ALIMENTOS FUNCIONALES

 

Soybean oligosaccharides. Potential as new ingredients in functional food

Oligosacáridos de la soja. Su potencial como ingredientes nuevos de los alimentos funcionales

 

 

I. Espinosa-Martos y P. Rupérez

Departamento de Metabolismo y Nutrición. Instituto del Frío (CSIC). Madrid, España.

Correspondencia

 

 

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ABSTRACT

The effects of maturity degree and culture type on oligosaccharide content were studied in soybean seed, a rich source of non-digestible galactooligosaccharides (GOS). Therefore, two commercial cultivars of yellow soybeans (ripe seeds) and two of green soybeans (unripe seeds) were chosen. One yellow and one green soybean seed were from intensive culture, while one yellow and one green soybean seed were biologically grown. Low molecular weight carbohydrates (LMWC) in soybean seeds were extracted with 85% ethanol and determined spectrophotometrically and by high performance liquid chromatography. LWC in soybean seeds were mainly: stachyose, raffinose and sucrose. Oligosaccharide content was not affected significantly, either by biological or intensive culture technique. On the contrary, significant differences in GOS content were found depending on ripeness degree of soybean seeds. Ripe yellow soybean seeds showed a higher oligosaccharide content (1.84-1.95%), than unripe green seeds (1.43-1.61%). Other LMWC content was also affected by ripeness degree, thus making that the relative percentage of GOS was higher in immature (47-53%) than in matured soybean seeds (21-34%). Moreover, in order to purify soybean GOS, biologically grown yellow soybean seeds with a higher GOS content were selected and a previously reported method was followed. Although the GOS containing fraction was enriched, the yield obtained was low and an effective purification was not achieved. According to these results, yellow soybean seeds seem to be a good source of GOS but, in order to improve their purification, simple methods must be further developed and evaluated.

Key words: Galacto-oligosaccharides. a-galactosides. Galactooligosaccharides. Soybean. Prebiotics.

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RESUMEN

En este trabajo se estudia cómo afecta el grado de madurez y el tipo de cultivo al contenido de oligosacáridos en la semilla de soja, que es una fuente rica en galactooligosacáridos (GOS) no digeribles. Para ello se eligieron dos variedades comerciales de habas de soja amarilla (semillas maduras) y dos de soja verde (semillas inmaduras). Una de las muestras de soja amarilla y otra verde provenían de cultivo intensivo; mientras que una semilla amarilla y otra verde se han producido mediante cultivo biológico. Los GOS, junto con otros azúcares de bajo peso molecular, se extrajeron con etanol al 85% y se determinaron espectrofotométricamente y por cromatografía líquida de alta eficacia. Los principales carbohidratos de bajo peso molecular detectados en la soja fueron: estaquiosa, rafinosa y sacarosa. Los resultados obtenidos muestran que las técnicas de cultivo, ya sea biológico o intensivo, no afectan de manera significativa al contenido en GOS. En cambio, el grado de madurez de las semillas sí pone de manifiesto diferencias significativas, observándose un mayor porcentaje de GOS en las semillas maduras (1,84-1,95%), que en las inmaduras (1,43-1,61%). El grado de madurez afecta también en mayor proporción al contenido de los otros azúcares de bajo peso molecular, lo cual hace que el porcentaje relativo de oligosacáridos sea mas alto en las semillas inmaduras (47-53%), que en las maduras (21-34%). Además, para evaluar un protocolo de aislamiento y purificación de oligosacáridos descrito en la bibliografía, se seleccionaron las semillas de soja amarilla de cultivo biológico por su mayor contenido en GOS y aunque se consiguió enriquecer la fracción que los contenía, las recuperaciones fueron bajas y no se observó una purificación efectiva. De acuerdo con estos resultados, la semilla de soja amarilla parece ser una buena fuente para la obtención de GOS, pero se deben seguir desarrollando y evaluando metodologías sencillas que permitan su purificación.

Palabras clave: Galacto-oligosacáridos. a-galactósidos. Galactoligosacáridos. Soja. Prebióticos.

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Introduction

Raffinose family oligosaccharides, α-galactosides or galactooligosaccharides (GOS) are non-digestible carbohydrates which naturally occur in different foods and legumes, such as soybeans. Humans do not possess the enzyme called α-galactosidase necessary for hydrolysing the linkage present in these oligosaccharides, so that they cannot be digested when consumed. Intact oligosaccharides reach the colon, where they are preferentially fermented by beneficial bifidogenic microorganisms that contain the enzyme¹. Fermentation of nondigestible oligosaccharides² results in production of gases such as carbon dioxide, hydrogen, methane, etc and of short chain fatty acids, which are interesting because of their prebiotic activity³ and associated health benefits⁴ . GOS potential as an ingredient of functional food⁵ 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 way⁶. Carbohydrates are the second largest component in soybeans. Soybean seed is a rich source of galactooligosaccharides⁷, namely raffinose and stachyose: raffinose is a trisaccharide containing galactose linked α-(1-6) to the glucose unit of sucrose; stachyose is a tetrasaccharide containing a galactose linked α-(1-6) to the terminal galactose unit of raffinose⁸. Other reported major sugar of soybeans is sucrose with lower amounts of the monosaccharides: fructose, rhamnose and arabinose; significant levels of glucose occurred only in immature seeds⁹. Non-digestible oligosaccharides are one of the most popular functional food components, particularly in Japan where different type of food grade oligosaccharides are produced^4,10.

In this work, the effect of maturity degree and type of culture on GOS content of soybean seeds was studied. Moreover, in order to evaluate a method of isolation and purification, yellow soybean seeds with higher GOS content were used.

 

Material and methods

Samples

Four commercial varieties of soybean seed (Glycine max) were used: Yellow from biological (YSBC) or intensive culture (YSIC) and green from biological (GSBC) or intensive culture (GSIC). Samples were milled to a particle size of less than 1.0 mm before analysis.

Residual moisture in these samples was determined by drying to constant weight at 105 ºC in an oven.

All determinations were performed at least in triplicate and they are reported on a dry matter basis.

Low molecular weight carbohydrate extraction

Low molecular weight carbohydrate (LMWC) from soybean seeds (400 mg) was extracted with ethanol (85% v/v; 40 mL). Extractions were performed in screw-capped tubes, at 50 ºC in a water bath with constant shaking for 1 h. After cooling to room temperature, samples were centrifuged (3,000 × g; 15 min) . Aliquots (10 mL) of supernatants containing LMWC were evaporated to dryness in an R-114 BUchi vacuum rotatory evaporator with a B-480 Büchi water bath and temperature not exceeding 50 ºC. The sample extracts were redissolved in Milli-Q water (1.5 mL) and passed through 0.45 µm filters for aqueous solutions (nonsterile, Tracer), just before high-performance liquid chromatography (HPLC) analyses.

Soluble sugars determination by colorimetric method

Total sugars in the ethanolic sample extracts (400mg/40 mL) were monitored spectrophotometrically by the anthrone method¹¹ with glucose as a standard.

Low-molecular-weight carbohydrate determination by HPLC 

LMWC samples (50 µL) were analysed by HPLC in an aqueous sample extract (400 mg/1.5 mL) on a Bio-Rad Aminex HPX-87P column (300 × 7.8 mm) with two Bio-Rad micro-guard cartridges (30 × 4.6 mm). The column was eluted isocratically with Milli- Q-filtered (0.45 µm) and degassed water at 85 ºC, with a flow rate of 0.6 mL/min^12-14. LMWC were identified by their retention times and quantified by comparison with known carbohydrate standards (inulin, stachyose, raffinose, glucose, fructose, xylose, arabinose and inositol from Sigma; sucrose from Merck). The following HPLC instruments were used: Kontron autosampler 360, Kontron ternary pump system 325, Waters differential refractometer R-401, Jones chromatography thermostatic oven. Kontron data system 450-MT2 and Hewlett-Packard deskjet 600 printer.

Isolation and purification of galactooligosaccharides

Yellow soybean seeds from biological culture (100 g) were extracted and purified according to the procedure of Gulewicz and coworkers¹⁵, slightly modified in our laboratory. In summary, seeds were soaked at 4 ºC for 12 h with constant shaking; the imbibed seeds were then extracted twice with 50% ethanol (v/v) at 40 ºC overnight. After decanting, supernatants from extractions were boiled for 10 min, combined together and concentrated on a rotatory evaporator at 50 ºC to a final volume of 25 mL. Oligosaccharides were precipitated with absolute ethanol, in a ratio of 1:10. The crude GOS precipitate was separated by centrifugation at 3,000 × g for 15 min and any ethanol residue was removed in a vacuum dessicator. GOS were dissolved in 25 mL of distilled water and were purified by filtration through diatomaceus earth and charcoal on a sintered glass funnel. The funnel was washed with 200 mL of distilled water and GOS were eluted with 70% ethanol (500 mL). The presence of GOS in the eluate was checked colorimetrically with phenol-sulfuric acid¹⁶ and glucose as a standard. Alcoholic solutions were concentrated to dryness on a rotatory evaporator. Purified GOS (3 g) were dissolved in 10 mL distilled water and applied into a Dowex 50WX8 cation exchange resin column (120 mm × 15 mm id) and washed with distilled water until GOS were not detected in the eluate. The presence of GOS was continuously monitored at 190 nm with a Pharmacia LKB-Uvicord VW 2251 detector. Twenty milliliter fractions were collected (Gradifrac-Pharmacia Biotech, Spain), appropriate GOS fractions were combined, concentrated and the acidic pH was adjusted to pH 7 with a freshly prepared alkali solution (4% calcium hydroxide), then boiled for 2 min and centrifuged. Supernatant containing purified GOS was evaporated to dryness. GOS purity was determined by HPLC in the fractions obtained from each purification step.

 

Results and discussion

Carbohydrate content in soybean seeds, as determined by HPLC and spectrofotometrically, is shown in table I. Ripe yellow soybean seeds had 2.5-3 times higher sugar content than unripe green seeds. In all samples, sugar content was slightly lower by HPLC than by colorimetric method. Other LMWC content was also affected by ripeness degree, thus making that the relative percentage of oligosaccharides was higher in immature (47-53%) than in matured soybean seeds (21-34%). Values of matured soybean seeds were similar to main carbohydrates (sucrose, stachyose and raffinose) in USA and Japan soybeans, which ranged from 9.3 to 10.9% dry weight⁸.

As depicted in figure 1, LMWC is higher in matured yellow than in immmature green soybean seeds. Biological or intensive agricultural practices did not influence the content of low molecular weight carbohydrate in soybean seeds.

Ripe yellow seeds showed a higher galactooligosaccharide content (1.84-1.95%), than unripe green soybean seeds (1.43-1.61%) . Seeds with a different degree of maturity (green versus yellow) exhibited significant differences in overall composition and GOS content (table I) . In green soybeans, GOS weremainly identified as stachyose with negligible amounts of raffinose. In yellow soybeans, almost equal amounts of stachyose and raffinose were found Minor monosaccharides detected in green and yellow soybean seeds were xylose and the sugar alcohol inositol (table I) . Main LMWC detected are in agreement with Van der Riet et al.⁹. who reported that the major sugars of soybeans are sucrose, stachyose and raffinose.A considerable variation in oligosaccharide content among varieties of soybeans has been reported1. Monosaccharides present in defatted soybean meal included fructose, rhamnose and arabinose, while significant levels of glucose occurred only in immature seeds⁹.

Isolation and purification of GOS from biologically grown yellow soybean seeds was performed following the protocol of Gulewicz and col.¹⁵. In order to standardize and minimize errors, the issue was carried out four times and results obtained in the best experiment are shown in table 1I. An efficient extraction of GOS was performed and an enrichment in GOS (2.78%) with respect to the sugars extracted (7.70 %) was obtained, when compared to the YSBC contents measured by HPLC (see table I). Less than a half of the material was recovered after precipitation with ethanol (9.23 g), in which GOS amount was 1.39 g and total sugar 2.78 g (table II). The crude GOS precipitate had the appearance of a sticky gum and its recovery was difficult. To determine the distribution of GOS and other sugars between supernatant and sediment, an aliquot was collected for HPLC analysis. In this step, a part of the sucrose was removed and an enrichment in GOS content was detected: 50.0% vs 36.1% in the previous step.

In order to minimize GOS losses, only half the amount of distilled water (100 mL) was used to wash the funnel. Besides, the rest of the protocol was applied to both fractions, the aqueous and the alcoholic, because in previous experiments all the sugars, GOS included, were removed with the washing water.

The recovery of filtration represented the sum of both fractions, because of HPLC analysis showed that the higher amount of GOS was in the aqueous fraction. Because of that, values of recovery and GOS percentage were similar for the precipitation and filtration steps. Gulewicz and col.¹⁵ sign the filtration as a determinant step in the removal of sucrose and coloured substances, but no evidence was found in our issue that allowed us to think that sucrose and soy galactooligosaccharides could have a differential behaviour. In fact, both had a similar distribution between the two eluents: water and 70% ethanol. The ion exchange chromatography of crude GOS was monitored at 190 nm, a wavelength that permitted to follow the elution of GOS. From 4.25 g of recovered material, 1.39 g was total sugars of which GOS amounted to 0.6 g. Due to the resin composition, this step removed charged substances and that supposed a slight purification, but the relation between total sugars and GOS did not change. Recovery and GOS percentage are represented in figure 2 and although during purification there was a slight enrichment of the GOS-containing fraction, oligosaccharide recovery after precipitation, filtration and ion exchange chromatography of soybean extracts was low and no effective purification was observed at the final step.

In the same way, Kim et al⁷ optimised the conditions for oligosaccharide extraction and evaluated an ultrafiltration system for the purification of galactooligosaccharides from defatted soybean meal. Their main conclusion is that the system used shows more efficiency in the removal of protein than in the concentration of oligosaccharides, and no different distribution of GOS and sucrose is observed.

 

Conclusions

GOS content of soybean seeds has been shown to vary with the degree of maturity. Immature green seeds contain less amount of GOS than fully matured yellow soybean seeds. Biological or intensive agricultural practices do not influence GOS content of soybean seeds.

Yellow soybean seeds are a good source of GOS. Nevertheless, simple methods which allow their effective purification must still be developed.

 

Acknowledgement

This work was supported by project AGL2002- 03221-ALI from the Spanish Ministry of Science and Technology. I.E-M. acknowledges the research project for her predoctoral scholarship.

 

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Correspondencia:
Pilar Rupérez
Dpto. de Metabolismo y Nutrición
Instituto del Frío, CSIC
C/ José Antonio Novais, 10
Ciudad Universitaria
28040 Madrid

Recibido: 22-VI-2005.
Aceptado: 23-XI-2005.