ORIGINAL ARTICLE

 

Structural characterization of fructans from Agave fourcroydes (Lem.) with potential as prebiotic

 

Caracterización estructural de los fructanos de Agave fourcroydes (Lem.) con potencialidades como prebiótico

 

 

Yanelys García-Curbelo,^I Mercedes G. López,^II R. Bocourt,^I Erika Collado,^II Nereyda Albelo,^I Odalis Nuñez,^I

^IDepartamento de Ciencias Biofisiológicas, Instituto de Ciencia Animal, Apartado Postal 24, San José de las Lajas, Mayabeque, Cuba.
^IIDepartamento de Biotecnologia y Bioquímica, Centro de Investigaciones y Estudios de Avanzada del IPN. Campus Guanajuato, Apartado Postal 629, Irapuato, Guanajuato, 36 500 México.

 

 

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ABSTRACT

The use of prebiotics, like fructans, increases in animal production. They can be used as growth promoting additives because they benefit health and productive performance. Agave fourcroydes shows high concentrations of these compounds. Besides, structural characteristics of these carbohydrates may influence on animal response. The objective of this study was to determine structural characteristics of fructans present in Agave fourcroydes from Cuba, with potential as prebiotic in animal feeding. An amount of 10 stems of Agave fourcroydes were used, which belonged to the Empresa Henequenera «Eladio Hernández León», located in Matanzas province, Cuba. For structural characterization, thin layer chromatography, gas chromatography coupled to a mass spectrometry and anion-exchange chromatography of high resolution coupled to an amperometric detector were used. From the obtained results, a complex mixture of oligosaccharides, which present few ramifications and neo-kestose series, insulin (GFn) and inulo-nose (Fn), is proposed as structure for fructans of Agave fourcroydes. Structural characteristics of these carbohydrates are essential for animal nutrition. Fructans of Agave fourcroydes are considered as prebiotic candidate for being natural sources of oligofructans, estimated as the best substrates for acid-lactic bacteria.

Key words: chemical stuctures, Agave fourcroydes, fructan, prebiotic.

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RESUMEN

En la producción animal se incrementa la utilización de prebióticos, como los fructanos, que pueden desempeñarse como aditivos promotores del crecimiento por los beneficiosos que representan para la salud y el comportamiento productivo. El Agave fourcroydes presenta grandes concentraciones de estos compuestos. Además, las características estructurales de estos carbohidratos pueden influir en la respuesta animal. El objetivo de este estudio fue determinar las características estructurales de los fructanos presentes en el Agave fourcroydes de Cuba, con potencialidades de uso como prebiótico en la alimentación animal. Se utilizaron 10 tallos de Agave fourcroydes, de la empresa henequenera «Eladio Hernández León», ubicada en la provincia de Matanzas, Cuba. Para la caracterización estructural, se aplicó la cromatografía en capa delgada, cromatografía de gases acoplada a espectrometría de masas y la cromatografía de intercambio aniónico de alta resolución acoplada a un detector de pulso amperométrico. A partir de los resultados obtenidos, se propone como estructuras para los fructanos de Agave fourcroydes una mezcla compleja de oligosacáricos, que presenta pocas ramificaciones y series neokestosa, insulina (GFn) e inulo-nose (Fn). Las características estructurales de estos carbohidratos son de gran importancia para la nutrición animal. Por ser fuentes naturales de oligofructanos, estimados como mejores sustratos para las bacterias ácido-lácticas, los fructanos de Agave fourcroydes se consideran como candidato prebiótico.

Palabras clave: estructuras químicas, Agave fourcroydes, fructano, prebiótico.

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INTRODUCTION

The use of prebiotic additives increases in animal nutrition because they benefit animal health and productive performance (Li and Kim 2013 and Zhao et al. 2013a).

Prebiotics are defined as nutritional ingredients, which are selectively fermented by the beneficial intestinal biota and modify their composition and metabolic activity, and, this way, improve health of the host (Roberfroid et al. 2010). Fructans, which are among the most studied compounds for these purposes, present a great structural diversity that influence on their prebiotic response (García-Curbelo 2010 and Ivarsson 2012).

Plants belonging to Agavaceae family are among those that synthesize and store fructans (Roberfroid 2007). Agave fourcroydes is grown in Cuba for obtaining fiber from its leaves. However, their stems constitute industry waste and remain without use in the field (Calixto and Mazorra 2008). Studies carried out by García-Curbelo et al. (2009) demonstrated the presence of high concentrations of fructans in stems of Agave fourcroydes. Under these conditions, obtaining these carbohydrates from this organ and knowing their structural characteristics are very important for biological evaluation of a new food additive for cattle rearing.

The objective of this study was to characterize chemical structures present in Agave fourcroydes from Cuba, with potential as prebiotic for animal nutrition.

 

MATERIALS AND METHODS

Biological material. Through the method of diagonals, 10 stems of Agave fourcroydes were collected, which were considered as industry wastes. Agave fourcroydes was grown in lands of Empresa Henequenera «Eladio Hernández León», located in Matanzas, Cuba. The soil was Lithosol type (data from Instituto de Geofísica, Matanzas). Collected agaves were 8 years old and the scape was not emerged from. Plant sampling was carried out in dry season and collection was performed during early hours in the morning. Stems used were those with 6.30 kg of mean weight. Fructans extraction was performed according to the methodology of López et al. (2003).

Thin layer chromatography (TLC). The thin layer chromatography was performed to the dry extract of Agave fourcroydes, according to the method of López et al. (2003), and it was compared to commercial fructans: Raftilose P95 (95 % of oligosaccharides with â bonds (2-1), linear), Raftiline HP (inulin polysaccharides of high purity) from ORAFTI brand and the mixture of standards: 1-kestose (Megazyme), nystose (Megazyme), kestopentaose (DP5, Megazyme), fructose (Fruc Sigma), glucose (Gluc Sigma) and sucrose (Sac Sigma).

Solutions of 20 mg. mL^-1 were prepared with deionized water and a 1 µL of each solution was applied with small Hamilton syringes in analytic plaques of silica gel with aluminum stand (10 x 10 cm, Alldrich). The chromatological test was performed in four stages, with a system of solvents of butanol:propanol:water (3:12:4, v/v/v). Plaques were dried at room temperature and were sprinkled a reactive of aniline-diphenylamine-phosphoric acid in an acetone basis (Anderson et al. 2000). Then, plaques were put into an oven at 100 ºC for five minutes.

The compounds within the sample of A. fourcroydes were identified after comparing its retention factor (Rf) with the standard mixture and commercial products Raftilose P95 and Raftiline HP.

The Rf was calculated using the following formula:

Rf= Distance from application point to stain/distance before solvent

Gas chromatography coupled to a mass spectrometry. A total of 10 mg of dry extract of Agave fourcroydes and of commercial fructans Raftilose P95 and Raftiline HP were weighed. The extract was located in 4 mL reactivials. An amount of 500 µL of dimethyl sulfoxide (DMSO, Sigma) was added and was shaken for two hours, until achieving a total dissolution of the simple. The method of Mancilla (2006) was used for deriving carbohydrates into partially methylated alditol acetate.

In order to identify derivates using gas chromatography coupled to a mass spectrometry, derived samples were dissolved in 2 mL of CH₂CL₂. For chromatographic separation, 1 µL was injected in a gas chromatographer (Hewlett Packard 5890 Series II) coupled to a mass spectrometer (Hewlett Packard 5972 Series). A capillary column of phenyl methyl silicon at 5 % (HP 1-DM, 25 m x 0.31 mm x 0.52 µm thick) and helium (He) as carrier gas were used, at a flow speed of 20 mL.min^-1 and a pressure of 7 PSI. Initial temperature of the oven was

80 ºC for three minutes and followed a slope of temperature of 5 ºC.min^-1 up to 160 ºC; 0.5 ºC.min^-1 up to 180 ºC and 14 ºC.min^-1 - 280 ºC. Total time was 60 min. Temperature of the injector was 270 ºC and that of the detector was 300 ºC. Identification of derivates was carried out according to their mass pattern.

Anion-exchange chromatography of high resolution coupled to an amperometric detector. Solutions at 1 % were prepared in water milli-Q from the dry extract of Agave fourcroydes and commercial fructans Raftilose P95 and Raftiline HP. These samples were filtered using a sieve of 0.22 micras of porosity. The methodology proposed by Shiomi (1991) was used. Identification of some compounds was determined with the standards: kestose, nystose and kestopentaose (DP5).

 

RESULTS AND DISCUSSION

After analyzing the results of thin layer chromatography (figure 1), there was a presence of stains in A. fourcroydes, which corresponded to glucose/fructose (Gluc/Fruc), sucrose (Sac), inulobiose (F2), 1-kestosa (1-Kes), nystose and kestopentaose. In addition, it was demonstrated that fructans of dry extract have a low level of polymerization, as well as the commercial prebiotic Raftilose P95, although with a different chromatographic profile. The stain belonging to those of high level of polymerization, like in the case of the commercial prebiotic Raftiline HP, did not appear. This characteristic is important for evaluating A. fourcroydes as a prebiotic candidate because oligosaccarides are more used for obtaining these additives (Kanakupt et al. 2011).

In A. fourcroydes, there was a different stain, which was identified according to its Rf like neo-kestose (6 G-Kes), which was identified according to similar chromatographic studies (Cairns et al. 1999). These structures may contribute to a higher growth and metabolic activity of beneficial intestinal microorganisms (Gutiérrez 2013). Besides, they are compounds different from the inulin present in commercial prebiotics (Quiminet 2014).

The presence of 1-Kes, F2 and 6G-Kes indicates that fructans within dry extract of A. fourcroydes may be divided into different series: GFn, which is characteristic from Cichorium intybus (common chicory) and Helianthus tuberosus (Jerusalem artichoke) (van Laere and van den Ende 2002); Fn, structures without glucose molecule (Roberfroid 2005); and neo-fructan series, which was found in Asparagus officinalis (asparagus) and Allium cepa (onion) (Ritsema et al. 2004). This composition characterizes A. fourcroydes as a mixture of different structures. Studies of Bathia and Nandra (1979) showed different results. These authors identified fructans of inulin type in this genre, while García-Albornoz (2006) only reported presence of inulin and polyfructans in studies with Agave fourcroydes in Yucatán.

In the analysis of gas chromatography coupled to a mass spectrometry, which is essential for determining chemical bonds of derivates of fructans to partially methylated alditol acetate of A. fourcroydes, there were differences regarding commercial fructans Raftilose P95 and Raftiline HP (figure 2).

Peaks 1 and 2 corresponded to 2.5 Di-O-acetyl-2-deuterium-1, 3, 4, 6-tetra-O-methyl-D-mannitol and 2.5 Di-O-acetyl-2-deuterium-1, 3, 4, 6-tetra-O-methyl-D-glucitol, derived from reduction of terminal fructose within fructans (â-D-Fruc-t). Peak 3 belonged to terminal unit of á-D- glucopyranose (á-D-Gluc-t): 1, 5-Di-O-acetyl-(1-deuterium)-2, 3, 4, 6-tetra-O-methyil-glucitol. The base fragment of this molecule is m/z 101, unlike the base fragment m/z 129, which is a characteristic of Fru residues.

Peak 4 was identified as a derivate corresponding to mannitol configuration of derivate â (2-6)- D- fructofuranose: 2, 5, 6-Tri-O-acetyl-(2-deuterium)-1, 3, 4-tri-O-methyl-mannitol. This peak represents â (2-6) bonds. This bond has the lowest proportion among the samples.

Peak 5 indicates the presence of â (2-1) bonds, given by 1, 2, 5 Tri-O-acetyl-(2-deuterium)-3, 4, 6-tri-O-methyl-mannitol. There was no evidence of separation of glucitol epimer, and, consequently, peak 6 contains glucitol configurations for both bond types: 2, 5, 6-Tri-O-acetyl-(2-deuterium)-1, 3, 4-tri-O-methyl-glucitol + 1, 2, 5-Tri-O-acetyl-(2-deuterium)-3, 4, 6-tri-O-methyl-glucitol. Peak 7 confirms the presence of neo-fructans due to the presence of an internal glucose: 1, 5, 6-Tri-O-acetyl-(1-deuterium)-2, 3, 4-tri-O-methyl-glucitol and peak 8 corresponds to ramifications 1, 2, 5, 6-Tetra-O-acetyl-(2-deuterium)-3, 4-di-O-methyl-hexitol (-glucitol and mannitol).

From chromatographic profiles of fructans of A. fourcroydes, it can be considered that there is a presence of internal glucose in these plants, which is a characteristic of neo-fructan series, as well as â (2-1) bonds and, in lower amount, â (2-6) bonds. In addition, the presence of few ramifications was confirmed.

Results demonstrate structural complexity of these compounds, which could be a characteristic of Agavaceae family, taking into account the study reports of López et al. (2003) in Agave tequilana Weber var. Azul. These authors identified fructans, â (2-1) bonds and â (2-6) bonds with abundant ramifications. The presence of complex structures was also confirmed in fructans of Urginea maritima (Spies et al. 1992), so they present similarities in their structure.

The presence of fructans with different â bonds within the dry extract of Agave fourcroydes makes possible to avoid their degradation by digestive enzymes of host and to be used as energy sources by lactobacilli and bifidobacteria, which are beneficial microorganisms of the gastrointestinal tract (Gibson and Roberfroid 2008).

Figure 3 shows the chromatographic profiles obtained by anion-exchange chromatography of high resolution of fructans of A. fourcroydes from Cuba (Raftilose P95 and Raftiline HP). According to Pavis et al. (2001), this method allows to know the distribution of fructans within the plant.

In chromatograms belonging to Raftilose P95 and Raftiline HP, there were fructans from inulin series, with well defined peaks (figure 3 b and c). However, when analyzing chromatographic profiles of dry extract of Agave fourcroydes, there was a great structural diversity. Only kestose, nystose y kestopentaose (DP5) were identified and when they were compared to the standards, a great difference between the peaks of the analyzed samples was confirmed. Fructans of A. fourcroydes were more complex due to the presence of more peaks, so carbohydrates from inulin (GFn), neo-kestose and inulo-nose (Fn) series may be present. This analysis established the structural difference between fructans of A. fourcroydes and inulin-type prebiotics.

Chromatological profile for fructans from dry extract of A. fourcroydes showed natural oligosaccharides, which confirms the results from thin layer chromatography. This chromatogram differed to that reported for Agave tequilana, Agave potatorum and Agave angustifolia by studies of Mancilla (2006), who stated fructans with high level of polymerization in the structure.

From the analysis of results, a complex mixture of oligosaccharides, which presents structures with few ramifications and Fn, GFn and neo-fructan series (figure 4), was proposed as structures for.

Structural characteristics of fructans of Agave fourcroydes confirmed in this research make them be very important for animal nutrition, and to be evaluated as prebiotic candidate because they constitute natural sources of oligofructans, which are considered as the best substrates for acid lactic bacteria regarding those with high level of polymerization (Gibson and Roberfroid 2008). Besides, fructans of Agave fourcroydes have different â-type bonds that make them indigestible by digestible enzymes from the superior part of the gastrointestinal tract of monogastric animals and, therefore, can be used as energy source for beneficial microorganisms from the caecum and colon of the host. This influenced on a better health state and on productive performance of the animal (Huyghebaert et al. 2011 and Zhao et al. 2013b).

These determinations allowed to perform the structural characterization of fructans from Agave fourcroydes, which are not from inulin type, and they constitute a polydisperse mixture of oligofructans. Due to their properties, fructans of Agave fourcroydes can be used as prebiotic in animal nutrition.

 

ACKNOWLEDGEMENTS

Thanks to the Empresa Henequenera «Eladio Hernández León» for the material provided for this study. Thanks also to the Centro de Investigaciones y Estudios de Avanzada del IPN, in Guanajuato, Mexico, for lending their facilities for the development of experiments.

 

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Received: December 12, 2014
Accepted: December 23, 2014

 

 

Yanelys García-Curbelo, Departamento de Ciencias Biofisiológicas, Instituto de Ciencia Animal, Apartado Postal 24, San José de las Lajas, Mayabeque, Cuba. Email: [e-mail]