Oligosaccharides in legume grains
Rezica Sudar*1, Tihomir Čupić1, Tea Horvatski Zivalov2, Aleksandra Sudarić1,
1Agricultural
2Pharmacies
Institute Osijek, Juzno predgrade, 17, 31000 Osijek, Croatia
Joukhadar, Dr.Franje Tuđmana 3, 10431 Sveta Nedjelja, Croatia
*Corresponding author:
Phone: +38531515570
Fax: +38531515579
E-mail address: rezica.sudar@poljinos.hr
ABSTRACT: Oligosaccharides from legume species produced in few regions of Croatia were
investigated. Eight traditionally cultivated legumes were analyzed: common bean (Phaseolus vulgaris
L.), faba bean (Vicia faba L.), lentil (Lens culinaris Medic.), white lupin (Lupinus albus), chickpeas
(Cicer arietinum L.), cowpea (Vigna unguiculata L.), soybean (Glycine max (L.) Merr.) and grass pea
(Lathyrus sativus).The soluble saccharides (monosaccharides, sucrose, raffinose and stachyose) were
extracted with water and separated by high performance liquid chromatography. Saccharides were
identified by their retention time and quantified by peak area using external standard procedure.The
results showed that the total water soluble saccharides content ranged from 3.71% to 7.5%, and
oligosaccharides represented 36.38% to 69.29% of the total saccharides in investigated dry legume
grains. Stachyose was the main oligosaccharide in all grains, except for chickpeas, in which the main
oligosaccharide was raffinose. Different amounts of sucrose and small amount of glucose, galactose
and fructose were present in all legume grains. Significant difference in total saccharides, raffinose
and stachyose content was found between investigated legume species. The discriminant analysis
showed that legume species can clearly classify according to content of water soluble saccharides.
According to obtained results, the investigated legumes can be considered a good source of
oligosaccharides.
Key words: legume species, oligosaccharides, HPLC separation
INTRODUCTION
Oligosaccharides are functional food ingredients that have great potential to improve the
quality of food. Oligosaccharides have been associated with many health-promoting
functions, which had been identified in many clinical studies, such as promoting the growth of
Bifidobacterium in human intestine and balance of intestinal bacteria, modulation the immune
response, inhibition of cancer and tumour and stimulation of mineral absorption (Mesina,
1999).
Legume grains are a rich source of galactooligosaccharides, namely raffinose and
stachyose: raffinose is a trisaccharide containing a galactose linked -(1-6) to the glucose unit
of sucrose; stachyose is a tetrasaccharide containing a galactose linked -(1-6) to the terminal
galactose unit to raffinose. Humans cannot digest these oligosaccharides because they do
not possess the enzyme α-galactosidase necessary for hydrolysing the linkage present in
oligosaccharides in consumed food. Intact oligosaccharides reach the colon, where they are
preferentially fermented by beneficial bifidogenic microorganisms that contain the enzyme.
Fermentation of nondigestible oligosaccharides results in production of gases and short
chain fatty acids, which are interesting because of their prebiotic activity associated with
health benefits (Roberfroid and Slavin, 2000). Oligosaccharides potential as an ingredient of
functional food makes the search for new sources interesting, as well as the development of
methods that allow its isolation and purification in a simple and effective way (Guillon and
Champ, 2002).
The objective of this study was to investigate the composition of oligosaccharides in legume
species produced in few regions of Croatia.
MATERIAL AND METHODS
Eight traditionally cultivated legumes: common bean (Phaseolus vulgaris L.), faba bean
(Vicia faba L.), lentil (Lens culinaris Medic.), white lupin (Lupinus albus), chickpeas (Cicer
arietinum L.), cowpea (Vigna unguiculata L.), soybean (Glycine max (L.) Merr.) and grass
pea (Lathyrus sativus) were used for analysis.
To prepare a sample for analysis, one gram of ground legume was weighed into a test tube
and mixed with water. The tube was placed horizontally on a shaker for 20 min at room
temperature. After cooling, sample was centrifuged and 5 mL of the clear supernatant was
transferred into a new test tube. A total of 7 mL of acetonitrile was added to precipitate
soluble proteins and incubated at room temperature for two hours. The mixture was then
centrifuged and the supernatant was collected and evaporated to dryness in a rotatory
evaporator. The dried extract was disolved in water and passed through 0.45 µm syringe
filter, just before high performance liquid chromatography (HPLC) analyses. The separation
was carried out by Perkin-Elmer High-Performance Liquid Chromatography system series
200 equipped with degasser, isocratic pump, oven, refractive index detector and TotalChrom
Navigator (HPLC software). The separation was performed on MetaCharb Ca Plus column
(300 x 7.8), thermostated at 900C. 20 L aliquot was injected onto the column and eluted
with deionized water of flow rate of 0.5 mL/min. A standard solution composed of stachyose,
raffinose, sucrose, glucose, galactose and fructose at concentration of 1; 2; 2.5; and 3
mg/mL respectively. Saccharides from aqueous sample extract were identified by their
retention time and quantified by peak area using external standard procedure.
Oligosaccharides were representeed as the sum of raffinose and stachyose.
One-way analysis of variance (ANOVA) and multiple comparisons (Duncan’s post hoc test)
were used to evaluate the significant difference of the data at P < 0.05. Comparative
analyses of legume were performed using linear discriminant analysis. All statistical analyses
were performed using statistical-graphic system "Statistica" version 7.0 (Stat Soft software
Inc., Tulsa, OK, USA).
RESULTS AND DISCUSSION
The composition of water soluble saccharides in legume grains, determined by HPLC, is
shown in Fig.1 and 2. The results indicate that the legume grains contain oligosaccharides,
disaccharide and monosaccharides.
Figure 1. Concentrations (%, dm) of stachyose, raffinose and sucrose in legume grain
Figure 2. Concentrations (%, dm) of glucose, galactose and fructose in legume grain
Most dominant oligosaccharide was stachyose. Among legume species, high level of this
oligosaccharide was found in cow pea (3.93%) and white lupin (3.73%), and the least in faba
bean (0.93%). Stachyose has been reported as main oligosaccharide in most legumes, i.e.
lentils, chickpeas, common bean and white lupin (Kueo et al., 1988; Guillon and Champ,
2002; Berrios et al., 2010; Hou et al., 2006). The content of stachyose detected in this
legume was within the range 2.21-3.23 % reported by Pilar (1998). Wide range of stachyose
was found in soybean (Wilcox and Shibles, 2001; Kim et al., 2003; Espinosa-Martos and
Ruperez, 2006; Giannoccaro et al., 2008). Hou et al. (2010) analyzed worldwide soybean
germplasm and found stachyose content ranged from 0.2 to 69.6 mg/g and several samples
contained less than 10 mg/g. Raffinose amount was highest in chick pea (2.13%) and lowest
in faba bean (0.35%), common bean (0.38%) and chick pea (0.39%). Sucrose, as only
disaccharide found, was the second most abundant water soluble saccharide, with amounts
ranging from 0.80% (grass pea) to 3.00% (soybean). In the soybean, sucrose is dominant
sugar ranging from 3% to 10% and it is responsible for the sweet taste of soy food
(Giannoccaro et al., 2008). Small amounts of monosaccharides: glucose, galactose and
fructose (1%) have also been found (Fig. 2). Besides small but different amounts of glucose
and fructose, it is interesting to note that galactose, not previously detected in legume, was
found in all species.
Figure 3. Concentration (%, dm) of total saccharides and oligosaccharides in legume grain
The content of total water soluble saccharides ranged from 3.71% (gras pea) to 7.50% (white
lupin), and oligosaccharides represented 36.38% to 69.29% of the total saccharides in
investigated dry legume grains (Fig. 3). In this study, the results of total water soluble
saccharides and oligosaccharires in legume species were in agreement with some published
data (Ruperez, 1998; Han and Baik, 2006). The analyses of the saccharides profiles
indicated that high total saccharides content is attributed to high content of sucrose and
oligosaccharides.
Table 1. Results of ANOVA for total water soluble saccharides in legume species
EFFECT
DF
Stach
Raf
Suc
Glc
Gal
Fru
Total sacch
Total oligo
Total
75
0.84
0.33
0.52
0.00
0.00
0.01
2.09
0.87
Blocks
3
0.00
0.00
0.01
0.00
0.00
0.00
0.04
0.01
Species
7
8.38**
3.43**
4.63**
0.01**
0.02**
0.05**
17.79**
7.99**
Residual
65
0.07
0.01
0.10
0.00
0.00
0.00
0.50
0.15
13.10
16.30
18.10
29.90
28.01
16.06
12.90
11.41
C.V.
Analysis of variance showed significant difference (p0.05) between investigated legume
species for: stachyose, raffinose, sucrose, monosaccharides, total saccharides and
oligosaccharides content (Tab. 1).
Individual saccharides content as the variables in discriminant analysis were used for
classification and differentiation of legume species.
Table 3. Results of cannonical discrimant analysis on legume species
Variable
Stachyose
Raffinose
Sucrose
Glucose
Galactose
Fructose
Variation %
Total variation %
Discriminate
r>±0,40
Can1
-0,17
0,61
0,12
-0,01
0,01
0,09
54,65
54,65
Raffinose
Pooled with cannonical structure
Can2
0,48
0,31
-0,06
-0,08
0,02
-0,57
26,01
81,66
Stachyose
Fructose
Can3
-0,47
-0,19
-0,45
-0,14
-0,43
-0,57
16,32
97,98
Sucrose
Galactose
All multivariate difference tests between legume groups were significant on the 0.001 level
and the credibility test indicated significance of the first three out of six newly created
canonical variables. The first three variables explained 97.98 % of the total variance (Table
3.) with the square canonical correlation percentage of R2= 80.51%. The first canonical
variable (54.66%) had the strongest legume group discrimination through raffinose influence.
The raffinose content (0.61) proved to be a trait that discriminates population the most and
clearly differentiates legume species. In the second canonical variable stachyose and
fructose dominated. These are variables which are similar in influence but of different
directions.
CONCLUSION
In investigated legume grain wide variation were found among individual and total water
soluble saccharides. Stachyose was the main oligosaccharide in all grains, except for
chickpeas, in which the main oligosaccharide was raffinose. Sucrose was the second most
abundant water soluble saccharide, while glucose, galactose and fructose were presented in
small quantities. According to obtained results, the investigated legumes can be considered
a good source of oligosaccharides for functional food. The discriminant analysis showed that
the legume species can be clearly classified according to content of water soluble
saccharides.
ACKNOWLEDGMENT
This research was supported by SEERANET 168 (Sustainable preservation of indigenous
South East European legumes and their traditional food and feed products) project. We wish
to thank the project Continued genetic improvement of soybean by modern breeding
strategies (073-0730489-0344) for the financial support.
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