inulin pharma

8/9/2019 Inulin Pharma

1/24


2/24


3/24

Review Paper

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 of

which inhibit growth of colon cancer cells.Butyrate has multiple actions that may contribute to its anti-cancer effects including inhibition 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, presumably mediated 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 suppressing tumours may be mediated by its enhancementof gut immune function (37). Notably, inulin

has been shown to exert immuno-modulatory effects 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 capacity of peripheral blood mononuclear cells toproduce interferon gamma (39). Inulin-fed ratshad a higher number and proportion of dendritic cells in gut Peyer's patches, andgreater ex vivosplenocyte 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 of short 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 containing fructo-oligosaccharides enhanced influenza-specific delayed-type hyper-sensitivity responses in mice receiving influenza

vaccination (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, fasting glucose, fructosamine, HbA1c and insulinresistance as measured by homeostatic modelassessment (HOMA-IR) (46). Gastric emptying

was also significantly delayed in the groupreceiving inulin-enriched pasta. At least part of this favourable metabolic effect may bemediated by an effect of inulin on productionof a glucagon-like peptide (GLP)-1 mentioned

above.

Dietary inulin has also been shown to increasecalcium and magnesium absorption and bonemineralisation in young adolescents, primarily through 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 requiring treatment 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 enabling patient preferred mechanisms of admi-nistration, such as oral, rather than injectedroutes. Inulin is generally biochemically inert,non-toxic and can form hydrogels. These

This Journal is © IPEC-Americas Inc J. E xc ipients and Food Chem.1 (3) 2010 - 29


4/24

Review Paper

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 drug delivery 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 of diseases that show a peak in symptoms in theearly morning (9, 50, 51). Inulin is an ideal

vehicle 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 by colonic bacteria, releasing the drug payload.

While inulin hydrogels might be ideal for drug transport 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 subsequently cross-linked by free radical polymerisation (9,50, 51). Hydration of these dry materials occursrelatively quickly in a manner independent of the degree of cross-linking, until a certain value(~15% of fructosyl units substituted) isreached, whereupon further cross-linking leadsto increased swelling time (50). Exposure to

varying 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 relatively small (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 irradiation

with 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 the

outside of the microspheres. For inulin andinulin acetate 58-62% of the drug was released within the first 5 minutes whereas for modifiedinulin acetate only 32% was released within thefirst 15 minutes (31). After this time the drug

was released slowly by diffusion forapproximately 1 day, after which the erosion of the microspheres led to quicker release untilcomplete after 3.5 - 4 days (31).

Use of inulin as a stabiliser

Protein and peptide drugs, including monoclonal antibodies, are increasingly important as human therapeutics. A drawback to these drugs is their lability, particularly inaqueous solution, which may limit their shelf life. Drying is a way to avoid this, oftenrequiring use of a protective agent to prevent



5/24

Review Paper

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 prevent

denaturation. 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 a

protective agent in the freeze-drying of proteinand peptide based drugs include a high glasstransition temperature, low hygroscopicity, low crystallisation rate and a lack of reducing groups (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). 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 - and -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 inactivated

whole organisms. This poor immunogenicity can 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 of inulin require a thorough understanding of itschemistry and physico-chemical behaviours

which 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 by microorganisms 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 Aspergillus family (70, 71), although whether inulin isproduced natively by these fungi is debatable asthey have no significant natural source of sucrose (23).

Chemically described asá-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



6/24

Review Paper

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 chemistry the polymer can undergo is the chemistry of theconstituent sugar, even if some avenues of reactivity 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 ascyclic forms, in solution the free sugars have a

very 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, the

ring 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 opening and 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 andfructose

Figure 1 Inulin Polymer



7/24

Review Paper

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 and

water combining to make oxygen andformaldehyde (81). The manner in which theformaldehyde intermediates combine dictates

whether 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 of glycosidic bonds (77, 81, 82). This bondprevents the chemistry involved in the ring opening 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, but

which also provides an inert starting group inthe biosynthesis of inulin (Figure 3).

Polymerisation through the anomeric hydroxylgroup, as is the case for inulin, similarly protects 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 of the hydroxyl groups attached to each ring.

These alcohol groups are generally relatively inert, but can undergo a number of organic

reactions 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 readily undergoing nucleophilic substitution anddehydration reactions. Other reactions of alcoholic groups include esterification reactions

with 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 of both fructose and glucose components andattached to C1 of the terminal fructose moiety are more reactive than the other secondary hydroxyl groups making selective substitutionpossible (9).

Figure 3 Starting materials of the biosynthesis of inulin



8/24

Review Paper

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 of glucose and fructose, and by extension inulin,also makes supramolecular chemistry significant, with non-covalent interactions withother similarly polar molecules easily achieved.

The chemistry of inulin polymers andoligomers

Inulin is biosynthesized from a starting molecule of sucrose (Figure 3), explaining thepresence of the single glucose residue in thepolymer (84). Enzymes then progressively transfer 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, forming the 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 of inulin 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 long chains (23). In plants, 1-kestose is also used as asubstrate for chain elongation with new fructose being added, not from sucrose, butfrom 1-kestose or other nascent polymers using a 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 polymer with â (2-1) linking glycosidic bonds. Poly-merisation in this way means each incoming monomer 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 ostensibly non-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 energy storage by various plants, and this inertness of the 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 under

such conditions the enzyme that usually elongates the inulin chains can begin to addfructose to fructose monomers creating fructose only chains that have a reducing end(23). This is why significant quantities of inulinare found that have no glucose protecting groups 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 primary hydroxyl 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



9/24


10/24

Review Paper

occur from the reducing end of an inulin chainthat does not contain a protecting glucosemoiety (2, 77, 94, 95). Given that commercially available inulin usually contains only a relatively small 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 by internal 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 another

example of this chemistry. Significantly thischemistry has been used to identify the amountof glucose-free chains, so that calculations of Degree 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 only intermolecular 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 of inulin 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 of different structures (13, 28, 66, 76, 77, 82, 97,98), which along with the high polydispersity of inulin and rotational ability of the primary hydroxyl groups, complicates the structuralanalysis of inulin (7, 76, 77). Nevertheless, many inulin 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 suitability

for 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 slightly basic pH and room temperature. Oftenchromatographic techniques have beencombined, utilising the optimal separation of each of the processes (90, 99). General

weaknesses of chromatography for inulinanalysis are that it is only useful for puresamples, DP can be unreliable as differentfractions are known to run together, lack of resolution for higher molecular weights and thelack of standards for calibration (17).

High Performance Liquid Chromatography

Standard HPLC techniques have effectively separate low molecular weight oligomers of



11/24

Review Paper

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 likely to induce hydrolysis, thus affecting accuracy (19, 86). Further, these columns often usechemical functionalities, such as amino groups,and gel size that allow combinations of separation 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 of inulin 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 conformational

variation 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 rm a n c e a n i o n e x c h a n g echromatography

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 of inulin have been variously separated by HPEAC, 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 of acetate concentration (65, 99), and the RI

detector introduces the added complexity of maintaining a constant refractive index for thesolvent as the concentration of acetateincreases. The benefit is that the PAD and RIdetector responses can be compared to allow better 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 by the 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 few glucose terminated oligomers are created, thuspresenting minimal interference in thedevelopment of fructose oligomer standards(1). The fructose oligomers were separated by size exclusion chromatography and the DPdetermined by mass spectroscopy before use as

standards 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 of glucose and fructose, which can be separatedeasily using HPLC (1, 21, 93, 101, 102). Thisprocess, described as end group analysis, usesassumptions that can have their accuracy eroded by the presence of fructose, glucose andsucrose monomers in the initial material, as wellas, glucose-free chains of inulin. The effect of these interferants can be minimised by



12/24


13/24

Review Paper

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 of inulin 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 fructosyl

unit (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 cases

via 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 fromethanol yields the â form while water yieldsinulin 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 thirdform ( ) that was largely insoluble at body temperature. This property allows a range of strong biological effects (12, 13, 15). A fourthisoform, (56) which is even more active hasnow been identified. These isoforms are all

soluble at concentrations of 10-15% w/w attemperatures below 75°C, enabling their ready purification 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 inthe sequence â -á- -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 spontaneously assume a characteristic molecular packing structure, 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 isoformsillustrated by the changes in turbidity of dilute suspensions(


14/24

Review Paper

conformations both thermodynamically andkinetically favoured. The structural implicationsof the existence of such isoforms are of considerable 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 isoform of inulin has been exploited(56) to form a range of much smaller particlesizes. In this method the ä particles are partially dissolved, when they break into many very small fragments that in turn act as crystallisationmicronuclei to grow a larger number of particles from the same weight of inulin. Thenew particles are accordingly much smaller. By further 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, along with 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 of surfactant 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 by shearing 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 shearing techniques used more usually in industry due tothe generation of smaller, more narrowly distributed particle sizes (27). A range of other

variables 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 commercially relevant being in food production, where it isused as a low calorie, bulking agent, replacing fat, 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 drug delivery means that improvement to gelcharacteristics and to suitability of theirchemical nature have been made throughchemical modification (26, 31, 50, 51). Such

modifications include functionalisation with vinyl groups for subsequent cross-linking reactions to stabilise the gel structure (50, 51).Inulin gels have also been cross-linked by derivatisation 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 drying procedure is often amorphous (106, 107). Sucha structure is not kinetically in equilibrium



15/24

Review Paper

because of the rapid drying process (107), and when heated above the glass transitiontemperature the amorphous solid canreorganise into crystalline forms. The glasstransition temperature can also be lowered by

water 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 of amorphous 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 neighbouring particles, creating a liquid interface betweenthem. This sticks the particles together (108).Caked inulin is undesirable both as a raw material 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

g transition 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 easily identifiable by various techniques and may bealtered by a number of parameters such as theuse of plasticisers and curing.

Plasticising effect of water on inulin

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

g in T , caused when water is absorbed by amorphous 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 effectively mobilises the polymer chains, leading to thereorganisation and plasticising effect (65). For

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

g in 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 that

crystallisation 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 Scanning

Calorimetry (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 reordering possible in a hydrated sample (3). Meanwhile anamorphous sample exposed to such humidity

will only develop a single melting endotherm at~160ºC (3, 108).

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

water from samples crystallised by the



16/24

Review Paper

plasticising effect of water does not remove thecrystalline character (65). More specificinvestigations made by WAXS showedcrystallinity developing for amorphous inulin

when stored in humidity of 75% or above.Indeed, at 94% humidity greater crystallinity isdetected than that seen for a sample of precipitated 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 reordering involving the exclusion of water occurs athumidity lower than detectable using othertechniques.

Effect of Cu ring on inulin crystallisation

Many investigations have been conductedtrying to establish the effect of varying crystallisation regimes on the final make-up of the 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 melting endotherms are attributable to differentcrystalline forms (107), possibly the four basiccrystalline species previously described asá-, 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 cooling to room temperature. Initially in this case,again, only two endotherms between 40 and90ºC were observed using DSC, but only one

additional 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 likely that 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 of the recrystallisation such as cooling rate, inulinconcentration and average molecular weight of the 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 low molecular 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 long periods for structures crystallising at high andlow temperatures (66). Overall, it is clear fromthese results that inulin has a complex character

with multiple crystalline forms, the formationof which is determined by the kinetics andthermodynamics of the recrystallisation

procedure (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 ycrystallisation method and DP

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



17/24

Review Paper

depending on crystalline form and DP (103).Further investigation of this anomaly usedsamples crystallised by hydration and by precipitation with roughly equal crystallinecontent, both being established as themonohydrate (106). Heating the precipitatedform in WAXS studies created a new peak attributed 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 of bound 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 differing chain 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).SPECTROSCO PIC 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 of most 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 carbonof the fructose component, a proton usually separated from the other related protons sodiagnostically useful (75, 112). Efforts to reducethese issues have been attempted by improving resolution and enhancing signals of the NMR using 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 is

often used for polymeric forms (112, 114). Accuracy of quantification of NMR analysis canalso be improved by ensuring appropriateexperimental set-up relative to T1 relaxation

and 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 poor

with 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 of inulin 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



18/24

Review Paper

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 the variation 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 analyses

with 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 positioning and 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 of barium (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 complexing of inulin with Ba , was greatest on the2+chemical shifts for oligomers of DP 4 and 5. It

was attributed to these chain lengths having a

cyclic conformation in solution that closely matches 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 binding between 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, being detected 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 reducing ends in unhydrolysed samples (101, 120-127).For inulin, such quantification is important, notjust for determination of the stability of inulinsamples to reactions involving the reducing end, but also for accurate determination of molecular weight using end group analysis (17,94, 95, 101). Wight et al. (101) used tetrazoliumblue to quantify the amount of glucose-free

chains 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 containing other reducing sugars (102). In this case DNS

was used to first determine the reducing sugarcontent of a sample before the amount of inulin was quantified by hydrolysis of thepolymer by sulphuric acid in the presence of phenol (102). This research compared thecolorimetric results with HPLC methods andfound the HPLC method more precise andaccurate due to being less affected by interfering compounds (102).

CONCLUSION



19/24

Review Paper

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 solely the responsibility of the authors and do notnecessarily represent the official views of theNational Institutes of Health or the NationalInstitute of Allergy and Infectious Diseases.

REFERENCES

1. Ronkart S.N. , et al., Isolation and Identification of Inulooligosaccharides Resulting from InulinHydrolysis. Analytica Chimica Acta, 604: 81-87,2007.

2. Stevens C.V., Meriggi A. and Booten K., ChemicalModification of Inulin, a Valuable RenewableResource, and Its Industrial Applications.Biomacromolecules, 2: 1-16, 2001.

3. Ronkart S.N. , et al., Impact of the CrystallisationPathway of Inulin on Its Mono-Hydrate to Hemi-Hydrate Thermal Transition. Food Chemistry, 119:317-322, 2010.

4. Matusek A. , et al., Effect of Temperature and pHon the Degradation of Fructo-Oligosaccharides.European Food Research and Technology, 228:355-365, 2009.

5. Dan A., Ghosh S. and Moulik S.P., PhysicochemicalStudies on the Biopolymer Inulin: A CriticalEvaluation of Its Self-Aggregation, Aggregate-Morphology, Interaction with Water, and ThermalStability. Biopolymers, 91: 687-699, 2009.

6. Fuchs A., Potentials for Non-Food Utilization of Fructose and Inulin. Starch-Starke, 39: 335-343,1987.

7. Phelps C.F., The Physical Properties of Inulin

Solutions. Biochemical Journal, 1965.

8. BeMiller J.N., Steinheimer T.R. and Allen E.E., Alkaline Degrad ation of Inu lin and Its StructuralImplications. Clinical Chem istry, 13: 261-269, 1967.

9. Vervoort L. , et al., Inulin Hydrogels as Carriers forColonic Drug Targeting: I. Synthesis andCharacterization of Methacrylated Inulin andHydrogel Formation. Pharmaceutical Research, 14:1730-1737, 1997.

10. Hinrichs W.L.J., Prinsen M.G. and Frijlink H.W.,Inulin Glasses for the Stabilization of TherapeuticProteins. International Journal of Pharmaceutics,215: 163-174, 2001.

11. Korbelik M. and Cooper P.D., Potentiation of Photodynamic Therapy of Cancer by Complement:

The Effect of Gam ma-Inulin . British Journal Of Cancer, 96: 67-72, 2007.

12. Cooper P.D. and Carter M., The Anti-Melanoma Ac tiv ity of Inulin in Mice. Mo lecular Immunology,23: 903-908, 1986.

13. Silva D.G., Cooper P.D. and Petrovsky N., Inulin-Derived Adjuvants Efficiently Promote Both Th1and Th2 Immune Responses. Immunology and CellBiology, 82: 611-616, 2004.

14. Cooper P.D. and Steele E.J., Algammulin, a New Vaccine Ad juv ant Co mp ris ing Gamma InulinParticles Containing Alum: Preparation and in VitroProperties. Vaccine, 9: 351-357, 1991.

15. Cooper P.D. and Steele E.J., The Adjuvanticity of Gamma Inulin. Immunology & Cell Biology, 66:345-352, 1988.

16. Cooper P.D., McComb C. and Steele E.J., The Ad juv antic ity of Algammu lin, a New Vaccine Ad juv ant. V accine, 9 : 408-415, 19 91.

17. Coussement P., Inulin and Oligofructose as Dietry Fibre: Analytical, Nutritional and Legal Aspects, inCho S. , et al. (eds), Complex Carbohydrates inFood. Marcel Dekker, Inc, New York, pp 203-212,1999

18. Roberfroid M.B., Introducing Inulin-Type Fructans.British Journal Of Nutrition, 93: S13-S25, 2005.

19. Vanloo J. , et al., On the Presence of Inulin andOligofructose as Natural Ingredients in the W esternDiet. Critical Reviews In Food Science AndNutrition, 35: 525-552, 1995.

20. K oo H .-N . , et al., Inulin Stimulates NO Synthesis Via Activ ation of PK C- á and Protein TyrosineKinase, Resulting in the Activation of NF- b by IFN- ã-Primed Raw 264.7 Cells. The Journal of Nutritional Biochemistry, 14: 598-605, 2003.



20/24

Review Paper

21. Dysseler P. and Hoffem D., Determination of Inulin and Oligofructose in Food Products(Modified AOAC Dietry Fibre Method), in Cho S. ,et al. (eds), Complex Carbohydrates in Food. MarcelDekker, Inc, New York, pp 213-227, 1999

22. Blecker C. , et al., Inulin: Its PhysicochemicalProperties and Technological Functionality. RecentRes Devel Agricultural & Food Chem, 5: 125-131,2001.

23. Franck A. and De Leenheer L., Inulin, inSteinbuchel A. , et al . (eds), Biopolymers,Polysacchar ides I I : Polysacchar ides f romEukaryotes. Wiley-VCH Verlag GmbH, Berlin, pp439-479, 2002

24. Reimer R.A. and McBurney M.I., Dietary FibreModulates Intestinal Proglucagon MessengerRibonucleic Acid and Postprandial Secretion of Glucagon-Like Peptide-1 and Insulin in Rats.Endocrinology, 137: 3948-3956, 1996.

25. Delzenne N.M., Cani P.D. and Neyrinck A.M.,Modulation of Glucagon-Like Peptide 1 andEnergy Metabolism by Inulin and Oligofructose:Experimental Data. The Journal of Nutrition, 137:2547S-2551S, 2007.

2 6. C astelli F. , et al., Differential Scanning Calorimetry Study on Drug Release from an Inulin-BasedHydrogel and Its Interaction with a BiomembraneModel: pH and Loading Effect. European Journalof Pharmaceutical Sciences, 35: 76-85, 2008.

27. Kim Y., Faqih M.N. and Wang S.S., Factors Affec ting Ge l Form ation of Inu lin . Carbohyd rat ePolymers, 46: 135-145, 2001.

28. Roberfroid M.B., Inulin-Type Fructans: FunctionalFood Ingredients. CRC Press, Boca Raton, 2005.

29. Blecker C. , et al., Kinetic Study of the AcidHydrolysis of Various Oligofructose Samples.

Journal of Agricultural and Food Chem ist ry, 50:1602-1607, 2002.

30. Petrovsky N., Novel Human Polysaccharide Ad juv ants with Dual Th1 and Th2 Poten tia ting Ac tiv ity. Vaccine, 24: S26-S29, 2006.

31. Poulain N. , et al., Microspheres Based on Inulin forthe Controlled Release of Serine ProteaseInhibitors: Preparation, Characterization and in

Vi tro Release. Journal of Controlled Release, 92 :27-38, 2003.

32. Fuchs A., Production and Utilization of Inulin PartII. Utilization of Inulin, in Suzuki M. andChatterton N.J. (eds), Science and Technology of Fructans. CRC Press, Inc, Boca Raton, pp 319-352,1993

33. Sealy L. and Chalkley R., The Effect of SodiumButyrate on Histone Modification. Cell, 14: 115-

121, 1978.

34. Taper H.S. and Roberfroid M., Influence of Inulinand Oligofructose on Breast Cancer and TumorGrowth. Journal of Nutrition, 1999.

35. Van Loo J. , et al., The Syncan Project: Goals, Set-up, First Results and Settings of the HumanIntervention Study. British Journal Of Nutrition,93: S91-S98, 2005.

36. R after J. , et al., Dietary Synbiotics Reduce CancerRisk Factors in Polypectomized and Colon CancerPatients. American Journal of Clinical Nutrition, 85:488, 2007.

37. Roller M., Rechkemmer G. and Watzl B., PrebioticInulin Enriched with Oligofructose in Com bination

with the Probiotic s Lacto bacil lus Rham nosus andBifidobacterium Lactis Modulates IntestinalImmune Functions in Rats. Journal of Nutrition,134: 153, 2004.

3 8. De Medina F.S .n . , et al., Non-Absorbable Glucids(Active Hexose Correlated Compound, Inulin andFructooligosaccharides) Exert Immunomodulatory Effects and Induce Differentiation in SeveralIntestinal Cell Types That Are Independent of

Their Prebiot ic Actions. Proceed ings of theNutrition Society, 69: E241, 2010.

39. Roller M . , et al., Consumption of Prebiotic InulinEnriched with Oligofructose in Combination withthe Probiotics Lactobacillus Rhamnosus andBifidobacterium Lactis Has Minor Effects onSelected Immune Parameters in Polypectomisedand Colon Cancer Patients. British Journal Of Nutrition, 97: 676-684, 2007.

40. Ryz N.R., Meddings J.B. and Taylor C.G., Long-Chain Inulin Increases Dendritic Cells in the Peyer'sPatches and Increases Ex Vivo Cytokine Secretionin the Spleen and Mesenteric Lymph Nodes of Growing Female Rats, Independent of Zinc Status.British Journal Of Nutrition, 101: 1653-1663, 2009.

41. Seifert S. and Watzl B., Inulin and Oligofructose:Review of Experimental Data on ImmuneModulation. Journal of Nutrition, 137: 2563S-2567,2007.

42 . B enyacoub J. , et al., Feeding a Diet Containing aFructool igosacchar ide Mix Can EnhanceSalmonella Vaccine Efficacy in Mice. Journal of Nutrition, 138: 123-129, 2008.

43. Vos A.P. , et al ., A Specific PrebioticOligosaccharide Mixture Stimulates Delayed-TypeHypersensitivity in a Murine Influenza VaccinationModel. International Immunopharmacology, 6:1277-1286, 2006.

44. Vos A.P. , et al., Dietary Supplementation of Neutraland Acidic Oligosaccharides Enhances Th1-Dependent Vaccination Responses in Mice.



21/24

Review Paper

Pediatric Allergy And Immunology, 18: 304-312,2007.

45. M eijer s B.K .I. , et al., P-Cresyl Sulfate SerumConcentrations in Haemodialysis Patients AreReduced by the Prebiotic Oligofructose-EnrichedInulin. Nephrology Dialysis Transplantation, 25:219-224, 2010.

46. Russo F. , et al., Metabolic Effects of a Diet withInulin-Enriched Pasta in Healthy Young

Vo lun tee rs. Cu rrent Pharma ceu tical Design, 16:825-831, 2010.

4 7. A bram s S .A . , et al., A Combination of PrebioticShort- and Long-Chain Inulin-Type FructansEnhances Calcium Absorpt ion and BoneMineralization in Young Adolescents. Am J ClinNutr, 82: 471-476, 2005.

48. Coudray C., Demigne C. and Rayssiguier Y., Effectsof Dietary Fibers on Magnesium Absorption in

An imals and Humans. Journal of Nutr ition, 133: 1-4, 2003.

49. Abram s S.A. , et al., An Inulin-Type FructanEnhances Calcium Absorption Primarily Via anEffect on Colonic Absorption in Humans. Journalof Nutrition, 137: 2208-2212, 2007.

5 0. Vervoort L . , et al., Inulin Hydrogels. I. Dynamic andEquilibrium Swelling Properties. International

Journal of Pharm aceutics, 1 72: 127-135, 1998.

5 1. Vervoort L . , et al., Inulin Hydrogels. II. In VitroDegradation Study. International Journal of Pharmaceutics, 172: 137-145, 1998.

52. Rossi S. , et al., Stabilization of the RestrictionEnzyme EcoRI Dried with Trehalose and OtherSelected Glass-Forming Solutes. Biotechnology Progress, 13: 609-616, 1997.

53. Slade L. and Levine H., Beyond Water Activity:Recent Advances Based on an Alternative

Approach to the Asses sment of Food Qua lity andSafety. Critical Reviews in Food Science &Nutrition, 30: 115-360, 1991.

54. Cooper P.D. and Carter M., Anti-Complementary Ac tion of Polym orph ic "Solubil ity Form s" Of Particulate Inulin. Molecular Immunology, 23: 895-901, 1986.

55. Cooper P.D., Vaccine Adjuvants Based on GammaInulin, in Powell M.F. and Newman M.J. (eds),

Vaccine Design: The Subunit Approach. PlenumPublishing, New York, pp 559-580, 1995

56. Cooper P.D. and Petrovsky N., New PolymorphicForm of Inulin and Uses Thereof, Organization

W.I.P . WO 2006/024100 A 1 (2006)

57. Petrovsky N. and Aguilar J.C., Vaccine Adjuvants:Current State and Future Trends. Immunology and

Cell Biology, 2004.

58. Petrovsky N., Freeing Vaccine Adjuvants fromDangerous Immunological Dogma. Expert Review of Vaccines, 7: 7-10, 2008.

59. Audibert F.M. and Lise L.D., Adjuvants: CurrentStatus, Clinical Perspectives and Future Prospects.Immunology Today, 14: 281-284, 1993.

60. Aguilar J.C. and Rodriguez E.G., Vaccine Ad juv ants Revis ited. Vaccine, 25: 3 752-3 762, 2007.

61. D eol H .S. , et al., The Influence of Gamma Inulinand Algammulin on the Immune Response inSheep to a Recombinant Antigen of Taenia Ovis.

Vaccine , 13: 4 29-43 3, 1995.

62. Brewer J.M., (How) Do Aluminium Adjuvants Work ? Im munolo gy Letters, 102: 10-15 , 2006.

63. Rosen F.S., Steiner L.A. and Unanue E.R.,Dictionary of Immunology. The Macmillan PressLtd., London, 1989.

64. Carpita N.C., Housley T.L. and Hendrix J.E., New Features of Plant-Fructan Structure Revealed by Methylation Analysis and Carbon-13 N.M.R.-Spectroscopy. Carbohydrate Research, 217: 127-136, 1991.

65. Ronkart S. , et al., Determination of PhysicalChanges of Inulin Related to Sorption Isotherms:

An X-Ray Diffr action, Modulat ed Diffe rentialScanning Calorimetry and Environmental Scanning Electron Microscopy Study. CarbohydratePolymers, 63: 210-217, 2006.

6 6. Hébette C.L .M . , et al., Complex Melting of Semi-Crystalline Chicory ( Cichorium Intybus L .) RootInulin. Carbohydrate Research, 310: 65-75, 1998.

67. Blecker C. , et al., Characterisation of DifferentIn u l i n S am p l es b y DS C : In f l uence o f Polymerisation Degree on Melting Temperature.

Journal of Thermal An alysis and Calo rim etry, 71 :215-224, 2003.

6 8. Tim merm an s J.W. , et al., Quantitative-Analysis of the Molecular-Weight Distribution of Inulin by Means of Anion-Exchange HPLC with Pulsed

Am perometr ic De tec tion. Journal of CarbohydrateChemistry, 13: 881-888, 1994.

69. Wolff D. , et al., Globular Shape of High Molar M assInulin Revealed by Static Light Scattering and

Visco metry . Polymer, 41: 8009-8016, 2 000.

70. K urakake M. , et al., Two Types of â -Fructofuranosidases from Aspergillu s Oryza e Kb .

Journal of Ag ricult ural and Food Ch em istry , 56 :591-596, 2007.

71 . K itam ura S . , et al., Conformation of (2 1)-â -D-Fructan in Aqueous Solution. International Journal



22/24

Review Paper

of Biological Macromolecules, 16: 313-317, 1994.

72. Andre I. , et al., Molecular and Crystal Structures of Inul in f rom Elect ron Di ff ract ion Data .Macromolecules, 29: 4626-4635, 1996.

73. Grube M. , et al., Infrared Spectra of Some Fructans.Spectroscopy, 16: 289-296, 2002.

74. André I. , et al., Single Crystals of Inulin.International Journal of Biological Macromolecules,18: 195-204, 1996.

75. Oka M. , et al., Studies on the Conformational Aspec ts of Inul in Ol igomers. Chemical &Pharmaceutical Bulletin, 40: 1203-1207, 1992.

76. French A.D., Accessible Conformations of the â -D-(2 1)-Linked and -(2 6)-Linked D-FructansInulin and Levan. Carbohydrate Research, 176: 17-29, 1988.

77. French A.D., Chemical and Physical-Properties of Fructans. Journal Of Plant Physiology, 134: 125-136, 1989.

78. Zimeri J.E. and Kokini J.L., The Effect of MoistureContent on the Crystallinity and Glass Transition

Temperature of Inulin . Carbohyd rate Polym ers, 48 :299-304, 2002.

79. Praznik W. and Beck R.H.F., Application of Gel-Permeation Chromatographic Systems to theDetermination of the Molecular-Weight of Inulin.

Journal Of C hrom atography, 348: 187-19 7, 1 985.

80. Koch K. , et al., Influence of Harvest Date on InulinChain Length Distribution and Sug ar Profile for SixChicory ( Cichorium Intybus L) Cultivars. Journal of the Science of Food and Agriculture, 79: 1503-1506, 1999.

81. Kuszmann J., Introduction to Carbohydrates, inLevy D.E. and Fügedi P. (eds), The OrganicChemistry of Sugars. Taylor & Francis G roup, BocaRaton, pp 2006

82. French A.D. and Waterhouse A.L., ChemicalStructure and Characteristics, in Suzuki M. andChatterton N.J. (eds), Science and Technology of Fructans. CRC Press, Inc, Boca Raton, pp 41-82,1993

83. Shallenberger R., Intrinsic Chemistry of Fructose.Pure and Applied C hemistry, 50: 1409-1420, 1978.

84. Wack M. and Blaschek W., Determination of theStructure and Degree of Polymerisation of Fructansfrom Echinacea Purpurea Roots. CarbohydrateResearch, 341: 1147-1153, 2006.

85. D e Bruyn A . , et al., Isolation and Identification of O - â - D - F r u c t o f u r a n o s y l - ( 2 1 ) - O - â - D -Fructofuranosyl-(2 1)-D-Fructose, a Product of the Enzymatic Hydrolysis of the Inulin from

Cichorium Intybus . Carbohydrate Research, 235: 303-308, 1992.

86. De Leenheer L. and Hoebregs H., Progress in theElucidation of the Composition of Chicory Inulin.Starch-Starke, 46: 193-196, 1994.

87. Biermann C.J., Hydrolysis and Other Cleavages of Glycosidic Linkages in Polysaccharides. Advancesin Carbohydrate Chemistry and Biochemistry, 46:251-271, 1988.

88. Shu C.-K., Flavor Components Generated fromInulin. Journal of Agricultural and Food C hemistry,46: 1964-1965, 1998.

89. L'Hom me C . , et al., Kinetics of Hydrolysis of Fructooligosaccharides in Mineral-Buffered

Aqu eo us So lu ti on s: In fluence of pH an d Temperature. Journal of Agricultural and FoodChemistry, 51: 224-228, 2003.

90. Heyraud A., Rinaudo M. and Taravel F.R., Isolationand Character izat ion of Ol igosacchar idesContaining D-Fructose from Juices of the

Jerusalem Artichoke - Kinetic Constan ts for Acid-Hydrolysis. Carbohydrate Research, 128: 311-320,1984.

91. Edward J., Stability of Glycosides to AcidHydrolysis: A Conformational Analysis. Chemistry and Industry, 1102-1104,

92. Moiseev Y.V., Khalturinskii N.A. and Zaikov G.E., The Mechanism of the Acid-Cata lysed Hydroly sisof Polysaccharides. Carbohydrate Research, 51: 39-54, 1976.

93 . R aessler M . , et al., Determination of Water-Extractable Nonstructural Carbohydrates, Including Inulin, in Grass Samples with High-Performance

An ion Exchange Ch romato graphy and Pulsed Am perometr ic De tection . Journal of Agriculturaland Food Chem istry, 56: 7649-7654, 2008.

94. BeMiller J.N., Isolation and Hydrolysis of Alkali-Stable Inulin. Carbohydrate Research, 21: 154-155,1972.

95. Courtin C.M., Van den Broeck H. and Delcour J.A.,Determination of Reducing End Sugar Residues inOligo- and Polysaccharides by Gas-LiquidChromatography. Journal of Chromatography A,866: 97-104, 2000.

96. Dickinson E. and McClements D.J., Advances inFood Colloids. Blackie Academic & Professional,Glasgow, 1996.

97. Marchessaul t R .H. , et al., Conformation andCrystalline Structure of (2 1)-â -D-Fructofuranan(Inulin). Canadian Journal Of Chemistry-RevueCanadienne De C himie, 58: 2415-2422, 1980.

98. Liu J.H., Waterhouse A.L. and Chatterton N.J., Do



23/24

Review Paper

Inulin Oligomers Adopt a Regular Helical Form inSolution. Journal of Carbohydrate Chemistry, 13:859-872, 1994.

9 9. Tim merm an s J. , et al., The Interaction of InulinOligosaccharides with Ba2+ Studied by H NMR 1

Spectroscopy. Journal of Carbohydrate Chemistry,16: 213-230, 1997.

100. Bancal P., Gibeaut D.M. and Carpita N.C., Analy tical Methods for the Dete rm ina tion of Fructan Structure and Biosynthesis, in Suzuki M.and Chatterton N.J. (eds), Science and Technology of Fructans. CRC Press, Inc, Boca Raton, pp 84-118, 1993

101. Wight A.W. and Van Niekerk P.J., Determinationof Reducing Sugars, Sucrose, and Inulin in Chicory R o o t b y H i g h - P e r f o r m a n c e L i q u i dChromatography. Journal of Agricultural and FoodChemistry, 31: 282-285, 1983.

102 . Wang H . , et al., A Comparative Study of High-Performance Liquid Chromatography andColorimetric Method for Inulin Determination.European Food Research and Technology, 230:701-706, 2010.

103. Heyer A.G. , et al., Structure of the Enzymatically Synthesized Fructan Inulin. Carbohydrate Research,313: 165-174, 1998.

104. Katz J.R. and Weidinger A., Polymorphism of Substances of High Molecular Weight. II.

Am orphous and Crystall ine Inul in. Recueil des Travaux Ch imique des Pays-B as et de la Belgique,50: 1133-1137, 1931.

105. McDonald E.J., The Polyfructosans and Difructose Anhydr ides. Advances in Carbohyd rate Ch em istry,2: 253-277, 1945.

106. Ronkart S.N. , et al., Effect of Water Uptake on Am orphous Inu lin Propert ies . Food Hy drocolloid s,23: 922-927, 2009.

107. Ronkart S . , et al., Characterization of the PhysicalState of Spray-Dried Inulin. Food Biophysics, 2: 83-92, 2007.

108. Ronkart S.N. , et al., Impact of the Crystallinity onthe Physical Properties of Inulin During WaterSorption. Food Biophysics, 4: 49-58, 2009.

109. Apostolov A.A. , et al., DSC and TGA Studies of theBehavior of Water in Native and CrosslinkedGelatin. Journal of Applied Polymer Science, 71:465-470, 1999.

110. Fielden K.E. , et al., Thermal Studies on theInteraction of Water and Microcrystalline Cellulose.

Journal Of Pharm acy And Pharm acology, 40: 674-678, 1988.

111. Van Halbeek H., NMR Developments in Structural

Studies of Carbohydrates and Their Complexes.Current Opinion in Structural Biology, 4: 697-709,1994.

112. Gidley M.J., Quantification of the StructuralFeatures of Starch Polysaccharides by N.M.R.-Spectroscopy. Carbohydrate Research, 139: 85-93,1985.

113. Bock K., Pedersen C. and Pedersen H., Carbon-13Nuc l ea r M agn e t i c R es onan ce Da t a fo rOligosaccarides. Advances in CarbohydrateChemistry and Biochem istry, 42: 193-225, 1984.

114. Jarrell H.C. , et al., Manifestation of Anomeric Form,Ring Structure, and Linkage in the C-N.M.R.13

Spectra of Oligomers and Polymers Containing D-Fructose - Maltulose, Isomaltulose, Sucrose,Leucrose, 1-Kestose, Nystose, Inulin, and GrassLevan. Carbohydrate Research, 76: 45-57, 1979.

115. Seymour F.R. , et al., C-Nuclear Magnetic-13

Resonance Spectra of Compounds Containing â -D-Fructofuranosyl Groups or Residues. CarbohydrateResearch, 72: 57-69, 1979.

116. Shimamura A. , et al., Structural Determination of D-Fructans from Streptococcus-Mutans , Serotype-B,Serotype-C, Serotype-E and Serotype-F Strains, by

C-NMR Spectroscopy. Carbohydrate Research,13

165: 150-154, 1987.

117. Peng J., Lynen F. and Sandra P., Characterization of Inulins by Capillary Electrophoresis with LaserInduced Fluorescence Detection. Journal of HighResolution Chromatography, 23: 656-658, 2000.

118. DuBois M. , et al., Colorimetric Method forDetermination of Sugars and Related Substances.

An aly tical Ch em ist ry, 28: 350-35 6, 1956.

119. Messineo L. and Musarra E., SensitiveSpectrophotometric Determination of Fructose,Sucrose, and Inulin without Interference from

Aldohexoses , Aldopento ses, and Ke top entoses .International Journal of Biochemistry, 3: 691-699,1972.

120. Somogyi M., Notes on Sugar Determination. Journal of B iological C hemistry, 195: 19-2 3, 1952.

121. Miller G.L., Use of Dinitrosalicylic Acid Reagentfor Determination of Reducing Sugar. AnalyticalChemistry, 31: 426-428, 1959.

122. Anthon G. and Barrett D., Determination of R e d u c i n g S u g a r s w i t h 3 - M e t h y l - 2 -B e n z o t h i a z o l i n o n e h y d r a z o n e . A n a l y t i c a lBiochemistry, 305: 287-289, 2002.

123. Waffenschmidt S. and Jaenicke L., Assay of Reducing Sugars in the Nanomole Range with 2,2'-Bicinchoninate. Analytical Biochemistry, 165: 337-340, 1987.



24/24

Review Paper

124. Garcia E . , et al., Assessment of Endo-1,4- â -D-Glucanase Activity by a Rapid Colorimetric Assay Using Disodium 2, 2'-Bicinchoninate. Journal of Food Biochemistry, 17: 135-145, 1993.

125. Shahine S. and Mahmoud R., IndirectSpectrophotometric Determination of Reducing Sugars and Ascorbic Acid Using Phenolphthalin asReagent. Microchimica Acta, 1980.

1 26. H u R . , et al., Reducing Sugar Content inHemicellulose Hydrolysate by DNS Method: ARevisit. Journal of Biobased Materials andBioenergy, 2: 156-161, 2008.

127. Prado F.E. , et al., A Simple and Sensitive Methodfor Determining Reducing Sugars in Plant Tissues.

Applica tion to Quantify the Sugar Content inQuinoa ( Chenopodium Quinoa Willd.) Seedlings.Phytochemical Analysis, 9: 58-62, 1998.

inulin pharma

Documents