Analytical and Bioanalytical Chemistry

, Volume 405, Issue 13, pp 4591–4605

Innovative analytical tools to characterize prebiotic carbohydrates of functional food interest

Authors

    • Department of ChemistryUniversity of Parma
  • Claudia Lantano
    • Department of ChemistryUniversity of Parma
  • Antonella Cavazza
    • Department of ChemistryUniversity of Parma
Review

DOI: 10.1007/s00216-013-6731-6

Cite this article as:
Corradini, C., Lantano, C. & Cavazza, A. Anal Bioanal Chem (2013) 405: 4591. doi:10.1007/s00216-013-6731-6

Abstract

Functional foods are one of the most interesting areas of research and innovation in the food industry. A functional food or functional ingredient is considered to be any food or food component that provides health benefits beyond basic nutrition. Recently, consumers have shown interest in natural bioactive compounds as functional ingredients in the diet owing to their various beneficial effects for health. Water-soluble fibers and nondigestible oligosaccharides and polysaccharides can be defined as functional food ingredients. Fructooligosaccharides (FOS) and inulin are resistant to direct metabolism by the host and reach the caecocolon, where they are used by selected groups of beneficial bacteria. Furthermore, they are able to improve physical and structural properties of food, such as hydration, oil-holding capacity, viscosity, texture, sensory characteristics, and shelf-life. This article reviews major innovative analytical developments to screen and identify FOS, inulins, and the most employed nonstarch carbohydrates added or naturally present in functional food formulations. High-performance anion-exchange chromatography with pulsed electrochemical detection (HPAEC-PED) is one of the most employed analytical techniques for the characterization of those molecules. Mass spectrometry is also of great help, in particularly matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), which is able to provide extensive information regarding the molecular weight and length profiles of oligosaccharides and polysaccharides. Moreover, MALDI-TOF-MS in combination with HPAEC-PED has been shown to be of great value for the complementary information it can provide. Some other techniques, such as NMR spectroscopy, are also discussed, with relevant examples of recent applications. A number of articles have appeared in the literature in recent years regarding the analysis of inulin, FOS, and other carbohydrates of interest in the field and they are critically reviewed.

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Keywords

FructooligosaccharidesHigh-performance anion-exchange chromatography with pulsed electrochemical detectionInulinMatrix-assisted laser desorption/ionization time of flight-mass spectrometryPrebiotics

Introduction

Today, foods are not intended to only satisfy hunger and to provide necessary nutrients for humans; consumers are increasingly aware of the relationship between diet, health, and disease prevention with the aim of improving their physical and mental well-being [1].

On the basis of the health criterion and on foods with specific health-enhancing characteristics, there has been growing interest in so-called functional food, generally used to refer to a food, similar to a conventional food, that is consumed as part of the usual diet which either provides physiological benefits or reduces the risk of chronic disease beyond basic nutritional functions [2]. The increasing demand for such functional foods can be explained by the increasing cost of health care, the steady increase in life expectancy, reduction of disease risk through prevention, and improvement of quality of life and well-being contributing to increased healthy longevity [2].

Food fibers are a heterogeneous group of components which include a mixture of plant carbohydrates, both oligosaccharides and polysaccharides, such as cellulose, hemicelluloses, pectic substances, gums, resistant starch, fructans, xylooligosaccharides (XOS), and galactooligosaccharides (GOS) that may be associated with lignin and other noncarbohydrate components (e.g., polyphenols, waxes, saponins, cutin, phytates, resistant protein) [3]. As recently summarized by the European Food Safety Authority [4], dietary fiber was originally defined in 1972 by Trowell as “that portion of food which is derived from cellular walls of plants which are digested very poorly by human beings.” Furthermore, many different definitions for dietary fibers have been proposed, as recently reviewed by Mann and Cummings [5]. Dietary fibers can be subdivided into soluble and insoluble types. Analytical methods for the determination of dietary fibers include nonenzymatic and enzymatic–gravimetric methods, as well as enzymatic chemical methods, as recently reviewed by Elleuch et al. [6]. The most employed analytical methods to determine dietary fiber values, according to the current EU and/or Codex Alimentarius Commission definitions, are the AOAC 985.29 and AOAC 991.43 methods The AOAC 991.43 method distinguishes between insoluble and soluble high molecular weight dietary fiber, whereas in the AOAC 985.29 method the total high molecular weight dietary fiber, being the sum of insoluble and soluble fractions, is measured directly. Recently, Westenbrik et al. [7] provided an overview of the available AOAC International analytical methods for total dietary fiber and its fractions.

All these methods are recommended analytical methods for nutritional labeling analysis with the purpose of stating the dietary fiber content on food labels, but they are inappropriate for the determination of low molecular weight dietary fibers such as inulin, fructooligosaccharides (FOS), GOS, XOS, soybean oligosaccharides, isomaltooligosaccharides, and polydextrose.

The appearance of modern promising techniques, more accurate and advanced, resulted in great development of research in the sector, allowing the investigation of complex mixtures and the study of several properties depending on the chemical structure.

In this review, an evaluation of the strengths, weaknesses, and notable features regarding the characterization of FOS, inulin, and other prebiotic fibers with use of high-performance anion-exchange chromatography (HPAEC) with pulsed electrochemical detection (PED) and matrix-assisted laser desorption/ionization (MALDI) time-of flight (TOF) mass spectrometry (MS) is presented. Furthermore, an overview of the most recent analytical methods employed for the characterization of other oligosaccharides and polysaccharides of relevance to functional food is given.

Prebiotic carbohydrates

Food and food ingredients that promote the growth or activity of a limited number of bacterial species for the benefit of health of the host are defined as prebiotics [8]. These food ingredients are normally restricted to certain carbohydrates, particularly oligosaccharides, such as FOS, inulin, GOS, XOS, soybean oligosaccharides, and isomaltooligosaccharides [9, 10].

Low molecular weight dietary fibers transit through the stomach and small intestine, where they are neither absorbed nor degraded, and reach the colon, where are fermented by resident bacteria, promoting their proliferation and thus improving health of the host [8]. Furthermore, they also affect the blood sugar level to a lesser extent than other carbohydrates, and then they are suited as a constituent in a diabetic diet.

Nonstarch oligosaccharides and polysaccharides such as FOS and inulin are not digested in the small intestine, but may be fermented in the colon into healthy short-chain fatty acids such as acetate, propionate, and butyrate.

The fructans, inulin, and oligofructose, which are known to possess many of the physiologic properties of dietary fiber, are usually not listed as dietary fiber because they do not precipitate in 78 % ethanol as prescribed in the AOAC International methods for dietary fiber. From scientific evidence, today there is agreement that such resistant oligosaccharides should be included in the dietary fiber complex.

FOS are fructans with a degree of polymerization (DP) up to 10 and are, together with inulin, one of the most important ingredients used in the formulation of functional foods, particularly those claiming prebiotic properties.

Fructans are widely distributed as carbohydrate storage polymers in the vegetative tissue of many families of plants, in bacteria, and in fungi. Their distribution depends on the plant source, harvesting date, and also extraction and postextraction processes. FOS such as 1-kestose (GF2), nystose (GF3), and 1F-fructofuranosyl nystose (GF4) represent a major class of fructan oligosaccharides.

Oligofructose is highly soluble and has advantageous technological properties closely related to those of sugar and glucose syrups, such as increased viscosity, leading to improved body and mouthfeel properties. Therefore, despite its moderate sweetness, it is used as a sugar replacement.

Inulin, in addition to the prebiotic effect, has a bland flavor and a fat-like texture. It develops a gel-like structure when thoroughly mixed with water or other aqueous liquid, forming a gel with a white creamy appearance and a spreadable texture. Therefore, it can be incorporated into various food preparations to replace sugar and fat, and is an interesting ingredient to provide structure in low-fat or zero-fat food products [11, 12]. Commercially available inulin is usually obtained by aqueous extraction from several members of the family Compositae, such as Jerusalem artichoke (Helianthus tuberous), artichokes, chicory, dahlias, and dandelions.

Different analytical techniques have been applied to the analysis of inulin and oligosaccharides. A list of recent applications for the analysis of several kinds of samples is given in Table 1.
Table 1

Application of different analytical techniques to the analysis of inulin and oligosaccharides in several kinds of samples

Technique

Analytes

Sample

Authors, year

HPAEC-PED

Fructans

Plant tissues

Liu et al. [27], 2011

HPAEC-PED

Prebiotic polysaccharides

Potato pulp

Thomassen et al. [81], 2011

HPAEC-PED

Fructooligosaccharides and inulin

Burdock roots

Ishiguro et al. [25], 2010

Hydrolysis–HPAEC-PED

Inulin

Chicory

van Arkel et al. [31], 2012

HPAEC-PED/GC

Arabinoxylans

Wheat flour

Virkki et al. [74], 2008

HPAEC-PED/ESI-MS/NMR

Polysaccharides, xylooligosaccharides

Gram husk and wheat bran

Madhukumar and Muralikrishna [63], 2010

HPAEC-PED/1H NMR/ESI-MS/NMR

Fructans

Agave americana

Ravenscroft et al. [49], 2009

HPLC-RI/HPAEC-PED/MALDI-TOF-MS/GC–MS

Water-soluble carbohydrates

Agave tequilana

Arrizon et al. [48], 2010

HPAEC-PED/GC-MS

Fructooligosaccharides

Banana

Ghedini Der Agopian et al. [58], 2008

HPAEC-PED/MALDI-TOF-MS

Fructooligosaccharides and inulins

Fermented milk, cooked ham, food ingredients

Borromei et al. [52], 2009

HPAEC-PED/MS

Fructooligosaccharides and inulooligosaccharides

Fermented milk

Borromei et al. [19], 2009

HPAEC-PED/MALDI-TOF-MS/NMR

Fructopyranose oligosaccharides

Fermented beverage of plant extract

Okada et al. [45], 2010

HPAEC-PED/MALDI-TOF-MS/NMR

Oligosaccharides

Fermented beverage of plant extract

Okada et al. [46], 2011

HPAEC-PED/CE-LIF/MALDI-TOF-MS

Glucomannan oligosaccharides

Konjac

Albrecht et al. [53], 2009

HPAEC-PED/MALDI-TOF/TOF-MS

Arabinooligosaccharides

Sugar beet

Zaidel et al. [43], 2011

HPAEC/MALDI-TOF/TOF-MS

Arabinooligosaccharides and oligosaccharides

Sugar beet

Holck et al. [72], 2011

MALDI-TOF-MS/HPAEC-PAD/NMR

Arabinooligosaccharides

Sugar beet

Westphal et al. [73], 2010

HPAEC-PED/MALDI-TOF-MS/1H NMR

Fructans

Wheat milling fractions

Haská et al. [51], 2008

HPAEC-PED/GC-MS

Inulin

Jerusalem artichoke

Bach et al. [28], 2012

HPAEC-PED/RI/ELSD

Fructooligosaccharides

Standards

Forgo et al. [82], 2012

HPLC

Fructooligosaccharides

 

Vankova and Polakovic [83], 2010

FTIR/TLC/HPLC-RI

Xylooligosaccharides

Corn cobs

Samanta et al. [71], 2012

HPLC-IR

Fructooligosaccharides

Yacon roots

Campos et al. [84], 2012

HPLC-RI

Oligosaccharides

Olive tree

Cara et al. [85], 2012

HPLC–ELSD

Fructooligosaccharides

Onion

Downes et al. [86], 2010

HPLC–ELSD

Inulin-type oligosaccharides

Root of Morinda officinalis

Yang et al. [64], 2011

HPLC-ELSD

Inulin

Milk

Kristo et al. [65], 2011

LC-ESI-MS

Inulin

Jerusalem artichoke

Matias et al. [87], 2011

HILIC-MS

Galactooligosaccharides

Standards

Hernández-Hernández et al. [68], 2012

MS (negative-ion mode)

Oligosaccharides

Barley, malt, beer

Fabrik et al. [67], 2012

GC-FID/GC-MS

Inulin

Honey

Ruiz-Matute et al. [59], 2010

GC-FID

Fructooligosaccharides

Thai plants

Judprasong et al. [60], 2011

ESI-MS/1H NMR

Xylooligosaccharides

Wheat bran

Manisseri and Gudipati [88], 2010

NMR/MALDI-TOF/ESI-MS

Fructooligosaccharides

Roots and leaves of Stevia rebaudiana

de Oliveira et al. [89], 2011

13C NMR

Garlic fructans

Aged garlic extract

Chandrashekar et al. [62], 2011

HPSEC/FTIR, DSC/XRD

Pectic oligosaccharides

Beet

Combo et al. [90], 2012

GC-MS/IR/HPSEC-MALLS-RI

Tea polysaccharide

Green tea

Guo et al. [91], 2011

DSC differential scanning calorimetry, ELSD evaporative light scattering detection, ESI electrospray ionization, FID flame ionization detection, FTIR Fourier transform IR, GC gas chromatography, HPAEC high-performance anion-exchange chromatography, HPLC high-performance liquid chromatography, HPSEC high-performance size-exclusion chromatography, LC liquid chromatography, LIF laser-induced fluorescence, MALLS multiangle laser-light scattering, MALDI matrix-assisted laser desorption/ionization, MS mass spectrometry, PED pulsed electrochemical detection, RI refractive index, TLC thin-layer chromatography, TOF time of flight, XRD X-ray diffraction

HPAEC-PED: analytical tool in FOS and inulin characterization

HPAEC-PED is a powerful analytical tool in carbohydrate separation owing its ability to separate all classes of alditols, amino sugars, monosaccharides, oligosaccharides, and polysaccharides.

Anion-exchange chromatography is not a technique commonly associated with the analysis of neutral carbohydrates. However, many carbohydrates are weak acids with pKa values in the range of 12–14 and, consequently, at high pH their hydroxyl groups are partially or totally transformed into oxyanions, allowing this class of compounds to be selectively eluted as anions by HPAEC in a single run.

Under alkaline conditions, carbohydrates are readily separated by highly efficient anion-exchange columns, where the order of increasing retention is correlated with decreasing pKa. Anion-exchange chromatography on high-pH-resistant polymeric-based strong-anion-exchange columns specifically tailored for carbohydrate analysis allows selective elution of carbohydrates, where the most important parameters influencing the separations are the number of hydroxyl groups, anomerism, positional isomerism, and the DP.

Retention of oligosaccharides on HPAEC column is not directly predictable by extrapolating the hierarchy of monosaccharide hydroxyl acidities to oligosaccharides. For members of homologous series of oligosaccharides, the capacity factors increases in a regular and predictable manner with chain length. However, besides chain length and saccharide composition, linkage position is expected to affect the chromatographic retention of oligosaccharides.

Corradini et al. [13] showed that separation by HPAEC of 12 disaccharides was strongly affected, besides their acidity, by the accessibility of oxyanions to the functional groups of the anion-exchange column. This effect has been observed in particular for the glucobioses trehalose, isomaltose, gentiobiose, nigerose, and maltose, which are disaccharides composed of two d-glucosyl residues, differing only in the configuration of their glycosidic bonds.

In HPAEC, detection of the selectively eluted carbohydrates is performed by pulsed amperometric detection (PAD), which is also referred to by the more generic name of “pulsed electrochemical detection” (PED). The sensitivity of PED of alditols, monosaccharides, oligosaccharides, and polysaccharides at gold electrodes is maximized under alkaline conditions (pH > 12), which are also the chromatographic conditions required in HPAEC.

Pulsed potential waveform optimization to determine the appropriate detection potential in HPAEC-PED is usually performed by cyclic voltammetry; the choice of the potential and time parameters in PED waveforms has been extensively reviewed [14, 15]. Improvement of long-term reproducibility of PED of carbohydrates has been achieved by applying a quadruple-potential waveform to the working gold electrode in a flow-through detector cell, instead of a triple-pulse potential waveform [16]. With the new selected waveform, negative cleaning and positive activation potentials maintains a clean and active gold electrode surface without causing electrode corrosion, with its consequent recession. Recession of the working gold electrode increases the volume between the working electrode and the counter electrode, decreasing the linear velocity of flow over the surface of the working electrode. This causes decreased transport of analyte to the electrode and consequent decreased detector response.

The proposed quadruple-potential waveform maximizes the signal-to-noise ratio, resulting in minimum detection limits similar to those obtained by employing the triple-potential waveform, but greatly improving long-term reproducibility.

The compatibility of electrochemical detection with gradient elution coupled with the high selectivity of the anion-exchange stationary phases allows mixtures of simple sugars, oligosaccharides, and polysaccharides to be separated with high resolution in a single run. Figure 1 depicts the separation of a commercially available inulin product containing glucose, fructose, sucrose, and over 40 linear homologous fructans which are selectively eluted under a step gradient program employing sodium hydroxide and sodium acetate as the mobile phase.
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Fig. 1

High-performance anion-exchange chromatography pulsed electrochemical detection (HPAEC-PED) profile of a commercially available inulin extracted from chicory root. CarboPac PA100 column, elution by a step gradient of sodium hydroxide and sodium acetate

The main limitation of HPAEC-PED is the difficulty in performing peak identification because of the lack of commercial standards for chains with high DP. Peak assignment is usually based on a generally accepted assumption that each eluted peak represents a chain with one more unit than the chain represented by the previous peak. In other words, the retention time of a homologous series of carbohydrates increases as the DP increases.

HPAEC-PED also has limits concerning the quantitative approach because of the variability of the detector response with increasing retention time, since the detector response decreases for longer unit chains. This means that the peak area in HPAEC-PED does not directly reflect the correspondent carbohydrate amount. Some authors suggested that a possible explanation for the declining PED detector response is that the ratio of the number of the most acidic hydroxyl groups on the reducing end to the number of the other hydroxyl groups in a molecule is larger for short chains than for longer ones, leading to a more effective oxidation at the gold electrode for the former [17, 18].

Differences in slope were observed for calibration curves built by injecting different concentrations of a commercial mixture of oligosaccharides containing compounds having different DP [19]. Some ways to calculate the amount of each compound taking into account the different detector responses have been proposed [19, 20].

However, it is difficult to determine the DP of a nonlinear series without available standards as several peaks may correspond to the same DP. These analyses can be complemented with MALDI-TOF-MS, which is able to measure the molecular weight and length profiles of oligosaccharides and polysaccharides and provide structural information on them.

Applications of HPAEC-PED

A recent review of HPAEC-PED employed in food analysis shows the potentiality of this technique in carbohydrate analysis, and reports many applications in the field of food research [21]. Another review, by Raessler et al. [22], focused on the same technique employed in the determination of carbohydrates in plant samples. In many cases HPAEC-PED methods have been shown to allow the determination of both FOS and inulin added as functional food ingredients in food products on a routine basis [21].

Corradini et al. [23, 24] monitored the degree of fermentation of fructans by comparing the HPAEC elution patterns of the oligosaccharides before and after fermentation. The fermentation of the oligosaccharides was evaluated from the shift in molecular weight and from the lowered amount of polymer left in the supernatant after fermentation. The experimental results demonstrated that, when studied in vitro, most Bifidobacterium strains are not able to ferment the long inulin chains.

Furthermore, production of short-chain fatty acids was monitored during fermentation by co-electroosmotic capillary electrophoresis and indirect UV detection. Production of short-chain fatty acids was much higher in cultures containing short-chain oligosaccharides than in those containing inulin at high DP. During this process, an increase in the bacterial biomass was observed. FOS and inulin greatly affected the production of short-chain fatty acids in fecal cultures, and butyrate was the major fermentation product of inulin, whereas mostly acetate and lactate were produced from FOS.

Ishiguro et al. [25] studied the variation of total FOS, inulooligosaccharides (IOS), and inulin in burdock rots (Arctium lappa L.) stored under different temperatures. The carbohydrates investigated were separated on a CarboPac PA1 column (Dionex) and detected in PED mode. Under the conditions applied, FOS and IOS were separated in less than 25 min (see Fig. 2). Total FOS increased progressively at all storage temperatures, whereas the increase in IOS was much higher at 0 °C than at 15 or 20 °C.
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Fig. 2

HPAEC chromatogram of fructooligosaccharides and inulooligosaccharides extracted from burdock roots stored for 28 days at 0 °C. 4a 1F-(1-β-d-fructofuranosyl)2 sucrose, 5a 1F (1-β-d-fructofuranosyl)3 sucrose, 6a 1F-(1-β-d-fructofuranosyl)4 sucrose, 7a 1F-(1-β-d-fructofuranosyl)5 sucrose, 8a 1F-(1-β-d-fructofuranosyl)6 sucrose, 9a 1F-(1-β-d-fructofuranosyl)7 sucrose. (From Ishiguro et al. [25])

Raccuia and Melilli [26] demonstrated by HPAEC-PED that Cynara cardunculus accumulates inulin in large amounts in roots, which can be used as functional food ingredient. Addition of FOS as a prebiotic ingredient to probiotic food and beverages has been demonstrated to have several benefits. Recently, an HPAEC-PED method was proposed which permits the evaluation of FOS and IOS added as functional ingredients to fermented milk. Quantitative determination was performed by employing calibration curves built by adding a known amount of the ingredient fiber, whose oligosaccharides present in the product are not commercially available as standards [19].

Liu et al. [27] developed an HPAEC-PED method to study changes in fructan levels in plant tissues. Similarly, an HPAEC-PED method was developed for the determination of inulin in Jerusalem artichoke tubers to investigate the effects of harvest time and variety on the content of these functional carbohydrates [28].

Long-chain inulins are less soluble, and they have the ability to form inulin microcrystals when sheared in water or milk. These crystals are not discretely perceptible in the mouth, but can interact to form a smooth creamy texture and provide a fat-like mouth sensation. Correlation of the gel structure with a creamy appearance of aqueous suspension of inulins with the composition and the chemical structure was studied by Chiavaro et al. [29]. The results obtained permitted Chiavaro et al. to verify that the chemical structure of inulins having different DP significantly affected not only the ability to form a gel, but also the textural and thermal properties. By HPAEC-PED characterization it was possible to hypothesize that the different hardness of inulin gels is dependent on the different distribution of both the oligosaccharide and the polysaccharide fractions. Textural and thermal profiles of the gels were also investigated, and it can be concluded that the long-chain inulin fraction could be used as fat substitute in the preparation of low-fat products on the basis of the capacity of the inulin polymer chains to form crystals that may interact and produce gel-like structures, entrapping the solvent and providing a texture with a mouth sensation similar to that of fat. A review regarding texture and rheological and sensory properties of food fortified with inulin was published very recently [30].

Very recently, van Arkel et al. [31] reported a study on the regulation of inulin metabolism and the processes that determine the chain length and inulin yield throughout the chicory growing season. Quantitative analysis was done HPAEC-PED using mannitol as an internal standard for glucose, fructose, and 1-kestose determination, whereas the concentration of FOS with DP higher than 4 was estimated using the response coefficients of 1-kestose as a reference, as described in an earlier article [32] about inulin production and its developmental modulation in commercial crops, maize and potato.

Low molecular weight GOS obtained by enzymatic digestion of galactans from potato pulps were characterized by Michalak et al. [33]. Separation and quantitation of the potentially prebiotic oligosaccharides obtained was performed by HPAEC-PED using a previously optimized method [34] which was slightly modified in the elution gradient program.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A single-method analytical technique is an unreasonable goal for analysis and characterization of carbohydratea.

Soft ionization techniques such as MALDI has been used for linkage and sequence determination of oligosaccharides and can be considered an effective tool for the structural characterization of carbohydrates, offering precise results, analytical versatility, and very high sensitivity [35].

In MALDI-MS the ionization of the analyte is assisted by mixing the analyte molecules with a matrix compound, usually a small organic molecule, that can be activated by the absorption of laser energy and mediates the generation of ions. The sample and the matrix are co-crystallized onto a solid-phase target, usually a metal plate. With use of a pulsed laser directed onto the sample/matrix crystals, activated matrix and analyte molecules are vaporized and released into the vacuum of the ion source. Desorption of molecules from the solid-phase target and transition into the gas phase is followed by ionization, usually by proton transfer between analyte and matrix molecules. The activated matrix can be either the proton donor or the proton acceptor, generating positively or negatively charged analyte ions. In most cases, H+ and Na+ adducts are observed.

Briefly, samples are deposited on a smooth metal surface and desorbed into the gas phase as the result of the pulsed laser beam impinging on the surface of the sample. Ions are produced in a short time, corresponding approximately to the duration of the laser pulse, and in a very small spatial region corresponding to that portion of the solid matrix and sample which absorbs sufficient energy from the laser to be vaporized.

Since MALDI is a pulsed ion source, having the ability to ionize very large molecules, it is often coupled to a discontinuous mass analyzer such as a TOF mass analyzer. A TOF spectrum is obtained by measuring the time elapsed from the laser pulse to the detection of the various ions that have propagated through the field-free region and by measuring the intensity of the detector signal. Resolution and peak widths can be improved using a TOF mass analyzer with a reflectron. With use of MALDI-TOF devices with a reflectron configuration, mass resolutions of about 10,000 and mass accuracies higher than about 30 ppm can be routinely achieved.

The benefit of MALDI-MS for analysis of oligosaccharides and polysaccharides results predominantly from the formation of singly charged molecular ions, which leads to a simple mass spectrum for polymer distribution analysis. The absence in carbohydrates of a basic site inhibits protonation in MALDI-MS. However, carbohydrates ionize by adduction of metal ions, such as sodium and potassium cations. Alkali metal salts of Li+, Na+, K+, and Cs+ can be used as cationizing agents to enhance the ionization efficiency. MALDI-TOF-MS of nonderivatized oligosaccharides was first achieved using 3-amino-4-hydroxybenzoic acid as the matrix, but better results regarding reproducibility and higher signal-to-noise ratio can be achieved using 2,5-dihydroxybenzoic acid as the matrix [35]. The choice of matrix must be adapted to the properties of the analyte, and in the recent years several other matrices have also been reported to improve sensitivity with a concomitant improvement in resolution [3638]. Derivatization methods for oligosaccharides have also been developed with the aim of increasing the sensitivity of MALDI-MS analysis [35].

In studies regarding the determination of carbohydrate structure, knowledge of the sugar sequence, the type of linkage between the monosaccharides, and the anomeric configuration is of high importance. Application of the postsource decay (PSD) technique for MALDI-TOF-MS provides information related to carbohydrate sequence and branching. However, other configurations with MALDI ion sources and MS/MS capabilities have been proposed [39].

The capability to confirm glycan structures by MS2 analysis was proposed by Guillard et al. [40], who explored the potential of MALDI combined with a linear ion trap mass analyzer.

Applications of MALDI-TOF-MS

The molecular weight of FOS, IOS, and fructans regardless of their branching structure has been evaluated by MALDI-TOF-MS [41, 42]. In some applications MALDI-TOF-MS has been used successfully for molecular sizing, permitting characterization of the molar mass giving the maximum DP, and easy verification of the average DP from the registration of a signal for every chain present in a sample of arabinooligosaccharides [43]. Wang et al. [44] analyzed enzymatically debranched maltooligosaccharides of corn and barley starch by MALDI-TOF-MS, where chain length profiles of hydrolyzed amylopectin revealed structural information about the parent polysaccharides

As discussed already, in MALDI-MS the matrix must be adapted to the properties of the oligosaccharides and polysaccharides to be characterized. Improvement of sample preparation might increase the reproducibility, thus facilitating better response. Consequently, sample preparation is a crucial step, and matrices are usually prepared using 2,5-dihydroxybenzoic acid, as well as α-cyano-4-hydroxycinnamic acid and 2,4,6-trihydroxyacetophenone. Lithium trifluoroacetate, sodium trifluoroacetate, potassium trifluoroacetate, cesium trifluoroacetate, lithium hydroxide, sodium hydroxide, potassium hydroxide, cesium hydroxide, lithium chloride, sodium chloride, potassium chloride, and cesium chloride can be employed as the cationizing agents.

Fructopyranose oligosaccharides, which are able to be selectively used by beneficial gut bacteria such as Bifidobacterium adolescentis and Bifidobacterium longum, were isolated from fermented beverage of plant extract and their structures were confirmed using methylation analysis, MALDI-TOF-MS, and NMR measurements [45]. The DP of the isolated saccharides was established using MALDI-TOF-MS in which spectra were measured in positive ion mode with 2,5-dihidroxybenzoic acid as the matrix. MALDI-TOF-MS data confirmed that only two disaccharides and two trisaccharides were produced during fermentation of the beverage of plant extract. The structural confirmation of these saccharides was done according to 1H and 13C NMR analyses, and the subsequent complete assignment of 1H and 13C NMR signals of the four saccharides was done using 2D NMR techniques. Furthermore the oligosaccharide fraction, isolated from fermented beverage of plant extract using carbon–Celite chromatography, was investigated by HPAEC-PED, and it was verified that the disaccharides and trisaccharides studied were present in the fermented beverage, but were absent in the unfermented beverage. Consequently, it was confirmed that they were produced during fermentation of the beverage plant extract. In a successive article [46], the same authors reported the isolation, from fermented beverage of plant extract, of two oligosaccharides containing an α-fructofuranoside linkage and a fructopyranoside residue; their structures were confirmed by methylation analysis MALDI-TOF-MS, and NMR measurements.

The characterization of Agave tequilana fructans is important for the elaboration of functional foods and drug delivery systems [47]; analytical techniques such as gas chromatography (GC–MS), NMR spectroscopy, and MALDI-TOF-MS have been widely used to elucidate the molecular structures of fructans in agave plants [41]. Arrizon et al. [48] investigated the oligosaccharide and polysaccharide composition of Agave tequilana fructans by linkage analysis with GC–MS and MALDI-TOF-MS methods. They showed that Agave tequilana fructans are a complex mix of branched neofructans with β(2→1) and β(2→6) linkages, with a DP ranging from 3 to 29. The composition of water-soluble fructans obtained by high-performance liquid chromatography (HPLC), HPAEC-PED, MALDI-TOF-MS, and GC–MS was compared. MALDI-TOF-MS analysis showed differences in oligosaccharide distribution as a function of plant age. The richest concentration of low molecular weight fructans was observed in younger plants (2 years old), which exhibited the highest levels of free monosaccharide and fructans with DP of 3–6, with potential application as prebiotics. Fructans in agave were also characterized by structural studies conducted using NMR and electrospray ionization (ESI) MS techniques [49].

Linde et al. [50] isolated and characterized two variants of the extracellular enzyme from Xanthophyllomyces dendrorhous having transfructosylation activity. Structural analysis of the oligosaccharides obtained (neokestose and neonystose), with potentially improved prebiotic properties, was performed using a combination of 1H, 13C, and 2D NMR techniques and by MALDI-TOF-MS in which the matrix generating positively charged ions was 2,5-dihidroxybenzoic acid doped with sodium iodide.

Characterization and quantification of fructans in milling fractions of wheat was proposed by Haská and et al. [51]. The molecular weight distribution of wheat fructans was evaluated by HPAEC-PED and MALDI-TOF-MS, whereas 1H NMR spectroscopy and enzymatic hydrolysis were used for identification of fructans.

The complementarity of HPAEC-PED and MALDI-TOF-MS to characterize fructans (FOS and inulins) having different DP was reported by Borromei et al. [52]. The two techniques were employed to characterize commercially available FOS and inulin usually employed as ingredients in functional food formulations. MALDI-TOF-MS and HPAEC-PED provided complementary information on structural characterization of oligosaccharides, as demonstrated by comparing chromatographic profile d in Fig. 3 with the corresponding MALDI-TOF-MS spectrum reported in Fig. 4, in which it is shown that, as for mixtures of large carbohydrate polymers, in MALDI-MS the ionization response drops off with increasing molecular weight.
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Fig. 3

Chromatographic profiles of standard solutions of Raftilose® (a), Frutafit® TEX (b), Frutafit® IQ(c), and Raftiline® (d). (From Borromei et al. [52])

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Fig. 4

Matrix-assisted laser desorption/ionization mass spectrometry spectrum of a standard solution (1.0 mg mL-1) of Raftiline® (scale expanded from m/z 37,59 onwards). 3-Aminoquinoline was used as the matrix. (From Borromei et al. [52])

Albrecht et al. [53] suggested the application of capillary electrophoresis coupled with laser-induced fluorescence detection and MALDI-TOF-MS techniques to elucidate structural characteristics of konjac glucomannan oligosaccharides derived from the tuber of Amorphophallus konjac C. Koch, which is native to Southeast Asia and seems to be a promising prebiotic substrate. The results showed that capillary electrophoresis is able to detect acetylated forms, whereas HPAEC-PED cannot give any information about them.

Xylans are the most abundant hemicelluloses in most plants. XOS are considered as novel prebiotic food ingredients. Enzymatic hydrolysis of xylans is of considerable relevance in food applications such as cereal processes regarding exploitation of xylans as a carbohydrate source for fermentation of xylose.

HPAEC and MALDI-TOF-MS measurements, with the use of other analytical techniques such as high-performance size-exclusion chromatography, Fourier transform IR spectroscopy, and NMR spectroscopy, were performed to characterize XOS extracted from Eucalyptus wood hemicelluloses under suitable operational conditions, such as autohydrolysis or hydrothermal processing, and then purified by a concentration–diafiltration–concentration sequence [54]. In vitro fermentation experiments were conducted to verify their prebiotic applications.

Nabarlatz et al. [55] characterized partially O-acetylated XOS isolated from almond shells by autohydrolysis as well as their deacetylated form, by MALDI-TOF-MS and NMR spectroscopy. The almond shell XOS comprised a mixture of partially O-acetylated neutral and acidic oligomers derived from the 4-O-methylglucuronoxylan-type polymers.

The characterization of XOS by MALDI-TOF-MS was also proposed by Cano and Palet [56]. Determination of structural isomers of xyloglucan oligosaccharide by post-source decay (PSD) MALDI-TOF-MS has also been proposed [57].

Other analytical techniques

GC-MS and NMR spectroscopy have been mainly used to obtain structural information about fructans, FOS, inulin, and other prebiotic carbohydrates. Those techniques can be used to assess both the level and the composition of these carbohydrates in plant tissues, as well as in food products.

Some works with the aim of comparing different techniques have been realized. GC-MS was found to be more sensitive than HPAEC-PED by Ghedini Der Agopian et al. [58], who detected small amounts of FOS in banana samples, whereas HPAEC-PED was found to have higher precision.

High-fructose inulin syrups, which consist of a mixture of oligosaccharides of various chain lengths made up of fructose units linked by β(2→1) bonds with a final single glucose unit, can be used as an inexpensive sweetener in honey adulteration. Ruiz-Matute et al. [59] proposed a GC-MS method for the detection of adulteration of honey with high-fructose inulin syrups. GC–MS analyses were performed for qualitative purposes, and 1-kestose, inulobiose, and inulotriose were quantified in honey. Inulotriose was not detected in any of the honey samples analyzed and was eluted in a region of the chromatogram where disaccharides and trisaccharides naturally present in honey were not eluted. On other hand, high concentrations of inulotriose were found in all high-fructose inulin syrups tested, and consequently the presence of this trisaccharide can be proposed as a good marker for detecting adulterations.

GC–MS is useful for the characterization and quantitation of low molecular weight carbohydrates (monosaccharides, disaccharides, and trisaccharides), although a previous derivatization step is mandatory for their analysis.

With the aim of identifying potential food sources of inulin, Judprasong et al. [60] determined FOS content by GC in 47 varieties of plants, distributed in five food groups.

As reported earlier, NMR analyses have been widely used to elucidate molecular structures of carbohydrates. NMR spectroscopy is a promising technique reported to be of help in the analysis of foodstuff. In a recent review by Mannina et al. [61], a rich series of metabolites identified by 1H NMR spectroscopy, including fructans, inulin and FOS, investigated in aqueous solution of foodstuffs is reported. Chandrashekar et al. [62] isolated both high molecular weight (more than 3.5 kDa) and low molecular weight (less than 3 kDa) fructans from aged garlic extract. The structures of purified high molecular weight and low molecular weight fructans from aged garlic extract were determined by 1H NMR and 13C NMR spectroscopy and revealed that both types have (2→1) β-d-fructofuranosyl bonds linked to a terminal glucose at the nonreducing end and β-d-fructofuranosyl branching on the backbone. 1H and 13C NMR spectra were recorded using a 500-MHz NMR instrument equipped with a dual probe in the Fourier transform mode at 20 °C. The samples were prepared in dimethyl-d6 sulfoxide to a concentration of 20 mg/mL. The results obtained demonstrated that both high molecular weight and low molecular weight fructans displayed mitogenic activity and activation of macrophages, including phagocytosis. 1H NMR spectroscopy was used by Madhukumar and Muralikrishna [63] to elucidate the structures of purified oligosaccharides; chemical shifts in the region from δ 3.00 to δ 5.00 ppm characteristic of α-linked arabinofuranoside as well as β-linked xylopyranoside residues were observed. By the analytical technique employed, both Bengal gram husk and wheat bran were found to be a rich sources of arabinoxylans, from which bioactive XOS were obtained by driselase treatment. In detail, the structural characterization of the purified XOS obtained from Bengal gram showed that they are mainly composed of pentasaccharides and trisaccharides, whereas those obtained from wheat bran were found to composed of hexasaccharides and tetrasaccharides, with varying ratios of arabinose and xylose. The purified oligosaccharides were also characterized by ESI-MS analysis. Furthermore, it was verified that the probiotic B. adolescentis NDRI 236 readily used these purified XOS. The prebiotic activity experiments showed that XOS from wheat bran have much more potential than Bengal gram oligosaccharides.

NMR studies and specific enzymatic hydrolysis, in combination with MALDI-TOF-MS, were used to study structural features of (1→4)-β-galactan extracted from bast fiber peels.

As for other techniques, several authors have proposed HPLC systems equipped with an evaporative light scattering detector to separate FOS and inulin of interest in the preparation of functional food or naturally present in food matrices. These chromatographic methods were employed using cyclodextrin-bonded columns [64] or ion exclusion monosaccharide columns [65]. The methods involved hydrolyzing inulin with the enzyme inulinase, and determination of released fructose and glucose by HPLC coupled with evaporative light scattering detection, using water as the mobile phase.

Moreover, Raessler [66] recently summarized the current methods of determination by HPLC techniques of nonstructural carbohydrates in plant samples.

Recently, HPLC coupled with multistage MS (MSn) was proposed by Fabrik et al. [67] for provision of structural information. They showed fragmentation of [M−H] or anion adducts produced in negative-ion-mode MS, offering an alternative to positive-ion mode MS as the fragmentation patterns to provide more structure-related information. In parallel to analysis in positive-ion mode, maltotetraose, stachyose, and nystose were analyzed in negative-ion mode.

Analytical determination of other prebiotic carbohydrates

GOS are mainly disaccharides, trisaccharides, and tetrasaccharides synthesized from lactose, via enzymatic transgalactosylation catalyzed by β-galactosidases. Characterization of different GOS has generally been done by the combination of a great variety of analytical methods (methylation analysis followed by GC–MS, NMR spectroscopy, HPAEC–PED–MS, ESI–MS) with previous fractionation of the oligosaccharides (yeast treatment, size-exclusion chromatography, hydrophilic interaction liquid chromatography). Complex carbohydrates can frequently be separated using hydrophilic interaction liquid chromatography. Hernández-Hernández et al. [68] characterized three commercial prebiotic GOS mixtures by hydrophilic interaction liquid chromatography coupled with MS.

Martínez-Villaluenga et al. [69] reported a study on the determination of GOS present in 14 fermented milk samples. The HPAEC-PED method they developed and validated was applied to yogurts, yogurts containing bifidobacteria, and ready-to-drink yogurts containing Lactobacillus casei, giving comprehensive information about the total and individual content of GOS in commercial fermented milks. The chromatographic profiles of GOS present in traditional yogurt, yogurt containing bifidobacteria, and ready-to-drink yogurts containing L. casei are reported in Fig. 5.
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Fig. 5

HPAEC–pulsed amperometric detection chromatograms of commercial yogurts (A), yogurts containing bifidobacteria (B), and ready-to-drink yogurts containing Lactobacillus casei (C). Analysis was done using a 250 mm × 4-mm inner diameter CarboPac PA1 column. The compounds identified are indicated as 1 3-galactobiose, 2 6′-galactosyl-lactose, 3 3-galactosylglucose, and 4 3′-galactosyl-lactose. Products marked with an X are galactooligosaccharides that were not identified. (From Martínez-Villaluenga et al. [69])

An interesting line of research has been conducted on the possibility of extracting useful products from plant biomass. A review by Deutschmann and Dekker [70] offers a wide variety of methods to obtain compounds of interest such as bioactive ingredients of food and health products from XOS. The article describes the processing of XOS from lignocellulosic materials rich in xylan on an industrial scale by chemical and enzymatic methods; the latter is preferred in the food industry because of the lack of undesirable side reactions and products. The information available does not provide an exact explanation about the bioactive effects of XOS. Their fermentation results in the acidification of the colonic contents and the formation of short-chain fatty acids that serve as fuels in different tissues and that may play a role in the regulation of cellular processes. The scheme shown in Fig. 6 shows the pathways to various xylan-based chemical products that can be potentially obtained from agro-industrial lignocellulosic materials.
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Fig. 6

Pathways towards xylan-based products from lignocellulosic materials. (From Deutschmann and Dekker [70])

Samanta et al. [71] analyzed XOS extracted from corn cob by HPLC with refractive index detection, using a Zorbax amino-bonded-phase column and elution with a mixture of acetonitrile and water.

Holck et al. [72] performed a complex study involving different separative techniques in order to establish the composition of sugar beet pulp arabinooligosaccharides. Several fractions were obtained on the basis of feruloyl substitution and chain length. The bifidogenic activity of the different chains was tested. This work also provided a comparison between the ferulated and the corresponding nonferulated chains. Structural characterization of XOS and glucosaccharides extracted from rice husks was performed by Westphal et al. [73] employing HPAEC-PED, MALDI-TOF-MS, and NMR techniques. Analysis by Fourier transform IR spectroscopy to allow gross characterization of carbohydrate and other components was also performed.

Some authors have investigated the composition of arabinoxylans, the predominant cell-wall polysaccharides occurring in wheat flour. A comparison of different wheat flours has also been made evaluating the effect of processing on the content and the structure of water-extractable arabinoxylans [74].

A carbohydrate commonly considered as a potential nutraceutical is chitosan, the most abundant marine mucopolysaccharide, having versatile biological properties such as biocompatibility, biodegradability, and a nontoxic nature. Chitosan is a deacetylated derivative of chitin, commonly found in the exoskeleton or cuticles of many invertebrates and in the cell walls of most fungi and some algae. It has antioxidant, hypocholesterolemic, antimicrobial, and anti-inflammatory activities, and has been extensively applied for encapsulation of active agents, such as antioxidants and vitamins, in a polymer matrix to protect them from the surrounding medium or processing conditions, and to control the release [7578].

Yang et al. [79] extracted and purified oligosaccharides from soy sauce lees; their structural characteristics were identified by GC, whereas a prebiotic effect was observed on both Lactobacillus bulgaricus and Streptococcus thermophilus.

Concluding remarks

As is well known, carbohydrates are difficult to detect when analyzed by the common HPLC techniques because they lack a chromophore. Consequently, it is common practice to form a derivative at the reducing terminus to allow the compounds to be detected by UV absorption or fluorescence. On the other hand, in basic solutions, all carbohydrates are negatively charged oxyanions, which can be separated by HPAEC as anions, without prederivatization, with each carbohydrate yielding a single peak easily detected by PED in PAD mode.

The advantage of detection with PED for carbohydrates is that no precolumn or postcolumn derivatization is necessary. Sample preparation is also simplified because only oxidizable analytes will be detected by PED, and the sensitivity for carbohydrates is orders of magnitude greater than that of possible contaminant species. Unlike refractive index detectors, PED is not sensitive to changes in mobile phase composition and consequently is by far the most widespread detection mode used for carbohydrate analysis by HPLC. On the other hand, one of the remaining problems of HPAEC-PED is the difficulty to predict the elution order of oligomers, since in most instances no standards are available.

As previously reported, long-chain inulin samples are polydisperse with respect to molecular size, and the identification by HPAEC-PED of their GFn sequence can be performed on the assumption that the retention time of GFn increases according to the DP, and that each successive peak represents an oligomer with an extra fructose. Furthermore, it is more difficult to predict the elution order of inulooligofructose and GFn if they coexist in the same chromatogram. This means that in most cases, the HPAEC technique is only able to provide a carbohydrate “fingerprint.” As a conclusion, HPAEC-PED has been demonstrated to be valid for the reliable separation and sensitive detection of FOS and inulin having a wide polymerization range. The use of pellicular polymeric anion-exchange columns coupled with PED can be considered the most selective and sensitive analytical tool to achieve separation of complex carbohydrates.

MALDI-TOF-MS is a faster analytical technique than HPAEC-PED. It is particularly valuable for carbohydrates because it allows underivatized as well as derivatized carbohydrates to be examined. This technique can provide valuable information on several aspects of structural analysis, such as the determination of sequence, branching, and linkages. However, for better isomer differentiation, MALDI-MS can be best combined with HPAEC-PED as described. Although in MALDI-MS the ionization response drops off with increasing molecular weight, for mixtures of large carbohydrate polymers, the use of MALDI for neutral oligosaccharide analysis has advantages over the use of ESI. Furthermore, MALDI mass spectra of oligosaccharides and polysaccharides are normally dominated by a single ion corresponding to the protonated molecule, and multiply charged ions are rare, even for polysaccharides at high DP. On other hand, the advantage of MALDI in terms of ionization response has to be balanced against the disadvantages of the metastable fragmentation that is caused by the higher internal energies imparted to the ions resulting from ESI.

Quantitative information achievable from MALDI spectra is often regarded to be limited. In any case, application to quantitative analysis requires careful optimization of the experimental parameters. In particular, sample preparation (e.g., choice of matrix compound, concentration, solvents, and crystallization conditions) is critical and must be optimized in order to reduce the variability introduced during this step [80].

As reported in this critical review, it has been shown that MALDI mass spectra of FOS, inulins, GOS, and other carbohydrates of interest in the development of functional foods provide important compositional information. The ability to rapidly obtain accurate and highly resolved mass information with relative ease of sample preparation suggests the potential of MALDI-TOF-MS to become a powerful routine tool in the analysis of carbohydrates.

Furthermore, MALDI-TOF-MS-PSD represents an equivalent to the conventional (low-energy collisionally activated decomposition) MS/MS technique. As is known, with reflectron TOF mass spectrometers, it is in theory possible to obtain structural information on a selected quasimolecular ion by mass analysis of daughter ions resulting from in-flight fragmentation of the parent ion. Consequently, by MALDI-TOF-MS-PSD detailed sequential structure analysis of highly branched oligosaccharides could be achieved. In conclusion, MALDI-TOF-MS is able to detect the mass-to-charge ratio of carbohydrates, giving spectra within minutes and providing extensive oligosaccharide structural information, resulting in a powerful tool to characterize oligosaccharides and polysaccharides of prebiotic interest.

NMR spectroscopy can also be a very powerful tool for the characterization and identification of oligosaccharides and polysaccharides, revealing structural information on such analytes at the atomic level. However, the high costs involved, the high purity of the samples, and the technical expertise required mean that NMR spectroscopy is used less than it probably should be.

MS, on the other hand, is a more affordable and universal method of detection, providing valuable information on the identity of analytes at the molecular level.

The numerous applications regarding the wide variety of samples reviewed in this article show the usefulness and the success of modern analytical techniques in the investigation of the carbohydrates employed in the development of functional foods. The complementarity between the techniques summarized has been demonstrated to be of great help in the studies performed, and will be precious for progress of knowledge in this field.

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