17], which only give a global measure
of condensed tannins. We have found no data in the

Fig. 1mChromatograms at 280 nm of raw (a), germinated (b) and
fermented (c) lentils. H1 Protocatechuic acid, 2 protocatechuic alde-
hyde, 3 gentisic acid, 4 p-hydroxybenzoic acid, 5 B3 [(+)-catechin-
(4a?8)-(+)-catechin], 6 B1 [()-epicatechin-(4b?8)-(+)-catechin], 7
T2 [()-epicatechin-(4b?8)-()-epicatechin-(4b?8)-(+)-catechin], 8
p-hydroxybenzoic aldehyde, 9 (+)-catechin, 10 vanillic acid, 11 deri-
vative of p-coumaric, 12 T3 [()-epicatechin-(4b?6)-()-epicatechin-
(4b?8)-()-epicatechin], 13 B2 [()-epicatechin-(4b?8)-()-epicate- using calibration curves of the standards. Derivatives of p-coumaric
chin], 14 p-hydroxyphenylpropionic acid, 15 vanillin, 16 C1 [()-
epicatechin-(4b?8)-()-epicatechin-(4b?8)-()-epicatechin], 17 p-
coumaric acid, 18 T4 (tetramer whose structure was not completely
determined), 19 tryptophol, 20 ferulic acid, 21 B5 [()-epicatechin-
(4b?6)-()-epicatechin], A D, G, P, unknown phenolic compounds
(see text)
literature about variations in the levels of individual con-
densed tannins or any other phenolic compounds during
legume processing. However, since the characteristics of
phenolic compounds mentioned above, i. e. their ability to
interact with nutrients and antioxidant properties, are
strongly influenced by their chemical structure, analysis
and characterization of individual phenolic compounds is
required. This paper reports the identification and quantifi-
cation of procyanidins, a group of condensed tannins
formed by (+)-catechin and ()-epicatechin, and other
low-molecular-weight phenolic compounds in lentils before
291
and after being subjected to germination and fermentation
processes.
Materials and methods
Plant material. Lentils (Lens culinaris, var. vulgaris) were purchased at
a local market.
Germination. Germination of lentils was carried out as described by
Vidal-Valverde et al. [11]. Seeds (25 g) were soaked in distilled water
(125 ml) at room temperature and shaken every 30 min. After 6 h, the
water was drained and the lentils transferred to a separating funnel and
kept in the dark at 20 C to germinate for 6 days. The seeds were
moistened with water every 24 h and carefully shaken and drained. The
germinated seeds were ground and freeze-dried.
Fermentation. Naturally fermented lentils were obtained following the
conditions reported by Tabera et al. [17]. Seeds (150 g) were ground in
a ball mill. Suspensions of lentil flour in sterilized tap water were
aseptically prepared in 1 l of water; they fermented spontaneously at
35 C for 4 days, without aeration, in a stirred fermentation unit.
Samples were collected and freeze-dried before analysis.
Analytical procedures. For analysis of individual phenolic compounds,
5 g of raw, fermented and germinated lentils was macerated for 12 h at
room temperature, three times with 20 ml of a solution of acetone and
water (70 : 30 v/v). The three combined macerates were concentrated to
35 ml using a rotatory evaporator. The concentrated solution was
extracted four times with ethyl acetate (20 ml). The organic solutions
were combined, dried for 30 min with anhydrous sodium sulphate,
filtered and evaporated to dryness. The residue was dissolved in 1 ml
of a mixture of methanol and water (50 : 50, v/v) and 50 ml was injected
onto the HPLC apparatus.
HPLC analysis was carried out with a Waters (Milford, Mass.,
USA) system equipped with a Model 991 photodiode-array detector
(DAD). The column was a reversed-phase Nova Pak C18
(30 cm 3.9 mm I.D.) with 4 mm packing (Waters). Elution of phenolic
compounds was carried out with an acetonitrile-water gradient [19].
Detection was performed by scanning from 210 to 400 nm.
Chromatographic peaks were checked for peak purity and identifica-
tion was achieved by comparing retention times and data of spectral
parameters (wavelengths of the spectrum maxima, width of the con-
vexity interval [20] and min-max distance [19] with those of standards
of low-molecular-weight phenolic compounds [20], or those of pre-
viously purified procyanidins and related compounds [19], or those of
previously purified hydroxycinnamic acid derivatives [21]. Quantita-
tive determinations were carried out by peak height measurements,
were quantified using the calibration curves of p-coumaric acid.
Procyanidins were quantified as (+)-catechin.
Samples were prepared and analysed in triplicate.
Results and discussion
Composition of lentils in terms of phenolic compounds
The following phenolic compounds were identified in the
HPLC chromatograms of raw and processed lentils (Fig. 1).
1. Benzoic acids and aldehydes: protocatechuic acid, p-
hydroxybenzoic acid, vanillic acid, gentisic acid, proto-
catechuic aldehyde, vanillin and p-hydroxybenzoic al-
dehyde.
2. Phenyl-propionic acids: p-hydroxyphenylpropionic acid.
3. Benzyl alcohols: tryptophol.
292
Table 1mComposition of raw, germinated and fermented lentils in terms of phenolic compounds (mg/g of dry material).a (t Traces, nd not
detected)
no. Phenolic compound Quantity (mg/g dry material) in lentils:

Raw Germinated Fermented

1 Protocatechuic acid 0.36+0.04 0.02+0.01 1.10+0.08
2 Protocatechuic aldehyde 0.13+0.02 t 0.12+0.03
3 Gentisic acid nd nd 14.96+0.90
4 p-Hydroxybenzoic acid 1.48+0.10 0.68+0.09 7.39+0.61
5 B3 [Catechin-(4?8)-catechin] 8.65+0.69 t 4.03+0.31
6 B1 [Epicatechin-(4?8)-catechin] 4.44+0.31 nd 4.25+0.29
7 T2 [Epicatechin-(4?8)-epicatechin-(4?8)-catechin] 3.42+0.25 nd 0.25+0.04
8 p-Hydroxybenzoic aldehyde nd 0.06+0.02 nd
9 (+)-Catechin 7.31+0.54 nd 17.53+1.09
10 Vanillic acid nd nd 0.23+0.03
11 Derivative of p-coumaric acid 0.74+0.09 nd nd
12 T3 [Epicatechin-(4?6)-epicatechin-(4?8)-epicatechin] 1.30+0.11 t 0.93+0.07
13 B2 [Epicatechin-(4?8)-epicatechin] 5.66+0.41 nd 1.03+0.09
14 p-Hydroxyphenylpropionic acid nd 0.15+0.03 3.90+0.25
15 Vanillin 0.12+0.02 0.58+0.08 nd
16 C1 [Epicatechin-(4?8)-epicatechin-(4?8)-epicatechin] 0.22+0.04 nd nd
17 p-Coumaric acid 7.51+0.62 0.65+0.08 1.95+0.16
18 T4 (Procyanidin tetramer) 0.32+0.03 1.03+0.08 0.58+0.07
19 Tryptophol nd nd 2.70+0.25
20 Ferulic acid 0.06+0.02 0.09+0.03 t
21 B5 [Epicatechin-(4?6)-epicatechin] nd 1.21+0.10 nd
a Data are mean of triplicate analyses

4. Hydroxycinnamic acids and derivatives: p-coumaric
acid, a derivative of p-coumaric acid and ferulic acid.
5. Flavan-3-ol and procyanidins: (+)-catechin, the procya-
nidin dimers B1 [()-epicatechin-(4b?8)-(+)-catechin],
B2 [()-epicatechin-(4b?8)-()-epicatechin], B3 [(+)-
catechin-(4a?8)-(+)-catechin] and B5 [()-epicate-
chin-(4b?6)-()-epicatechin], the procyanidin trimers
C1 [()-epicatechin-(4b?8)-()-epicatechin-(4b?8)-(
)-epicatechin], T2 [()-epicatechin-(4b?8)-()-epicate-
chin-(4b?8)-(+)-catechin], and T3 [()-epicatechin-
(4b?6)-()-epicatechin-(4b?8)-()-epicatechin], and
the procyanidin tetramer T4 (whose structure was not
completely determined).
Procyanidin-type compounds [(+)-catechin, dimers B3,
B2 and B1, timer T2] were found in the greatest quantities in
raw lentils (Table 1). The sum of all these compounds in
raw lentils was lower than the value of total condensed
tannins as determined by colorimetric methods [11, 17];
this is partly due to the fact that the highly polymerized
tannins are not extracted from lentils by ethyl acetate, but
do give a colorimetric response. In addition, global deter-
minations are expressed as standards [cyanidin equivalents
for the HCl/butanol assay, and (+)-catechin equivalents for
the vanillin assay] that can have a different response in the
colorimetric method compared to that of the tannin com-
pounds in lentils. Free p-coumaric acid was also found in
large amounts, whereas other cinnamic and benzoic acids
were found in at least fivefold lower quantities.
Changes in the composition in terms of phenolic
compounds during germination
The phenolic composition of lentils qualitatively changed
during germination (Fig. 1 b). On the one hand, (+)-catechin
and the main procyanidins in raw lentils (B3, B2, B1, T3, T2
and C1) were not detected in the germinated samples
(Table 1). A significant decrease in the quantity of free
and esterified p-coumaric acid was also observed. On the
other hand, the highest peak arising from the analysis of
germinated lentils at 280 nm corresponded to an unidenti-
fied compound (named A in Fig. 1 b), which was not
detected in raw lentils. Spectral analysis of peak A showed
a maximum at 277.2 nm with a shoulder at 310 nm. The
spectral parameters of the first band (22.9 nm for width of
the convexity interval, and 26 nm for min-max distance)
suggested that it could correspond to a procyanidin trimer
[19] linked to p-coumaric acid, which was responsible for
the shoulder band [20]. Escribano-Bailon [22] assigned the
structure of p-coumaroyl-(+)-catechin to a peak with a
similar spectrum in the chromatogram of grape seeds. B5
[procyanidin with a (4b?6) linkage], an unknown com-
pound (named P in Fig. 1 b) showing a procyanidin-type
spectrum, and a series of unknown compounds (named G in
Fig 1 b) showing procyanidin gallate-type spectra [19] were
also detected in germinated lentils, but not in raw lentils. In
addition, the only procyanidin tetramer (T4) found in raw
lentils was found in noticeably increased quantities after
germination.
A small increase in the content of benzoic aldehydes
(vanillin and p-hydroxybenzoic aldehyde this latter com-
pound was not detected in raw lentils) was observed during
lentil germination, whereas benzoic acids (protocatechuic
and p-hydroxybenzoic acids) showed contrasting behaviour.
p-Hydroxyphenylpropionic acid was also detected in ger-
minated samples, but at a lower level (Table 1).
During germination, endogenous enzymes are activated,
which can modify the composition of lentils in terms of
phenolic compounds. It has been reported that during

germination procyanidins and catechins could condense to
give more highly polymerized forms through the activation
of polyphenoloxidase [23]. This could explain the observed
decrease in the levels of (+)-catechin and procyanidin
dimers and trimers found in the present work. The presence
of the tetramer T4 in germinated lentils could also be due to
this polymerization mechanism.
Changes in the composition of lentils in terms of phenolic
compounds during fermentation
Fermentation led to a general increase in the HPLC-DAD
response of low-molecular-weight phenolic compounds
(Fig. 1 c). Thus, fermented lentils exhibited the highest
content of an individual phenolic compound among the
samples analysed, i. e. 17.53 mg/g of dry material for (+)-
catechin, which represents more than twofold its content in
raw lentils (Table 1). Gentisic acid (2,5-dihydroxybenzoic
acid), a compound which was not detected in raw lentils,
was also present at a high level in fermented lentils. Also
found in significant quantities were p-hydroxybenzoic acid
(almost fivefold its value in raw lentils), p-hydroxyphenyl-
propionic acid and tryptophol (these two latter did not occur
in raw lentils). The other main peaks in the chromatogram
of fermented lentils corresponded to three unknown com-
pounds, named B D in Figure 1 c. The spectrum of peak B
showed a band only at 273.3 nm (with a width of the
convexity interval of 31.7 nm and a min-max distance of
24.7 nm), which suggests that it could correspond to a
gallate of a procyanidin tetramer [19]. Comparisons of the
spectra of peak C (with a maximum at 275.9 nm, a width of
convexity interval of 22.1 nm and a min-max distance of
27.3 nm) and peak D (with a maximum at 292.8 nm, a width
of the convexity interval of 30.2 nm and a min-max distance
of 42.9 nm) with those of standards [20] indicates that both
peaks could correspond to low-molecular-weight phenolic
compounds, the former to a simple phenol, and the latter to a
phenol with a group conjugated with the aromatic ring [20].
Concerning procyanidins, the dimer content varied se-
lectively during fermentation: whereas B1 did not exhibit
any changes, the concentrations of B2 and B3 decreased
twofold. The quantities of the trimers T2 and T3 also
decreased: a small increase in the quantity of tetramer T4
was observed, although it was lower than those reportedly
present in germinated samples.
As in germinated samples, the amounts of p-coumaric
and its derivative decreased markedly with fermentation.
However, the amounts of minor benzoic acids (protocate-
chuic and vanillic acids) increased slightly during fermen-
tation, but not during germination (Table 1).
Natural fermentation is a microbial process in which
different enzymatic systems, not only from the micro-
organisms but also from the seed, can act. The increment
in the quantity of (+)-catechin observed cannot be totally
attributed to the hydrolysis of dimers since no ()-epica-
techin (which also forms the procyanidin molecules) was
detected in fermented lentils. Other mechanisms, such
as hydrolysis of larger (+)-catechin precursors or selective
293
()-epicatechin degradation, may be involved and thus
cause these results. Tryptophol has been associated with
fermentation processes [24]. Gentisic and p-hydroxyphenil
propionic acids could be formed by the action of micro-
organisms on phenolic compounds. Gentisic acid is be-
lieved to originate from p-coumaric acid [25], which may
be the reason why this cinnamic acid decreases during
fermentation, as found in this study.
Total-content assays, such as those for condensed tan-
nins and catechins, can be useful analytical tools for
determining global changes in the composition of phenolic
compounds in lentils, but studies of individual phenolic
compounds are required if the processes occurring during
germination and fermentation are to be understood in
greater depth. Since the chemical structure of phenolic
compounds strongly influences their physiological proper-
ties, such as their ability to join proteins and their antiox-
idant properties, greater knowledge of their behaviour
during these processes is of paramount importance. In
general, the extent of procyanidin-protein interactions in-
creases with the degree of procyanidin polymerization and
the rate of galloylation [4, 26]. In this sense, germination
may enhance the ability of lentil procyanidins to interact
with food proteins since it has been shown that procyanidin
polymerization and gallate formation occur during this
process. A similar conclusion could be drawn for lentil
fermentation, but to a lesser extent.
Flavonoids and cinnamic acids are considered to be
primary antioxidants and they act as free radical acceptors
and chain breakers in foods [27, 28]. The position and the
degree of hydroxylation is of primary importance in deter-
mining the antioxidant activity of phenols. For flavonoids,
the antioxidant activity decreases with p-quinol4o-dihy-
droxylation4p-hydroxylation structures of the B-ring of
flavonoids. Hydroxylated cinnamic acids are more effective
than their benzoic acid counterparts; p-hydroxy substitution
enhances antioxidant activity, whereas the presence of
methoxy groups reduces that activity. In model systems
that simulate the oxidation processes leading to atheroscle-
rosis and heart disease [29, 30], flavan-3-ols, their gallates
and procyanidins show the highest antioxidant activity
among the different phenolic structures, even higher than
that of a-tocopherol. The monomers catechin and epicate-
chin, the procyanidin dimers B2 and B8, and trimer C1 have
greater activity than other procyanidin dimers (B3, B4 and
B6) and trimer C2 [30]. It might be expected, therefore, that
fermented lentils have a higher antioxidant potential than
raw lentils due to their high content of (+)-catechin.
In summary, this paper shows that germination and
fermentation lead to significant changes in the phenolic
compound content and composition of lentils which could
modify the nutritional value of this legume.
AcknowledgementsmThe authors thank Dr. C. Vidal, Dr. J. Tabera and
Dr. J. Frias for their collaboration in sample preparation and helpful
discussion, and to Mr. Luis Pinal for technical assistance. This study
was supported by Spanish Comision Interministerial de Ciencia y
Tecnologa ALI 91-1092-CO2-01.
294
References
1. Savage GP (1988) Nutr Abstr Rev 5: 319 343
2. Van der Poel AFB, Huisman J, Saini HS (1993) Recent advances
of research in nutritional factors in legume seeds. Wageningen
Pers, Wageningen
3. Butler LG (1989) Sorghum polyphenols. In: Cheeke PR (ed)
Toxicants of plant origin. CRC, Boca Raton, Fla., pp 95 121
4. Hagerman AE, Butler LG (1981) J Biol Chem 256: 4494 4497
5. Quesada C, Bartolome B, Nieto O, Gomez-Cordoves C, Hernan- 20. Bartolome B, Bengoechea ML, Galvez MC, Perez-Ilzarbe FJ,
dez T, Estrella I (1996) J Food Protect 59: 185 192
6. Garrow JS, James WPT (1993) Human nutrition and dietetics.
Churchill Livingstone, Edinburgh, p 179
7. Rajalakshmi D, Narasimhan S (1995) Food antioxidants: sources
and methods of evaluation. In: Madhavi DL, Deshpande SS,
Salunke DK (eds) Food antioxidants: technological, toxicological
and health perspectives. Dekker, New York, pp 65 157
8. Aruoma OI (1994) Food Chem Toxicol 7: 671 683
9. Hsu D, Leung HK, Finney PL, Morad MM (1980) J Food Sci 45:
87 92
10. Urbano G, Lopez-Jurado M, Hernandez J, Fernandez M, Moreu
MC, Frias J, Diaz-Pollan C, Prodanov M, Vidal-Valverde C (1995)
J Agric Food Chem 43: 1871 1877
11. Vidal-Valverde C, Frias J, Estrella I, Gorospe MJ, Ruiz R,
Bacon J (1994) J Agric Food Chem 42: 2291 2295
12. Ruiz RG, Price K, Rose M, Rhodes M, Fenwick R (1996) Z
Lebensm Unters Forsch 203: 366 369
13. Reddy NR, Pierson MD, Sathe SK, Salunkhe DK (1982) Crit Rev
Food Sci 17: 335 370
14. Ragaee SM, El-Banna AA, Damir AA (1986) Alexandria Sci Exch
7: 217 224
15. Vidal-Valverde C, Frias J, Prodanov M, Tabera J, Ruiz R,
Bacon J (1993) Z Lebensm Unters Forsch 197: 449 452
16. Reddy NR, Salunke DK (1989) Fermentation. In: Salunke DK,
Kadam SS (eds) CRC Handbook of world food legumes: nutri-
tional, chemistry, processing technology and utilization, vol. III.
CRC, Boca Raton, Fla., pp 177 218
17. Tabera J, Frias J, Estrella I, Villa R, Vidal-Valverde C (1995) Z
Lebensm Unters Forsch 201: 587 591
18. Kozlowska H, Honke J, Sadowska J, Frias J, Vidal-Valverde C
(1996) J Sci Food Agric 71: 367 375
19. Bartolome B, Hernandez T, Bengoechea ML, Quesada C, Gomez-
Cordoves C, Estrella I (1996) J Chromatogr A 723: 19 26
Hernandez T, Estrella I, Gomez-Cordoves C (1993) J Chromatogr
A 655: 119 125
21. Bengoechea L, Hernandez T, Quesada C, Bartolome B, Estrella I,
Gomez-Cordoves C (1995) Chromatographia 41: 94 98
22. Escribano-Bailon MT (1993) PhD Thesis. University of Sala-
manca, Salamanca, Spain
23. Hathway DE, Seakins JWT (1957) Biochem J 67: 239 244
24. Sapis JC, Ribereau-Gayon P (1969) Ann Technol Agric 18:
207 219
25. Kawakami H (1981) Kent T, Higuchi T, Chang H (eds) In: Lignin
biodegradation: microbiology chemistry, and potential applica-
tions, vol II. CRC, Boca Raton, Fla., p 103
26. Ricardo-da-Silva JM, Cheynier V, Souquet JM, Moutounet M
(1991) J Sci Food Agric 57: 111 125
27. Shahidi F, Janitha PK, Wanasundara PD (1992) CRC Food Sci
Nutr 32: 67 103
28. Thompson LU (1994) CRC Food Sci Nutr 34: 473 497
29. Vinson JA, Dabbagh YA, Serry MM, Jang J (1995) J Agric Food
Chem 43: 2800 2802
30. Teissedre PL, Frankel EN, Waterhouse AL, Peleg H, German JB
(1996) J Sci Food Agric 70: 55 61