Ana səhifə

Oligosaccharides in legume grains


Yüklə 55.58 Kb.
tarix25.06.2016
ölçüsü55.58 Kb.
Oligosaccharides in legume grains

Rezica Sudar*1, Tihomir Čupić1, Tea Horvatski Zivalov2, Aleksandra Sudarić1,



1Agricultural Institute Osijek, Juzno predgrade, 17, 31000 Osijek, Croatia

2Pharmacies 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

C.V.




13.10

16.30

18.10

29.90

28.01

16.06

12.90

11.41

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

Pooled with cannonical structure




Can1

Can2

Can3

Stachyose

-0,17

0,48

-0,47

Raffinose

0,61

0,31

-0,19

Sucrose

0,12

-0,06

-0,45

Glucose

-0,01

-0,08

-0,14

Galactose

0,01

0,02

-0,43

Fructose

0,09

-0,57

-0,57

Variation %

54,65

26,01

16,32

Total variation %

54,65

81,66

97,98

Discriminate

Raffinose

Stachyose

Sucrose

r>±0,40




Fructose

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.



REFERENCES


  1. Berrios J. De J., Morales P., Cámara M., Sánchez-Mata M.C. (2010). Carbohydrate composition of raw and extruded pulse flours. Food Research International, 43, 531–536b.

  2. Espinosa- Martos I., Ruperez P. (2006). Soybean oligosaccharides. Potential as new ingredients in functional food. Nutr Hosp, 21, 92-96.

  3. Giannoccaro E., Wang Y.J., Chen P. (2008). Comparation of two systems and an enzymatic method for quantification of soybean sugars. Food Chem, 106, 324-330.

  4. Guillon F., Champ M. M.-J. (2002). Carbohydrate fractions of legumes: uses in human nutrition and potential for health. British Journal of Nutrition, 88, S293–S306.

  5. Han I.H., Baik B.K. (2006). Oligosaccharide content and composition of legumes and their reduction by soaking, cooking, ultrasound, and high hydrostatic pressure. Cereal Chem, 83(4), 428–433.

  6. Hou A. et al. (2009). Genetic variability of seed sugar content in worldwide soybean germplasm collections. Crop Sci, 49,903–912.

  7. Khattab R.Y., Arntfield S.D. (2009). Nutritional quality of legume seeds as affected by some physical treatments 2. Antinutritional factors. LWT - Food Sci and Tech, 42, 1113-1118.

  8. Kim S., Kim W., Hwang K. (2003). Optimization of the extraction and purification of oligosaccharides from defatted soybean meal. Int J Food Sci Tech, 38, 337-344.

  9. Kuo T. M., Vanmiddlesworth J. F., Wolf, W. J. (1988). Content of raffinose oligosaccharides and sucrose in various plant seeds. J Agric Food Chem, 36, 32-36.

  10. Messina M.J. (1999). Legumes and soybeans: overview of their nutritional profiles and health effects. Am J Clin Nutr, 70 (3), 439S-450S.

  11. Rupérez P. (1998). Oligosaccharides in raw and processed legumes. Z Lebensm Unters Forsch, 206,130-133.

  12. Roberfroid M.B., Slavin J. (2000). Nondigestible oligosaccharides. Crit Rev Food Sci Nutr, 40,461-480.

  13. Wilcox J.R., Shibles R.M. (2001) Interrelationships among seed quality attributes in soybean. Crop Sci, 41, 11-14.


Verilənlər bazası müəlliflik hüququ ilə müdafiə olunur ©atelim.com 2016
rəhbərliyinə müraciət