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Nutrition 21 (2005) 230 –239
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Basic nutritional investigation
Nutritional assessment of raw and germinated pea (Pisum sativum L.)
protein and carbohydrate by in vitro and in vivo techniques
Gloria Urbano, Ph.D.a,*, María López-Jurado, Ph.D.a, Sławomir Frejnagel, Ph.D.b,
Elena Gómez-Villalvaa, Jesús M. Porres, Ph.D.a, Juana Frías, Ph.D.c,
Concepción Vidal-Valverde, Ph.D.c, Pilar Aranda, Ph.D.a
b
a
Departamento de Fisiología, Instituto de Nutrición, Universidad de Granada, Granada, Spain
Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn, Poland
c
Instituto de Fermentaciones Industriales (CSIC), Madrid, Spain
Manuscript received January 1, 2004; accepted April 2, 2004.
Abstract
Objective: We assessed the effect of germinating Pisum sativum L. variant Arvense cv. Esla for 3
and 6 d in darkness on the chemical composition and nutritive utilization of protein and carbohydrates.
Methods: Nutritional assessment of protein and carbohydrates was based on chemical analysis of
raw and germinated pea flours and in vitro and in vivo rat balance methodologies.
Results: Germination caused a notable decrease in ␣-galactoside content and significant increases
in sucrose, glucose, and fructose. The ratio of available starch to total starch increased as a
consequence of processing. The content of vitamin B2 increased significantly, whereas no significant change was observed in vitamin B1 content in germinated peas. Protein digestibility assessed
with an in vivo technique (apparent digestibility coefficient) or as the percentage of dialyzable
nitrogen increased significantly as a result of germination in contrast to what was observed with the
in vitro pH-drop methodology. Daily food intake, nitrogen absorption and balance, percentage of
retained versus absorbed nitrogen, protein efficiency ratio, and the index of available carbohydrates were
significantly improved by germination for 3 d and significantly decreased by germination for 6 d.
Conclusions: Germination of pea seeds for 3 d significantly improves palatability of these seeds
and the nutritive utilization of protein and carbohydrates. © 2005 Elsevier Inc. All rights reserved.
Keywords:
Peas (Pisum sativum L.); Germination; Nutritional assessment; Protein; Carbohydrates; In vitro; In vivo
Introduction
Peas are of great nutritional importance due to their high
content of protein, complex carbohydrates, dietary fiber,
minerals, vitamins, and antioxidant compounds. Although
peas are widely used in animal nutrition [1], human consumption of peas is lower than that of other traditionally
more accepted pulses [1,2]. Nevertheless, in recent years,
the wealth of nutrients available from the pea and its beneficial functional properties have prompted increasing interThis research was funded by projects ALI 96-0480, AGL 2001-2301,
and AGL 2002-02905 ALI from the Spanish CICYT.
* Corresponding author. Tel.: ⫹34-958-243885; fax: ⫹34-958-248959.
E-mail address: [email protected] (G. Urbano).
0899-9007/05/$ – see front matter © 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.nut.2004.04.025
est and demand for this legume for the food preparation
oriented to geriatric and infant nutrition [3].
Germination is a technologic application widely used for
its ability to decrease levels of antinutritional factors present
in legume seeds and improve the concentration and availability of their nutrients [4,5]. However, germination is also
a very active and complex metabolic process that may alter
the chemical, structural, and organoleptic properties of the
seed meal, thus decreasing its nutritive value [6,7].
Among the different techniques used to assess the nutritive utilization of protein, in vitro methodologies are frequently used to predict the potential digestive utilization of
protein in different foods and feed ingredients [8,9]. These
techniques have the advantages of simplicity, reproducibility, speed, and lower costs. Compared with in vitro tech-
G. Urbano et al. / Nutrition 21 (2005) 230 –239
niques, in vivo methodologies are expensive and time consuming and require specialized equipment and personnel;
conversely, they allow the researcher to control the physiologic or pathologic status of the experimental animal and
offer numerous data related to food intake and metabolic
utilization that are not available when using in vitro methods. Therefore, it seems important to compare and correlate
the different in vitro and in vivo methodologies, particularly
when the food processing conditions are known to cause
considerable changes in the chemical structure and composition of the meal, as is the case with processes such as
germination [6].
This study examined the effect of germination during
short (3 d) or prolonged (6 d) experimental periods on the
chemical composition and nutritive utilization of protein
and carbohydrates from Pisum sativum L. variant Arvense
cv. Esla in growing rats. In addition, we studied the effect of
the germination process on the digestive utilization of protein, seeking to correlate an in vivo balance technique with
two in vitro methodologies, pH-drop [10] and nitrogen
dialyzability [11].
Materials and methods
Diets
Raw pea flour
Raw pea flour (RP; Pisum sativum L. variant Arvense cv.
Esla) was obtained from the germ plasm collection of Valladolid (Valladolid, Spain).
Germination
The process was carried out on a semi-pilot scale. Pea
seeds, 500 g, were soaked in 2500 mL of 0.7% sodium
hypochlorite solution for 30 min at room temperature. Seeds
were drained, washed to neutral pH, and then soaked in
distilled water for 5 h 30 min. Imbibed seeds were germinated at a pilot scale by layering them over a moist filter
paper continuously watered by capillarity in a seed germinator (G-120 Snijders, Tilburg, The Netherlands) for 3 d
(G3DNL) and 6 d (G6DNL) without light at 20°C and 99%
relative humidity. Sprouted seeds were freeze dried and
grounded to pass through a 0.18-mm sieve for chemical and
biological analyses. All experimental diets were supplemented with 5% olive oil before feeding to animals.
Chemical methods
Moisture content of the different pea diets was determined by drying to constant weight in an oven at 105 ⫾
1°C, and pH and titratable acidity were determined as described by Barampama and Simard [12] and Frias et al. [13].
Titratable acidity was expressed as milliequivalents of
NaOH per 100 g of dry matter (DM). Total nitrogen was
determined according to Kjeldahl’s method. Crude protein
231
was calculated as nitrogen ⫻ 6.25. Insoluble nitrogen and
soluble protein and non-protein nitrogen were measured
according to the methodology described by Periago et al.
[14].
Determination of available soluble sugars and ␣galactosides
Analysis of glucose, fructose, sucrose, and ␣-galactosides (raffinose, stachyose, and verbascose) was carried out
by high-performance liquid chromatography according to
the method described by Frías et al. [15].
Determination of total and available starch levels
Total and available starch levels for samples were determined according to the method of Vidal-Valverde et al. [16]
by a procedure based on total enzyme digestion of starch to
glucose for 3 h and 30 min, respectively, and starch content
was calculated by multiplying the glucose content by 0.9.
Determination of vitamins B1 and B2
A single extraction procedure for vitamins B1 and B2
was carried out according to the method of Vidal-Valverde
et al. [17]. These vitamins were quantified by high-performance liquid chromatography as described previously [18].
Trypsin inhibitor activity determination
Trypsin inhibitor activity (TIA) was determined as described in Vidal-Valverde et al. [17].
Sodium dodecylsulfate polyacrylamide gel electrophoresis
Proteins were extracted from the pea flours with 50 mM
phosphate buffer, pH 7.8, containing 1% sodium dodecylsulfate (SDS) and 1% ␤-mercaptoethanol. SDS polyacrylamide gel electrophoresis (SDS-PAGE) was done according
to the method of Laemmli [19]. The final concentration of
acrylamide in the running gel was 15%. Gels were fixed and
stained with 0.2% Coomassie brilliant blue R-250 in methanol:acetic acid:water (5:4:1 v/v/v). Equal amounts of nitrogen were loaded in each lane. The mixture of molecular
weight markers (Merck, Darmstadt, Germany) consisted of
cytochrome C (12.3 kDa), myoglobin (16.9 kDa), carboanhydrase (30 kDa), ovalbumin (42.7 kDa), albumin (66.25
kDa), and ovotransferrin (78 kDa).
Endoprotease activity assay in gel
Pea flours (2 g) were extracted with 20 mL of 50 mM
sodium acetate (pH 4.7) containing 1 mM ethylene-diaminetetra-acetic acid and 2 mM cysteine for 1 h at 4°C. The
procedure described by Jameel et al. [20] was used to detect
endoprotease activity in SDS-PAGE (12%) copolymerized
with 0.20% (w/v) gelatin. Equal amounts of nitrogen (12.5
␮g) were loaded in each lane. Gels were run at constant
current (12 mA) for 4.5 h at 4°C. After electrophoresis, SDS
was removed by incubating gels in 2% Triton X-100 for 60
min at room temperature. Gels were then incubated overnight at 45°C in 50 mM sodium acetate (pH 4.94), 2 mM
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G. Urbano et al. / Nutrition 21 (2005) 230 –239
cysteine, and 1% Triton X-100 or in 50 mM phosphate
buffer (pH 6.75), 2 mM cysteine, and 1% Triton X-100.
Endoproteolytic activities were developed by staining the
gels with 0.2% Coomassie brilliant blue R-250 in methanol:
acetic acid:water (5:4:1 v/v/v) and appeared as white regions as against a dark blue background.
In vitro protein digestibility
Two in vitro methods were tested. The pH-drop multienzyme system described by Hsu et al. [10] and the in
vitro method of Miller et al. [11] that uses a pepsin
digestion period of 2 h followed by pH equilibration and
pancreatin digestion for another 2 h coupled with equilibrium dialysis (Molecular Weight Cut Off [MWCO]
12 000 Da). Dialyzable nitrogen was expressed as a percentage of the total amount of nitrogen in each digestion
vial, assuming that the dialyzable component had equilibrated across the dialysis membrane by the time the
dialysis bag was removed at the end of the digestion
period.
intake (expressed as dry weight), body weight gain, protein
efficiency ratio (weight gain in grams per day/protein intake
in grams per day), index of available carbohydrates (weight
gain in grams per day/total intake of available carbohydrates
in grams per day), apparent digestibility coefficient (ADC),
nitrogen retention (nitrogen balance), and percentage of
nitrogen retention versus nitrogen absorption (%R/A).
ADC ⫽ [(I ⫺ F) ⁄ I] ⫻ 100
balance ⫽ I ⫺ (F ⫹ U)
%R ⁄ A ⫽ 兵[I ⫺ (F ⫹ U)] ⁄ (I ⫺ F)其 ⫻ 100
where I represents intake, F represents fecal excretion, and
U represents urinary excretion
Statistics
Data were subjected to multifactor analysis of variance
with Statgraphics Statistical Graphics 5.0 (Statistical Graphics Corporation, Rockville, MD, USA).
Biological methods
Results
Experimental design and diet
We used a biological balance technique that recorded
changes in body weight and food intake and then calculated
nitrogen intake and fecal and urinary nitrogen excretion.
Three 10-d experiments, in which raw or germinated peas
were the only food source, were carried out. During the first
3 d of experiments, rats were allowed to adapt to the diet
and experimental conditions, and the main experimental
period comprised the next 7 d, during which body weight
and food intake were recorded and feces and urine were
collected for analysis.
The RP assayed had an average total nitrogen content of
4.05 ⫾ 0.01 g of nitrogen/100 g of DM, 14.1% of which
corresponded to soluble non-protein nitrogen, 75.8% of
which corresponded to soluble protein nitrogen, and the
remaining 10.1% was insoluble at the basic pH conditions
used for extraction (Table 1). Total nitrogen content increased gradually with increasing germination periods, resulting in significant differences among the three pea flours
tested. Insoluble nitrogen content decreased significantly
during the germination period (32% and 19% decreases in
pea flour germinated for 3 d and 6 d, respectively, versus
RP). The content of soluble protein nitrogen was significantly decreased by 6 d of germination, with no significant
differences being found between 3-d germinated flour and
RP. Soluble non-protein nitrogen increased progressively
with increasing germination periods (1.7-fold and 2.8-fold
in G3DNL and G6DNL, respectively, versus RP).
Fig. 1A shows changes in the SDS-PAGE pattern of
proteins from the RP and germinated pea flour used in the
present study. RP exhibited high-density bands corresponding to the storage proteins convicilin (70 kDa), vicilin (12 to
30 to 35 kDa), ␣-legumin (39 to 40 kDa), and ␤-legumin
(20 to 25 kDa). Germination for 3 or 6 d led to severe
hydrolysis of pea storage proteins, which was reflected in
the disappearance or loss of density from the main polypeptide bands and the appearance of a smear of lower-molecular-weight polypeptides that was particularly evident after
6 d of germination. Fig. 1B shows changes in the protease
activity of pea flours as a result of germination. Hardly any
activity was detected in the RP, whereas a significant increase in endoproteolytic activity could be observed for the
pea flours germinated for 3 and 6 d.
The pH of RP was 6.45, which decreased to 6.27 and 6.05
Animals
In each experiment we used 10 young, albino, Wistar rats
(five male and five female). Growing animals (recently
weaned), with an initial body weight of 100 ⫾ 1.5 g, were
housed from day 0 of the experiment in individual stainlesssteel metabolic cages designed for the separate collection of
feces and urine; cages were located in a room with a 12-h
light, 12-h dark period, a temperature of 21 ⫾ 2°C, and an
appropriate ventilation system. Throughout the experimental period all rats had free access to doubly distilled water,
and the diet was consumed ad libitum. At the end of the
experimental period, animals were anesthetized with CO2
and killed by decapitation. The liver and the longissimus
dorsi muscle were collected for analysis. Rats were handled
at all times in accordance with current European regulations
regarding laboratory animals.
Biological indices
The following indices and parameters were determined
for each group according to the formulas given below:
G. Urbano et al. / Nutrition 21 (2005) 230 –239
233
Table 1
Effect of germination on nutrient and antinutritional factor content of peas*
Diets
RP
N (g/100 g of DM)
Total
Insoluble
Soluble protein
Soluble non-protein
Available soluble sugars (g/100 g of DM)
Fructose
Glucose
Sucrose
Total available soluble sugars
Starch (g/100 g of DM)
Total
Available
Resistant
Total available sugars
Vitamins (mg/100 g of DM)
Vitamin B1
Vitamin B2
␣-Galactosides (g/100 g of DM)
Raffinose
Stachyose
Verbascose
Total
Trypsin inhibitor activity (TIU/mg of DM)
G3DNL
G6DNL
4.05 ⫾ 0.01a
0.41 ⫾ 0.02b
3.07 ⫾ 0.03b
0.57 ⫾ 0.01a
4.31 ⫾ 0.01b
0.28 ⫾ 0.02a
3.08 ⫾ 0.02b
0.95 ⫾ 0.01b
4.50 ⫾ 0.03c
0.33 ⫾ 0.01a
2.70 ⫾ 0.03a
1.57 ⫾ 0.06c
NDa
NDa
1.73 ⫾ 0.14a
1.73 ⫾ 0.14a
0.05 ⫾ 0.01b
0.09 ⫾ 0.01b
2.51 ⫾ 0.03b
2.65 ⫾ 0.03b
0.12 ⫾ 0.01c
0.34 ⫾ 0.02c
3.05 ⫾ 0.12c
3.52 ⫾ 0.08c
42.65 ⫾ 0.58c
38.70 ⫾ 1.21b
3.95 ⫾ 0.65b
40.43 ⫾ 0.36b
39.73 ⫾ 0.16b
37.81 ⫾ 0.45b
1.93 ⫾ 0.29a
40.45 ⫾ 0.20b
37.49 ⫾ 0.44a
34.94 ⫾ 0.49a
2.55 ⫾ 0.06a
38.46 ⫾ 0.16a
0.73 ⫾ 0.01a
0.15 ⫾ 0.01a
0.76 ⫾ 0.02a
0.27 ⫾ 0.01b
0.74 ⫾ 0.02a
0.28 ⫾ 0.02b
0.56 ⫾ 0.03c
2.24 ⫾ 0.06c
2.39 ⫾ 0.10b
5.19 ⫾ 0.13c
8.69 ⫾ 0.01a
0.21 ⫾ 0.02b
0.07 ⫾ 0.01b
NDa
0.28 ⫾ 0.02b
8.74 ⫾ 0.01a
0.08 ⫾ 0.02a
NDa
NDa
0.08 ⫾ 0.02a
9.65 ⫾ 0.14b
DM, dry matter; G3DNL, peas germinated for 3 d in darkness; G6DNL, peas germinated for 6 d in darkness N, nitrogen; ND, not detected; RP, raw peas
* Values are mean ⫾ standard deviation (n ⫽ 3). The same superscript letter in the same row indicated no significant difference (P ⬍0.05).
in the G3DNL and G6DNL diets, respectively. Conversely, the
titratable acidity expressed as milliequivalents of NaOH/100 g
of DM increased from 3.37 in the RP diet to 7.04 and 10.14 in
the G3DNL and G6DNL diets, respectively.
Total ␣-galactoside content was significantly decreased
by the germination process (94.6% and 98.4% in G3DNL
and G6DNL, respectively). Decreases in raffinose and
stachyose content were 63% and 97%, respectively, in the
G3DNL diet and 86% and 100%, respectively, in the
G6DNL diet compared with the RP diet. No detectable
levels of verbascose were observed after either germination
period.
The content of sucrose increased by 1.5- to 2-fold in the
G3DNL and G6DNL diets compared with RP. Under the
experimental conditions of the present study, levels of fructose and glucose, monosaccharides that were not detected in
Fig. 1. (A) Sodium dodecylsulfate polyacrylamide gel electrophoresis of proteins extracted from raw and germinated pea flours. An equal amount of legume
nitrogen (1.45 ␮g) was loaded in each lane. (B) Protease staining after sodium dodecylsulfate polyacrylamide gels copolymerized with 0.20% gelatin; 12.5
␮g of nitrogen was loaded per well. The gel was incubated in 50 mM of phosphate buffer (pH 6.75), 2 mM cysteine, and 1% Triton X-100 overnight at 45°C.
Both gels are representative of three independent analyses. Lane 1, raw pea flour; lane 2, flour made from peas germinated in darkness for 3 d; lane 3, flour
made from peas germinated in darkness for 6 d. M, molecular weight markers.
234
G. Urbano et al. / Nutrition 21 (2005) 230 –239
Table 2
Nutrient and antinutritional factor intake*
Diets
Intake (g of dry matter)
Intake (g/100 g of rat body weight per day)
Protein (g)
Available soluble sugars (g)
Total starch (g)
Resistant starch (g)
Available starch (g)
Total available sugars (g)
Vitamin B1 (mg)
Vitamin B2 (mg)
Total ␣-galactosides (g)
Trypsin inhibitor activity (TIU)
RP
G3DNL
G6DNL
10.72 ⫾ 0.20b
9.08 ⫾ 0.24a
2.71 ⫾ 0.05b
0.19 ⫾ 0.00a
4.57 ⫾ 0.09b
0.42 ⫾ 0.01c
4.15 ⫾ 0.08b
4.33 ⫾ 0.08b
0.08 ⫾ 0.00b
0.02 ⫾ 0.00a
0.56 ⫾ 0.01c
93 148 ⫾ 1775b
14.23 ⫾ 0.42c
10.19 ⫾ 0.29b
3.83 ⫾ 0.11c
0.38 ⫾ 0.01c
5.65 ⫾ 0.17c
0.27 ⫾ 0.01b
5.38 ⫾ 0.16c
5.76 ⫾ 0.17c
0.11 ⫾ 0.00c
0.04 ⫾ 0.00b
0.04 ⫾ 0.00b
124 388 ⫾ 3638c
8.55 ⫾ 0.25a
8.52 ⫾ 0.19a
2.41 ⫾ 0.06a
0.30 ⫾ 0.01b
3.21 ⫾ 0.09a
0.22 ⫾ 0.01a
2.99 ⫾ 0.09a
3.29 ⫾ 0.10a
0.06 ⫾ 0.00a
0.02 ⫾ 0.00a
0.01 ⫾ 0.00a
82 536 ⫾ 2396a
G3DNL, peas germinated for 3 d in darkness; G6DNL, peas germinated for 6 d in darkness RP, raw peas
* Values are mean ⫾ standard error of the mean (n ⫽ 10) per rat per day. The same superscript letter in the same row indicated no significant difference
(P ⬍0.05).
RP, increased significantly to 0.05 and 0.09 g/100 g of DM,
respectively, in the G3DNL diet and to 0.12 and 0.34 g/100
g of DM, respectively, in the G6DNL diet. RP had an
average total starch content of 42.65 ⫾ 0.58 g/100 g of DM,
90.7% of which corresponded to available starch and 9.3%
of which corresponded to resistant starch. Germination resulted in a significant decrease of total and resistant starch
content, thus increasing the proportion of available starch
with respect to total starch (4.9% and 6.8% increases in
G3DNL and G6DNL, respectively).
Germination caused a significant increase in levels of
vitamin B2 of the two germinated pea flours tested (80%
and 87% increments for 3 and 6 d of germination, respectively). Vitamin B1 content was not affected by the germinating conditions and remained similar to that of RP.
No difference for TIA was observed as a result of germination for 3 d when compared with RP but did increase
significantly after 6 d.
Food intake, expressed as grams per rat per day, increased significantly in animals fed the diet of peas germinated for 3 d compared with animals fed RP and decreased
significantly in animals fed the diet of peas germinated for
6 d in comparison with the former two groups (Table 2).
Intake of protein, total available sugars, and vitamins B1
and B2 was related to food intake and the amount of nutrients present in the diet. Therefore, the highest intake of
protein, total available sugars, and vitamins B1 and B2 was
found in animals fed the G3DNL diet.
Intake of ␣-galactosides decreased significantly with
longer germination periods, whereas the intake of total
starch, available sugars, and TIA increased significantly in
the group fed the G3DNL diet and decreased significantly in
the group fed the G6DNL diet with respect to the group fed
the RP diet.
In vitro protein digestibility assessed by the pH-drop
technique decreased slightly (P ⬍ 0.05) with longer germination periods compared with the RP diet (2.3% and 7.9%
in G3DNL and G6DNL, respectively; Fig. 2). In vitro protein digestibility, calculated as the percentage of dialyzable
nitrogen, was significantly improved as a result of germination (from 59.5% in the RP diet to 67.2% and 70.9% in
the G3DNL and G6DNL diets, respectively), and there were
no significant differences between germination periods. In
vivo protein digestibility assessed by the ADC increased
significantly in groups fed germinated pea diets compared
with the group fed the RP diet, and there were no differences between germinated pea groups.
Nitrogen absorption in absolute values was significantly
higher in the group fed the diet of peas germinated for 3 d
than in those fed the other two diets (Table 3). Metabolic
utilization of nitrogen assessed as a balance or %R/A was
higher among rats fed the G3DNL diet than among those
Fig. 2. In vitro and in vivo protein digestibilities of RP (gray bars), G3DNL
(white bars), and G6DNL (hatched bars) diets. Each bar represents the
mean ⫾ standard error of the mean (n ⫽ 3 for in vitro; n ⫽ 10 for in vivo).
Those bars not sharing a common letter are significantly different (P ⬍
0.05). ADC, apparent digestibility coefficient; G3DNL, peas germinated
for 3 d in darkness; G6DNL, peas germinated for 6 d in darkness; RP, raw
peas.
G. Urbano et al. / Nutrition 21 (2005) 230 –239
235
Table 3
Digestive and metabolic utilization of nitrogen*
Diets
Daily N intake (mg/rat per day)
Daily total fecal N (mg/rat per day)
Daily urinary N (mg/rat per day)
Daily absorbed N (mg/rat per day)
ADC (%)
Balance (mg/rat per day)†
%R/A‡
RP
G3DNL
G6DNL
433.71 ⫾ 8.26b
78.17 ⫾ 3.32b
239.11 ⫾ 9.50a
355.54 ⫾ 8.90a
81.91 ⫾ 0.84a
116.42 ⫾ 8.73a
32.71 ⫾ 2.23a
613.12 ⫾ 17.93c
91.14 ⫾ 2.52c
311.54 ⫾ 12.44b
521.98 ⫾ 17.64b
85.04 ⫾ 0.54b
210.44 ⫾ 8.22b
40.37 ⫾ 1.03b
384.90 ⫾ 11.17a
61.23 ⫾ 2.64a
218.79 ⫾ 8.10a
323.67 ⫾ 9.55a
84.09 ⫾ 0.53b
104.88 ⫾ 6.23a
32.39 ⫾ 1.71a
ADC, apparent digestibility coefficient; G3DNL, peas germinated for 3 d in darkness; G6DNL, peas germinated for 6 d in darkness N, nitrogen; %R/A,
percentage of nitrogen retention versus nitrogen absorption; RP, raw peas
* Values are mean ⫾ standard error of the mean (n ⫽ 10). The same superscript letter in the same row indicated no significant difference (P ⬍ 0.05).
†
Balance ⫽ N intake ⫺ (fecal N ⫹ urinary N).
‡
%R/A ⫽ [balance/(N intake ⫺ fecal N)] ⫻ 100.
fed the RP and G6DNL diets, and there was no significant
differences between the two latter experimental groups.
Weight gain (grams per day), growth efficiency coefficient (protein efficiency ratio), and index of available carbohydrates were significantly improved among animals fed
the diet of peas germinated for 3 d compared with those that
fed the diet of RP or peas germinated for 6 d. There were no
differences between the latter two diets (Fig. 3).
No significant differences were found in water or nitrogen content in the liver and longissimus dorsi muscle of the
animals given the different diets (Fig. 4).
Discussion
Total nitrogen content of the peas used for the present
study was within the range of values found in the literature
Fig. 3. Weight gain and nutritive utilization coefficients of protein in rats
fed RP (gray bars), G3DNL (white bars), and G6DNL (hatched bars) diets.
Each bar represents the mean ⫾ standard error of the mean of 10 Wistar
rats. Those bars not sharing a common letter are significantly different (P
⬍ 0.05). G3DNL, peas germinated for 3 d in darkness; G6DNL, peas
germinated for 6 d in darkness; IAC, index of available carbohydrates;
PER, protein efficiency ratio; RP, raw peas.
[21]. The insoluble nitrogen residue has been described as
proceeding from non-covalent interactions or from disulfide
bonds between different proteins [21]. The decrease in the
level of insoluble nitrogen as a result of the germination
process found in the present experiment was similar to that
reported by Wanasundara et al. [22] in flax seeds.
Under our experimental conditions, the drastic decrease
in ␣-galactoside content was similar to that described by
other investigators in germinated legume seeds such as
cowpea [23] and lentil [24]. Monteze Guimaraes et al. [25]
suggested that germination contributes to the hydrolysis of
galacto-oligosaccharides into mono- or disaccharides that
are used as an energy source or hydrocarbon skeleton for the
biosynthesis of macromolecules necessary for embryonic
development during the early stages of germination. The
hydrolysis of galacto-oligosaccharides is catalyzed by ␣-galactosidase. This enzyme is not found in appreciable
amounts in resting seeds and becomes active at the onset of
germination. Nnanna and Phillips [23] and Monteze Guimaraes et al. [25] observed that ␣-galactosidase activity reach
maximum values within 3 d of germination in cowpeas;
under our experimental conditions, the enzyme also remained active after 3 d, as demonstrated by the decreased
␣-galactoside content (71.4%) from day 3 to day 6 of
germination.
Total starch content in peas underwent a significant decrease during germination, similar to that observed after
germination of cowpeas [6] and lentils [24]. ␣-Amylase
inhibitors in peas (0 to 16.8 IU/kg of DM) [4] can interfere
with starch digestion. However, Alonso et al. [4] and Sathe
et al. [26] reported a significant decrease in the levels of
␣-amylase inhibitors during germination.
As a result of the germination process, legume seeds
undergo considerable metabolic changes in their carbohydrate storage, which includes starch and oligosaccharides of
the raffinose family that are hydrolyzed, causing a concomitant increase in levels of reducing sugars [27]. This is the
reason for the great increment in available soluble sugars,
236
G. Urbano et al. / Nutrition 21 (2005) 230 –239
mination (1.8-fold), whereas no further increment was observed after 6 d, in agreement with what has been described
in the literature for peas and lentils [30,31]. The thiamin
content of peas was not modified after a prolonged germination period of 6 d, similarly to the findings of Prodanov et
al. [31] in lentils and in contrast to what has been described
for peas by Sierra and Vidal-Valverde [30] who found
decreased levels of thiamin during the germination period.
The TIA content of RP used for the present experiment
was within the range of values found in the literature for the
pea [21]. The influence of germination on TIA depends on
the type of legume and on the conditions and duration of the
germination process [32]. Thus, some investigators have
found significant decreases in the TIA content of germinated beans [33], whereas others have found no substantial
variations in TIA levels of beans [34] or lentils [35] after
germination. Under our experimental conditions, the TIA of
peas was not affected by 3 d of germination but increased
slightly with 6 d of germination due to concentration caused
by the loss of DM.
Intake
Fig. 4. (A) Water composition (%) and (B) nitrogen composition (%DM)
of liver and LDM of rats fed RP (gray bars), G3DNL (white bars), and
G6DNL (hatched bars) diets. Each bar represents the mean ⫾ standard
error of the mean of 10 Wistar rats. Those bars not sharing a common letter
are significantly different (P ⬍ 0.05). DM, dry matter; G3DNL, peas
germinated for 3 d in darkness; G6DNL, peas germinated for 6 d in
darkness; LDM, longissimus dorsi muscle; N, nitrogen; RP, raw peas.
mainly sucrose, observed after 6 d of germination. Our
results are in agreement with those of Balasaraswathi and
Sadasivam [28] who described a gradual increase in the
amount of reducing sugars from day 0 to day 5 in sunflower
seeds and with those of Frias et al. [24] who observed a
significant increase in levels of sucrose after 4 d of germination of lentils. These mono- or disaccharides are more
easily absorbed by the small intestine of monogastrics.
The proportion of available versus total starch improved
as a consequence of the germination process. These results
are in agreement with those described by Vidal-Valverde
and Frias [27] for lentils and indicate that starch could be
better used nutritionally. Reddy et al. [29] suggested that
germination of legumes alters their starch properties by
increasing gelatinization capacity and solubility. These
changes improve accessibility of the starch granule to digestive enzymes, thus improving its digestibility.
Under our experimental conditions, we observed a considerable increment of riboflavin content after 3 d of ger-
The duration of the germination period affected the daily
food intake of the experimental animals. The increase in
food intake by the animals fed the diet of 3-d germinated
peas may have been due to the decreased ␣-galactoside
content and to an increment in the amount of palatable
carbohydrates such as the available sugars sucrose and fructose derived from the hydrolysis of ␣-galactosides and the
predigestion of starch. In addition, supplementation of 4%
olive oil to the diet may have improved its aroma and flavor.
The significant decrease in food intake after 6 d of
germination (G6DNL), even compared with the RP diet,
could be attributed to changes in food texture or properties
such as hardness, elasticity, or chewiness [6,36], to the
appearance of compounds that may cause the loss of the
peas’ organoleptic characteristics [23,37], to changes in the
acidity of the diet, and to the appearance of free non-protein
amino acids with neurotoxic effects or anorexigenic compounds that could affect the nutritional value and acceptability of the food [7,38,39]. Under our experimental conditions, the influence of these compounds that tended to
decrease the palatability of the G6DNL diet was greater
than that of the increase in soluble palatable sugars and of
the total absence of ␣-galactoside oligosaccharides.
In vitro and in vivo protein digestibility
Results obtained with the multienzyme technique of pHdrop seem to indicate that the RP diet had the highest
protein digestibility, followed by the G3DNL diet (2.2%
decrease) and the G6DNL diet (7.9% decrease). These results could be due to the appearance of peptides that are
resistant to proteolytic enzymes [34]. Further, the pH-drop
technique did not take into account the higher titratable
G. Urbano et al. / Nutrition 21 (2005) 230 –239
acidity of germinated diets, which is caused by predigestion
of storage protein that takes place during germination and is
reflected in the observed decrease of soluble protein nitrogen in addition to the increment in the levels of soluble
non-protein nitrogen composed of free amino acids, nucleic
acids, purine and pyrimidine bases, polyamines, alkaloids,
and small peptides. These results agree with the decreased
density of polypeptide bands corresponding to the seed
storage proteins that was observed in the protein gel as a
result of increasing germination periods and the increment
in endoprotease activity of the germinated pea flours. The
greater buffering capacity of the germinated pea diets interfered with the pH-drop and would be responsible for the
lower protein digestibility value obtained. In contrast, the
percentage of dialyzable nitrogen showed a higher digestibility of legume protein after 3 and 6 d of germination and
maintained a good correlation with the enhanced hydrolysis
of legume storage proteins caused by a higher endoprotease
activity present in the germinated pea flours, which was
active over a wide pH range (4.79 to 6.79), and the increased levels of soluble non-protein nitrogen. These results
showed a trend similar to those obtained by the in vivo
technique (ADC). Predigestion of pea protein caused by
germination and the decrease in tannin content [40]
achieved by the process could be responsible for the significant improvement in the digestive utilization of nitrogen
because TIA was not affected. Nevertheless, the in vivo
method showed a much lower increase in digestive utilization when compared with the in vitro dialyzability system
assayed, and it produced a much higher value (Fig. 2),
overall indicating that the rat’s digestive system is well
able to cope with the protein in a non-germinated pea
flour diet.
In vivo digestive utilization of protein from the peas used
in the present experiment was high and within the range of
values found in the literature [41], similar to that reported
for faba beans [42], and higher than that of lentils [43].
Metabolic utilization of protein and carbohydrates
The highest nutritive utilization of nitrogen was obtained
for the group fed the diet of 3-d germinated peas versus the
groups fed the RP and 6-d germinated pea diets. This is
due to the higher daily food intake but also to better
metabolic utilization of nitrogen (%R/A) obtained with
the G3DNL diet. All these factors produced a higher level
of nitrogen retention with which to respond to growth and
structural repair necessities because weight gain was significantly higher in this experimental group than in the
others.
The protein efficiency ratio, which measures the relation
between weight gain and protein consumed, was significantly higher among rats given peas germinated for 3 d than
among rats fed raw or 6-d germinated peas. Within the
G3DNL group, the higher food intake, the good metabolic
utilization of protein, and the better nutritive utilization of
237
carbohydrates would be responsible for improved nitrogen
%R/A. Animals did not use protein to fulfill energetic needs
as was the case with the other two diets. Further, peas
germinated for 3 d without light provided an amount of
vitamins B1 and B2 that was higher than the nutrient requirements of the growing rat [44] and that contributed to
the improved nutritive utilization of carbohydrates.
Germination for 6 d caused a sharp decrease in food
intake and, hence, in net absorption and retention of nitrogen. In addition, it did not improve the metabolic utilization
of protein or carbohydrates, as shown by the percentage of
retained versus absorbed nitrogen, protein efficiency ratio,
and index of available carbohydrates. The caloric malnutrition generated by decreased daily food intake in the group
of animals fed the G6DNL diet may have been responsible
for these findings.
The nitrogen contents in the muscle and liver of the
animals given the diets of raw peas and of germinated peas
were similar in all cases and might have been related to the
size of the tissues. This would indicate that, for rats in the
range of body weights used in the present study, variations
will appear in the size of the different tissues but not in their
composition.
In conclusion, we recommend short germination periods
of 3 d without light, which are optimal for improving the
organoleptic and nutritional properties of peas. These germination periods are sufficient to produce an appreciable
decrease in the factors responsible for flatulence, thus increasing food intake and improving the utilization of protein
and available carbohydrates. Under our experimental conditions, the percentage of dialyzable nitrogen was a better
index to predict the potential digestibility of protein because
it more accurately reproduced the physiologic process of
digestion at acidic pH with pepsin followed by pH adjustment and digestion with pancreatic enzymes at alkaline pH
that takes place in the animal. Nevertheless, digestive utilization of protein was found to be more efficient in the
digestive tract of the animal. The multienzyme technique of
pH-drop may be useful to assess the digestibility of proteins
that have not been predigested because this would release
free amino acids or peptides with buffering capacity that
interfere with the pH-drop on which this methodology is
based. Compared with these in vitro methodologies, the
biological assay is more complete in providing information
about food intake, weight gain, and the metabolic utilization
of nitrogen by the animals as complementary results to the
digestive utilization of protein.
Acknowledgments
The authors thank Rosa Jiménez and Dr. Carmen Lluch
Plá for skillful technical assistance and literature support.
238
G. Urbano et al. / Nutrition 21 (2005) 230 –239
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