Levan-Application and Perspectives

8/11/2019 Levan-Application and Perspectives

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Levan: Applications and Perspectives

Soon Ah Kang, Ki-Hyo Jang, Jeong-Woo Seo, Ki Ho Kim, Young

Heui Kim, Dina Rairakhwada, Mi Young Seo, Jae Ok Lee, Sang Do

Ha, Chul-Ho Kim and Sang-Ki Rhee

consists of --fructofuranosyl residues linked

predominantly through -(2,6) as 6-kestose ofthe basic trisaccharide, with extensive branchingthrough -(2,1) linkages. In contrast, inulin iscomposed of --fructofuranose attached by-(2,1) linkages (Han 1996).

Levan is a diversely distributed component,particularly in plants, yeasts, fungi and bacteria(Jang et al., 2002). Levans produced in grasses(Dactylis glomerata, Poa secunda, and Agropyroncristatum) are present as storage carbohydrates

in the stem and leaf sheaths, and are degradedin the later stages of-the growing season to pro-vide plants with carbohydrates for grain filling(Pollock and Cairns, 1991). Levan is also con-tained in wheat and barley (Hordeum vulgare),fungi (Aspergillus sydawi and A. versicolor), andin yeasts in trace amounts (Han, 1990).

Levan is naturally present in various foodproducts, and thus, is regularly consumed in verysmall amounts by humans. However, it has been

relatively ignored as a functional food ingredientdue to its limited resources and very low content,until now. Furthermore, it has been argued thatunlike inulin, levan might not be useful as acarbon source for animals and humans due to itshigh molecular weight and branched structure(Marxet al., 2000, Yamamotoet al., 1999). Tishypothesis is supported by observations based onin vitroexperiments, where lactic acid-producing-bacteria (LAB) did not utilize levan as a carbonsource.

Although industry has been interested inthe diverse aspects of this fructose homopolymerover the last few decades, the application of

Abstract

Levan, a homopolysaccharide which is composedof -fructofuranosyl residues joined by -2,6with multiple branches by -2,1 linkages hasgreat potential as a functional biopolymer infoods, feeds, cosmetics, and the pharmaceuti-cal and chemical industries. Levan can be usedas food or a feed additive with prebiotic andhypocholesterolaemic effects. Levan is alsoshown to exert excellent cell-proliferating, skinmoisturizing, and skin irritation-alleviating

effects as a blending component in cosmetics.Levan derivatives such as sulphated, phosphated,or acetylated levans are asserted to be anti-AIDSagents. In addition, levan is used as a coating ma-terial in a drug delivery formulation. In addition,levan has a number of industrial applicationssuch as a surfactant for household use due to itsexcellent surface-active properties, a glycol/levanaqueous two-phase system for the partitioning ofproteins, etc.

However, there are some limitations for theindustrial applications of levan due to its weakchemical stability of in solution and the complexprocess to purify levan. Once the limitationsare solved, the market for levan will graduallyincrease in the various fields.

IntroductionFructan, one of the most highly distributedbiopolymers in nature, is a homopolysaccharidecomposed of -fructofuranosyl residues joinedby -(2,6) and -(2,1) linkages. wo types offructan, inulin and levan, are distinguishable bythe type of linkage present. Chemically, levan

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Kang et al.146 |

levan is relatively less intense than inulin-typefructan, as a result of limited sources and theabsence of large production methods. Yet, eversince large scale levan production was success-fully achieved by levansucrase from geneticallyengineered Escherichia coli (Song et al., 1994),

there has been renewed interest in the potentialindustrial applications of levan and its derivativesfor agriculture, cosmetic, food ingredient, foodadditive, feed additive, medicinal, and industrialuses (Clarkeet al., 1997; Kimet al., 1998; Vijnand Smeekens, 1999; Rhee et al., 2000); and inaddition, a good source of pure fructose and di---fructofuranose dianhydride IV (DFA IV)(Saito and omita, 2000; Songet al., 2000). In aprevious review, we described the biotechnologi-

cal production of levan and its related enzymes(Rheeet al., 2002). Here we describe the biologi-cal functions and applications of levan.

Applications of levan

In food

Prebiotic effect of levan and its applicationsLevan has numerous demonstrated uses in foods

(Han, 1990). Dry levan powder is white andnon-hydroscopic, and dissolves slowly in hot orcold water. Levan is non-toxic, non-mutagenic,odourless, tasteless, and a soluble dietary fibre;and its hydrolysates particularly help improve gutfunction. Gibson and Roberfroid (1995) defineda prebiotic as a nondigestible and non-absorbablefood ingredient in the upper portion of thegastrointestinal tract that beneficially affects thehost by selectively stimulating the growth and/

or activity of one, or a limited number, of bac-teria in the colon, which can improve the hostshealth. Terefore, nondigestible carbohydrates,especially inulin oligosaccharides, are classifiedas authentic prebiotics. Both inulin and inulinoligosaccharides are utilized by Bifidobacteriumspecies in vivo (Gibson and Roberfroid, 1995).Generally, fructo-oligosaccharides indicate-2,1--fructans, with degrees of polymerizationvarying between 2 and 20 (oligofructose) and2060 (inulin). Levan is a homopolysaccharidecomposed of -fructofuranosyl residues joinedby -2,6 and -2,1 linkages (as the major link-

ages), of which molecular weights reach severalmillion daltons, with multiple branches.

Te physiological effects of levan are depend-ent on its size and linkage type, and the fermenta-bility of levan is therefore an important issue. Invitrostudies testing the abilities of various genera

to ferment levan and levan oligosaccharides havebeen performed on pure cultures that includeBifidobacterium adolescentis, B. longum, B. breve,B. pseudocatenulatum, Lactobacillus plantarumandPediococcus pentosaceus(Marxet al., 2000; Kanget al., 2002b). An enrichment of the tested strainswas found with the levan oligosaccharides.

Te in vitrofermentation properties of levan(originating from Erwinia herbicolaas the control)and levan type exopolysaccharides (originating

from Lactobacillus sanfranciscensis) were studiedusing human faeces as an inoculate (Belloet al.,2001). An enrichment of Bifidobacteriumspecieswas found with the levan type exopolysaccha-rides, but not for levan. Te problem with thisapproach is that levan can be hydrolysed bygastric acids. o demonstrate in vitro the resist-ance to acid (stomach) and enzymatic (saliva,pancreatic, and small intestinal) hydrolysis, twofructans (chicory inulin and Zymomonas mobilis

levan) and their oligosaccharides were dissolvedin appropriate media (saline or buffer solution).At pH 1.0, the initial fructose release rate dif-fered and was rapidly hydrolysed in the order oflevan oligosaccharides, inulin oligosaccharides,levan, and inulin. At pH 4.5, 7, and 14, either noor small amounts of fructose were found in thefour samples (Kanget al., 2006b). When 0.4 mlof 5% levan was incubated with 0.2 ml of artificialgastric juice (2 g of NaCl, 7 ml of concentrated

HCl, per 1 l) at 37C, the product of hydrolysis(fructose) was found to be approximately 10%(after 4 h of incubation).

A possible explanation for the fermentationof levan could be that its acid hydrolysis in thestomach produces smaller sized levan or levan-oligosaccharides, which are subsequently fullyutilized by lumen bacteria. In in vivoexperimentswith rats, the concentration of total short-chainfatty acids (SCFAs) in the caecum was two timeshigher with the levan diet as compared to thecontrol diet, mainly due to the formation of ac-etate and butyrate (Janget al., 2002). When therats were fed inulin-type oligosaccharides at 6%


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of the dietary weight, the butyrate formation ratewas higher in the inulin-type oligosaccharide fedrats than in those fed xylooligosaccharides, withacetate being the primary SCFA followed bybutyrate (Campbellet al., 1997). It is well knownthat dietary fibre reaches the large intestine and

is fermented by the colonic microflora, producingSCFAs, hydrogen, carbon dioxide, and biomass(opping et al., 2001). Tis fermentative proc-ess dominates human large bowel function, andprovides a means whereby energy is obtainedfrom the carbohydrates not digested in the smallbowel through the absorption of SCFAs. TeseSCFAs limit the growth of harmful lumen bac-teria and are an important energy source for thehost (oppinget al., 2001).

About 510% of SCFAs are excreted in thefaeces, while 9095% are absorbed. Butyrateis mainly used by colonocytes as maintenanceenergy. Propionate is metabolized in the liver,whereas acetate serves as a metabolic substrateboth for the liver (5070%) and peripheraltissues (Hoverstad et al., 1982; Henning et al.,1972; Roediger, 1980; Dankert et al., 1981;Remesy and Demigne, 1983).

Te ingestion of levanheptaose was shown

to increase the faecal counts of endogenousbifidobacteria by a factor of 10, without affectingLactobacillussp. (Kanget al., 2000). Te amountof butyrate as well as -fructosidase activity wereincreased, whereas total aerobes and pH werereduced in rats fed levanheptaose diets as com-pared to those on the control diet.

Although the identity of the bacteriaresponsible for levan fermentation remainsunclear, the above concepts suggest that levan

might be degraded and then fully fermented bylumen bacteria in the ceacum and colon. Even athigh-intake doses, no significant amounts of Z.mobilis levan have been detected in the faeces.Food-grade levan is being produced commerciallyby a number of companies, including RealBiotechCo., Ltd (www.realbio.com), Chungnam, Korea,and Advance Co., Ltd (www.advance.jp), okyo,

Japan.

Effects of levan on lipid parameters and itsapplicationsTe intake of high-molecular weight levan [1and 5% (w/v)] reduced serum total cholesterol in

a dose dependent manner, while serum triglycer-ides were not affected by levan (1%) (Yamamotoet al., 1999). A high-fat diet (40% of calories asfat) caused obesity in rats by increasing bodyweight and fat accumulation (Honget al., 2001);however, the oral administration of 2% levan

reduced adiposity as well as serum lipids (Kanget al., 2002a).Energy intake and energy expenditure

are closely regulated processes, as reflected byrelatively stable body weight in the presence oflarge daily fluctuations in energy intake. Lipid ac-cumulation by adipose tissue depends on plasmalipids that are derived from hepatic lipogenesisand absorbed from the diet (Griffenet al., 1992).Terefore, hepatic lipogenesis and the export of

lipids are crucial steps linked to adipose tissuelipid accretion. Decreases of serum triglyceridesin animals are shown to result from the reductionof very low-density lipoprotein-triglyceride se-cretion and the inhibition of hepatic lipogenesisby the reduced activity and gene expression oflipogenic enzymes (Koket al., 1996; Delzenneetal., 2001). Additionally, with the development ofhigh-fat diet-induced insulin resistance, which ischaracterized by hyperinsulinaemia, lipogenesis

is elevated in the liver, which further exacerbatesthe accumulation of excess visceral fat, increas-ing the serum free fatty acid level (Commins etal., 1999). Peroxisome proliferation-activatedreceptors (PPARs) are transcription factors thathave important effects on lipid homeostasis, byregulating the expression of genes involved inlipid metabolism (Escher and Wahli, 2000).

Leptin is a hormone secreted by adipocytes(Griffen et al., 1992). By acting as a satiety fac-

tor and increasing energy expenditure, leptin,which is regulated by neuropeptide Y (NPY)in the hypothalamus, plays a major role in bodyweight homeostasis (Griffenet al., 1992). Whenthe NPY level is decreased, leptin increasessympathetic activation, thermogenesis, and themetabolic rate (Daubioulet al., 2004; Mortensenet al., 2002), thereby increasing energy expendi-ture as well as adaptive thermogenesis in brownadipose tissue (Steinberg et al., 2002). Tesupplementation of 110% levan was able todecrease adiposity and postprandial lipidaemiain high-fat diet-induced obese rats, through the


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enhancement of uncoupling protein gene expres-sion (Kanget al., 2004; Kanget al., 2002b).

Te mRNA expressions of hepatic fattyacid synthase and acetyl CoA carboxylase, whichare the key enzymes in fatty acid synthesis,were down-regulated by levan. Hepatic PPAR

mRNA expression was up-regulated dose-dependently by levan, which may have resultedfrom the inhibition of lipogenesis and stimula-tion of lipolysis, accompanied with the regulationof hepatic lipogenic enzyme and PPAR geneexpression (Fig. 6.1) (Kanget al., 2006a).

In human studies, levan diet administration(5.56 g, twice a day) for 23 months reducedbody weight and body fat mass (Kang et al.,2003; Leeet al., 2003).

In Korea, levan is available at markets asa functional food in various forms, including

chewing gum and powder.

Effect of levan on the intestinal absorption ofminerals and its applicationsIn Europe, Asia, and the United States, currentdietary calcium intake is far below Recommended

Figure 6.1 mRNA expression of hepatic enzymes and peroxisome proliferation-activated receptor(PPAR)in rats fed experimental diets. Quantitative RT-PCR was used for the mRNA determination. Levels of mRNAwere calculated as percentage values of the N diet group. Values are mean SE (n= 4). Different lettersindicate significant difference (P


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Daily Allowance (RDA) levels. In South Korea,average calcium consumption is 500600 mg/day for adults and only 400 mg/day for peopleolder than 65 years, whereas the Korean RGAis 700 mg/day (Te Korean Nutrition Society,2000).

Osteoporosis is a major public healthproblem for elderly women around the world. Inpostmenopausal women, true intestinal calciumabsorption is reported to range between 28% and32% (Van denet al., 1999). Recent estimates sug-gest that 30% of postmenopausal white womenin the United States, and 23% of women overage 50 in European countries, have osteoporosis(Cooperet al., 1996). Te stimulation of mineralabsorption (Ca, Mg, and Fe) by inulin ingestion

has been repeatedly demonstrated in animalfeeding studies (Ohta et al., 1993, 1994; Levratet al., 1991; Lopez et al., 2000). Furthermore,inulin and its oligofructose were found to in-crease bone density in rats (Lopez et al., 2000;Roberfroidet al., 2002). Yet, data relative to levanand osteoporosis, in animals or humans, have notbeen published. In some studies, the intake oflevan increased divalent cation (Zn, Mg, and Fe)(Kimet al., 2004) and calcium (Noet al., 2007)

absorption in rats. However, these results needto be clarified by further human study.

Applications of levan for beveragesOne way to improve the rheological propertiesand functional characteristics of dairy products,such as yogurt, is the addition of stabilizers. Teapplication of levan in the food industry includeuses as a stabilizer, an emulsifier, a formulationaid, surface-finishing agent, an encapsulating

agent, and a carrier of flavours and fragrances(Han, 1990). Te exopolysaccharides (EPS)produced by lactic acid bacteria can be dividedinto three major groups: levan, glucans, andheteropolysaccharides (Monsan et al., 2001).EPS has been used to improve consistency andviscosity as well as to avoid whey separation inyogurts. Also, for the production of fermentedmilk products and cheeses, EPS-producing lacticacid bacteria are used in starter cultures (Mar-shall and Rawson, 1999).

Te solution properties of levan have alsobeen studied (Kasapiset al., 1994), demonstrat-ing that the viscosity of a levan solution is stable

to heating and sodium chloride. In addition, theviscosity of levan is influenced by acidic condi-tions (pH 2), but stable in the range of pH 410(Kimet al., 1998).

Levan is useful in terms of its water-holdingcapacity. At greater than 1% (w/v) concentration,

a levan solution will show film-forming charac-teristics on an appropriate smooth surface. A lowconcentration levan solution (below 1%) can alsobe used in coatings.

In human studies, non-sensitive and sensitivepersons can consume 30 g or more of inulin/dayand 10 g of inulin/day, respectively (Absolonneetal., 1995). However, levan at a dose of 10 g/dayfor 3 months in sensitive volunteers led to somesigns of intestinal discomfort, including flatulence

and intestinal noise (Kanget al., 2003).Te enzymatic or chemical hydrolytic prod-

ucts of levan may be used in the food industryas sweetener or dietary fibre, for example ultra-high-fructose syrups (UHFS) and -(2,6)-linkedfructofuranosyl oligosaccharides (Han, 1990).

In feed

Massive amounts of information exist withregards to the significance of levan in modulating

various functions of the human body. Its effectsinclude anti-tumour activity (Yoonet al., 2004),immunostimulating activity (Xu et al., 2006),lipid metabolism (Yamamoto et al., 1999), andprebiotic activity (Belloet al., 2001). But there isinadequate information available for the potentialvalue of levan in livestock, poultry, and animalnutrition and health.

Effect on levan on intestinal microflora

Te colonic microflora are of crucial importancewhen considering the role of feed ingredients inhealth and disease, since the various physiologi-cal effects of such compounds influence theiractivities. Te carbohydrate chain length provesto be another important factor. Roberfroidet al.(1998) demonstrated that the long chain fraction(DP 1060) in chicory inulin is fermented atleast twice as slowly as the oligofructose fraction.Tis implies that in the intestine, degradationof the longer chains will be slower, which willresult in their arrival to the more distal parts ofthe intestine. Particularly, for animal feeds, wheredifferent types of animals have different degrees


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of bacterial associations, the chain length fac-tor could prove to be a valuable criterion whencomposing feedstuffs, matched with the specificrequirements of a certain type of animal (poultry,pigs, pets, and horses).

Te benefits of fructan ingestion arise

from its ability to increase the population ofindigenous Bifidobacteria and Lactobacilli inthe large bowel, which suppress the activity ofputrefactive bacteria and reduce the formationof toxic fermentation products (omomatsu,1994). Jang et al. (2002) fed SpragueDawleyrats a basal feed containing 7% levan, and foundthat the levan diet stimulated the growth of bifi-dobacteria in the caecum. Tese results indicatethat the intake of levan stimulated the growth of

lactic acid-producing bacteria in the caecum, andthereby improved the intestinal conditions of therats. Fructans may exert a health-promoting ef-fect by the production of SCFAs, which are endproducts of fructan fermentation. oppoing andClifton (2001) reported that dietary fibre andSCFA result in intestinal epithelial proliferationand increased intestinal function, thus decreas-ing colon cancer risk. Similar roles may exist forcompanion animals and livestock.

Effect of levan on fluid stoolsIn order to prevent and treat diarrh0ea, or fluidstools, which bring economic loss to livestockfarms, several antibiotics and microorganismshave been administered. However, with thelong-term use of antibiotics, tolerance may bedeveloped, and in the case of beneficial micro-organisms, adequate enough amounts cannotreach the large intestine due to simple death

by gastric acid. In relation to a patent (PC/KR00/01556), the effects of levan as a feed ad-ditive to improve evacuation, prevent diarrhoea,and promote animal growth were studied. In thisstudy, 90-day-old pigs were fed a fermented testfeed consisting of food wastes and levan. Tepigs were fed for varied time spans of 10 days, 20days, and 110 days. Te group fed the levan dietshowed improved faeces conditions in the shortadministration period, as well as weight increasesof 2.93.6%, compared with the control group.

Levan as an immunostimulant in aquacultureHigh yields in aquaculture involve intensivemanagement systems (Vandeputte, 2003), whereantibiotics, drugs, and chemicals are used to pre-vent fish diseases caused by environmental stressand other factors. However, these are found to

be effective only for a short time; in addition toenhancing the risk of their bioaccumulation inthe environment. On the other hand, the use ofimmunostimulants in aqua-feed is considered tobe safe and effective against various pathogens.Immunostimulants quickly activate non-spe-cific defence mechanisms to protect fish againstpathogens (Siwicki, 1994), and aquaculture nu-tritionists are making good efforts to explore newimmunostimulants for aquaculture practices.

Microbial levan is shown to have immunos-timulating properties. Dinaet al. (2007) studiedthe effects of dietary levan on the survival ofCyprinus carpiojuveniles fed a fish feed containinglevan, at concentrations ranging from 0.1 to 1.0%.One hundred per cent survival was obtainedwith 0.5% levan in the feed. But increasing theconcentration to 1% probably increased the an-tigenic load, leading to immunosuppression andthus reducing the protection efficiency. Tis is in

agreement with Anderson (1992) who proposedthat an inadequate amount of immunostimulantwill result in no protection, whereas too muchmay cause immunosuppression. Levan activatesthe non-specific phagocytes, which is importantfor reducing mortality in fish. Tis research hasbeen the only report submitted presenting dataon levan in aquaculture, which should be furtherexplored in more detail with varied species offish.

In cosmetics

Levan may be useful as an active ingredient in theformulation of cosmetics and pharmaceuticals, aswell as foods and feeds. And, from what we canascertain, the use of levan in cosmetics is scarce.However, we previously reported the results ofour study on the cosmeceutical properties oflevan produced by Zymomonas mobilis (Kim etal., 2005).

Stability of levan in ethanolIngredient stability is a key prerequisite for agood cosmetic formulation. In order to deter-


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mine the storage stability of levan at room tem-perature, 5C, and 40C, for a total of 45 days, itsturbidity and precipitation characteristics wereobserved. An aqueous levan solution (5% on asolid basis) was mixed with ethanol at differentconcentrations ranging from 10 wt% to 50 wt%.

From the observations made, the solubility ofthe levan solution in ethanol was so exceptionalthat precipitation or a cloudy solution were neverobserved.

Formation of nanoparticles by self-assemblyLevan partially forms nanoparticles by self-as-sembly in water. Te distribution of particle sizesof these self-forming levan nanoparticles wasdetermined in water and 20% aqueous ethanol

(by weight) via the laser light scattering method,on an ELS-8000 particle size analyser (OtsukaElectronics, Japan) with a 632.8 nm HeNe laser(10 mW) at 25C (Marquardt analysis method,100 repeated tests) (Fig. 6.2). Te mean particle

sizes of the complexes were 224.3 nm and 251.8nm, respectively.

Partially self-formed nanoparticles were alsoobserved by transmission electron microscopy(EM). Te image obtained by EM showedthe same results as the dynamic light scattering

described above, indicating self-formed nano-particles and their agglomerates existing in a sizerange of several hundred nanometres. Demel etal. (1998) reported that fructans strongly interactwith lipid model membranes, due to their highsolubility, which would indicate that hydroxylgroups are primarily available for interactionswith the surrounding water molecules, as well asmembrane phospholipid head groups (Demeletal., 1998). Vereyken et al. (2001) also reported

that fructans exhibited much stronger effectson different lipid systems than other polysac-charides. Tis phenomenon was attributed tothe fructans hydrophobic properties, which inturn are due to the additional CH2 groups in

Figure 6.2 Nanoparticles of levan. TEM micrograph (A) and the weight average size distribution (B) oflevan.

A

B


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furanosides, as compared to pyranosides, such asdextran (Vereykenet al., 2001). Considering theabove data, levan exhibits not only hydrophilicity,but also hydrophobicity, similar to a surfactant.Tis quality aids in explaining why levan tendsto form nanoparticles in water.

Cytotoxicity assayIn order to evaluate the cell viability and cyto-toxicity of levan [5% (w/w)], an M assay wascarried out in human fibroblast and keratinocytecell lines (Mosmann, 1983). Each of the cell lineswas inoculated on a 96-well plate supplementedwith 100 l of DMEM (Dulbeccos modifiedEagle medium) containing 10% FBS (fetal bo-vine serum, Gibco BRL), with a density of 104

cells per well. Te plates were then incubated for24 h at 37C in an atmosphere containing 5%CO2. After the addition of the levan solution, thecells were incubated for another 24 h. Te Msolution (100 l) was added to each well, andthen incubated for 4 h. Te quantity of formazanformed in the culture medium was determinedvia absorbance, measured at 570 nm with anELISA reader. Te cell viability was calculated asfollows;

Cell viability (%) = (OD570(sample)/OD570(control)) 100

where OD570(sample) refers to the absorbance at570 nm for the cells treated with levan or SLS(sodium lauryl sulphate), and OD 570(control)is the absorbance at 570 nm for the negativecontrol (non-treated cells). Levan exhibited nocytotoxicity in the human fibroblast cell line, up

to a relatively high concentration (100 g/ml).Moreover, levan exhibited a considerable cellproliferation effect in the keratinocyte cell line above 30% at high concentrations (>1 mg/ml).Tese results suggest levan is a safe ingredientthat can be used in cosmetics.

Cell protection test with three-dimensional(3-D) artificial skinTree-dimensional artificial skin was constructedaccording to a previously reported method(Kamolz and Luegmair, 2005; Ponec and Kem-penaar, 1995). After inducing skin irritation inthe 3-D artificial skin using 0.05% SLS, which

is widely used as a skin irritant (Ponec andKempenaar, 1995), 0.01 mg/ml and 0.05 mg/mlof levan solution were applied. Te cell viabilitywas compared to that of the 0.05% SLS used asa negative control (in the absence of levan). Televan demonstrated good cell protective effects

from the SLS-induced irritation in the 3-Dartificial skin, with more than 30% improvementin cell viability.

Anti-inflammation test with three-dimensionalartificial skinAn interleukin 1 release assay was carried outusing a human IL-1ELISA kit (Pierce, Rock-ford, USA), and E. coliderived recombinanthuman IL-1 was utilized as a standard. After

the human I-1 antibody was allowed to reactwith the sample at room temperature, the samplewas treated with biotin-conjugated second-ary antibody. Streptavidin-HRP (horseradishperoxidase) was applied for 30 min, followed bytreatment with trimethylbenzidine solution. Teabsorbance was then measured at 450 nm usingan ELISA reader.

Interleukin 1(IL-1) is known to functionas a proinflammatory mediator during intercellu-

lar signal transport, and is also shown to inducethe proliferation of certain cells, including oste-oblasts, monocytes, macrophages, keratinocytes,hepatocytes and fibroblasts, via stimuli such asinflammation or infection.

Furthermore, IL-1 is known to stimulatethe increase of arachidonic acid lipoxygenase me-tabolites, including leukotriene B4 and 5-, 12-,and 15-HEE, and also functions as a potentialinducer of reepithelialization in wounded skin

(Dykes et al., 1991). Terefore, measuring thequantity of IL-1secreted in the culture mediumis just one of many methods for evaluating theanti-inflammatory effects of levan.

After inducing primary skin irritation with0.05% SLS in 3-D artificial skin, 0.01 mg/ml and0.05 mg/ml of levan solution were applied, andsubsequently, the quantity of secreted interleukin1was measured. Te artificial skin treated with0.01 mg/ml and 0.05 mg/ml of levan exhibited areduced quantity of IL-1, as opposed to the ar-tificial skin that had not been treated with levan.

aking the above into account, the resultssuggest that levan exerts an emollient effect during


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skin irritation caused by irritants. However, theexact mechanism underlying this phenomenonis unclear. Despite the high molecular weight oflevan, it still exhibits cell proliferation and anti-inflammatory effects. Tis may be attributableto its penetrative ability, which is due to a much

smaller particle size distribution (ranging fromapproximately 170 to 300 nm) than that of otherpolysaccharides.

Moisturizing effect of levanIn order to evaluate the moisturizing effects oflevan, transepidermal water loss and water con-tent in the skin were assessed using a VapoMeterand Corneometer CM825.

Measurement of transepidermal water loss (TEWL)using a VapoMeterTe EWL measurements were sequentiallyrecorded at the application sites prior to treat-ment (Levin and Maibach, 2005). Tese initialmeasurements were used as pre-treatment con-trol values. Subsequently, 20 l of the sample wasapplied over a 4 cm2 area of the volar forearm(2 2 cm), followed by measurements taken atregular intervals for a total of 6 h.

In the EWL test, the results of the levanapplied to the skin were compared with the re-sults of 0.2% hyaluronic acid and distilled water.Te statistical analysis of this data revealed asignificant decrease in water loss on the skin as aresult of levan and hyaluronic acid application, asopposed to when distilled water was applied.

Measurement of skin moisture content using aCorneometer CM825

A skin hydration reading for each sample wasrecorded with a Corneometer CM825 (Cour-age Khazaka, West Germany). Tis equipmentconsists of a recording device, and an impedanceprobe that measures electrical conductivity onthe surface of the skin. Capacitance refers to thequantity of electric charges stored, and thus, ca-pacitance is proportional to the amount of waterin the skin, a factor that is commonly referred toas skin hydration. Basically, the higher the levelof skin moisture, the stronger the observed con-ductance signal will be (Leonardi et al., 2002).Baseline values were taken from 10 femalevolunteers between 22 and 37 years of age, using

40-mm-diameter circular test areas on both fore-arms. Tese panellists remained at rest in a room,at a temperature of 25C with 4555% relativehumidity, for the duration of the test. Ten, eachof the designated areas was treated with 10 lper circle of five different test formulations. Te

moisture contents of 0.2% levan and 0.2% hy-aluronic acid were measured with a CorneometerCM825. Te results showed the same tendencyas those mentioned above. Based on these data, itappears that levan exhibits a substantial moistur-izing effect.

In medicine

Anti-tumour activity of levan and its

applicationsLevan has certain biological activities, includingthe promotion of infection and necrosis, tumourinhibition and stimulation, and an increase in cellpermeability to cytotoxic agents (Leibovici andStark, 1985). Calazans et al. (1997) postulatedthat levan showed anti-tumour activity againstsarcoma 180 and Ehrlich carcinoma in Swissalbino mice, and hypothesized that the effectsmight have been associated with a specific mo-

lecular weight of the polymer.Aerobacterlevan, asan anti-tumour agent, was also studied (Leiboviciand Stark, 1986; Stark and Leibovici, 1986); andthe anti-tumour activity of Zymomonaslevan wasrelated to the specific class of molecular weight(Calazans et al., 2000). Te chemical structureand molecular weight of levan closely relate to itsbiological activities. Levan from Microbacterium(branching degree of 12.3%) was isolated andtreated with an inulinase, to modify its branch-

ing structure. Te sequential degrees of branch-ing obtained were 11.6%, 9.3%, 6.1% and 4.2%.As the branching degree of levan decreased, theanti-tumour activity on a stomach carcinoma cellline linearly decreased. In a hepatocellular carci-noma cell line, the anti-tumour activity rapidlydropped when the branching reached 9.3%, andthen slightly increased as the branching degree oflevan further decreased (Yoonet al., 2004).

Tere is limited information on levan fromin vivo studies on cancers. Among the variouscancers, colon cancer is the most important interms of frequency. Only 50% of those whodevelop colon cancer live longer than five years


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after diagnosis. Epidemiological studies indicatethat increased consumption of fruits, vegetables,and a high total dietary fibre intake, includ-ing inulin, reduce the risk of developing coloncancer. An animal study showed that the intakeof a 10% inulin diet significantly reduced the

number of colonic ACF (aberrant crypt foci) inazoxymethane-induced rats (Reddyet al., 1997).Te tumour-inhibitory effect of inulin seems tobe dose related (Franck and Leenheer, 2002). Itis likely that inulins protective effects proceed viathe selective modulation of microflora (increasesin bifidobacteria and decreases in bacteroidesand clostridia). Tis then changes the gut to amore favourable environment, which involvesincreases in lactic acid-producing bacteria, the

production of acidic compounds, modulationof beta-glucuronidase activity, and the produc-tion of SCFAs in the colon (Roberfroid, 2005;Franck and Leenheer, 2002). In the case of levan,the data available on gut health are as follows: itlowers caecal pH and beta-glucuronidase activ-ity, and increases the levels of bifidobacteria andSCFAs (Yamamotoet al., 1999; Janget al., 2002;

Jang et al., 2003; Kim et al., 2004; Kang et al.,2005). From these data, it is suggested that levan

intake may help inhibit carcinogenesis.

Applications of levan as a blood plasmavolume expanderBlood plasma volume expanders, includingdextran, can replace normal blood proteins inproviding osmotic pressure, to pull fluid from theinterstitial space into the plasma. Tis treatmentis useful for preventing shock from haemorrhage,burns, and surgery, as well as to reduce the risk of

thrombosis and embolisms. Te clinical dextransused are Dextran 40 and Dextran 70, which are40,000 and 70,000 Da average molecular weightfractions, respectively (Leathers, 2002). Appro-priately, levan has been suggested as a possibleblood plasma volume expander (Moffitt, 1995).

In industry

In addition to the use of levan in foods, feeds,medicines, and cosmetics, other rare uses of levanare reported in the chemical and biotech indus-tries. A PEG/levan two-phase liquid system wasreported to lead macroscopic phase-separation,which can be used to purify biological materi-

als of interest by selectively partitioning theminto one phase (Chunget al., 1997). Te PEG/levan two-phase system, which is made of 60%PEG (w/w) and 6.77% levan (w/w), can displayphase-separation phenomena with pectin, locustbean gum, and PEG the same as the PEG/dex-

tran system. Tis levan/PEG aqueous two-phasesystem has demonstrated good partitioning withsix model proteins: horse heart cytochrome c,horse haemoglobin, horse heart myoglobin, henegg albumin, bovine serum albumin, and hen egglysozyme.

Levan was also utilized to develop anenvironmentally friendly adhesive using itsadhesive properties (Combie et al., 2004).Levans tensile strength on aluminium, and

excellent shear strength on certain plastics, wastechnically competitive with many petrochemicalbased adhesives (Combie and Yavorsky, 2005).A company producing levan (Montana BiotechSE Inc.) has developed two forms of levan,including water-soluble and cross-linked levan.Te water soluble levan is useful for temporarybonds and certain indoor applications, and thecross-linked levan can be utilized as a water re-sistance adhesive for an extended time. Levan has

also been examined as a food adhesive (MontanaBiotech SE Inc.). Montana Biotech expects thatthe water resistant levan and cross-linked levanwill be used in the wood adhesive industry, andin biodegradable plastic production, respectively.Levan can also be used as water resistant film forfood preservation, in foundry applications, andfor shale stabilization in the oil drilling industry.Te adhesive strength, film-forming ability, andnon-toxicity of levan are its key properties, and

are comparable to petrochemical derivatives inmany applications (Combie and Yavorsky, 2005).

Levan was made into cohesive films bypressing levan/glycerol blends on a heatedcompression moulder with 2030 weightpercent (wt%) glycerol (Barone and Medynets,2007). Te efficient extrusion of the levan/glyc-erol blends, to incorporate glycerol into the levanstructure, required room temperature mixingand heated compression-moulding with at least35% glycerol.

Levan derivatives have also been developedby Montana Biotech, extending the applicationfield (Gunn et al., 2006). Te levan derivatives


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were made by adding cationic substituentsor non-cationic nitrogenous substituents to apolymer by reaction with at least a portion of thehydroxyl, hydroxyalkyl, or hydroxyl and hydroxy-alkyl groups of the levan. Tis derivatized levan isuseful as an ingredient in personal care products

such as hair conditioner, and may also be usefulin home and fabric care applications, in oilfieldapplications, and in emulsion polymerizationapplications.

Te Sugar Processing Research Institutehas also developed novel levan derivatives, whichhave predominant -(26) glycosidic link-ages between the -fructofuranoside monomericunits (Roberts and Garegg, 1998). Te companysynthesized sulphate, phosphate, and acetate

derivatives, which can be present in amountsfrom 0.2 to 3 sulphates, phosphates, or acetatesper fructose ring, respectively. Such levan deriva-tives may be used as inhibitors of smooth musclecell proliferation, anti-AIDS agents, excipients inmaking tablets, and as agents to transform waterinto a gel. Te company expects that the deriva-tives will be utilized for wound healing and otherdermatological uses, for subcutaneous packing inmedical or dental applications, and for veterinary

uses in treating inflamed udders and mastitis incattle. In addition, the company found that thelevan cross-linked by epichlorohydrin acts as afilm former, to form thin plastic films. A companyhas also developed fructan N-alkylurethanes,which have excellent surface-active propertiesand good biodegradability, and are suitable assurfactants to be used in household and indus-trial applications, e.g. as detergents, emulsifiers,emulsion stabilizers, foaming agents, foam stabi-

lizers, dispersants, and wetting agents (Stevensetal., 1999).

Levan can be used as a cryoprotectant forfreeze-preservation of animal cells and fish(Yoshimizu et al., 1996). Te gene responsiblefor microbial levan was introduced in plants, andtransgenic tobacco plants expressing levansucrasegenes from Bacillus subtilis (Pilon-Smits et al.,1995) or Z. mobilis (Park et al., 1999) showedincreased tolerance to drought and cold stress.Levan is able to minimize the effects of tem-perature variation during storage. Freezethawcycles often destroy the delicate texture offrozen deserts, because during unfavourable

storing conditions small ice crystals will migratetowards larger ice crystals during the thaw stage.Furthermore, microbial levan could be used as asoil conditioner, improving the germination ofvarious seeds (Gamalet al., 1974).

Levan is valuable as a specific substrate for

levan fructotransferase (LFase, EC 4.2.2.16),which is employed in the production of difructosedianhydride IV (DFA IV). DFA IV, a kind of acyclic disaccharide, is a non-digestive and non-fermentative subsaccharide that is not digestedin the animal body. In addition to being useful asa low-calorie sweetener, the subsaccharide plays arole in inhibiting tooth decay and as a productivefactor for Bifidusbacteria. It is also reported thatDFA is used as a mineral absorption factor in

the body (Sakuraiet al., 1997; Saito and omita2000).

Safety of levanLevan is recognized as a virtually non-toxicsubstance, and its safety has been verified bymany scientists (Vina et al., 1998; Calazans etal., 1997). Its LD50 and NOEL (no observedeffective level) are known to be 7.5 0.5 g/kgb.w. and 1.5 g/kg/day, respectively (Ministry of

Health report, Japan, 2000). Te safety of levan,a polysaccharide isolated from either sugar orraffinose-fermented products by B. subtilis, wasevaluated by the Department of Food Chemistry,Ministry of Health, Japan in 14 December 2000.Tey verified its safety by general toxicology testsand mutagenicity tests, and levan has been usedas a legal food additive in Japan.

In Korea, a levan that is a sugar-fermentedpolysaccharide from Z. mobilis, was ap-

proved as a food by the Korea Food and DrugAdministration (KFDA) in 2001, and is nowlisted in the Korea Health and Functional FoodCode of KFDA.

A levan with the trade name FRUCAN isapproved as a food and food additive, and mar-keted in many countries such as the USA, EU,

Japan, Australia, and New Zealand (ANZFA).

Relevant patents for levanMany applications for levan, including as a foodadditive, have been filed (able 6.1) (Cremeret al., 2006; Mobasseri et al., 2004; Park et al.,2005). It was claimed that levan from S. salivarius


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could be used as a food additive with a hypoc-holesterolaemic effect (Kazuoki et al., 1996). AGenerally Recognized as Safe (GRAS) gradeLactobacillus reuteri,which is capable of produc-ing levan and/or fructo-oligosaccharides, can beused as a probiotic (Van Hijumet al., 2004). Te

levan produced from another lactobacilli, L. san-franciscensis, can be used in human or pet foodproducts as well as cosmetic products (Vincentet al., 2005). Tis levan is also shown to exert ex-cellent cell-proliferating, skin moisturizing, andskin irritation-alleviating effects as a blendingcomponent in cosmetics (Asai and Chun, 2004;Kim et al., 2003). In one study, the beautifyingand whitening effects of levan were pronounced,such as a melanin production inhibitory effect,

tyrosinase activity inhibitory effect, and pigmen-tation inhibitory effect (Furukawa and suboi,2006). reatments of cosmetics or externallyused medicines composed of levan could be ex-pected to effectively prevent or improve variousskin disorders such as spots, freckles, sunburn,etc. An oral dose of levan, which was producedby B. natto, at approximately 120600 mg, pro-vided allergy inhibitory effects by reducing theproduction of IgE and IL-4, and by promoting

the production of IL-12, inhibiting the dif-ferentiation to T2 cells (Jo and Kikkai, 2006).Levan derivatives such as sulphated, phosphated,or acetylated levans were asserted to be anti-AIDS agents (Robert and Garegg, 1998). Levanwas used as a coating material in a colonic drugdelivery formulation to target the release of thedrug from a core to the intestine, particularlythe colon (Basit and Ibekwe, 2007). A fructanN-alkylurethane, which has excellent surface-

active properties in combination with goodbiodegradability, was patented as a surfactantfor household use and industrial applications bymeans of replacing a hydroxyl group of fructosewith an alkylaminocarbonyloxyl group (Stevenset al.,1999). A glycol/levan aqueous two-phasesystem that can substitute the glycerol/dextransystem was developed for the partitioning ofproteins (Rhee et al., 2000b). New applicationsof levan as food and feed additives (Rhee et al.,2000a), as well as for a raw material in the pro-duction of difructose dianhydride IV (Rhee etal., 2000c), have been developed. It was claimedthat novel ascorbic acid derivative compounds

showing improved stability to oxidation, wereprepared by transferring a fructosyl moiety fromlevan to ascorbic acid using levan fructosyltrans-ferase (Kimet al., 2005). A fructose derivative ofascorbic acid having anti-oxidant and skin whit-ening activity could be widely used in medicine,

functional foods, or cosmetics.

Limitations and perspectivesAs shown in the previous sections, levan has greatpotential as a functional biopolymer in foods,feeds, cosmetics, and the pharmaceutical andchemical industries. However, the use of levanhas not been practical due to scarce informationon its biological safety and polymeric propertiesrequired for industrial applications, and a lack

of economic means for large scale production.Te weak chemical stability of levan in solutionis one reason for its limited application. Levan iseasily partially hydrolysed, at acidic conditionsand/or high temperatures, to fructose and low-molecular-weight fructo-oligosaccharides, whichare easily deteriorated by microbial contaminantsat room temperature. Tus, it is difficult to pre-serve a levan solution at room temperature andto process it at high temperature. o extend the

industrial use of levan and its derivatives, process-ing technologies must be further developed.

Te purification process is one of the expen-sive steps in the large scale production of levan.Complex processes are required to purify levanfrom reaction mixtures that contain glucose,fructose, sucrose, and certain fructo-oligosaccha-rides. Another important factor is to get officialapproval for levan as a safe eating material fromthe food administrations of various countries;

presently, levan is approved as a food additive inonly a few countries.

Levan clearly has great potential as a func-tional biopolymer for use in various industries.Levan was initially industrialized as a food ad-ditive to support functional soluble fibre. Levanhas very diverse biological functions, as previ-ously stated, for the chemical industries and bio-industries. Once its aforementioned limitationsare solved, the market for levan will graduallyincrease within the various fields.


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