inulin - a versatile polysaccharide with multiple pharmaceutical

Review Paper

Inulin - a versatile polysaccharide with multiple pharmaceuticaland food chemical uses.

Thomas Barclay , Milena Ginic-Markovic , Peter Cooper , Nikolai Petrovskya a b,c c,d

Flinders University, Adelaide, Australia 5042a

Cancer Research Laboratory, ANU Medical School at the Canberra Hospital, Australian National University, Canberra, Australia 2605b

Vaxine Pty Ltd, Flinders Medical Centre, Adelaide Australia 5042c

Department of Endocrinology, Flinders Medical Centre, Adelaide, Australia 5042d

Received: 27 August 2010 Accepted: 10 October 2010

ABSTRACT

á-D-glucopyranosyl-[â-D-fructofuranosyl](n-1)-D-fructofuranoside, commonly referred to as inulin,is a natural plant-derived polysaccharide with a diverse range of food and pharmaceuticalapplications. It is used by the food industry as a soluble dietary fibre and fat or sugar replacement,and in the pharmaceutical industry as a stabiliser and excipient. It can also be used as a precursor inthe synthesis of a wide range of compounds. New uses for inulin are constantly being discovered,with recent research into its use for slow-release drug delivery. Inulin, when in a particulate form,possesses anti-cancer and immune enhancing properties. Given its increasing importance toindustry, this review explains how inulin's unique physico-chemical properties bestow it with manyuseful pharmaceutical applications.

KEY WORDS: Inulin, polysaccharide, fructose, excipient, vaccine, adjuvant

INTRODUCTION

á-D-glucopyranosyl-[â-D-fructofuranosyl](n-1)-D-fructofuranoside (inulin, shown in Figure 1)is a natural renewable polysaccharide resourcewith a significant number of diversepharmaceutical and food applications. In thefood industry it is used as a fat or sugarreplacement and soluble dietary fibre (1-5), butit also has important pharmaceuticalapplications, as an excipient or stabiliser, and asan injectable for clinical measurement of kidney

function (5-8). Inulin may also have utility as aslow release drug delivery medium (2, 6, 9) andas a stabiliser for protein and peptide-baseddrugs and vaccines (10). Additionally inulin hasinteresting biological effects, being a potentcomplement pathway activator when in aparticulate form and having anti-cancer (11, 12)and immuno-modulatory properties (13-16).

These pharmaceutical applications rely upon anumber of unique chemical and physicalproperties. Firstly, soluble inulin is largelybiochemically inert and non-toxic. Specifically,its â(2-1) glycosidic bonds make it indigestibleby humans and other higher animals that donot possess inulinase enzymes. Inulin is,

Corresponding author: Department of Endocrinology, Flinders Medicald

Centre/Flinders University, Bedford Park, Adelaide, South Australia,Australia 5042, Tel: 61-(0)8 82044572, Fax 61-(0)8 82045987,[email protected]

This Journal is © IPEC-Americas Inc J. Excipients and Food Chem. 1 (3) 2010 - 27

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however, digestible by certain microorganismsliving in the gut that have inulinase activityincluding lactobacilli (2, 17-23). This meansinulin can pass though the human digestivesystem relatively intact, until it reaches the largeintestine, where it is digested by bifidobacteria(20, 23). This encourages growth of a healthyintestinal micro flora that in turn producesimportant metabolic by-products includingbutyric and propionic acid, which suppresscolon cancer development. Inulin also inducesproduction of a glucagon-like peptide (GLP)-1hormone, an important endogenous stimulatorof insulin secretion and appetite suppressant(24, 25). This makes inulin a valuable probioticdietary fibre (2, 4, 5, 17-23). The indigestibilityof inulin also means it has relatively low foodenergy for humans and this, combined with itsbland to sweet taste, means that it can be usedas a low calorie bulking agent in food, replacingsugar, flour and fat (2, 13, 18, 23). Inulin's useas a bulking agent, in particular as a fatreplacement, is aided by its particular propertiesof water solubility. Parts of the molecularstructure, specifically the hydroxyl groups, aremore able to interact with water than otherparts. This provides inulin with some surfactantcharacter and it is able to form stable gels withwater at concentrations of 13-50% (22, 23, 26,27). These gels provide similar textualcharacteristics to fat, allowing it to be used toreplace fat, resulting in low fat foods that arepalatable and have good mouth feel (2, 18, 22,23, 28).

Further advantage can be taken of the solubilityof different molecular weights of inulins inwater. Inulin's solubility is closely related to thechain length of the polymer, and thus shorteroligomers are much more soluble than longchain polymers (22, 28, 29). This meansapplications can utilize either inulin's solubilityor its insolubility, depending on which polymerlength is utilised. For example, in the foodindustry it is generally required that the inulin isdissolved in processing to make the gels thatare important for texture and bulk (22). Incontrast, crystalline forms of inulin with lowsolubility in water activate the complement

system, relevant to its use as a vaccine adjuvant(13-16) or cancer treatment (11, 12). Thesemedicinal applications benefit from inulin'sbiochemical inertness and lack of toxicity in thehuman body (8). In kidney function testing,intravenously administered inulin is rapidlyexcreted by the kidney without beingmetabolised or reabsorbed in the renal tubulesenabling it to be used as a measure of glo-merular filtration (6, 7, 30). Inulin's rapidexcretion from the body in the urine means itcould potentially be used in drug delivery to theurinary tract (6). Further, inulin's ability to formgels, particularly when modified with cross-linking groups (9), also provides potential utilityas a drug delivery vehicle for water-solubledrugs to other parts of the body (9, 26, 31).

The biochemical properties of inulin are alsoutilised in the chemical industry, with microbialfermentation of inulin used to producealcohols, including ethanol (5, 6). More usually,though, it it is the basic chemical structure thatis more frequently taken advantage of, beingused itself, or as a source of the constituentsugars, as precursors in the production of anumber of chemicals such as glycerol, as acomponent in detergents, and for many lessprosaic species (6, 32).

In the optimisation of inulin applications, agood understanding of the physio-chemicalproperties of inulin polymers is required. Muchwork has been conducted into inulin over thelast century, but, given the diversity ofapplications, the literature is highly fragmented.It is the aim of this review to consolidate theknowledge of inulin's physio-chemicalproperties to assist future research of thisinteresting polymer.

POTENTIAL INULIN USES

Inulin's various uses have been addressed byprevious reviews of its chemical (2, 6), industrial(2, 6), food (2, 6, 18), and pharmaceutical app-lications (2, 5, 6, 18). This review will largelyfocus on the potential of new pharmaceuticalapplications of inulin.


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Therapeutic effects of dietary inulin

Dietary inulin inhibits development of coloncancers in animal models. Similar tumour-inhibitory effects are seen with fermentationproducts of inulin, particularly the short chainfatty acids butyric and propionic acids, both ofwhich inhibit growth of colon cancer cells.Butyrate has multiple actions that maycontribute to its anti-cancer effects includinginhibition of histone deacetylases (33). Theeffect of dietary inulin is not just seen locally oncolon tumours, as dietary inulin also suppressedmethylnitrosourea-induced mammary carcino-genesis in Sprague-Dawley female rats and, inaddition, the growth of muscle-implantedtumour cells (34). This indicates a systemic anti-tumour effect of dietary inulin, presumablymediated by one or more soluble mediatorsinduced by inulin. Several ongoing studies areseeking to confirm these findings in humans. Treatment of subjects with colon polyps withinulin plus probiotics resulted in reductions inDNA damage, colonocyte cell proliferation andfaecal water genotoxicity (35, 36). Part of thebeneficial effect of inulin in suppressingtumours may be mediated by its enhancementof gut immune function (37). Notably, inulinhas been shown to exert immuno-modulatoryeffects and induces differentiation in severalintestinal cell types independently to its effectson the gut flora (38). Dietary inulin has beenshown to have an immunomodulatory effect onthe gut, increasing secretory immunoglobulin(Ig) A and interleukin-10 production anddecreasing the oxidative burst activity of bloodneutrophils (37). It also increases the capacityof peripheral blood mononuclear cells toproduce interferon gamma (39). Inulin-fed ratshad a higher number and proportion ofdendritic cells in gut Peyer's patches, andgreater ex vivo splenocyte secretion of IL-2, IL-10 and interferon-gamma (40). Themechanisms of the immuno-regulatory effectsof dietary inulin may include indirect effectssuch as changes in the composition of theintestinal flora, and the enhanced production ofshort chain fatty acids with immuno-regulatory

actions (41). In mice dietary inulin enhancedthe response to an oral salmonella vaccineconsistent with an enhancing effect on mucosalimmunity (42). Similarly, mixtures containingfructo-oligosaccharides enhanced influenza-specific delayed-type hyper-sensitivityresponses in mice receiving influenzavaccination (43, 44).

Dietary inulin may reduce risk of cardiovasculardisease. A recently reported human trialshowed that dietary inulin reduced serumconcentrations of the proatherogenic molecule,p-cresyl sulphate, in haemodialysis patients (45).Inulin may also reduce cardiovascular risk by itsfavourable effect on plasma cholesterol andglucose levels. A recent study to investigate theeffects of dietary inulin in young healthy menfound a significant increase in HDL-cholesteroland reductions in total cholesterol/HDL-cholesterol ratio, serum triglycerides, fastingglucose, fructosamine, HbA1c and insulinresistance as measured by homeostatic modelassessment (HOMA-IR) (46). Gastric emptyingwas also significantly delayed in the groupreceiving inulin-enriched pasta. At least part ofthis favourable metabolic effect may bemediated by an effect of inulin on productionof a glucagon-like peptide (GLP)-1 mentionedabove.

Dietary inulin has also been shown to increasecalcium and magnesium absorption and bonemineralisation in young adolescents, primarilythrough an effect of increasing calciumabsorption in the large intestine (47-49).

Use of inulin for drug delivery

Drug delivery systems aim to maximise theexposure of a drug to the tissue requiringtreatment and can also be used to stabilise labiledrugs such as proteins and peptides. This helpsto minimise dosage, and reduce side effects andcost, while maximising efficacy and enablingpatient preferred mechanisms of admi-nistration, such as oral, rather than injectedroutes. Inulin is generally biochemically inert,non-toxic and can form hydrogels. These


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properties provide the opportunity for its use asa drug delivery vehicle (9). These factors areassisted by inulin's pharmacological inertia andready availability (31). Although inulin is able toform aqueous gels without any chemicalmodification, its use as a hydrogel in drugdelivery applications may require it to be cross-linked to improve gel stability, allowing morecontrolled drug release. An example is its use asan orally-delivered drug delivery systemtargeting the colon, to allow delayed absorptionof drugs that have adverse effects in thestomach (26) or to provide treatment ofdiseases that show a peak in symptoms in theearly morning (9, 50, 51). Inulin is an idealvehicle to deliver drugs to the colon as the â(2-1) glycosidic linkages are stable to theendogenous enzymatic action of the humandigestive system, but it is still broken down bycolonic bacteria, releasing the drug payload.While inulin hydrogels might be ideal for drugtransport to the colon, they must be stable tothe range of pH and ionic strength observed inthe human gastro-intestinal tract and the gelswelling characteristics must be such that thebulk of the drug is released during colon transit(50, 51).

Hydrogel stability can be increased by cross-linking and for inulin polymers this has beenachieved via functionalisation with vinylgroups. The vinyl groups are subsequentlycross-linked by free radical polymerisation (9,50, 51). Hydration of these dry materials occursrelatively quickly in a manner independent ofthe degree of cross-linking, until a certain value(~15% of fructosyl units substituted) isreached, whereupon further cross-linking leadsto increased swelling time (50). Exposure tovarying pH, matching those expected within thegastro-intestinal tract (GIT), shows thathydrogels are stable to the mildly acidic andbasic pHs expected within the human GIT (50).For the range of ionic strengths in the GIT theequilibrium degree of swelling was relativelysmall (50). Similarly, exposure to esterasesshowed no significant destructive effect uponthe inulin hydrogel, despite the ester bondsintroduced with the cross-linking groups. In

contrast, exposure to inulinases, like those usedby the bifidobacteria in the colon, leads toenzymatic degradation of the inulin hydrogel ata rate inversely related to the degree of cross-linking (50).

Cross-linking of inulin hydrogels has also beenachieved through functionalisation with bothmethacrylic anhydride and succinic anhydride,cross-linking triggered by subsequent irradiationwith ultra violet light (26). This cross-linkedinulin hydrogel, which was designed to deliverthe anti-inflammatory agent diflunisal to thegut, exhibited pH-sensitive swelling, withreduced swelling in acidic environments andincreased swelling in more neutral to alkalineenvironments of the intestine (26). The cross-linking did not prevent degradation of thehydrogel by inulinases (26).

Inulin, inulin acetate and modified inulinacetate microspheres have been used for thetransport of water soluble model drugs (31)with the modified inulin acetate microspheresbeing supramolecularly associated with 1,2-dodecanedicarboxylic acid. Encapsulation wassimilar for all three species, being maximised at65% (31). Drug release was initially quick as thedrug was solubilised when absorbed onto theoutside of the microspheres. For inulin andinulin acetate 58-62% of the drug was releasedwithin the first 5 minutes whereas for modifiedinulin acetate only 32% was released within thefirst 15 minutes (31). After this time the drugwas released slowly by diffusion forapproximately 1 day, after which the erosion ofthe microspheres led to quicker release untilcomplete after 3.5 - 4 days (31).

Use of inulin as a stabiliser

Protein and peptide drugs, includingmonoclonal antibodies, are increasinglyimportant as human therapeutics. A drawbackto these drugs is their lability, particularly inaqueous solution, which may limit their shelflife. Drying is a way to avoid this, oftenrequiring use of a protective agent to prevent


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loss of protein activity. Polysaccharides can actas protective agents, as they provide multiplehydroxyl groups, that can replace the hydrogenbond interactions as the water is removedduring drying (10). This helps to maintain theprotein's native conformation and preventdenaturation. For the protective effect to work,the protein or peptide must set the molecularorganisation, meaning that the polysaccharidemust not crystallise in the drying process (52).Furthermore, the molecular mobility must below after drying to prevent degeneration of theprotein structure in the dried product. For thisreason a glassy polymeric structure is best ableto protect the protein or peptide and retain thenative active conformation over time (53).Requirements for polysaccharides to act as aprotective agent in the freeze-drying of proteinand peptide based drugs include a high glasstransition temperature, low hygroscopicity, lowcrystallisation rate and a lack of reducinggroups (10). Inulin compared well to a controlpolysaccharide, trehalose, for these propertiesand was found to successfully protect alkalinephosphatase during drying, with longer inulinpolymer lengths being the most effective (10).

Vaccine adjuvant activity of specificinulin isoforms

Insoluble inulin particles, but not soluble inulin,activate complement via the alternativepathway (APC) (13, 15, 16, 54, 55). The APCforms a key part of the innate immune system,and particulate forms of inulin have beenshown to have vaccine adjuvant activity (13, 16,

55). g and ä-inulin isoforms, together referredto as microparticulate inulin (MPI), activatecomplement whereas earlier described moresoluble á- and â-inulin isoforms do not (13, 55,

56), explaining why these are the only g- and d-inulin that have shown vaccine adjuvantactivity.

A problem with modern vaccines based onrecombinant proteins or peptides, is their

relative lack of immunogenicity, compared toolder vaccines based on living or inactivatedwhole organisms. This poor immunogenicitycan be alleviated though the use of adjuvants(13, 30, 55, 57-62). Adjuvants are agents thatact non-specifically to increase the specificimmune response or responses to a co-administered antigen (55, 63). MPI is a goodcandidate to replace traditional aluminium-derived adjuvants because it stimulates bothhumoral (antibody-mediated) and cellular (Tcell-mediated) immunity to co-administeredantigens. Inulin's safety and lack of toxicity aremajor attributes favouring its use as a vaccineadjuvant (13, 15, 30, 54, 55).

CHEMICAL PROPERTIES OF INULIN

All the above pharmaceutical applications ofinulin require a thorough understanding of itschemistry and physico-chemical behaviourswhich will be discussed in detail below. Inulin isa natural storage polysaccharide of variousplants that are mostly, but not exclusively, partof the Compositae family including chicory,dahlia, and Jerusalem artichoke (2, 19, 20, 22,23, 55, 64-68). Inulin is also produced bymicroorganisms including a single knownnatural bacterial species, Streptococcus mutans (69).Other microorganisms that have been shown tobe able to produce inulin in the lab are fungalspecies, specifically members of the Aspergillusfamily (70, 71), although whether inulin isproduced natively by these fungi is debatable asthey have no significant natural source ofsucrose (23).

Chemically described as á-D-glucopyranosyl-[â-D-fructofuranosyl](n-1)-D-fructofuranoside,inulin is a polymer of fructans consisting of li-near chains of fructosyl groups linked by â(2-1)glycosidic bonds terminated at the reducing endby an á-D-(1-2)-glucopyranoside ring group(Figure 1) (5, 30, 54, 55, 64-66, 72-77). Gene-rally, plant inulins are found to have chainsincorporating 2-100 or more fructose units,chain length and polydispersity depending onplant species and the point in its life cycle (1, 2,6, 17, 19, 21-23, 28, 66, 74, 78-80). Microbial


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inulin has much larger degree of polymerisationranging from 10,000 to 100,000 (23).

The chemistry of fructose and glucose

To understand the chemistry of the polymericand oligomeric forms of inulin someunderstanding of the basic chemistry of theconstituent sugars is required. This is because,while the behaviour of polysaccharides is oftenquite different from that of its constituentmonosaccharides (81), much of the chemistrythe polymer can undergo is the chemistry of theconstituent sugar, even if some avenues ofreactivity are lost in polymerisation. Also, anunderstanding of the chemical decompositionof the polymeric forms, which requires anunderstanding of the how synthesis mightoccur from monomers, helps manage thedesired or undesired decomposition of inulinduring processing (29).

Inulin comprises a chain of fructose molecules

terminated at the reducing end with glucose.While fructose and glucose both crystallise as cyclic forms, in solution the free sugars have avery small equilibrium amount of an acyclicform, the formation of which creates a carbonylgroup (Figure 2) (81, 82). This carbonyl groupis reactive to hydroxyl groups and it is theintramolecular reaction between the carbonylgroup and a hydroxyl group present within thesugar that closes the ring, reforming thehemiacetal cyclic structure (81). For six carboncarbohydrates, like glucose and fructose, thering closing reaction can occur with more thanone of the hydroxyl groups, leading toisomerisation and multiple cyclic forms (81).Most often this ring structure has five or sixmembers. Seven-membered rings are alsopossible for glucose, though the six memberedrings are most highly favoured (81). Fructose ismore complex as it is crystallised as a 5-mem-bered ring or furanose, but mostly exists as a 6-membered ring or pyranose in solution (2, 83).Further complexity is provided as each of theserings has multiple conformations, as is also thecase for glucose. Additionally, the ring openingand closing reactions about the chiral anomericcarbon means that two stereoisomers oranomeric forms for each conformation of thesugar exist (77, 81, 82).The reactivity of the acyclic carbonyl group isnot just limited to intramolecular cyclisationsand provides a range of reactivity for the sugar

Figure 2 Cyclic and acyclic forms of glucose and

fructose

Figure 1 Inulin Polymer


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that is, to some extent, dependant on thecarbonyl form. Which form the carbonyl grouptakes depends on the way biosynthesis of thesugar occurs, initially with carbon dioxide andwater combining to make oxygen andformaldehyde (81). The manner in which theformaldehyde intermediates combine dictateswhether the carbonyl chemistry of the acyclicsugar is terminal to the linear chain, making analdehyde, or incorporated within the chain,making a ketone. Sugars that have an aldehydeacyclic form are known as an aldose, of whichglucose is an example. Fructose is a ketone inthe acyclic form and these types of sugars areknown as ketoses (81). The reactivity of thecarbonyl group is generally lower for ketonescompared to aldehydes as the acidic carbonylattached hydrogen in aldehydes makes themmuch more susceptible to oxidation reactions(82). Nonetheless, both carbonyl chemistriesare susceptible to reduction and addition-elimination reactions with nucleophiles.Another variance between ketoses and aldoses,which affects reactivity oppositely, is thatketoses generally form more constrained, lessstable ring structures. Consequently ketoseshave higher concentrations of the acyclic formin solution than aldoses, the higherconcentration increasing relative reactivity (82).

The complexity of structure and carbonylchemical reactivity of glucose and fructose canbe prevented by substitution through theanomeric hydroxyl group in the formation ofglycosidic bonds (77, 81, 82). This bondprevents the chemistry involved in the ringopening reaction, fixing the cyclic structure,including its stereochemistry (77), and notallowing the acyclic carbonyl chemistry likereduction reactions. As such, sugars substitutedhere are described as non-reducing (77, 82). Anexample of a non-reducing sugar is sucrose.Sucrose is a disaccharide that bridges theanomeric carbons of á-D-glucose and â-D-fructose (77) creating a non-reducing sugar thatis most notable for its use as table sugar, butwhich also provides an inert starting group inthe biosynthesis of inulin (Figure 3).

Polymerisation through the anomeric hydroxylgroup, as is the case for inulin, similarlyprotects the component sugars with glycosidicbonds (77) reducing the types of chemicalreactivity available to the polymer (thoughhydrolysis of the glycosidic bond is a new onethat is discussed in detail in the next section).The basic monomeric reactivity that is availableto the polymer revolves around the reactivity ofthe hydroxyl groups attached to each ring.These alcohol groups are generally relativelyinert, but can undergo a number of organicreactions in the presence of appropriatereagents. Both deprotonation and protonationcan occur in the presence of strong bases andacids respectively. These create much morereactive intermediates with deprotonatedderivatives becoming very strong nucleophilesand protonated intermediates readilyundergoing nucleophilic substitution anddehydration reactions. Other reactions ofalcoholic groups include esterification reactionswith carboxylic acids and oxidation reactions toform carbonyl chemistry, aldehydes andcarboxylic acids for primary alcohols andketones for the secondary alcohols. In general,the primary hydroxyl groups attached to C6 ofboth fructose and glucose components andattached to C1 of the terminal fructose moietyare more reactive than the other secondaryhydroxyl groups making selective substitutionpossible (9).

Figure 3 Starting materials of the biosynthesis of inulin


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Another form of chemistry that is brought toinulin by the chemical structure of theprecursor molecules is supramolecularinteraction. That is, the polarity of the oxygento hydrogen bond in the hydroxyl groups ofglucose and fructose, and by extension inulin,also makes supramolecular chemistrysignificant, with non-covalent interactions withother similarly polar molecules easily achieved.

The chemistry of inulin polymers andoligomers

Inulin is biosynthesized from a startingmolecule of sucrose (Figure 3), explaining thepresence of the single glucose residue in thepolymer (84). Enzymes then progressivelytransfer fructose from another sucrosemolecule to begin the inulin polymer chain.The attachment of the incoming fructose is tothe relatively reactive primary hydroxyl group(77) linked to the anomeric carbon through themethylene group at C1 of the fructose group inthe sucrose substrate. This reacts with theanomeric hydroxyl group of the fructose,cleaved from the incoming sucrose, formingthe glycosidic bond. This initially forms 1-kestose, essentially a sucrose molecule with anadditional fructosyl group attached (Figure 3).In the case of bacterial and fungal synthesis ofinulin additional fructose is then added to theterminal fructose in 1-kestose from sucrose,with the longest chain polymers being the bestacceptors leading to the quick synthesis of longchains (23). In plants, 1-kestose is also used as asubstrate for chain elongation with newfructose being added, not from sucrose, butfrom 1-kestose or other nascent polymers usinga separate enzyme to the one that forms 1-kestose (23). As with bacterial or fungalsynthesis, the longest chains are the mostefficient fructose acceptors, along with thestarting sucrose, which favours the productionof longer chains (23).

The biosynthesis of inulin builds a polymerwith â(2-1) linking glycosidic bonds. Poly-merisation in this way means each incomingmonomer becomes inert to carbonyl chemistry

upon the formation of the new glycosidic bond,locking the fructose units in the furanose form(77). The growing end of the nascent polymeris described as the terminal end, the other endof the chain being generally known as thereducing end. Of course, in the case of inulin,what would be a reducible end of polymerchain made purely from fructose is ostensiblynon-reducing due to the presence of theglucose end group. This is the reason that inulinis chemically relatively inert, although cleavageof the polymer chain at any of the glycosidicbonds will produce a reducing end that then issusceptible to the reactions of carbonyl groups(2, 8, 84). Interestingly, while the fructosyl unitsin the chain and the terminal fructose unit havefuranose form, when free of glucose thereducible end fructosyl group is in thepyranose, or 6-membered ring form (85). Inulin is enzymatically synthesised for energystorage by various plants, and this inertness ofthe glucose protected chain is valuable becausethe storage medium does not break downspontaneously. To access this energy whenrequired these plants have enzymes (inulinase)able to cleave fructose from the terminal end(23). Under appropriate conditions this can leadto high concentrations of fructose, and undersuch conditions the enzyme that usuallyelongates the inulin chains can begin to addfructose to fructose monomers creatingfructose only chains that have a reducing end(23). This is why significant quantities of inulinare found that have no glucose protectinggroups in samples isolated from their naturalsource and why there is such a largepolydispersity in chain length, despite thefidelity common to enzymatic synthetic systems(64).

Branching through reactions of the primaryhydroxyl group at C6, rather than through thetypical C1 group, has been reported for inulin,but it is usually at low levels in plants (1-4%)(19, 23, 28, 64, 86), and a little higher formicroorganisms (5-15%) (23, 69). Carpita et al.(28, 64) suggest this is not actually branching,

but that the detection of â(2®6) bonds is due


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to the presence of a small proportion of

â(2®6) ‘inulin’ synthesized by a differentenzyme (64). The basis for this claim is thatotherwise there must be two enzymes workingto make a branched polymer, as enzymaticsystems usually achieve such high fidelity (64).

Inulin hydrolysis

Inulin undergoes cleavage of the glycosidicbond though the addition of water in a processknown as hydrolysis (4, 23, 87). Eventually thisreaction will lead to the decomposition of thepolymer into the component monosaccharides(29). While inulin is relatively stable tohydrolysis at room temperature and neutral pH,the rate of this reaction can be increased byincreasing temperature and extremes of pH (4,23, 29, 88, 89).

The hydrolysis of inulin proceeds by rates thatvary depending on the properties of theglycosidic bond. In particular, the glucosyl-fructosyl bond is 4-5 times more resistant toacid hydrolysis than the fructosyl-fructosylbond (90). Also, terminal fructose units arecleaved more easily than internal ones (90),most likely due to the change of conformationrequired of the fructosyl group duringhydrolysis (91). This change is more easilyachieved by an end group than one internal tothe polymer (92). Despite these effects, forshort chain oligomers where solubility issuesand viscosity effects are negligible, the rate ofhydrolysis is directly proportional toconcentration of inulin (up to 40%/w) (29).This suggests that the hydrolysis follows firstorder or pseudo first order kinetics (29, 89).

At extremes of temperature, pH or both,decompos i t i on o f the hyd ro l y sedmonosaccharides can occur during thehydrolysis reaction (87, 88). Understanding thisis of importance when trying to establishdefinitive parameters for the hydrolysisreaction. Therefore, although neither glucoseor fructose are particularly susceptible to this,and relatively gentle conditions can be used inthe hydrolysis of inulin, factors for allowing the

correction of this effect have been developed(87). A way to avoid this monomericdecomposition altogether is to use methanolysisinstead of hydrolysis because the methylatedmonosaccharides are more stable (87).Excluding oxygen when hydrolysing with HClor trifluoroacetic acid also helps preventdecomposition of the monosaccharides (87).

Thermal hydrolysis of inulin

In the presence of water an increase intemperature leads to an increase in hydrolysisof inulin that follows first- or pseudo-first-orderkinetics at both neutral and acidic pHs (29, 89).The rate of hydrolysis of inulin at neutral pHcan be considered insignificant for processingtime frames up to ~ 60ºC, but can be more re-levant at higher temperatures (88, 89). Perhapsmore important is the effect that temperaturehas on hydrolysis of inulin in the acidicconditions often used in processing (4, 29).

Acid hydrolysis of inulin

Inulin is susceptible to acid hydrolysis due to itshigh energy content (77), the acid promotinghydrolysis by protonating the glycosidic oxygen,activating the leaving group (4, 89). Even indistilled water, where the dissolution of carbondioxide has reduced the pH marginally belowneutrality to less than 6.8, hydrolysis has beenobserved (7, 93). The hydrogen ion con-centration is found to affect the kinetics ofinulin hydrolysis in a first-order manner (29).The combination of the first- or pseudo-first-order kinetics of the hydrolysis of inulin withrespect to both acid concentration andtemperature (29, 89) have been used to showthat the hydrolysis of inulin follows theArrhenius rate law for temperatures, from 7 -130ºC, and pHs, from 2.0 - 4.2. These conditionsencompass those most commonly used ininulin processing (29).

Base hydrolysis of inulin

Base-induced hydrolysis of inulin occursthrough a carbonyl group, meaning it can only


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occur from the reducing end of an inulin chainthat does not contain a protecting glucosemoiety (2, 77, 94, 95). Given that commerciallyavailable inulin usually contains only a relativelysmall amount of reducing sugar such hydrolysisis generally slow. Nonetheless, during condi-tions of raised temperature, where the increasein thermally induced internal scission will formsmaller chains with reducing ends (8), base-induced hydrolysis can become significant (94).To avoid base hydrolysis of inulin at lowertemperatures, treatment with a reducing agent,such as sodium borohydride, can protect inulinas the terminal ketone is reduced to an alcohol(8).

Reducing chemistry of inulin

Inulin is ostensibly a non-reducing sugar, butthe existence of chains free of glucose, whethercreated by natural enzymatic processes or byinternal scission of the chains during hydrolysis,means that some small amount of reducibleinulin is invariably present (95). Such inulin canundergo reactions not accessible to non-reducing inulin, including, but not limited to,the basic hydrolysis described in the previoussection. Reduction of the acyclic reducing endketone to a secondary alcohol is anotherexample of this chemistry. Significantly thischemistry has been used to identify the amountof glucose-free chains, so that calculations ofDegree of Polymerisation (DP) based on ratiosof glucose to fructose become more accurate (95).

Supramolecular chemistry of inulin

Inulin is rich in hydroxyl groups that are able totake part in supramolecular interactions, inparticular hydrogen bonding. These interactionscan be both intermolecular and intramolecular,though modelling of the inulin structure basedon X-ray diffraction analysis of the solid formsuggests that crystalline inulin has onlyintermolecular hydrogen bonding betweenchains (72). The gelation of inulin is describedas particle gel in which three dimensionalnetworks form of aggregated colloidal particlesof inulin (28, 96). In this instance hydrogen

bonding between inulin particles is intrinsic togel formation and stabilisation.

PHYSICAL PROPERTIES OF INULIN

The polymeric structure of linear chains ofinulin resembles a polyethylene oxide back-bone, being made up of the anomeric C2carbon, the C1 carbon and its attached oxygenmolecule for each of the fructose units (Figure1). This means that only one atom of thepolymer backbone is attached to the fructosering making inulin unusually flexible inconformation for a polysaccharide. Thisconfers its ability to assemble into a range ofdifferent structures (13, 28, 66, 76, 77, 82, 97,98), which along with the high polydispersity ofinulin and rotational ability of the primaryhydroxyl groups, complicates the structuralanalysis of inulin (7, 76, 77). Nevertheless, manyinulin structures have slowly been elucidated.

Determination of degree of polymerisationand polydispersity

Some of the most important physical propertiesof inulin are the molecular weight of thepolymer and the polydispersity, because thesefactors have a large influence on its suitabilityfor various applications. Several chroma-tographic methods used for inulin are describedbelow. Preferably chromatography is done attemperatures and pH that will not increasehydrolysis (7, 19, 93), i.e. at neutral or slightlybasic pH and room temperature. Oftenchromatographic techniques have beencombined, utilising the optimal separation ofeach of the processes (90, 99). Generalweaknesses of chromatography for inulinanalysis are that it is only useful for puresamples, DP can be unreliable as differentfractions are known to run together, lack ofresolution for higher molecular weights and thelack of standards for calibration (17).

High Performance Liquid Chromatography

Standard HPLC techniques have effectivelyseparate low molecular weight oligomers of


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inulin up to a maximum of about DP 16 (17,19, 23, 68, 75, 86, 90, 100). Most usually thisanalysis used columns specifically designed forcarbohydrate analysis (19, 21, 75, 86, 90),requiring elution at pH and temperatures likelyto induce hydrolysis, thus affecting accuracy(19, 86). Further, these columns often usechemical functionalities, such as amino groups,and gel size that allow combinations ofseparation mechanisms such as ion exclusion,ion and ligand exchange, reversed phase,normal phase and even size exclusion. Thesecharacteristics are ideal for most carbohydrates,but the conformationally flexible backbone ofinulin means that in chromatography it behavesunusually for a polysaccharide so that simplereversed phase columns have been shown to besimilarly effective (68, 90, 100). Even with thesemore simple columns the conformationalvariation in inulin can cause problems. Forexample, elution from a C18 column gavefractions that clearly separate DP of 11-16,however, DP 8 and 9 eluted in the samefraction as did DP 6, 7 and 10 and DP from 2-5required separation using another technique(68). Indeed, in general most forms of HPLCare only capable of separating oligomeric formsof inulin and longer polymer chains elutetogether (100). One form of HPLC, however,does provide improved separation for a largerrange of DP, as follows.

H i g h p e r f o r m a n c e a n i o n e x c h a n g e

chromatography

High performance anion exchange chroma-tography (HPEAC) provides improved sensi-tivity and resolution in the analysis of poly-saccharides, compared to other types of HPLC,but also uses high salt concentrations, that canbe difficult to remove from the sugar afteranalysis (100). Different molecular weights ofinulin have been variously separated byHPEAC, most often using pulse amperometricdetectors (PAD) (1, 19, 21, 65, 68, 86, 93), butat least in one case a refractive index (RI)detector was used (99). PADs are usually usedbecause elution is mostly using a gradient ofacetate concentration (65, 99), and the RI

detector introduces the added complexity ofmaintaining a constant refractive index for thesolvent as the concentration of acetateincreases. The benefit is that the PAD and RIdetector responses can be compared to allowbetter quantitative analysis (99). HPAEC PADis able to provide improved separation of inulinoligomers and resolution of larger chains isbetter than for standard HPLC techniques (1).However, HPAEC PAD is still unable toresolve long chain polymers with much success,due to the lack of sensitivity for such species bythe PAD detector (1, 23).

One of the major issues for using HPAECPAD to determine inulin molecular weight hasbeen the lack of relevant standards, that is, itprovides qualitative, rather than quantitativeresults (1, 17, 23, 93). To this end Ronkart et al.(1) generated standards via hydrolysis of globeartichoke inulin by endo-inulinase into smalloligosaccharides (mostly 3 and 4 fructose units).Due to the large starting DP relatively fewglucose terminated oligomers are created, thuspresenting minimal interference in thedevelopment of fructose oligomer standards(1). The fructose oligomers were separated bysize exclusion chromatography and the DPdetermined by mass spectroscopy before use asstandards in HPAEC PAD (1). Neverthelesslonger chains have created problems andcomplete hydrolysis followed by end groupanalysis was required to quantify the longerchains.

Hydrolysis of inulin for determinationby HPLC

One way to improve the performance of HPLCis to hydrolyse the inulin first and then makedeterminations of the DP based on the ratio ofglucose and fructose, which can be separatedeasily using HPLC (1, 21, 93, 101, 102). Thisprocess, described as end group analysis, usesassumptions that can have their accuracyeroded by the presence of fructose, glucose andsucrose monomers in the initial material, as wellas, glucose-free chains of inulin. The effect ofthese interferants can be minimised by


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quantifying them before hydrolysis (21, 93,101), but even if this is accurately carried out,there will no separation by DP. Consequently,only an average measure of molecular weight isdetermined with no indication of polydispersity(93).

Gas chromatography

Gas chromatography (GC) has also been usedto determine the molecular weight of inulin,though only DP up to 9-10 can be volatilisedby silylation of extracted sugars, and longerchains require the hydrolysis of the inulinfollowed by end group analysis (17, 19, 23).Even to achieve the analysis of short chainoligomers requires the use of apolar columnscapable of heating to 440ºC (19, 23).

Linkage and branching of inulin has beendetermined by permethylation followed byreductive cleavage of the polymer and thenacetylation. Subsequent GC, with detection byeither flame ionisation (23) or massspectroscopy (64, 100), is able to separate andidentify the constituent sugars with varyingmethylation patterns, allowing branching to bedetermined. Similar techniques have also beenused to determine the quantity of reducingsugar in polysaccharides, by labelling thereducing end group through redox chemistryprior to hydrolysis (95).

Gel permeation chromatography

Gel Permeation Chromatography (GPC) orSize Exclusion Chromatography (SEC) has alsobeen used to determine the DP of inulin (54,79, 90, 103). The same issues arise as with otherchromatographic methods, with an inability toresolve larger polymers (maximum resolutionbeing up to a DP of about 10) and also withcalibration (79, 90, 98). Praznik et al. (79) triedto combat these problems by using polymal-totriose as a calibrant on a polyacrylamidecolumn. For higher molecular weights two dif-ferent columns in series were required, osten-sibly providing separation in the range of200-50,000 using the glucan series as a calibrant

(79). Though individual polymers can still notbe resolved by such analysis, at least the elutionis progressive, allowing reasonable calculationsof polydispersity (79, 103).

Microbial high molecular mass inulin

Very high molecular mass inulin, isolated frombacteria and fungi, has been analysed by GPCand found to have a small root-mean-squareradii of gyration with respect to their molecularmass, suggesting a compact molecular confor-mation (69, 71). Theoretical prediction and ex-perimental evidence both agree that a globularshape is likely (69). The globular shape is herepredicted to be created by branching (5-7%)and indeed variation between the branchingarchitectures of the bacterial and fungal inulinsare predicted by this work, despite both formshaving the same amount of branching (69). Aseparate theory suggests that if the inulin isessentially linear, due to limited branching, or ifthe branches are short then intramolecularsupramolecular associations between distantresidues can also cause the globular shape (71).

Molecular organisation of inulin

There is great variation in the molecularconformations for inulin for the shortestoligomers with DP of below about nine (75,98). It seems likely that some significantorganisation starts with oligomers of DP 4 and5 that are predicted to favour a cyclic (75, 99),or single helical structure (98). Oligomers ofDP 6-8 have organised though less welldetermined conformations (98), and this changein structure is responsible for the unusualsequence of chromatographic elution in thisDP range (discussed previously in the sectionon High Performance Liquid Chromatography)(23). This means the increase in chain lengththrough this range of DP provides somesignificant alterations to molecular packing suchthat the polarity exposed to solution and/or theshape of the oligomer changes sufficiently toaffect chromatography (75). Inulin oligomerswith a DP of 9 and above form a regular helicalstructure, argued both to be five- (75, 97) and


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six-fold helices (72). The right-handed six-foldhelical structure has generally become acceptednow. The helical structures of each chain areable to pack together into a range of isoforms,the nature of which is dependant on thekinetics and thermodynamics of formation.

Crystal structure of inulin

The more organised, crystalline structures ofinulin have been analysed by X-ray and electrondiffraction studies. This data has been used incombination with theoretical considerations (65,72, 74, 78, 97) to predict an isotactic arrangementof fructosyl units about the polymer backbone(97), as well as, the helical structure (72, 74, 97).Electron diffraction studies showed that helicalcrystalline inulin exists in two polymorphs, ahemihydrate and a monohydrate (72). Thehemihydrate form has one water molecule forevery two fructosyl subunits (72) and is createdby vacuum drying of the hydrated form,removing all the labile water molecules with theremaining water being securely bound in thecrystal structure (74). Due to the labile natureof the extra proportion of the water containedin the monohydrate the electron diffractionresults were less reproducible, but suggest thatit has one water molecule for each fructosylunit (72). It is the monohydrate that is likely tobe significant for medicinal aspects of inulin,and its crystal structure has been worked out asa unit cell containing two anti-parallel right-handed helices of six fructosyl residues perturn, made into a rigid packing structure by sixintermolecular hydrogen bonds, in some casesvia water molecules (72).

Crystalline isoforms of inulin

Perhaps the most useful property of inulin froma medicinal viewpoint is its occurrence inmultiple, distinct molecular packing structures(polymorphs or isoforms). Precipitation from

ethanol yields the â form while water yields ainulin at room temperature or lower (7, 54, 66,104, 105). Theoretical studies show that many

forms are allowable (69, 70). In practice it wasfound that both á- and â-forms were unstable

in water, soon progressing (54) to a novel third

form (g) that was largely insoluble at bodytemperature. This property allows a range ofstrong biological effects (12, 13, 15). A fourth

isoform, d (56) which is even more active hasnow been identified. These isoforms are allsoluble at concentrations of 10-15% w/w attemperatures below 75°C, enabling their readypurification for clinical use with minimalhydrolysis. They are conveniently distinguishedby their solubility temperatures in water (Figure4), representing a phase-shift as abrupt as amelting point.

The isoforms comprise an increasing series in

the sequence â-á-g-ä, in which lower isoformsare converted to higher ones at specifictemperatures, and all higher isoforms can bereturned to lower by complete dissolution andre-crystallisation. Thus the structures are for-med by reversible bonding rather thancovalently, and higher isoforms have a highermean DP (54). The isoforms spontaneouslyassume a characteristic molecular packingstructure, presumably determined by hydrogenbonding, when crystallised at specifictemperatures. This unusual behaviour isattributed to the flexible polyoxyethylene-likepolymer backbone (55), allowing multiple

Figure 4 The temperature solubilities of inulin isoforms

illustrated by the changes in turbidity of dilute suspensions

(<0.5 mg/mL) at different temperatures. The isoforms are

distinguished by several parameters that compare strengths

of intermolecular non-covalent bonding


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conformations both thermodynamically andkinetically favoured. The structural implicationsof the existence of such isoforms are ofconsiderable interest.

Inulin particles of the various isoformsnormally crystallise from water as ovoids of 1-10 µm diameter (14, 54) if the suspension isbriskly stirred. Particle size is determined by anumber of factors including temperature, inulinconcentration, ionic strength and rate of stir-

ring. The d isoform of inulin has been exploited(56) to form a range of much smaller particlesizes. In this method the ä particles are partiallydissolved, when they break into many verysmall fragments that in turn act as crystallisationmicronuclei to grow a larger number ofparticles from the same weight of inulin. Thenew particles are accordingly much smaller. Byfurther fragmenting the micro-nuclei while hotby shear stress (passing under pressure througha small orifice) or by ultrasonication, theparticle size is further reduced to form a widerange of sizes down to 100 nm in a mannercontrolled by the degree of shear stressintroduced. This is expected to expand thepotential uses of inulin.

Solubility and gelation of inulin

The variation in molecular conformation, alongwith DP, affects solubility and short chainoligomers of inulin are relatively soluble inaqueous solution being soluble at up to 80%concentration (28). Longer chain inulinpolymers are much less soluble and willprecipitate in the crystalline forms discussed inthe previous section. Due to a degree ofsurfactant character inulin can also gelateaqueous solutions from >25% inulin in waterfor shorter chains, and from >13% for longerchains by cooling a hot dissolved solution or byshearing inulin suspensions (22, 23, 27). Theseprocesses form a gel network of crystallineparticles of about 100 nm diameter thataggregate to form larger clusters of 1-5 µm (23,28), which trap a large amount of water in thenetwork. It has been shown that the thermalmethods used to produce inulin gels create

stronger and smoother gels than the shearingtechniques used more usually in industry due tothe generation of smaller, more narrowlydistributed particle sizes (27). A range of othervariables also affect the character of the finalgel including inulin concentration, pH, mole-cular weight and solvent (27, 28).

Inulin gels are important for several app-lications, currently the most commerciallyrelevant being in food production, where it isused as a low calorie, bulking agent, replacingfat, sugar and flour, while providing valuabledietary fibre (1, 3-5). Inulin gels imitate fatextremely well in terms of texture, mouth-feel,glossy appearance and balance of flavourrelease, with longer chain inulin polymersproviding the best results in terms of thesecharacteristics (23). Importantly, inulin exhibitssynergistic effects with most gelling agents,many being used in food production, togethercreating stronger gels than the sum of thecomponents (22, 23).

The prospective use of inulin gels in drugdelivery means that improvement to gelcharacteristics and to suitability of theirchemical nature have been made throughchemical modification (26, 31, 50, 51). Suchmodifications include functionalisation withvinyl groups for subsequent cross-linkingreactions to stabilise the gel structure (50, 51).Inulin gels have also been cross-linked byderivatisation with methacrylic anhydride andsuccinic anhydride, followed by UV irradiation.In this case gel swelling is pH sensitive, idealfor drug delivery to the colon (26).Supramolecular associations between inulinacetate and 1,12-dodecanedicarboxylic acidhave also been used to stabilise inulin gels fordrug delivery (31).

Amorphous inulin

Industrial inulin raw material produced by theconvenient and cost effective spray dryingprocedure is often amorphous (106, 107). Sucha structure is not kinetically in equilibrium


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because of the rapid drying process (107), andwhen heated above the glass transitiontemperature the amorphous solid canreorganise into crystalline forms. The glasstransition temperature can also be lowered bywater absorption, the water acting as aplasticizer for inulin (3).

Analysis of relative crystallinity of inulin

The state of inulin has significant effect on theutility and stability of the product, amorphousand semi-crystalline materials have the highestindustrial value (107, 108). Crystallisation ofamorphous and semi-crystalline inulin by waterabsorption or heating results in a reorganisationof molecular structure, leading to caking (3, 26,108). Caking of particulate inulin is due toextrusion of water which is no longer accom-modated in the more organised structure andthen absorbed onto the surface of neighbouringparticles, creating a liquid interface betweenthem. This sticks the particles together (108).Caked inulin is undesirable both as a rawmaterial and in final products, as thecrystalline/amorphous balance and processa-bility are changed (108). As such, this transitionbetween amorphous and crystalline inulins hasbeen extensively investigated.

One of the most important properties inunderstanding the relative crystalline oramorphous qualities of a polymer is the glass

gtransition temperature (T ). The glass transitionis an effect noticed in solid polymers with atleast some amorphous character in which thereis a reorganisation of polymer chains into moreordered crystalline forms at a temperaturebelow the melting point. This transition is easilyidentifiable by various techniques and may bealtered by a number of parameters such as theuse of plasticisers and curing.

Plasticising effect of water on inulin

gOne of the most investigated effects on the Tof inulin is the plasticising effect, or reduction

gin T , caused when water is absorbed byamorphous inulin (3, 65, 78, 106, 108). Water is

known to act as a plasticizer in hydrophilicpolymers such as gelatin (109). In suchpolymers, when reducing the temperaturebelow 0ºC, there is often water that is able tocrystallise (unbound water) and water thatcannot be crystallised (bound water) (109, 110).It is the uncrystallised water that effectivelymobilises the polymer chains, leading to thereorganisation and plasticising effect (65). For

ginulin this effect is significant with T goingfrom 110ºC to -10ºC as water content increasesfrom 0-15 g per 100 g of inulin in an essentiallylinear fashion (65). Crystallinity, as determinedby Wide Angle X-ray Scattering (WAXS) ana-lysis (see below), continues the trend observed

gin T showing that amorphous content disap-pears at 15.7 mg/100 mg and that the crystal-line character of inulin continues to increase upto 18.8 g of water to every 100 g of inulin (106).

g This means that when sufficiently wet, the T of

inulin can be depressed to the extent thatcrystallisation can occur at room temperature.Of course, given the multiple isoforms forcrystalline inulin, this process is not a simpleone.

The complexity of the crystallisation processdue to the plasticising effect of water has beeninvestigated using Differential ScanningCalorimetry (DSC). Semicrystalline andcrystalline samples often exhibit multipleendotherms for melting transitions between170ºC - 180ºC, due to the multiple crystallineisoforms of inulin. For humidity of 75% andabove for semicrystalline samples a third mel-ting endotherm develops at ~160ºC due toextra crystallinity induced by the reorderingpossible in a hydrated sample (3). Meanwhile anamorphous sample exposed to such humiditywill only develop a single melting endotherm at~160ºC (3, 108).

X-ray diffraction and scattering techniques havealso been used to investigate the plasticisingeffect of water on amorphous inulin (65, 78,106, 108). X-ray diffraction studies showed thatcrystallinity increases with increasing watercontent (65, 78, 106). Subsequent removal ofwater from samples crystallised by the


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plasticising effect of water does not remove thecrystalline character (65). More specificinvestigations made by WAXS showedcrystallinity developing for amorphous inulinwhen stored in humidity of 75% or above.Indeed, at 94% humidity greater crystallinity isdetected than that seen for a sample ofprecipitated crystals (108). The lower relativelevel of crystallinity observed for theprecipitated samples is attributed to ‘cross-linked’ crystalline domains, constraining thatportion of amorphous material caught betweenthem from reorganising into crystallinestructures. This constriction of the amorphousmaterial prevents the glass transition fromoccurring before melting, superimposing themelting and glass transition peaks (106, 108).

Visual inspection of amorphous inulin particlesby Environmental Scanning ElectronMicroscopy (ESEM) shows caking below 20ºCfor humidity between 59% and 75% (108). Thisindicates that some molecular reorderinginvolving the exclusion of water occurs athumidity lower than detectable using othertechniques.

Effect of Curing on inulin crystallisation

Many investigations have been conductedtrying to establish the effect of varyingcrystallisation regimes on the final make-up ofthe solid inulin. It has been found that wheninulin is dissolved in water at 96ºC, then cooledslowly to room temperature, and held at thistemperature for one hour, DSC shows twoendotherms for the dispersed mixture between40ºC and 90ºC that are indicative of crystallinemelting (66, 107, 108). After 48 hours curing atroom temperature two additional endothermsbetween 40ºC and 90ºC have reached maxi-mum intensity (66). It seems such endothermsare intrinsically related to the four endothermsalso found for the dry solid between 170ºC and180ºC in DSC analysis (90, 91). These meltingendotherms are attributable to differentcrystalline forms (107), possibly the four basiccrystalline species previously described as á-, â-, ã-and ä-isoforms (13, 54, 56). In another process

a solution was cooled from 96ºC to anintermediate curing temperature of 65ºC, whereit was held for 12 hours, before slowly coolingto room temperature. Initially in this case,again, only two endotherms between 40 and90ºC were observed using DSC, but only oneadditional endotherm formed in the sametemperature range after storage at roomtemperature (66). This endotherm occurs at thesame temperature of the highest temperatureendotherm for inulin recrystallised without thehot curing stage and has enthalpy equivalent tothe two highest endotherms found in thesample without hot curing (66). It seems likelythat the hot curing procedure strongly favourscrystallisation into only one of the hightemperature isoforms of inulin.

The various melting transitions for crystallineinulin induced by curing and plasticisers areadditionally affected by various parameters ofthe recrystallisation such as cooling rate, inulinconcentration and average molecular weight ofthe analysed inulin. Indeed, to a significantextent the varied crystallisation formsfractionate inulin by molecular weight.Unfortunately there is co-crystallisation at bothhigh and low temperatures with portions of lowmolecular weight material crystallising out withthe high molecular weight material at hightemperature and vice versa (66, 106). Despiteco-crystallisation, the character of the variouscrystalline forms differs, with Small Angle X-ray Scattering (SAXS) revealing different longperiods for structures crystallising at high andlow temperatures (66). Overall, it is clear fromthese results that inulin has a complex characterwith multiple crystalline forms, the formationof which is determined by the kinetics andthermodynamics of the recrystallisationprocedure (3, 66).E f f e c t o n c r y s t a l l i n e f o r m o f i n u l i n b y

crystallisation method and DP

As already stated, crystalline inulin created byhydration has a melting point of ~ 160C andthat created by precipitation melts between170ºC and 180ºC (66, 67, 103, 107, 108),


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depending on crystalline form and DP (103).Further investigation of this anomaly usedsamples crystallised by hydration and byprecipitation with roughly equal crystallinecontent, both being established as themonohydrate (106). Heating the precipitatedform in WAXS studies created a new peakattributed to the transition from themonohydrate to the hemihydrate. This samebehaviour did not occur for the amorphoussolid crystallised by hydration (3). TGA wasused to follow the same phenomenon, in whichsamples crystallised by hydration exhibited acontinuous mass loss from room temperatureto thermal degradation. In contrast, theprecipitated crystalline forms showed loss ofbound water mass up to 95ºC, where thethermogram levelled off before thermaldegradation. The onset of this plateaucorresponds to transition from monohydrate tohemihydrate as determined by WAXS (3).Furthermore, precipitated crystalline samplesdehydrated of labile water exhibit a mass loss at160-180ºC attributed to the release of waterfrom the hemihydrate crystals upon melting (3).These results show that there is a difference inthe mobility of the water in the crystals createdby different methods.

Another variation in the crystallisation of inulinis found between samples with differentmolecular weight. DSC of chains with differingchain length, separated by chromatography,shows an increasing melting point withincreasing chain length (67). These authorstheorize that for shorter chains the relativeamount of glucosyl end groups increasesincreasing the number of defects in the inulincrystal. As molecular weight increases so doesthe perfection of the crystal allowing for thickercrystals to grow, and a consequent increase inmelting temperature (67).SPECTROSCOPIC ANALYSIS OF INULIN

Nuclear Magnetic Resonance spectroscopy

Nuclear Magnetic Resonance (NMR)spectroscopy is an invaluable tool in theanalysis of organic compounds. Polysaccharides

are an example of such compounds for whichNMR analysis has been extensive (111-113),though the similar chemical environments ofmost of their detectable groups means thatshifts overlap each other to a large extent (75).This effect is worsened in the case of inulin dueto the lack of a proton on the anomeric carbon of the fructose component, a proton usuallyseparated from the other related protons sodiagnostically useful (75, 112). Efforts to reducethese issues have been attempted by improvingresolution and enhancing signals of the NMRusing a range of techniques. These includepulsed field gradients for suitably concentratedsamples (111), utilisation of 3-D COSY,TOCSY and NOESY to ameliorate the effectsof spectral overlap (111) and the use of hightemperatures, which makes for longeracquisition times but increases resolution andshifts the signal of the solvent to a lessinterfering higher field position (112). Solventselection is also important, deuterium oxidebeing able to resolve more peaks than

6deuterated dimethyl sulfoxide (DMSO-d ),

6though solubility issues mean that DMSO-d isoften used for polymeric forms (112, 114).Accuracy of quantification of NMR analysis canalso be improved by ensuring appropriateexperimental set-up relative to T1 relaxationand amelioration of nuclear Overhauser effectsby using a gated-decoupling pulse sequence(112).

Early experimentation seemed to focus oncarbon NMR (90, 113-116), probably becausethe resolution of proton NMR was too poorwith the relatively weak NMR machines thenavailable. Nonetheless, carbon NMR has beenimportant as it can differentiate inulin fromother polysaccharides (116) and explore substi-tution effects on the fructose ring (115, 116),being able to identify and quantify branching(64). It can also differentiate lower oligomers ofinulin up to about DP 4. This is because carbonshifts, particularly the fructose carbons closestto the polymer backbone, are sensitive tomolecular conformation of the backbone whichis dependant on DP (90, 98). Importantly muchof this differentiation of peaks remains for the


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first few fructose groups adjacent to the glucoseunit and do not coalesce as with some otherpolysaccharides.(98) This makes possibleanalyses based on these terminal groups (112).

A similar effect described above for thevariation in shifts for those carbons attachedclosest to the polymer backbone in fructoseunits positioned near the glucose unit is alsoobserved for the attached protons (75).Combining these one-dimensional analyseswith two-dimensional NMR has been used toaccurately allocate NMR shifts for inulin (75).This assignment has allowed conclusions aboutthe conformation of the structures of variousoligomers to be made based on the positioningand movement of shifts for specific protonsand carbons, predicting helical structures forlarger oligomers (75). Similarly, NMR for eacholigomer of DP from 2-12 was conducted insolutions increasing in the concentration ofbarium (II) cations (99). Addition of Ba2+

caused a change in chemical shift for severalprotons in the inulin chain that increasedlinearly with increasing salt concentration for agiven oligomer. This effect, due to complexingof inulin with Ba , was greatest on the2+

chemical shifts for oligomers of DP 4 and 5. Itwas attributed to these chain lengths having acyclic conformation in solution that closelymatches that of cycloinulo-hexaose, which isknown to strongly complex Ba (99). From2+

this it can be presumed that longer and shorteroligomers do not have the same ring-likeconformation (99).

Fluorescence spectroscopy

Inulin characterised by fluorescencespectroscopy after binding hydrophobic pyrenemolecules shows that the critical aggregationconcentration (cac) is from 0.07-0.08 mg/ml inaqueous solution (5). This is determined by thereduction in fluorescence during bindingbetween inulin and pyrene in solution. Incontrast, the pyrene molecules are less able tofind free hydrophobic binding spots in theaggregate and fluorescence remains high(5).The cac was marginally affected by the

presence of various salts in the solution (5).

Inulin samples from different sources wereanalysed by capillary electrophoresis, beingdetected by a fluorescent tag (117). Difficultiesare associated in not knowing how many timesa sugar group has been tagged as all thehydroxyl groups are susceptible to the reaction,the primary hydroxyls reacting faster (117).Even so, the authors concluded that inulinfrom different sources can be identified by thismethod (117).

Ultraviolet-visual spectroscopy

Though Ultraviolet-Visual (UV-Vis) spectros-copy is of limited direct analytical value forpolysaccharides, colorimetric response to redu-cing chemistry enables quantification of hydro-lysed polysaccharides (102, 118, 119). It alsoallows determination of the amount of reducingends in unhydrolysed samples (101, 120-127).For inulin, such quantification is important, notjust for determination of the stability of inulinsamples to reactions involving the reducingend, but also for accurate determination ofmolecular weight using end group analysis (17,94, 95, 101). Wight et al. (101) used tetrazoliumblue to quantify the amount of glucose-freechains in inulin samples so that end groupanalysis could be used to establish the DP fromthe HPLC of hydrolysed samples. Perhaps amore common reagent for the colorimetricdetection of reducing sugars is dinitrosalicylicacid (DNS) (102, 121, 126) that has also beenused to enable simple colorimetricmeasurement of inulin in mixtures containingother reducing sugars (102). In this case DNSwas used to first determine the reducing sugarcontent of a sample before the amount ofinulin was quantified by hydrolysis of thepolymer by sulphuric acid in the presence ofphenol (102). This research compared thecolorimetric results with HPLC methods andfound the HPLC method more precise andaccurate due to being less affected byinterfering compounds (102).

CONCLUSION


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Inulin is a natural polysaccharide with uniquephysicochemical properties that give it a rangeof uses in the food and pharmaceuticalindustry. A broad range of analytical tools hasbeen applied to inulin's characterisation. Anincreased understanding of the chemistry andbehaviour of inulin polymers has led toimportant new uses as a drug delivery vehicle,immuno-stimulator and vaccine adjuvant.

ACKNOWLEDGEMENTS

We particularly wish to thank FarukhKhambaty, our program officer at NIH, for hisgreat encouragement and support over the lastfive years. This work was supported by theNational Institute of Allergy and InfectiousDiseases, NIH (Grants U01-AI061142 andHHSN272200800039C). Its contents are solelythe responsibility of the authors and do notnecessarily represent the official views of theNational Institutes of Health or the NationalInstitute of Allergy and Infectious Diseases.

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