High-performance Anion-exchange Chromatography With Pulsed Amperometric Detection Research Articles

Novel rapid method for the characterisation of polymeric sugars from macroalgae

Laminarins are storage polysaccharides found only in brown seaweeds, specifically Laminarialaes and Fucales. Laminarin has been shown to have anti-apoptotic and anti-tumoural activities and is considered as a nutraceutical component that can positively influence human health. The structure is species dependent, generally composed of linear ß(1–3) glucans with intrachain β(1–6) branching and varies according to harvest season and environmental factors. Current methods for analysis of molar mass and DP length are technically demanding and are not widely available. Here, we present a simple inexpensive method which enables rapid analysis of laminarins from macroalgal biomass using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) without the need for hydrolysis or further processing. This is based on the linear relationship observed between log10 DP and retention time following separation of laminarins on a CarboPac PA-100 column (Dionex) using standard 1,3-β-d-gluco-oligosaccharides ranging in DP from 2 to 8. This method was applied to analyse laminarin oligomers in extracts from different species harvested from within the intertidal zone on Welsh rocky shores containing laminarin polymers with different ranges of DP. The degree of polymerisation and extrapolated molar mass agreed well with values estimated by LC-ESI/MSn analysis and those reported in the literature.

Chemical characterization of Pseudomonas veronii 2E soluble exopolymer as Cd(II) ligand for the biotreatment of electroplating wastes

The production, isolation, purification and characterization of soluble Extracellular Polymeric Substances (EPS) secreted by Pseudomonas veronii 2E were studied. Optimal production was obtained in medium M9-14 g l−1 glycerol, 25 °C. Spectrophotometric and potentiometric titration studies showed that EPS consisted on extracellular DNA, proteins and polysaccharides as the main components. DNase I, Proteinase K and further sodium deoxycholate treatments yielded similar proportions of lipopolysaccharides (LPSs) and exopolysaccharides (ExPs). ExP was subjected to total acid hydrolysis and then analyzed by High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD), showing the presence of fucose, galactosamine, glucosamine, galactose, glucose, mannose and glucuronic acid. Further treatment with oxalic acid confirmed the presence of a pyruvilated sugar. The ability of the secreted products to interact with Cd(II) was studied by anodic stripping voltammetry (ASV). LPS fraction contained the highest ligand concentration (279 ± 10 μmoles Cd(II) binding sites g LPS−1). This information is a valuable tool for the application of EPS in industrial wastewater treatment.

Validated HPAEC-PAD Method for the Determination of Fully Deacetylated Chitooligosaccharides.

An efficient and sensitive analytical method based on high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was established for the simultaneous separation and determination of glucosamine (GlcN)1 and chitooligosaccharides (COS) ranging from (GlcN)2 to (GlcN)6 without prior derivatization. Detection limits were 0.003 to 0.016 mg/L (corresponding to 0.4–0.6 pmol), and the linear range was 0.2 to 10 mg/L. The optimized analysis was carried out on a CarboPac-PA100 analytical column (4 × 250 mm) using isocratic elution with 0.2 M aqueous sodium hydroxide-water mixture (10:90, v/v) as the mobile phase at a 0.4 mL/min flow rate. Regression equations revealed a good linear relationship (R2 = 0.9979–0.9995, n = 7) within the test ranges. Quality parameters, including precision and accuracy, were fully validated and found to be satisfactory. The fully validated HPAEC-PAD method was readily applied for the quantification of (GlcN)1–6 in a commercial COS technical concentrate. The established method was also used to monitor the acid hydrolysis of a COS technical concentrate to ensure optimization of reaction conditions and minimization of (GlcN)1 degradation.

A novel analytical method for d-glucosamine quantification and its application in the analysis of chitosan degradation by a minimal enzyme cocktail

Enzymatic depolymerization of chitosan, a β-(1,4)-linked polycationic polysaccharide composed of d-glucosamine (GlcN) and N-acetyl-d-glucosamine (GlcNAc) provides a possible route to the exploitation of chitin-rich biomass. Complete conversion of chitosan to mono-sugars requires the synergistic action of endo- and exo- chitosanases. In the present study we have developed an efficient and cost-effective chitosan-degrading enzyme cocktail containing only two enzymes, an endo-attacking bacterial chitosanase, ScCsn46A, from Streptomyces coelicolor, and an exo-attacking glucosamine specific β-glucosaminidase, Tk-Glm, from the archaeon Thermococcus kodakarensis KOD1. Moreover, we developed a fast, reliable quantitative method for analysis of GlcN using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The sensitivity of this method is high and less than 50 pmol was easily detected, which is about 1000-fold better than the sensitivity of more commonly used detection methods based on refractive index. We also obtained qualitative insight into product development during the enzymatic degradation reaction by means of ElectroSpray Ionization-Mass Spectrometry (ESI-MS).

Multivalent sialylation of β-thio-glycoclusters by Trypanosoma cruzi trans sialidase and analysis by high performance anion exchange chromatography.

The synthesis of multivalent sialylated glycoclusters is herein addressed by a chemoenzymatic approach using the trans-sialidase of Trypanosoma cruzi (TcTS). Multivalent β-thio-galactopyranosides and β-thio-lactosides were used as acceptor substrates and 3'-sialyllactose as the sialic acid donor. High performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was shown to be an excellent technique for the analysis of the reaction products. Different eluting conditions were optimized to allow the simultaneous resolution of the sialylated species, as well as their neutral precursors. The TcTS efficiently transferred sialyl residues to di, tri, tetra and octa β-thiogalactosides. In the case of an octavalent thiolactoside, up to six polysialylated compounds could be resolved. Preparative sialylation reactions were performed using the tetravalent and octavalent acceptor substrates. The main sialylated derivatives could be unequivocally assigned by MALDI mass spectrometry. Inhibition of the transfer to the natural substrate, N-acetyllactosamine, was also studied. The octalactoside caused 82% inhibition of sialic acid transfer when we used equimolar concentrations of donor, acceptor and inhibitor.

Characterization of Pyrolytic Sugars in Bio-Oil Produced from Biomass Fast Pyrolysis

This study characterizes the pyrolytic sugars in three bio-oils (with a total sugar content range of 55.6–69.2 mg g–1 bio-oil) produced from biomass fast pyrolysis by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and two-dimensional 1H–13C heteronuclear single-quantum correlation–nuclear magnetic resonance (HSQC-NMR). Depending on bio-oil sample, glucose (mainly derived from cellulose) contributes ∼67–79 wt % of total pyrolytic sugars in the bio-oil, and the rest of the sugars are derived from hemicellulose. The majority (>96%) of pyrolytic sugars are present in the water-soluble fraction of bio-oil, mainly in the form of cellulose-derived anhydrosugars such as levoglucosan and cellobiosan. A small portion of hemicellulose-derived sugar structures are also found in the water-soluble fraction of bio-oil. Unlike six-carbon sugars (glucose, galactose, and mannose) which are mainly present as anhydrosugars (i.e., ∼ 79–86% on a carbon basis for glucose), a large ...

Physico-chemical properties and amino acid profiles of sap from Tunisian date palm

Date palm sap (Phoenix dactylifera L.), also known as “legmi”, is a fresh juice extracted from date palm trees. The present study aimed to elucidate the effects of collection time (at the beginning of the tapping period and after seven days of collection) on the amino acid profile and physico-chemical properties of date palm sap from both male and female trees. Dry matter, protein, amino acid, and sugar profiles were determined using the Kjeldahl method, High-Performance Liquid Chromatography (HPLC), and High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD), respectively. Date palm sap from both male and female trees contained high levels of carbohydrates. HPLC analysis showed that this fraction was dominated by sucrose in the sap sample from female trees compared to that from male trees. Male date palm sap was noted to exhibit lower dry matter content than female date palm sap but higher protein, total polyphenol, ash, and amino acid contents. While the major essential amino acids in the sap from male trees consisted of valine and threonine, they were represented by lysine and phenylalanine in sap samples from female trees. Further, Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis showed the presence of a proteinic band of 30 kDa only for the sap from male trees. Taken together, the sap from both male and female date palm trees had a number of properties that are highly valued by the functional food industry.

Profiling N-linked oligosaccharides from IgG by high-performance anion-exchange chromatography with pulsed amperometric detection.

Understanding and characterizing protein therapeutic glycosylation is important with growing evidence that glycosylation impacts biological efficacy, pharmacokinetics and cellular toxicity. Protein expression systems and reactor conditions can impact glycosylation, leading to potentially undesirable glycosylation. For example, high-mannose species may be present, which are atypical of human antibody glycosylation. Their presence in the Fc domain has been linked to increased serum clearance of immunoglobulin G (IgG) antibodies. High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) is an effective tool for determining glycans present in glycoprotein therapeutics. We report an improved HPAE-PAD method for IgG oligosaccharide separation. The neutral glycans are well resolved, including separation of high-mannose species from typical human IgG glycans. Oligosaccharide identification was performed by comparison to known standards in conjunction with selective exoglycosidase digestion of both standards and released glycans. Retention times (RTs) of known glycans were compared with the retention times of maltose, maltotriose and maltotetraose standards to define a retention index value for each glycan. These retention indices were used to aid identification of glycans from an example monoclonal antibody sample of unknown glycosylation. Method ruggedness was evaluated across duplicate systems, analysts and triplicate column lots. Comparing two systems with different analysts and columns, retention time precision relative standard deviations (RSDs) were between 0.63 and 4.0% while retention indices precision RSDs ranged from 0.27 to 0.56%. The separation is orthogonal to capillary electrophoresis-based separation of labeled IgG oligosaccharides.

Sugar Profile of Kernels as a Marker of Origin and Ripening Time of Peach (Prunus persicae L.).

Large amounts of fruit seeds, especially peach, are discarded annually in juice or conserve producing industries which is a potential waste of valuable resource and serious disposal problem. Regarding the fact that peach seeds can be obtained as a byproduct from processing companies their exploitation should be greater and, consequently more information of cultivars' kernels and their composition is required. A total of 25 samples of kernels from various peach germplasm (including commercial cultivars, perspective hybrids and vineyard peach accessions) differing in origin and ripening time were characterized by evaluation of their sugar composition. Twenty characteristic carbohydrates and sugar alcohols were determined and quantified using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC/PAD). Sucrose, glucose and fructose are the most important sugars in peach kernels similar to other representatives of the Rosaceae family. Also, high amounts of sugars in seeds of promising hybrids implies that through conventional breeding programs peach kernels with high sugar content can be obtained. In addition, by the means of several pattern recognition methods the variables that discriminate peach kernels arising from diverse germplasm and different stage of maturity were identified and successful models for further prediction were developed. Sugars such as ribose, trehalose, arabinose, galactitol, fructose, maltose, sorbitol, sucrose, iso-maltotriose were marked as most important for such discrimination.

The lactose and galactose content of milk fats and suitability for galactosaemia

IntroductionButter oil, ghee and butter are theoretically low in lactose. All three products are high in milk fat; butter oil and ghee contain approximately 99.3% fat and butter around 80% fat. We report the lactose and galactose content of butter, ghee, and butter oil and assess their suitability in a low galactose diet. MethodsA total of 12 samples (butter oil n=5, ghee n=5, butter n=2) were analysed by High-Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) technology used to perform lactose and galactose analyses. ResultsButter oil and ghee were consistently low in lactose and galactose. Butter oil analysis: lactose, 0.47 to 3.08mg/100g; galactose, <0.05 to 2.28mg/100g. Ghee analysis: lactose, <0.05 to 2.9mg/100g; galactose, <0.05mg to <1.0mg/100g. Butter analysis: lactose 685 to 688mg/100g; galactose, 1.3mg to 1.6mg/100g. ConclusionsButter oil (commonly used in food industry) and ghee contain minimal lactose and galactose and so are permitted in a UK galactosaemia diet. Butter is considered too high in lactose and is unsuitable in a low galactose diet.

Structural Features and Anti-coagulant Activity of the Sulphated Polysaccharide SPS-CF from a Green Alga Capsosiphon fulvescens.

Previously, we reported that the sulphated polysaccharides (SPS)-CF, a water-soluble polysaccharide isolated and purified from Korean green alga Maesaengi (Capsosiphon fulvescens, Chlorophyta), is a glucuronogalactomannan based mainly on the monosaccharide composition determined by high-performance liquid chromatography (HPLC) analysis after 1-phenyl-3-methyl-5-pyrazolone (PMP) labelling of sugars in the acid (trifluoroacetic acid (TFA)) hydrolyzates of SPS-CF, which showed mannose (55.4 mol %), galactose (25.3 mol %) and glucuronic acid (16.3 mol %) as major sugars (Na et al., Int Immunopharmacol 10:364-370, 2010). However, the results of the present study re-performed for monosaccharide composition of this polysaccharide using, in addition to HPLC of PMP-labelled sugars, other separation methods, i.e. high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), gas chromatography with flame ionising detection (GC-FID) and thin-layer chromatography (TLC), clearly demonstrated that the most prominent neutral monosaccharides of SPS-CF are xylose (38.6-49.4 mol %) and rhamnose (39.6-45 mol %), while mannose and galactose are present at a much lesser extent or in negligible amount. These extensive monosaccharide analyses, correlation nuclear magnetic resonance (NMR), electrospray ionization mass spectrometry (ESI-MS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) measurements confirmed the sulphated glucuronorhamnoxylan (ulvan) type of SPS-CF polysaccharide, whose backbone is composed of alternating sequence of 4-linked L-rhamnose-3-sulphate and D-xylose residues (ulvobiose U3s) carrying monomeric D-glucuronic acid or D-glucuronic acid-3-sulphate on O-2 of some L-rhamnose-3-sulphate units as the side chains. The SPS-CF exhibited significant in vitro anti-coagulant activity by which the activated partial thromboplastin time (aPTT) and thrombin time (TT) were significantly prolonged. The results of this study demonstrated that the ulvan SPS-CF isolated from Korean Maesaengi C. fulvescens can be considered a potential anti-coagulant agent.

Improved sample treatment for the determination of fructooligosaccharides in milk related products by liquid chromatography with electrochemical and refractive index detection

A simple and reliable method for the determination of the fructooligosaccharides (FOS) kestose (GF2), nystose (GF3), fructofuranosylnystose (GF4), in the presence of fructose, glucose and lactose in dairy products is proposed. The most relevant advantages of the proposed method are the simultaneous determination of the most common FOS in enriched products and a reduction of the time required for sample treatment since the method consists merely in addition of a precipitation solution for the removal of lipids and proteins. Furthermore, the method saves a substantial amount of reagents compared with other methods and sample manipulation is reduced. Two chromatographic separations are proposed. The first one is carried out on an amino phase column for liquid chromatography with refractive index detection (HPLC-RI) (concentration of analytes higher than 0.1mgmL−1) and the second one on an anion-exchange Carbopac PA-1 column for high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) (concentration of FOS lower than 0.1mgmL−1). The method was validated by recovery assays with spiked samples using matrix-matched calibration. The limits of quantification of the method ranged from 1.2 to 2.0µgmL−1 for HPAEC-PAD and from 140 to 200µgmL−1 for HPLC-RI, while inter- and intra-day variability was under 3.5% in all cases. The proposed method was applied to the determination of compounds in supplemented milk, infant formulas and milk related juices with good recoveries in all cases.

Phytases Improve Myo-Inositol Bioaccessibility in Rye Bread: A Study Using an In Vitro Method of Digestion and a Caco-2 Cell Culture Model

Preparations of 6-phytase A (EC 3.1.3.26) and phytase B (acid phosphatase, EC 3.1.3.2) were applied alone and combined in the preparation of dough to estimate their catalytic potential for myo-inositol liberation from rye flour in the breadmaking technology. The experimental bread samples were ground after baking and subjected to determination of myo-inositol bioavailability by an in vitro method that simulated digestion in a human alimentary tract, followed by measurements of myo-inositol transport through enterocyte- -like differentiated Caco-2 cells to determine its bioaccessibility. Myo-inositol content was measured by a high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) technique. The concentration of myo-inositol in the dialysates of control bread was 25.3 µg/mL, whereas in the dialysates of bread sample baked with 6-phytase A, the concentration increased to 35.4 µg/mL, and in the bread baked with phytase B to 64.98 µg/mL. Simultaneous application of both enzymes resulted in myo-inositol release of 64.04 µg/mL. The highest bioaccessibility of myo-inositol, assessed by the measurement of the passage through the Caco-2 monolayer was determined in the bread baked with the addition of 6-phytase A. Enzymatically modified rye bread, particularly by the addition of 6-phytase A, may be therefore a rich source of a highly bioaccessible myo- -inositol.

High performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) for the sensitive determination of hyaluronan oligosaccharides.

High performance anion exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) was optimized for the analysis of oligosaccharides derived from the extracellular matrix component hyaluronan. Using this sensitive approach, the separation of oligosaccharides consisting of two (molecular weight ca. 0.8 kDa) up to 25-30 (molecular weight: ca. 9.5-11.4 kDa) disaccharide moieties was possible. Standard oligosaccharides (comprising 2-4 repetitive disaccharides) were detectable at very low amounts of 0.2-0.3 pmol (20-30 nM). Including 10 min of column equilibration, a complex mixture of low molecular weight hyaluronan can be analyzed within 40 min. The HPAEC method was successfully applied to the study of the size-dependency of both the action of bovine testicular hyaluronidase (BTH) and the precipitation of hyaluronan by cetyltrimethylammonium bromide (CTAB), a physicochemical reaction often used for the determination of hyaluronan and hyaluronidase activity.

Neutral Pectin side chains of Amaranth (Amaranthus hypochondriacus) contain long, partially branched Arabinans and short galactans, both with terminal arabinopyranoses.

Amaranth is a pseudocereal of high nutritional value, including a high dietary fiber content. Amaranth dietary fiber was suggested to contain large amounts of neutral rhamnogalacturonan I side chains. In this study, endo-arabinanase and endo-galactanase were used to liberate arabinan and galactan oligosaccharides from amaranth fiber. The liberated oligosaccharides were identified by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and HPLC-MS(n) using standard compounds, which were isolated from amaranth, sugar beet, potato, and red clover sprouts and characterized by one- and two-dimensional NMR spectroscopy. It was demonstrated that insoluble amaranth arabinans have linear and branched areas, with the O-3 position being the dominant branching point. Minor amounts of branches at position O-2 and double substitution were also found. Amaranth arabinans were also demonstrated to contain terminal α-(1→5)-linked l-arabinopyranose units. In addition, it was evidenced that galactans from amaranth seeds are composed of β-(1→4)-linked d-galactopyranose units, which can also be terminated with l-arabinopyranose units. In direct comparison to structural elucidation of amaranth fiber by using methylation analysis, the advantage of the enzymatic approach over methylation analysis was demonstrated.

Impact of hydrolysis conditions on the detection of mannuronic to guluronic acid ratio in alginate and its derivatives

Alginate is a linear and acidic polysaccharide, composed of (1→4) linked β-d-mannuronic acid (ManA) and α-l-guluronic acid (GulA). The ratio of ManA to GulA (M/G) is one of the most important factors for the application of alginate and its derivatives in various areas. In this work, a robust and accurate method was developed to analyze M/G using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The impact of hydrolysis conditions on the release patterns of ManA and GulA from alginate and its derivatives was investigated. The release patterns of ManA and GulA need to be considered separately to obtain an accurate M/G. Several hydrolysis conditions were established that released ManA and GulA completely and maintained these saccharide residues intact. The proper M/G of alginates from different sources and its derivatives could then be calculated by integration of the corresponding ManA and GulA peaks.

The Lactose and Galactose Content of Cheese Suitable for Galactosaemia: New Analysis.

The UK Medical Advisory Panel of the Galactosaemia Support Group report the lactose and galactose content of 5 brands of mature Cheddar cheese, Comte and Emmi Emmental fondue mix from 32 cheese samples. The Medical Advisory Panel define suitable cheese in galactosaemia to have a lactose and galactose content consistently below 10mg/100g. A total of 32 samples (5 types of mature Cheddar cheese, Comte and "Emmi Swiss Fondue", an emmental fondue mix) were analysed by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) technology used to perform lactose and galactose analysis. Cheddar cheese types: Valley Spire West Country, Parkham, Lye Cross Vintage, Lye Cross Mature, Tesco West Country Farmhouse Extra Mature and Sainsbury's TTD West Country Farmhouse Extra Mature had a lactose and galactose content consistently below 10mg/100g (range <0.05 to 12.65mg). All Comte samples had a lactose content below the lower limit of detection (<0.05mg) with galactose content from <0.05 to 1.86mg/100g; all samples of Emmi Swiss Fondue had lactose below the lower limit of detection (<0.05mg) and galactose between 2.19 and 3.04mg/100g. All of these cheese types were suitable for inclusion in a low galactose diet for galactosaemia. It is possible that the galactose content of cheese may change over time depending on its processing, fermentation time and packaging techniques.

Mediterranean semi-arid plant Astragalus armatus as a source of bioactive galactomannan

A water-soluble polysaccharide fraction (WSPF) was extracted and purified from Astragalus armatus Lam. (Fabaceae), a spontaneous plant found in North Algerian Sahara. The structural features of WSPF were evaluated by colorimetric assays, Fourier transform infrared (FTIR) spectroscopy, high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and gas chromatography coupled to mass spectrometry (GC/MS). WSPF was a galactomannan-rich fraction, with a β-(1,4)-d-manp backbone with α-(1,6)-d-galp branches, and a Man:Gal ratio of 1.13. Besides, the anti-complement activity and the antioxidant potential (DPPH and hydroxyl radicals) of WSPF were evaluated. The results exhibited an anti-complementary activity, with an IC50 value of 0.39 mg/mL of the fraction, better than for heparin (0.58 mg/mL). Moreover, WSPF possessed a notable antioxidant activity on DPPH radical with an IC50 of 330 µg/mL.

Short communication: Amino trap column improves the separation of methylimidazoles, 5-hydroxymethyl-2-furaldehyde, and sugars in Maillard reaction

A simultaneous analysis of methylimidazoles, reducing sugars, and 5-hydroxymethyl-2-furaldehyde in the Maillard reaction was improved by use of an amino trap column. Analysis was carried out by using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) coupled with an amino trap column. The amino trap column was a useful tool to improve the separation of methylimidazoles, reducing sugars, and 5-hydroxymethyl-2-furaldehyde. This technique is useful for simultaneous analysis of methylimidazoles, reducing sugars, and 5-hydroxymethyl-2-furaldehyde in risk assessment for dairy products.

Prebiotic potential of Agave angustifolia Haw fructans with different degrees of polymerization.

Inulin-type fructans are the most studied prebiotic compounds because of their broad range of health benefits. In particular, plants of the Agave genus are rich in fructans. Agave-derived fructans have a branched structure with both β-(2→1) and β-(2→6) linked fructosyl chains attached to the sucrose start unit with a degree of polymerization (DP) of up to 80 fructose units. The objective of this work was to assess the prebiotic potential of three Agave angustifolia Haw fructan fractions (AFF) with different degrees of polymerization. The three fructan fractions were extracted from the agave stem by lixiviation and then purified by ultrafiltration and ion exchange chromatography: AFF1, AFF2 and AFF3 with high (3–60 fructose units), medium (2–40) and low (2–22) DP, respectively. The fructan profile was determined with high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), which confirmed a branched fructan structure. Structural elucidation was performed by Fourier Transform Infra-Red Spectroscopy. The AFF spectrum shows characteristic fructan bands. The prebiotic effect of these fractions was assessed in vitro through fermentation by Bifidobacterium and Lactobacillus strains. Four growth patterns were observed. Some bacteria did not grow with any of the AFF, while other strains grew with only AFF3. Some bacteria grew according to the molecular weight of the AFF and some grew indistinctly with the three fructan fractions.