The Book of Fructans

After more than 30 years, The Book of Fructans represents the first and most comprehensive coverage of fructans generated by pioneer glycoscientists from the field. It outlines the fundamentals of all fructan types, their terminology, chemical and structural-functional features, biosynthetic enzymes that make and break them, their presence and possible roles in nature, their evolutionary aspects and their microbial, enzymatic, and plant-based production. Additional sections cover the applications of fructans, specifically, the agro/chemical and biomedical applications, health, pharmaceutical and cosmetic applications, fructans in food and feed, fructan nanotechnology, the immunomodulatory and antiviral effects of fructans and the perspectives for fructans in circular economies and sustainable societies. Intended for scientists, entrepreneurs, academicians and students working in related fields, this book will be a useful resource for all who wish to learn more about these extraordinary carbohydrates.

• Combines all aspects of fructans in a single volume
• Covers fundamentals, applications and society
• Introduces ‘Fructans for Life’ concepts

• Language: English
• Publisher: Academic Press
• Release date: Mar 21, 2023
• ISBN: 9780323858083

Book preview

Preface

Dear Reader,

Almost 30 years after the previous book by Suzuki and Chatterton (1993), here we are with The Book of Fructans, consisting of 19 chapters written by dozens of excited fructan pioneers. Who are these people? Just like fructans, fructaneers are the rebellious furanoses in the world of pyranoses. Lev(an)orotatory to the light when other common sugars go dextrorotatory; flexible, open, and free minded people with no borders; soulmates driven by the same passion, unraveling the deep secrets and mysteries of fructans; out of the box thinkers; using all their powers to bring thrilling stories with knowhow, heart, and soul. It was a privilege for us, the editors, to work with such strong writing teams, doing their best despite the worldwide COVID crisis.

And dear reader, be aware that this book is more than just these 19 chapters. It is the story of a long journey that began with a mission to further lift fructans out of the shadows, positioning them better within the broad carbohydrate landscape and beyond; a story about how to bring fructans from the ordinary to the extraordinary, introducing them in the daily life of each citizen, from fundamentals to applications, from the lab to the kitchen and the living room.

We, the editors, are just two humble fructaneers trying to bring life to fructans with the Fructans For Life concept, as we detailed in the last chapter where all aspects are chained into the circle of life. We just hope that this book will serve as a guide to the next generation fructaneers and industrial fructan people, as well as for anyone interested in this topic. Let it be a source of inspiration for all, as it has been for us. Our questions about the true meaning of life shed light to our path into the unknowns of fructans and led us to the discovery of fructans in the third domain of life, the archaeal fructans. Vast number of chained questions followed. Endless discussions leading to even more questions. Why did fructans evolve multiple times during evolution? Why do fructans exist in so many forms? Are fructans the key to survive under extreme conditions when nothing else matters? What is the mysterious connection between fructans and salinity? Is it possible that fructans, like other 5-ring sugars, associate with the beginning of life in salty oceans? Maybe these extremely sticky fructans enabled the first life forms to adhere on the fractured seafloor? After all, there is a crack in everything and that's how the light gets in Leonard Cohen.

Are you ready to face your own questions and write your own story? Do you want to learn more on how fructans could become central in our future societies, focusing on circular concepts? Are you curious how a diverse array of fructans may be driving signaling concepts and symbiotic associations throughout the tree of life? Are you wondering how fructans may contribute to many industrial challenges and novel products? And how fructans may be stimulating plant, animal, and microbial health? Then hop on board and free your mind in the extraordinary world of fructans.

Much reading pleasure,

Ebru Toksoy Öner, the microbial fructaneer

Wim Van den Ende, the plant fructaneer

16.09.2022

Section I

The fundamentals

Outline

Chapter 1. Fructans: The Terminology

Chapter 2. Chemical and Structural-Functional Features of Fructans

Chapter 3. Macromolecular Properties of Fructans

Chapter 4. Fructan Enzymes in Microbes and Plants: Structure, Function, and Product Formation

Chapter 5. Evolutionary Aspects of the Fructan Syndrome

Chapter 6. Fructans: Physiology and Development in Plants

Chapter 7. The Role of Fructans in Stress Responses

Chapter 8. Relation of Plants with Other Kingdoms: the Unique Role of Fructans

Chapter 9. Traditional Fermented Foods: Introducing the Fructan Link

Chapter 1: Fructans: The Terminology

Lázaro Hernández ¹ , and Francisco J. Plou ²       ¹ Centro de Ingeniería Genética y Biotecnología (CIGB), Havana, Cuba      ² Instituto de Catálisis y Petroleoquímica, CSIC, Madrid, Spain

Abstract

Fructans are linear or branched oligo- and polysaccharides in which fructose constitutes the sole or major monosaccharide. Different types of fructans exist in plants or are produced by microorganisms. Fructan names should reflect the full complexity of the chemical structure rather than the source. The preferred way is to designate the fructan molecules in terms of their monosaccharide composition, linkage type(s), and degree of polymerization. This introductory chapter describes the historical context of fructan terminology and provides an updated glossary of fructan terms, including new designations and reformulated definitions.

Keywords

Fructan; Fructooligosaccharides; Galacto-fructan; Graminan; Inulin; Inulobiose; Kestose; Levan; Levanbiose; Neo-fructan

1. Introduction

With a history of over two centuries, fructan —fructose-based oligo and polysaccharides— research has left us with many trivial names, some of which were coined even before the discovery of fructose. Fructans were first isolated from plants and early classified according to their source while microbial fructans were named using other arbitrary criteria. Many historical fructan designations became obsolete and other ones have been modified so as to reflect the full complexity of the chemical structure. The preferred way is to designate the fructan molecules in terms of their monosaccharide composition, linkage type(s), and degree of polymerization (DP). This chapter describes the historical context of fructan terminology, provides an updated glossary of fructan terms, including new designations, and reformulates the fructan definition proposed in 1993 by Waterhouse & Chatterton in the emblematic book Science and Technology of Fructans (Waterhouse & Chatterton, 1993). Basic concepts and specific terminologies are discussed.

2. The history of fructan terminology

Nowadays, we know fructans as fructose-containing oligo- and polysaccharides mostly from microbial or plant origin. However, since the isolation of plant fructans occurred before fructose was discovered and coined (Dubrunfaut, 1847; Miller, 1857), it is not surprising that early classifications were based on the source rather than the chemical structure. «A peculiar substance» isolated from a boiling water extract of Inula helenium (Asteraceae) (Rose, 1804) was named «inulin» (Thomson, 1818). Six decades later, the fructans recovered from rye grains (Müntz, 1878) and Phleum pratense (Ekstrand & Johanson, 1887, pp. 3310–3317) were called «graminin» and «phlein», respectively. Waterhouse and Chatterton (1993) proposed the name «graminan» to replace the original graminin, perhaps to avoid confusion with graminin A, a toxic metabolite produced by the phytopathogenic fungus Cephalosporium gramineum (Van Wert & Fulbright, 1986). The term «agavin» was first proposed by Mancilla-Margalli and Lopez (2006) to designate the branched neo-fructans recovered from Agave and Dasylirion species (Mancilla-Margalli & Lopez, 2006). Inulin, graminan, phlein (current recommended name levan), and agavin cover the four representative types of fructans isolated from different plant species. The other more than 50 names that appeared in old literature to designate plant fructans are not in current use (Suzuki, 1993).

Referring to microbial fructans, «lävulan» was the first name used to describe the gum recovered from molasses in the sugar beet industry (Lippmann, 1881). At the turn of the twentieth century, Greig-Smith (1902) introduced the term «levan» for the levorotation of polarized light and properties analogous to dextran, a bacterial polymer already known at that time (Greig-Smith, 1902). For years, all bacterial fructan polysaccharides were designated as levan, but such a designation did not consider the current classification criterion of having a predominant presence of the β-(2→6) fructosyl-fructose linkages (Waterhouse & Chatterton, 1993). Nowadays, it is known that high DP levan is widely spread in bacteria of different habitats but it is not the only type of microbial fructan polysaccharide (Toksoy Öner et al., 2016). Several Gram-positive bacteria and few archaea produce high DP fructans that have mostly or exclusively the β-(2→1) fructosyl-fructose linkages (Hernández et al., 2019; Kırtel et al., 2018, 2019).

The development of chromatographic methods had a crucial impact on the isolation and structural characterization of fructan oligosaccharides. In 1952, a sweet-tasting trisaccharide consisting of two fructose units and one glucose was termed «kestose», a name derived from the town of Keston, UK (Whalley, 1952). In the next two years, three isomeric kestoses (isokestose, kestose, and neokestose) were identified after incubating yeast or mold invertases with sucrose at high concentrations (Albon et al., 1953; Bacon & Bell, 1953; Gross et al., 1954). Later, the dissimilar terms «bifurcose» (from bifurcation) (Schlubach, 1961, pp. 291–315) and «nystose» (Binkley & Altenburg, 1965) were assigned to a DP 4 branched and a DP 4 linear sucrose-containing fructan, respectively. The original names isokestose (later termed 1-kestose, currently 1-kestotriose) and kestose (later termed 6-kestose, currently 6-kestotriose) became obsolete, while the terms neokestose, bifurcose, nystose, and even fructosyl-nystose are still used in the scientific literature. The recommended names for neokestose, bifurcose, nystose, and fructosyl-nystose are 6G-kestotriose, (1&6)-kestotetraose, 1,1-kestotetraose, and 1,1,1-kestopentaose, respectively (Lewis, 1993; Waterhouse & Chatterton, 1993).

3. The fructan definition

Systematic means are currently used for naming most carbohydrates. However, general nomenclature systems do not address the complexity of each field, which often creates ambiguities. In the carbohydrate nomenclature system, the term for a polysaccharide (glycan) composed of a single type of monosaccharide residue is obtained by replacing the ending «-ose» of the monosaccharide name by «-an» (McNaught, 1857). Examples of established usage of the «-an» ending are xylan for polymers of xylose, mannan for polymers of mannose, and galactan for polymers of galactose. Cellulose, starch, and dextran are glucans, as they are composed of glucose. In another generic definition, polysaccharides are defined as carbohydrates that contain 10 or more monomeric units (Cummings & Stephen, 2007). However, the fructan term comprises both oligosaccharides (DP 2–9) and polysaccharides (DP ≥ 10). Unlike starch and cellulose, plant fructans often have a wide DP distribution with a rather low and variable average value. For instance, commercial chicory inulin is a mixture of linear chains ranging from 2 to 60 monosaccharides. The average size of the inulin chains in chicory roots is influenced by the action of fructan 1-exohydrolases (1-FEHs) and the harvesting period (Van Laere & Van den Ende, 2002).

A definition of fructan, which diverges from the standard usage of the «-an» ending (DP ≥ 10), was recommended at the First and Second International Symposia on Fructan, held in Bonn (1988) and Wales (1992), respectively (French, 1989; Lewis, 1993; Waterhouse & Chatterton, 1993). The authors defined fructan as «any compound in which one or more fructosyl-fructose linkages constitute a majority of the linkages». This definition fails to cover all short-chain fructans. For instance, the sucrose-containing trisaccharide neokestose (current recommended name 6G-kestotriose) contains an internal glucose unit connected to two fructose units and thus lacks fructosyl-fructose linkages. We propose to update the fructan definition as follows:

Fructan: Any compound in which two or more fructose residues are bound by glycosidic linkages and constitute a majority of the monomeric composition. Material encompassed in this definition may or may not contain other monosaccharides, most often glucose.

Thus, fructans include disaccharides, oligosaccharides, and polysaccharides. Oligofructans (DP 2–9) are often called fructooligosaccharides (FOSs) or oligofructoses depending on the enzymatic production process. Current commercial FOSs are synthesized via sucrose fructosylation, while oligofructoses are the products of the partial degradation of long-chain fructan substrates. FOSs are not capable of acting as a reducing agent because they have the anomeric carbon (C2) of the two or more fructose units and the anomeric carbon (C1) of the glucose unit tied up in the glycosidic bonds (Ghazi et al., 2006). Commercial oligofructose mixtures are currently produced by treating plant inulin with endo-inulinases, and thus contain several shorter linear chains ending with a reducing fructose unit and one chain with the terminal nonreducing glucose unit (Ávila-Fernández et al., 2011; Yang et al., 2016).

4. Specific fructan-related terms

In nature, sucrose [β-D-fructofuranosyl-(2→1)-α-D-glucopyranoside] is the preferred precursor of de novo fructan synthesis acting both as the fructosyl donor and acceptor. The glycosidic linkage normally occurs at one of the two primary hydroxyls (OH-1 or OH-6) of the fructose unit or the primary hydroxyl (OH-6) of the glucose unit. The transfructosylation of the three trisaccharides (1-kestotriose, 6-kestotriose, and 6G-kestotriose) at their primary OH groups generates hypothetically nine linear or branched tetrasaccharides, some of which have not been isolated so far. A wider variety of pentasaccharides can be generated in the next fructosylation cycle, and so on.

Raffinose [α-D-galactopyranosyl-(1→6)-β-D-glucopyranosyl-(1→2)-β-D-fructofuranoside] is another precursor of de novo fructan synthesis in microorganisms. It has a sucrose moiety with the terminal fructose in its composition and a galactose bound to the glucose part of sucrose. Like it is the case for sucrose, raffinose can act as a donor and acceptor of fructosyl moieties for levansucrase (EC 2.4.1.10) and inulosucrase (EC 2.4.1.9) from bacterial origin (Andersone et al., 2004; Díez-Municio et al., 1887; Visnapuu et al., 2011) and β-fructofuranosidase (EC 3.2.1.26) and fructosyltransferase (EC 2.4.1.9) from fungal origin (Montilla et al., 2011). The tetrasaccharide stachyose (galactosyl-raffinose) has also been used as a sole substrate to form galactose-containing fructans (proposed name galacto-fructans) of varied DP (Masaru et al., 2002; Montilla et al., 2011).

Galacto-fructan: Fructan that contains one or more galactose units in its composition but fructose remains to be the dominant monosaccharide. Material encompassed in this definition may or may not contain other monosaccharides, most often glucose.

Galacto-fructans are synthesized by fructosyl- or galactosyl transfer reactions. The fructosylation of raffinose at its terminal fructose results in the synthesis of fructosyl-raffinose, which is a nonreducing galactofructan. The galactosylation of a fructan can also yield a galacto-fructan.

The acceptor promiscuity of some enzymes involved in fructan biosynthesis offers alternatives for the fructosylation of other carbohydrates (xylose, fucose, arabinose, ribose, mannose, maltose, isomaltose, lactose, cellobiose, and maltotriose) producing a broad spectrum of hetero-oligofructans (Hernández et al., 2019; Kırtel et al., 2018; Tieking et al., 2005; Visnapuu et al., 2011).

Based on the position and complexity of the bonds that connect the monomeric units, natural fructans can be classified into four main groups:

Inulin: Fructan of any origin that has mostly or exclusively the β-(2→1) fructosyl-fructose linkage. Linear inulin implies β-(2→1) fructosyl-fructose linkages exclusively. Branched inulin has a minority presence of the β-(2→6) fructosyl-fructose linkages. The disaccharide inulobiose is the shortest inulin-type fructan. The trisaccharide 1-kestotriose (former name 1-kestose) containing a terminal glucose is the shortest nonreducing inulin-type fructan.

Levan: Fructan of any origin that has mostly or exclusively the β-(2→6) fructosyl-fructose linkage. Linear levan implies β-(2→6) fructosyl-fructose linkages exclusively. Branched levan has a minority presence of the β-(2→1) fructosyl-fructose linkages. The disaccharide levanbiose is the shortest levan-type fructan. The trisaccharide 6-kestotriose (former name 6-kestose) containing a terminal glucose is the shortest nonreducing levan-type fructan.

Graminan: Fructan that contains both the β-(2→1) and β-(2→6) fructosyl-fructose linkages in similar percentages or significant amounts generally forming a branched structure. The trisaccharide graminotriose is the shortest and the only linear graminan-type fructan. The tetrasaccharide (1&6)-kestotetraose (former name bifurcose) containing a terminal glucose is the shortest nonreducing branched fructan.

Neo-fructan: Fructan that contains a glucose moiety between two fructose units which can be extended by the β-(2→1) and/or β-(2→6) fructosyl-fructose linkages. Neo-fructans are formed on a 6G-kestotriose (former name neokestose) backbone where elongations occur on both sides of the molecule. Neo-fructans are nonreducing carbohydrates. Depending on the predominant position and complexity of the fructosyl-fructose linkages, neo-fructans can be classified into three types: neo-inulin, neo-levan, and agavin.

d.1. Neo-inulin: Also called «inulin neoseries», has two β-(2→1)-linked fructose chains attached on either end of a core sucrose molecule. A minority presence of the β-(2→6) fructosyl-fructose linkages is allowed.

d.2. Neo-levan: Also called «levan neoseries», has two β-(2→6)-linked fructose chains attached on either end of a core sucrose molecule. A minority presence of the β-(2→1) fructosyl-fructose linkages is allowed.

d.3. Agavin: Also called «branched neo-fructan», has two fructose chains attached to the sucrose core. The β-(2→1) and β-(2→6) fructosyl-fructose linkages are both present in significant amounts, forming a highly branched structure.

The usage of inulin makes no distinction in the source of the β-(2→1)-linked fructan. By contrast, the terms levan and phlein have been historically used to distinguish between the β-(2→6)-linked fructan produced by bacteria and plants, respectively. As recommended by Lewis (1993), levan should be used as a general term and the use of phlein should be avoided (Lewis, 1993).

Bacterial levan and inulin are the largest fructans in nature, with a DP ranging from 10⁴ to 10⁶ and often contain branches (Banguela & Hernández, 2006). The percentages of the β-(2→1) linkages forming the branches in bacterial levan or the β-(2→6) linkages at the branching points of bacterial inulin are relatively low. Therefore, high DP fructans of bacterial origin are not recommended to be referred to as graminan, a name derived from the plant context. However, this does not mean to say that very highly branched microbial fructans to be discovered in the future may be considered as graminans, perhaps leading to a reconsideration of the terminology once again.

5. The oligofructan nomenclature system

The combination of enzymatic methods and modern chromatographic techniques has permitted the isolation and structural characterization of complex mixtures of oligofructans produced in vivo or in vitro via substrate fructosylation or hydrolysis. Two subgroups of oligofructans differing in their composition and chemical reactivity are distinguishable. Oligofructans of the Fn subgroup contain fructose units exclusively and the anomeric carbon (C2) of one terminal fructose is free conferring reducing properties. Oligofructans of the GFn subgroup, known as kesto-n-oses, contain sucrose and have nonreducing properties because all the anomeric carbons (C1 of glucose and C2 of fructoses) are forming glycosidic bonds.

Based on the position of the fructosyl-fructose linkages, the Fn oligofructans can be classified into three types: inulo-n-ose, levan-n-ose, and gramino-n-ose.

Inulo-n-ose: Reducing fructofuranosyl-only fructan of DP 2–9 that has all β-(2→1) linkages forming a linear-chain inulin. For instance: inulobiose [O-β-D-fructofuranosyl-(2→1)-β-D-fructofuranose]. Chemical Abstracts Service (CAS) number: 470-58-6.

Levan-n-ose: Reducing fructofuranosyl-only fructan of DP 2–9 that has all β-(2→6) linkages forming a linear-chain levan. For instance: levanbiose [O-β-D-fructofuranosyl-(2→6)-β-D-fructofuranose]. CAS number: 17669-60-2.

Gramino-n-ose: Reducing fructofuranosyl-only fructan of DP 3–9 that contains both β-(2→1) and β-(2→6) linkages forming a graminan. We propose the name graminotriose for the branched trisaccharide O-β-D-fructofuranosyl-(2→1)-β-D-fructofuranosyl-(2→6)-β-D-fructofuranose observed in chicory roots (Timmermans et al., 2001).

The GFn oligofructans were named kesto-n-oses by Waterhouse and Chatterton (1993). The term kesto-n-ose is updated as follows:

Kesto-n-ose: Nonreducing oligofructans (DP 3–9) that contain sucrose (end standing or internally). The C1 position of the glucose unit and the C2 positions of the two or more fructose units are all forming glycosidic bonds.

In the above defined terms, inulo-n-ose, levan-n-ose, gramino-n-ose, and kesto-n-ose each unit-length series uses a consistent name (inulin-, levan-, graminan-, or kesto-) plus the appropriate Greek root designation (n) for each DP and the -ose ending. Examples are inulobiose, levantriose, graminotetraose, kestopentaose, etc.

Waterhouse and Chatterton (1993) and Lewis (1993) proposed a branch numbering system that complements the kesto-n-ose nomenclature. Since, in the common naturally occurring fructans, linkages in every case involve the reducing group of fructose (position C2), it is unnecessary to include this universal piece of information in shorthand notation. In the three natural kestotrioses, the linkages are between the reducing C2 position of one fructose and sucrose at either the - C1 or C6 position of the fructose or the C6 position of the glucose. Thus, the linkages from the sucrose to each successive fructose are designated by the numbers 1-, 6-, or 6G-. In this sense, 1-kestotriose reflects its β-(2→1) fructosyl-fructose linkage, 6-kestotriose reflects its β-(2→6) fructosyl-fructose linkage, and 6G-kestotriose reflects its β-(2→6) fructosyl-glucose linkage (Fig. 1.1).

1-Kestotriose: O-β-D-fructofuranosyl-(2→1)-β-D-fructofuranosyl-(2→1)-α-D-glucopyranoside. Equivalent to 1-kestose (former name) and isokestose (obsolete). CAS number: 470-69-9.

Figure 1.1  Structure of the three naturally occurring kestotriose isomers. Current names according to the nomenclature proposed by Waterhouse and Chatterton (1993) with former names in parentheses.

6-Kestotriose: O-β-D-fructofuranosyl-(2→6)-β-D-fructofuranosyl-(2→1)-α-D-glucopyranoside. Equivalent to 6-kestose (former name) and kestose (obsolete). CAS number: 562-68-5.

6G-Kestotriose: O-β-D-fructofuranosyl-(2→6)-α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside. Equivalent to neokestose (former name). CAS number: 3688-75-3.

The compounds in each unit-length series are named by listing the linked fructoses from the sucrose out. Linear chains are simply a list of the nonreducing hydroxyl linkage numbers found at each juncture. For instance, the commercial inulin-type FOSs mixture comprises 1-kestotriose, 1,1-kestotetraose, and 1,1,1-kestopentaose.

1,1-Kestotetraose: O-β-D-fructofuranosyl-(2→1)-β-D-fructofuranosyl-(2→1)-β-D-fructofuranosyl-(2→1)-α-D-glucopyranoside. Equivalent to nystose (former name). CAS number: 13133-07-8.

1,1,1-Kestopentaose: O-β-D-fructofuranosyl-(2→1)-β-D-fructofuranosyl-(2→1)-β-D-fructofuranosyl-(2→1)-β-D-fructofuranosyl-(2→1)-α-D-glucopyranoside. Equivalent to fructosyl nystose (former name). CAS number: 59432-60-9.

In the case of kesto-n-oses with chains attached to both glucose and fructose, the fructosyl chain is numbered first, and at other branches, the 1-linked branch is named first. At points of branching, each branch would be separately named and separated by the word and (&). If a linkage is described before the branch, then a semicolon (;) designates the branch point. When describing the name orally, one would interject the word then at the branch to indicate that there will be two subsequent chains (Lewis, 1993; Waterhouse & Chatterton, 1993). Sucrose-containing oligofructans (DP < 10) with three or more branches should not be named using this system. Instead, they should be referred to as short-chain graminan if the glucose unit is positioned at one end, or short-chain agavin if the glucose unit is between two fructose units.

Fig. 1.2 shows the structures of the main linear and branched kestotetraoses occurring in nature. Representative examples are 1,1-kestotetraose (described above), 6,6-kestotetraose and (1&6)-kestotetraose.

6,6-Kestotetraose: O-β-D-fructofuranosyl-(2→6)-β-D-fructofuranosyl-(2→6)-β-D-fructofuranosyl-(2→1)-α-D-glucopyranoside.

(1&6)-Kestotetraose: O-β-D-fructofuranosyl-(2→1)-O-[β-D-fructofuranosyl-(2→6)]-β-D-fructofuranosyl-α-D-glucopyranoside. Equivalent to bifurcose (former name). CAS number: 3568-31-8.

Branched FOSs can be formed and elongated in different ways. In the case of (1&6)-kestotetraose, the two enzymatic possibilities are the β-(2→6) transfer of the fructose moiety from the donor to the internal fructose unit of the acceptor 1-kestotriose or the β-(2→1) transfer of the fructose moiety from the donor to the internal fructose moiety of the acceptor 6-kestotriose.

Examples of branched FOSs with DP 5–6 isolated from plants are (1,1&6)-kestopentaose, (1&6,6)-kestopentaose, (6;1&6)-kestopentaose, (1,1,1&6)-kestohexaose, (1&6,6,6)-kestohexaose, (6;1&6,6)-kestohexaose, and (6;1,6&6)-kestohexaose (Chatterton et al., 1993).

6. Diagrammatic representation of fructans

Several diagrammatic representations of branched fructans have been proposed (Bancal et al., 1992; Lewis, 1993; Waterhouse & Chatterton, 1993). The most common representation is that proposed by Waterhouse and Chatterton (1993) and discussed by Lewis (1993), in which the β-(2→1) linkages between fructosyl moieties are drawn vertically, and the β-(2→6) linkages horizontally. The 6G bonds between fructosyl and glucosyl units (e.g. in 6G-kestotriose) are also drawn vertically. We propose to adapt the convention of Waterhouse and Chatterton (1993) to a more general and broadly used diagrammatic representation of carbohydrates.

Figure 1.2  Structure of common naturally occurring kestotetraoses. Current names according to the nomenclature proposed by Waterhouse and Chatterton (1993) with former names in parentheses when available.

Figure 1.3  Diagrammatic representation of several branched oligofructans of different complexity drawn following the convention of Waterhouse and Chatterton (1993) (left) and the Symbol Nomenclature for Glycans (SNFG) rules (right).

The Symbol Nomenclature for Glycans (SNFG) facilitates the efficient communication of carbohydrate structures and has become widely accepted by the glycobiology community. It is based on a graphic representation of monosaccharides and complex glycans employing various colored-coded, geometric shapes and short text additions (Varki et al., 2015). The SNFG is hosted by the National Center for Biotechnology Information (NCBI) on the NCBI-Glycans Page (www.ncbi.nlm.nih.gov/glycans/snfg.html). Several changes have been recently made to this page to update the rules for depicting glycans (particularly for nonmammalian organisms) and to avoid ambiguities for complex glycan structures (Neelamegham et al., 2019). Fig. 1.3 shows examples of branched sucrose-containing oligofructans (kesto-n-oses) of different complexity drawn following both the convention of Waterhouse and Chatterton (1993) and the SNFG rules.

7. Conclusions

Fructan research has a history that covers two centuries. Early classifications of fructans were based on criteria that did not reflect the full complexity of the chemical structure. Consequently, several names were often assigned to a fructan of similar properties recovered from different plant species while levan was the name given for years to all fructan polysaccharides of microbial origin. Nowadays, available technical and systematic means allow naming fructans in terms of their monomeric composition, linkage type(s), and DP. This chapter provides a glossary of recommended fructan terminology, proposes new designations, and redefines the term fructan itself as any oligosaccharide (DP 2–9) or polysaccharide (DP ≥ 10) in which fructose is the predominant monosaccharide but not necessarily the only one. The reformulated fructan definition leaves no doubt that 6G-kestotriose (former name neokestose) is a genuine fructan, despite lacking fructosyl-fructose linkages.

References

1. Albon N, Bell D.J, Blanchard P.H, Gross D, Rundell J.T. Kestose: A trisaccharide formed from sucrose by yeast invertase. Journal of the Chemical Society (Resumed). 1953:24–27. doi: 10.1039/jr9530000024.

2. Andersone I, Auzina L, Vigants A, Mutere O, Zikmanis P. Formation of levan from raffinose by levansucrase of Zymomonas mobilis. Engineering in Life Sciences. 2004;4(1):56–59. doi: 10.1002/elsc.200400006.

3. Ávila-Fernández A, Galicia-Lagunas N, Rodríguez-Alegría M.E, Olvera C, López-Munguía A.Production of functional oligosaccharides through limited acid hydrolysis of agave fructans. Food Chemistry. 2011;129(2):380–386. doi: 10.1016/j.foodchem.2011.04.088.

4. Bacon J.S.D, Bell D.J. A new trisaccharide produced from sucrose by mould invertase. Journal of the Chemical Society (Resumed). 1953:2528–2530. doi: 10.1039/jr9530002528.

5. Bancal P, Carpita N.C, Gaudillere J.P. Differences in fructan accumulated in induced and field‐grown wheat plants: An elongation‐trimming pathway for their synthesis. New Phytologist. 1992;120(3):313–321. doi: 10.1111/j.1469-8137.1992.tb01071.x.

6. Banguela A, Hernández L. Fructans: From natural sources to transgenic plants. Biotecnologia Aplicada. 2006;23(3):202–210.

7. Binkley W.W, Altenburg F.W. Nystose, a fructosyl-1-kestose. The International Sugar Journal. 1965;67:110–112.

8. Chatterton N.J, Harrison P.A, Thornley W.R, Bennett J.H. Structure of fructan oligomers in cheatgrass (Bromus tectorum L.). New Phytologist. 1993;124(3):389–396. doi: 10.1111/j.1469-8137.1993.tb03829.x.

9. Cummings J.H, Stephen A.M. Carbohydrate terminology and classification. European Journal of Clinical Nutrition. 2007;61:S5–S18. doi: 10.1038/sj.ejcn.1602936.

10. Díez-Municio, Herrero M, Rivas Muñoz, Jimeno M.L, Moreno F.J. Synthesis and structural characterization of raffinosyl-oligofructosides upon transfructosylation by Lactobacillus gasseri DSM 20604 inulosucrase. Applied Microbiology and Biotechnology. 1887;100:3310–3317. doi: 10.1007/s00253-016-7405-z.

11. Dubrunfaut A.P. Sur une propriété analytique des fermentations alcoolique et lactique, et sur leur application à l’étude des sucres. Annales de Chimie et de Physique. 1847;21:169–178.

12. Ekstrand A.G, Johanson C.J. Zur Kenntniss der Kohlehydrate. Berichte Der Deutschen Chemischen Gesellschaft. 1887;20(2):3310–3317. doi: 10.1002/cber.188702002242.

13. French A.D. Chemical and physical properties of fructans. Journal of Plant Physiology. 1989;134(2):125–136. doi: 10.1016/S0176-1617(89)80044-6.

14. Ghazi I, Fernandez-Arrojo L, Gomez De Segura A, Alcalde M, Plou F.J, Ballesteros A. Beet sugar syrup and molasses as low-cost feedstock for the enzymatic production of fructo-oligosaccharides. Journal of Agricultural and Food Chemistry. 2006;54(8):2964–2968. doi: 10.1021/jf053023b.

15. Greig-Smith R. The gum fermentation of sugar cane juice. Proceedings of the Linnean Society of New South Wales. 1902;26:589–625.

16. Gross D, Blanchard P.H, Bell D.J. neoKestose: A trisaccharide formed from sucrose by yeast invertase. Journal of the Chemical Society (Resumed). 1954:1727–1730. doi: 10.1039/JR9540001727.

17. Hernández L, Quintero Y, Musacchio A. Microbial exopolysaccharides: Current research and developments, Chapter 6: Fructan biosynthesis in bacteria. In: Özlem Ateş Duru. 2019:133–164. doi: 10.21775/9781912530267.

18. Kırtel O, Lescrinier E, Van den Ende W, Toksoy Öner E. Discovery of fructans in archaea. Carbohydrate Polymers. 2019;220:149–156. doi: 10.1016/j.carbpol.2019.05.064.

19. Kırtel O, Menéndez C, Versluys M, Van den Ende W, Hernández L, Toksoy Öner E. Levansucrase from Halomonas smyrnensis AAD6T: First halophilic GH-J clan enzyme recombinantly expressed, purified, and characterized. Applied Microbiology and Biotechnology. 2018;102(21):9207–9220. doi: 10.1007/s00253-018-9311-z.

20. Lewis D.H. Nomenclature and diagrammatic representation of oligomeric fructans—a paper for discussion. New Phytologist. 1993;124(4):583–594. doi: 10.1111/j.1469-8137.1993.tb03848.x.

21. Lippmann E.O. Ueber das Lävulan, eine neue, in der Melasse der Rübenzuckerfabriken vorkommende Gummiart. Berichte Der Deutschen Chemischen Gesellschaft. 1881:1509–1512. doi: 10.1002/cber.188101401316.

22. Mancilla-Margalli N.A, Lopez M.G. Water-soluble carbohydrates and fructan structure patterns from Agave and Dasylirion species. Journal of Agricultural and Food Chemistry. 2006;54(20):7832–7839. doi: 10.1021/jf060354v.

23. Masaru I, Takayuki M, Ben A.Y, Kazuo I, Noshi M. Mechanism of synthesis of levan by Bacillus natto levansucrase. Journal of Applied Glycoscience. 2002;49(2):229–237. doi: 10.5458/jag.49.229.

24. McNaught A.D. Elements of chemistry: Theoretical and practical, Part III. Advances in Carbohydrate Chemistry and Biochemistry. 1857;52:47–177.

25. Miller W.A. Elements of chemistry: Theoretical and practical, Part III. John W. Parker and Son; 1857.

26. Montilla A, Olano A, Martínez-Villaluenga C, Corzo N. Study of influential factors on oligosaccharide formation by fructosyltransferase activity during stachyose hydrolysis by Pectinex ultra SP-L. Journal of Agricultural and Food Chemistry. 2011;59(19):10705–10711. doi: 10.1021/jf202472p.

27. Müntz A. Sur la maturation de la graine du seigle. Comptes rendus de l'Académie des Sciences. 1878;87.

28. Neelamegham S, Aoki-Kinoshita K, Bolton E, Frank M, Lisacek F, Lütteke T, O'Boyle N, Packer N.H, Stanley P, Toukach P, Varki A, Woods R.J.Updates to the symbol nomenclature for glycans guidelines. Glycobiology. 2019;29(9):620–624. doi: 10.1093/glycob/cwz045.

29. Rose V. Uber eine eigenthiimliche vegetablische Substanz. Neues Allgemeines Journal Der Chemie. 1804;3.

30. Schlubach H.H. Der Kohlenhydratstoffwechsel im Roggen und Weizen. Springer Science and Business Media LLC; 1961 doi: 10.1007/978-3-7091-7156-1_6.

31. Suzuki M. History of fructan research: From Rose to Edelman. In: Suzuki M, Chatterton N.J, eds. Science and Technology of fructans. CRC Press; 1993:21–39.

32. Thomson T. A system of chemistry. 5th London ed., Abraham Small.; 1818.

33. Tieking M, Kühnl W, Gänzle M.G. Evidence for formation of heterooligosaccharides by Lactobacillus sanfranciscensis during growth in wheat sourdough. Journal of Agricultural and Food Chemistry. 2005;53(7):2456–2461. doi: 10.1021/jf048307v.

34. Timmermans J.W, Slaghek T.M, Iizuka M, Van den Ende W, De Roover J, Van Laere A. Isolation and structural analysis of new fructans produced by chicory. Journal of Carbohydrate Chemistry. 2001;20(5):375–395. doi: 10.1081/CAR-100105711.

35. Toksoy Öner E, Hernández L, Combie J. Review of Levan polysaccharide: From a century of past experiences to future prospects. Biotechnology Advances. 2016;34(5):827–844. doi: 10.1016/j.biotechadv.2016.05.002.

36. Van Laere A, Van den Ende W. Inulin metabolism in dicots: Chicory as a model system. Plant, Cell and Environment. 2002;25(6):803–813. doi: 10.1046/j.1365-3040.2002.00865.x.

37. Van Wert S.L, Fulbright D.W. Pathogenicity and virulence of Cephalosporium gramineum is independent of in vitro production of extracellular polysaccharides and graminin A. Physiological and Molecular Plant Pathology. 1986;28(3):299–307. doi: 10.1016/S0048-4059(86)80072-8.

38. Varki A, Cummings R.D, Aebi M, Packer N.H, Seeberger P.H, Esko J.D, Stanley P, Hart G, Darvill A, Kinoshita T, Prestegard J.J, Schnaar R.L, Freeze H.H, Marth J.D, Bertozzi C.R, Etzler M.E, Frank M, Vliegenthart J.F.G, Lütteke T, … Kornfeld S. Symbol nomenclature for graphical representations of glycans. Glycobiology. 2015;25(12):1323–1324. doi: 10.1093/glycob/cwv091.

39. Visnapuu T, Mardo K, Mosoarca C, Zamfir A.D, Vigants A, Alamäe T. Levansucrases from Pseudomonas syringae pv. tomato and P. chlororaphis subsp. aurantiaca: Substrate specificity, polymerizing properties and usage of different acceptors for fructosylation. Journal of Biotechnology. 2011;155(3):338–349. doi: 10.1016/j.jbiotec.2011.07.026.

40. Waterhouse A.L, Chatterton N.J. Glossary of fructan terms. In: Suzuki M, Chatterton N.J, eds. Science and Technology of fructans. CRC Press; 1993:1–7.

41. Whalley. Kestose and sugar losses. International Sugar Journal. 1952;54.

42. Yang J.K, Zhang J.W, Mao L, You X, Chen G.J. Genetic modification and optimization of endo-inulinase for the enzymatic production of oligofructose from inulin. Journal of Molecular Catalysis B: Enzymatic. 2016;134:225–232. doi: 10.1016/j.molcatb.2016.10.020.

Chapter 2: Chemical and Structural-Functional Features of Fructans

Mercedes G. López     Biotechnology and Biochemistry, Centro de Investigación y de Estudios Avanzados del IPN, Unidad Irapuato, Guanajuato, Mexico

Abstract

This chapter summarizes the basic features of fructan molecules. Fructans are special storage carbohydrates since they are found in only about 15% of higher plants. Fructans (DP > 9) and fructooligosaccharides (FOSs; DP 2-9) are always found as polydisperse mixtures. These two classes of carbohydrates are made exclusively of glucose and fructose units, linked by either β(2 → 1) and/or β(2 → 6) linkages. It is known that fructan structures are species-specific (e.g., inulin and agavin). To establish fructan structures, more than one analytical technique is usually required. Among the most helpful are thin-layer chromatography, high-performance anionic-exchange chromatography coupled to pulse amperometric detection, nuclear magnetic resonance, enzymatic kits, infrared spectroscopy, and gas chromatrography-mass spectrometry. The knowledge of a specific fructan structure is key to set up or propose a biological or functional role; therefore, we must extend our efforts to unravel the detailed structural features of these fascinating molecules.

Keywords

Agavin; Analytical techniques; DP; Fructans; Fructooligosaccharides; Inulin; Isomerization; Species; Structure

1. Introduction

Fructans are reserve carbohydrates synthesized by 15% of flowering plants (Hendry, 1993). Approximately 45,000 species, represented mainly by highly evolved plant families accumulate these types of carbohydrates, which are also found in fungi, algae, and several bacteria (Vijn & Smeekens, 1999) and very recently in archaea (Kirtel et al., 2019). Fructans have been defined as a dispersed mixture of fructose polymers derived from sucrose (Ritsema & Smeekens, 2003; Vijn & Smeekens, 1999). They can be linear or branched; most are soluble in water and also most, but not all, have a nonreducing character (Banguela & Hernández, 2006). Plant fructans are structurally more diverse than bacterial fructans, largely due to the type of linkage between fructose units. Fructans are commonly classified based on their linkage-type, followed by their degree of polymerization (DP) and/or their natural source. Further details on the terminology of fructans and some of their derivatives are provided in Chapter 1 of this book.

Fig. 2.1 shows the four main fructan groups inulin, levan, graminan, and agavin besides the fructan neoseries and reducing Fn-type fructans. Fructans are frequently classified based on their linkage-type, but they can also be classified based on their DP and branching degree. For instance, inulin-type fructans are named fructooligosaccharides (FOS) if they have a DP 2-9, and termed inulin if they have a DP ≥ 10. Another example is a-FOS, which refers to agavin-type fructans with a DP 2-9 (Huazano-García & López, 2018).

2. Natural sources of fructans

Fructans were first isolated from Inula helenium by Rose in approximately 1804, and in 1818 Thomson called them inulin. Inulin is mainly found in plants from the Asteraceae family, such as chicory (Cichorium intybus), artichoke (Cynara scolymus), Jerusalem artichoke (Helianthus tuberosus), and Dahlia (Dahlia variabilis) (Van Laere & Van den Ende, 2002). Between 1891 and 1893, Tenet found that the complete hydrolysis of these polydisperse class of carbohydrates resulted in only fructose and some glucose (Suzuki, 1993). Among the inulin species, the smallest member with a terminal glucose is called 1-kestotriose (DP 3), and one of the larger representatives has a DP close to 200 (Edelman & Jefford, 1968; Lewis, 1993). Levan contains β(2 → 6) linkages instead of β(2 → 1) linkages, with some possible degree of branching. They are more common in bacteria than in plants and usually have a DP larger than thousands of monomeric fructose units. The smallest member with terminal glucose in this group is called 6-kestotriose. Fructans in cereals are known as graminan, and they are also called mixed fructans because they have both β(2 → 1) and β(2 → 6) types of bonds. They are found in wheat (Triticum aestivum) and barley (Hordeum vulgare) (Pavis et al., 2001). The structural backbone in this group is termed (1&6)-kestotetraose or bifurcose (Pollock & Cairns, 1991; Van den Ende et al., 1996). Lastly, agavin is the most recently reported fructan (Mancilla-Margalli and López, 2006) which is found in several agave species. They are highly branched and complex fructans with both β(2 → 1) and β(2 → 6) linkage types, and they have branches on the main β(2 → 1) chain, also containing an internal glucose.

Figure 2.1  Representative structures of inulin, levan, graminan, agavin, neoseries and Fn-series. Linear and branched fructan structures.

3. Fructans are unique carbohydrates

Being carbohydrates, fructans have chemical and structural functions and features very similar to other carbohydrates, such as the number of carbons (monosaccharides, disaccharides, and oligosaccharides or polysaccharides), the ring type, the number of hydroxyl groups, other functional groups such as carbonyl type (aldose or ketose), DP and linkage type, among others. However, and independently of their high structural similarities, they have a diverse physical and chemical features and highly unique biological characteristics. I refer to an extraordinary introductory book on carbohydrates, their fundamentals and functions (Varit et al., 2015).

The chemical formula of a compound is commonly used to represent its structure and also some information about its function. It is usually assumed that comparable chemical structures have similar or related functions, but proposing a function based on a chemical structure alone is not an easy task because small details such as the orientation of a functional group, whether it is small or large, and the presence of –OH or –CH2–CH3 can have a profound effect on that compound's biological function (French & Waterhouse, 1993). As well as being the most abundant polymers in nature, carbohydrates are the most complex and diverse, essentially due to the elevated number of hydroxyl groups and the many configurations that they can develop into a molecule. This feature makes all carbohydrates hard to physically separate from each other, consequently rendering their identification and characterization within a mixture a difficult task. For instance, the arrangement of five different amino acids can give more than a 100 different molecules, but the arrangement of five different monosaccharides may give more than a few thousand of different molecules.

4. The building blocks of fructans

As carbohydrates, fructans exhibit many homogeneous chemical, structural, and physical properties but reviewing all carbohydrate properties that are not directly related to fructans is beyond the scope of this chapter.

Figure 2.2  Some basic chemical and structural-functional features of carbohydrates, specifically fructans.

Fig. 2.2 presents the most relevant chemical-structural features of a carbohydrate, e.g., an oligosaccharide in this case.

A few highlights of the chemical and structural-functional features of fructans are:

Ring type: Monosaccharides can adopt mostly 5 or 6 member rings, but with several types of conformations.

Anomer type: Once a ring is formed, the anomericity property shows either a α or β form.

Linkage type: Carbohydrates can possess many types of linkages between monomeric units, but fructans can only present two types β(2 → 1) and/or β(2 → 6).

Sequence: Unraveling the sequence of the monosaccharides in an oligosaccharide or polysaccharide is not a simple assignment to perform.

Residue number: In this oligosaccharide, there are 4 terminal residues that may confer different properties to this molecule. The more terminal fructose units, the more available sites for enzymes.

DP: This is equal to 13 (1 glucose and 12 fructoses) but the branching should be noted.

Ratio of monosaccharides: The proportion of glucose and fructose units in a fructan molecule provides information about its size and type. Fn-series fructans contain no glucose.

Stable isotope ratio: This is a highly sophisticated way of determining the natural origin of a molecule based on the biochemical photosynthetic pathway of a plant.

The main building blocks are the monosaccharides, glucose and fructose, all derived from the disaccharide sucrose precursors (Fig. 2.2). Mild acid hydrolysis of plant extracts releases the hexoses glucose and fructose, but these do not explain the fructans' physicochemical properties depending on the carbonyl nature (aldose or ketose), chirality, ring type of the monosaccharides, linkage type, branching degree and sequence.

Research on both oligosaccharide and polysaccharide fructans has increased in the last two decades, mainly due to their applications in food products as prebiotics and to some extent as a popular soluble dietary fiber. A new fructan named AAP70-1 isolated from Anemarrhena asphodeloides is highly branched (as agavin) with a long chain on the 6G position (Zhang et al., 2020). Apart from agavin and AAP70-1, not much research has been devoted to the structural diversity and biological function of novel fructan types, suggesting that much remains to be discovered in this field in the coming decades.

5. Fructan structures

This chapter aims to enlighten the reader with the basic information necessary to establish a chemical structure of a fructan in a pure fructan extract or at least to propose a representative structure of a fructan molecule present in an plant or bacterial extract. Several different analytical tools are highlighted in this chapter because they all provide different pieces of information on the structure of a fructan sample at a different qualitative or quantitative level and in some cases, at the level of their identification and characterization. These data allow the prediction of a structure for a known (or even an unknown) fructan molecule. In general, it is essential to use a combination of at least two or three of these tools to correctly predict a fructan structure, certainly in the case of a brand new fructan.

The chemical structures of fructans as well as some of their features determined via a wide range of powerful analytical techniques are discussed. A variety of analytical tools is available to study fructans. Even those that do not look like very complex molecules (such as inulin) possess intrinsic complexity, due to their carbohydrate nature and the fact that fructans usually occur as a mixture and not as a single molecule. The chemical structures of fructans like FOS, oligosaccharides, and/or polysaccharides can be correlated to some specific functional activity. Monosaccharides and disaccharides are always present in fructan extracts and can be analyzed using one or two analytical techniques, but more than two are usually required to determine the putative chemical structures of short and long fructans.

The existence of several techniques allows to choose between them taking into account the costs and time required for the analyses. Sometimes a single analytical tool can provide rapid answers to some questions when the question is what kind of fructan am I dealing with? However, one needs more than one or two types of analysis to generate a putative chemical structure and correlate it with a putative biological function. Some of the most common and useful analytical techniques for determining the chemical structures of fructans are thin layer chromatography (TLC), gas chromatography coupled to mass spectrometry (GC-MS), nuclear magnetic resonance (NMR), high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), Fourier transformed infrared (FT-IR) spectroscopy, enzymatic assays, matrix-assisted laser desorption ionization (MALDI), and stable isotopes, to mention some.

Not all these tools are essential in all situations. The type or types of tools required will depend on the kind of information needed, be it qualitative, quantitative, or structural. These different types of information will be discussed in the next section while referring to some examples. Extraction of a fructan of interest requires a certain degree of purity prior to a compositional analysis.

6. Fructan extraction

Fructans can be extracted or isolated from an original plant or bacterial source in many different ways. In brief, the biological material is lyophilized, weighed, and extracted with either hot water or a mixture of ethanol and water at least three consecutive times. The extracted phases are mixed together, concentrated, and passed through a column of anhydrate sodium sulfate (drying agent). In some cases, the extracts are also passed through ionic exchange resins. The extracts are then lyophilized and stored in a desiccator prior to their use (Goto et al., 1995; López et al., 2003). Other methods such as ultrasound (Chemat et al., 2011) and sonication (Wang & Weller, 2006) can also be used. Water alone or water/solvent mixtures can be used to extract fructans (López et al., 2003; Lingyun et al., 2007). Regardless of the way in which they are obtained, the extracts are usually lyophilized, kept dry, and used in partial or complete characterization analyses, and/or quantitative or qualitative analyses.

7. Fructan identification and characterization

Irrespective of their potential, sophistication, or innovativeness, all analytical tools have associated technical challenges, and no single method alone can elucidate the whole chemical-structural features needed to subsequently determine correlations with biological activity.

7.1. Glucose, fructose, and sucrose determination

Glucose, fructose, and sucrose are usually present in a fructan extract and can be measured in numerous different ways such as the traditional chemical (Dubois et al., 1956) and Lane-Eynon methods (AOAC, 968.28). Many commercially available enzyme assay kits are also used for the purpose to determine total or specific carbohydrates. The most commonly used approaches are quantitative transformation and determination of the initial or formed product or just the determination of a carbohydrate concentration. These include the use of enzymatic kits (K-SUFRG analytical test kit) from Megazyme International (Ireland) or Boehringer Mannheim and/or HPAEC-PAD.

7.2. Thin-layer chromatography

Two excellent books, Hahn-Deisntrop (2006) and Reich and Schibli (2006) contain all the basic principles of this type of chromatography, describing this separation techique in great detail.

The first procedure that is carried out to know the structural characteristics of a fructan extract is usually TLC due to its low cost, sample size, and time. TLC of fructans has been used very successfully for decades (Cains et al., 1999; Itaya et al., 2002; Kanaya et al., 1978; Leroy et al., 2010; Shiomi, 1992; Sims et al., 1992), in conjunction with a visualizing reagent made up of aniline/diphenylamine/phosphoric acid in acetone (Anderson et al., 2000) (Fig. 2.3).

Fig. 2.3 represents a general TLC run of S-Mix which contains a mixture of glucose (G), fructose (F), sucrose (S), 1-kestotriose (1K), 1,1-kestotetraose (N), and 1,1,1-kestopentaose (DP5 inulin), followed by linear standards such as raftilose (RSE: Fn-series type: 2F, 3F, 4F) and raftiline (RNE: inulin type) obtained from chicory roots (commercial brands), onion (O) and agave (A). Overall, this TLC presents a great deal of information about four different fructan samples, that is, their building blocks. Also, the developed colors are very informative because glucose-containing fructans tend to be bluish and fructans without glucose (fructose-only type) are very reddish, like RSE. At the top of the runs, glucose is the blue spot on the front of the solvent and the red spot corresponds to fructose. Assignments in this TLC are possible using the S-Mix. Other assignments can be made by prior knowledge of the basic fructan components and the position of the spots, as well as of some known isomers such as 6G-kestotriose or neokestose (NK).

This TLC suggests the presence of some different compositions or isomers (Fig. 2.3A). On initial inspection agavin (A) looks like inulin, but some extra spots can be seen on the TLC suggesting that there are additional components present. In a TLC, the complexity of a fructan extract can be inferred based on the number of spots present, and their different colors. Fig. 2.3B shows FOS, the same S-Mix and MOS (standard ladder of G and some maltooligosaccharides). This MOS mixture does not provide a reddish color because there are no ketoses present in their structure; these oligosaccharides contain only glucose units. It is worth mentioning that the development color in the TLC runs might depend on the preparation of the developer reagent and the developing temperature. The Rf, retention factor, is a particular or unique descriptor or value that each spot (molecule) has in a TLC run and can contribute to the identification of a molecule. In fructans, this value is not very useful because the TLCs are executed several times with the same mobile phase or with a different one, this will not allow comparing Rf values from other reported articles.

Figure 2.3  Thin layer chromatography (TLC) of some typical fructans. TLC of standards and some samples (A) and FOSs (fructooligosaccharides) and MOSs (maltooligosaccharides) (B).

There are many studies of fructans with excellent TLC data. For instance, Salimas et al. (2016) produced useful TLCs data, but some others that are less clear have also been published (López-Molina et al., 2005). TLC has many more amazing applications and another extremely useful way to apply TLC in fructan field research is to follow the activity or progression of fructosyl transferase and/or fructan hydrolase reactions, because their products can be easily seen, and they can even be semiquantified. This technique is not time-consuming, inexpensive, practical, and not as sophisticated as compared to others, but it is very informative.

7.3. Ascertaining profiles via HPAEC-PAD

As any other type of chromatography, HPAEC-PAD is a separating tool. Its usefulness lies in the pKa property of the carbohydrate hydroxyl groups that fluctuate between 12 and 13, which are ideal for separation in an alkaline solution. This type of chromatography is highly appreciated because it has a wide range of applications for separating a very diverse variety of carbohydrates, including fructans. The separation and quantification of carbohydrates of different chemical structures, for example, from monosaccharides to polysaccharides, requires different stationery and mobile phases. This is an easy task when it comes to a sample extract composed of only one type of carbohydrate, either small or large; however, when a sample is composed of a mixture of them, as it is the case with fructans, there are only two excellent stationary phases, CarboPac-PA100 and 200, capable of separating most of the components present in a single sample when all the components are linear (Fig. 2.4).

A good program to separate fructans is using a solvent gradient of an alkaline solution made up of (a) NaOH 0.15 M, (b) NaOH 0.15/CH3COONa 0.5 M and (c) distilled H2O (DIONEX ICS-300, guard- and analytical-column CarboPac-PA100 from Dionex, CA, USA). The potentials used are E1 (440 ms), E2 (20 ms), E3 (20 ms) and E4 (60 ms) and +0.1, −2.0, +0.6 and −0.1 V, respectively (Arrizon et al., 2010; Bancal et al., 1993; Mancilla-Margalli & López, 2006; Mellado-Mojica & López, 2012; Ritsema et al., 2003; Shiomi et al., 1991)

Figure 2.4  High performance anion exchange chromatography-pulse amperometric detector (HPAEC-PAD) of (A) several representative fructans and (B) inulin and agavin isomers with a DP of 8.

Fig. 2.4 depicts typical chromatographic profiles of a standard mix (G, F, S, 1K, N, and DP5 inulin), RSE, RNE, and agavin. It is clear that this tool is much more sensitive and reliable than TLC because one can easily distinguish the different DPs present in the samples as well as the highest DP and the types of fructans—with the exception of the large DP from the agave fructans which are highly branched and exhibit very dispersed DPs. It is relevant to mention that the determination of carbohydrates by HPAEC-PAD is a fast and accurate method without the need for any type of derivatization (pre- or postdetection) which is necessary for many other chromatographic technologies such as HPLC or GC-MS.

RSE and RNE in Fig. 2.4A to an extent serve as standards, because their well-known profiles of a short DP inulin sample (RSE) and a long DP inulin sample (RNE) are unequivocal. Notably, each sample contains a little of the other species. These two samples help to establish that agavin is not composed of either sample, but contain a small amount of both, because the agavin profile differs markedly from the two-fructan samples together. In contrast, Fig. 2.4B depicts a very close comparison of fructan HPAEC-PAD profiles, specifically long DP inulin from Cichorium intybus and long DP agavin from Agave tequilana in this case. To illustrate the complexity of agavin compared to inulin, a fructan with a DP of 8 can be used. In raftiline, there are only two molecules with a DP of 8, but in agavin there are at least 10 different molecules with this DP. Unfortunately, to date there is no combination of stationary phase and/or mobile phase methods capable of separating all these components (isomers) from an extract, to convert these mountains into single peaks. At the beginning of a chromatographic run, short DP components can be easily distinguished, even in an agavin extract, but as the DP increases the number of isomers increases so they can no longer be separated. Another advantage of HPAEC-PAD over TLC is that in most cases small DP components within an extract can be determined quantitatively, if there are available standards. In this case, the type of detector is very relevant because for the quantification, the oxidation/reduction, of a carbohydrate must be carried out in a fraction of milliseconds. Therefore, the detector (gold electrode) must be cleaned very frequently to obtain reliable and reproducible results despite its high sensitivity (0.1 nmol).

A recent review (Matros et al., 2019) presents a relevant number of examples from the plant fructan context, using HPAEC-PAD and other chromatographic techniques, liquid and gas chromatography, coupled to a variety of detectors for the separation and quantification of these carbohydrates.

7.4. Fructan quantification via enzymatic assays

Different methods can be used to quantify the total percentage of fructans in a fructan extract. A good commercial kit for determining the total fructan content in different samples is that produced by Megazyme International. This assay is based on the hydrolysis of polymers (fructans) present in a sample using various enzymes, but only after other carbohydrates present in the extract or sample such as monosaccharides and disaccharides have been removed (mainly glucose, fructose, and sucrose). These simple sugars are always present in fructan extracts regardless of the origin or methods used. Physically removing these simple sugars is very costly. The test is based on the hydrolysis of all fructans to fructose via the action of an exo-inulinase, and at the end of the hydrolysis, the reducing sugars (monosaccharides) are determined as such by using the hydrazide of the p-hydroxybenzoic acid. However, often such quantifications for branched or complex fructan molecules cannot be correctly acquired using these types of kits because they were primarily designed to quantify linear fructans such as inulin and/or