Přednáška 1

Imobilizované biologické systémy

ImmobilizedImmobilizedBiocatalystsBiocatalysts

Doc. Ing. Jiří SAJDOK,CSc.

1.1.IntroductionIntroduction

WorkingWorking Party on Party on AppliedApplied BiocatalystsBiocatalystswithinwithin thethe EuropeanEuropean FederationFederation ofofBiotechnology: Biotechnology: ““ImmobilizedImmobilized biocatalystsbiocatalystsare are enzymesenzymes, , cellscells, , oror organellesorganelles ((ororcombinationscombinations ofof themthem) ) whichwhich are in a are in a statestate thatthat permitspermits theirtheir reusereuse””

P. B. P. B. PoulsenPoulsen, Enzyme , Enzyme MicrobMicrob. . TechnolTechnol. . 55 (1983) 304(1983) 304 –– 307 307

HistoricalHistorical BackgroundBackground::

( 1823 ( 1823 vinegarvinegar productionproduction, , sludgesludge, , attachmentattachment to to equipmentequipment ))50s 50s –– 60s : 60s : immobiliimmobilizzationation of enzymesof enzymes( 1916 Nelson ( 1916 Nelson –– GriffinGriffin: invertase : invertase adsads.on .on charcoalcharcoal1948 1948 SunmerSunmer: : jackjack beanbean urease)urease)1950 1950 –– 1970: 1970: intensiveintensive investigationsinvestigations on on immobilizedimmobilized enzymesenzymes andand

otherother proteinsproteins( e.g.( e.g.antigensantigens --> affinity chromatography > affinity chromatography ))

1969 1969 –– firstfirst industrialindustrial apptappt..ofof immobilizedimmobilized enzymeenzymeOpticalOptical resolutionresolution ofof DL DL aminoacidsaminoacids withwith immobilizedimmobilized aminoamino acylaseacylase

( ( ChibataChibata etet alal. ). )SinceSince 1960 1960 investigationsinvestigations on on immobilizedimmobilized cellscellsIndustrialIndustrial applicationsapplications ofof immobilizedimmobilized microbialmicrobial cellscells::19731973 L L –– asparticaspartic acidacid --EscherichiaEscherichia colicoli ((aspartaseaspartase ))1974 L 1974 L -- malicmalic acidacid –– BrevibacteriumBrevibacterium ammoniagenesammoniagenes( ( fumarasefumarase))19821982 L L –– alaninalanin –– PseudomonasPseudomonas dacunhaedacunhae ( L( L--aspartateaspartate ββ --

decarboxylasedecarboxylase ))

IntroductionIntroduction

BiocatalystsBiocatalysts dissolveddissolved in in aqueousaqueous bufferbuffersolutionssolutionssolublesoluble oror nativenative enzymesenzymes, , cellscells, cell , cell partsparts, , orororganellesorganelles..IImmobilizedmmobilized, , fixedfixed, , oror insolubilizedinsolubilized enzymesenzymes, , cellscells, , etcetc., ., denotedenote biocatalystsbiocatalysts thatthat are are boundbound to to a support. a support. carriercarrier, support, support, , oror matrixmatrixcrosscross--linkinglinking agent, agent, bifunctionalbifunctional agentagent, , oror carriercarrieractivatoractivator. .

MembrMembráánovnovéé bbíílkovinylkoviny

2.2.2.2. MethodsMethods ofof Enzyme Enzyme ImmobilizationImmobilization

1.1. modifiedmodified intointo a a waterwater--insolubleinsoluble formform,,

2.2. retainedretained by by anan ultrafiltrationultrafiltration membranemembrane insideinside a a reactorreactor, , oror

3.3. boundbound to to anotheranother macromoleculemacromolecule to to restrictrestrict theirtheirmobility.mobility.

ImobilizationImobilization TechniquesTechniques

FigureFigure 1.1. ClassificationClassification ofof enzyme enzyme immobilizationimmobilizationmethodsmethods

EncapsulationEncapsulation ofof EnzymeEnzyme

2.2.1.2.2.1. CarriersCarriers for Enzyme for Enzyme ImmobilizationImmobilization

1. 1. LargeLarge surfacesurface area area andand highhigh permeabilitypermeability2. 2. SufficientSufficient functionalfunctional groupsgroups for enzyme for enzyme attachmentattachmentunderunder nondenaturingnondenaturing conditionsconditions3. 3. HydrophilicHydrophilic charactercharacter4. 4. WaterWater insolubilityinsolubility5. 5. ChemicalChemical andand thermalthermal stabilitystability6. 6. MechanicalMechanical strengthstrength7. 7. HighHigh rigidity rigidity andand suitablesuitable particleparticle formform8. 8. ResistanceResistance to to microbialmicrobial attackattack9. 9. RegenerabilityRegenerability10. 10. ToxicologicalToxicological safetysafety11. 11. LowLow oror justifiablejustifiable priceprice

TableTable 2. 2. ChemicalChemical classificationclassification ofof matrixesmatrixes usedused for for enzyme enzyme immobilizationimmobilization

Synthetic polymers

Synthetic materials

Polystyrene Nonporous glass

Polyacrylates and poly-Controlled pore

glass

methacrylatesControlled pore

metal

Polyacrylamide oxides

Hydroxyalkylmethacrylates Metals

Glycidyl methacrylates

Maleic anhydride polymers

Vinyl and allylpolymers

Polyamides

Natural polymers MineralsPolysaccharides Attapulgite clays

Cellulose Bentonite

Starch Kieselghur

Dextran Pumice stone

Agar and agarose

Alginate

Carrageenan

Chitin and chitosan

Proteins

Collagen

Gelatin

Albumin

Silk

Carbon materials

(activated carbon)

AgarAgarAgar, Agar, alsoalso calledcalled agaragar--agar, agar, kantenkanten, , ororgelosegelose, , isis thethe oldestoldest knownknown gelgel--formingforming

polysaccharidepolysaccharide

DiscoveredDiscovered in in thethe 17th 17th centurycentury in Japan in Japan andandconsumedconsumed for 200 for 200 yearsyears, agar , agar isis extractedextractedfromfrom certaincertain marinemarine redred algaealgae ofof thethe classclassRhodophyceaeRhodophyceae mainlymainly fromfrom GelidiumGelidium andand

GracilariaGracilaria species, species, growinggrowing essentiallyessentially alongalongthethe coastscoasts ofof MoroccoMorocco, , SpainSpain, Portugal, Chile, , Portugal, Chile,

Japan Japan andand KoreaKorea

OOriginrigin ofof seaweedseaweed extractsextracts ——generalgeneral classificationclassification

OOriginrigin ofof seaweedseaweed extractsextracts ——generalgeneral classificationclassification

aa Species Species ofof economiceconomicsignificancesignificancebb ContainsContains onlyonly componentcomponentmentionedmentionedcc ContainsContains predominantlypredominantlyunderlinedunderlined componentcomponent

AgarAgarKoch Koch andand PetriPetri in 1882 in 1882 -- medium in medium in whichwhich to to growgrow bacteriabacteriano no betterbetter solidifyingsolidifying agent in agent in microbiologicalmicrobiological media has media has beenbeen foundfound

microbiologicalmicrobiological, , biotechnologicalbiotechnological, , andand public public healthhealth laboratorieslaboratories, , andand ananimportantimportant colloidcolloid in in otherother industriesindustries

permittedpermitted gellinggelling, , stabilizingstabilizing, , andand thickeningthickening agent for agent for foodfood applicationsapplications, , authorizedauthorized in in allall countriescountries withoutwithout limitationslimitations ofof dailydaily intakeintake

((confectioneryconfectionery, , bakerybakery, , pastrypastry, , beveragebeverage, , saucessauces, , wineswines, , spreadsspreads, , spicesspices andandcondimentscondiments, , meatsmeats andand fishesfishes, , dairydairy, , jamsjams, , etcetc.).)

ApartApart fromfrom itsits abilityability to to gelifygelify aqueousaqueous solutionssolutions andand produceproduce gel gel withoutwithout thethesupport support ofof otherother agentsagents, agar , agar cancan alsoalso bebe usedused as a safe as a safe sourcesource ofof dietarydietaryfiberfiber sincesince itit isis not not digestibledigestible by by thethe humanhuman body.body.

AgarAgar

FlowFlow sheetsheet ofof traditionaltraditionalagar agar extractionextraction

ExtractionExtractionPurificationPurificationDehydratationDehydratation

StructureStructure ofof agaragaragaroseagarose

1,41,4--linkedlinked 3,63,6--anhydroanhydro-- aa--ll--galactosegalactose alternatingalternatingwithwith 1,31,3--linkedlinked bb--dd--galactosegalactose

AgaropectinAgaropectinrepeatingrepeating unit as unit as agaroseagarose, , somesome ofof thethe 3,63,6--anhydroanhydro--ll--galactosegalactose residuesresidues cancan bebereplacedreplaced withwith ll--galactosegalactosesulfatesulfate residuesresidues andand thethe dd--galactosegalactose residuesresidues are are partiallypartially replacedreplaced withwith thethepyruvicpyruvic acidacid acetalacetal 4,64,6--OO--(1(1--carboxyethylidenecarboxyethylidene))--dd--galactosegalactose

AgarAgar

AgarAgar

Agar cell Agar cell wallswalls ofof redredseaweedsseaweeds: : DD--galactosegalactose3,63,6--anhydroanhydro--LL--galactosegalactosefewfew sulfatesulfategroupsgroups

AgarAgarAgar Agar consistsconsists ofof a a mixturemixture ofof agaroseagarose andand agaropectinagaropectin. . AgaroseAgarose isis a a linearlinear polymer, polymer, ofof molecularmolecular weightweight aboutabout120,000, 120,000, basedbased on on thethe --(1(1®®3)3)--bb--DD--galactopyranosegalactopyranose--(1(1®®4)4)--3,63,6--anhydroanhydro--aa--LL--galactopyranosegalactopyranose unit; unit; thethe major major differencesdifferences fromfrom carrageenanscarrageenans beingbeing thethe presence presence ofof LL--3,63,6--anhydroanhydro--aa--galactopyranosegalactopyranose ratherrather thanthan DD--3,63,6--anhydroanhydro--aa--galactopyranosegalactopyranose unitsunits andand thethe lacklack ofof sulfatesulfategroupsgroups. . AgaropectinAgaropectin isis a a heterogeneousheterogeneous mixturemixture ofofsmallersmaller moleculesmolecules thatthat occuroccur in in lesserlesser amountsamounts. . TheirTheirstructuresstructures are are similarsimilar butbut slightlyslightly branchedbranched andand sulfatedsulfated, , andand theythey maymay havehave methyl methyl andand pyruvicpyruvic acidacid ketalketalsubstituentssubstituents. . TheyThey gel gel poorlypoorly andand maymay bebe simplysimplyremovedremoved fromfrom thethe excellentexcellent gellinggelling agaroseagarose moleculesmolecules by by usingusing theirtheir chargecharge. . TheThe qualityquality ofof agar agar isis improvedimproved by by alkalinealkaline treatmenttreatment thatthat convertsconverts ofof anyany LL--galactosegalactose--66--sulfatesulfate to 3,6to 3,6--anhydroanhydro--LL--galactosegalactose..

AgarosaAgarosa

ProcessingProcessing to to removeremove SOSO3 3 NaBHNaBH4/4/--OHOH

MacropourusMacropourus, , hydrophylichydrophylicComercialyComercialy availabilityavailabilityChemicallyChemically stablestableLowLow nonnon--specificspecific bindingbindingResistentResistent to MOto MO

AgarosaAgarosa

AgaroseAgarose

AgaroseAgarose moleculesmolecules havehave molecularmolecular weightsweightsaboutabout 120,000, 120,000, TheThe gel network gel network ofof agaroseagarosecontainscontains double double heliceshelices formedformed fromfrom leftleft--handedhandedthreefoldthreefold heliceshelices. These double . These double heliceshelices are are stabilizedstabilized by by thethe presence presence ofof waterwater moleculesmoleculesboundbound insideinside thethe double double helicalhelical cavitycavity [[508508]. ]. ExteriorExterior hydroxyl hydroxyl groupsgroups allowallow aggregationaggregation ofof upupto 10,000 to 10,000 ofof these these heliceshelices to to formform suprafiberssuprafibers..

AgarosaAgarosa

ZlepZlepššeneníí mechanických vlastnostmechanických vlastnostíí

ProkProkřříížženeníí napnapřř. . epichlorhydrinemepichlorhydrinem

GellingGelling mechanismmechanism

HysteresisHysteresis ofof 1.51.5 % agar % agar gelsgels

ThreeThree equatorialequatorial hydrogenhydrogenatomsatoms ofof thethe 3,63,6--anhydroanhydro-- aa--ll--galactosegalactose residueresidue are are responsibleresponsible for for constrainingconstrainingthethe moleculemolecule soso as to as to formform a a helixhelix withwith a a threefoldthreefold screwscrewaxisaxis

Gel formation mechanism in aqueous agar solutions

QuickQuick SolubleSoluble AgarAgar

Comparison ofproductionprocesses oftraditional agar and QSA

Patent manufacturing process without any chemical or geneticmodifications

AlginateAlginate

familyfamily ofof linearlinear 1,41,4--linkedlinked αα--L L guluronogulurono-- ββ--DD--mannuronansmannuronans ofof widelywidely varyingvaryingcompositioncomposition andand sequentialsequential structuresstructures

CommercialCommercial preparationspreparations are are usuallyusuallydesignateddesignated as as alginatesalginates andand includeinclude alginicalginicacidacid, , itsits saltssalts, , andand derivativesderivatives

OOriginrigin ofof seaweedseaweed extractsextracts ——generalgeneral classificationclassification

OOriginrigin ofof seaweedseaweedextractsextracts ——generalgeneralclassificationclassification

aa Species Species ofof economiceconomicsignificancesignificancebb ContainsContains onlyonlycomponentcomponent mentionedmentionedcc ContainsContains predominantlypredominantlyunderlinedunderlined componentcomponent

Flow sheet for Flow sheet for the productionthe production

of sodium of sodium alginatealginate

1

1

1

AlginateAlginate

AlginatesAlginates are are linearlinear unbranchedunbranched polymerspolymers containingcontaining ββ--(1(1--4)4)--linkedlinked DD--mannuronicmannuronic acidacid ((MM) ) andand αα--(1(1--44))--linkedlinked LL--guluronicguluronic acidacid ((GG) ) residuesresidues. . AlthoughAlthough these these residuesresidues are are epimersepimers (D(D--mannuronicmannuronic acidacid residuesresidues beingbeingenzymaticallyenzymatically convertedconverted to Lto L--guluronicguluronic afterafter polymerizationpolymerization andand onlyonly differdifferatat C5, C5, theythey possesspossess veryvery differentdifferent conformationsconformations; D; D--mannuronicmannuronic acidacid beingbeing4C14C1 withwith diequatorialdiequatorial linkslinks betweenbetween themthem andand LL--guluronicguluronic acidacid beingbeing 1C4 1C4 withwith diaxialdiaxial linkslinks betweenbetween themthem. . BacterialBacterial alginatesalginates are are additionallyadditionally OO--acetylatedacetylated on on thethe 2 2 andand//oror 3 3 positionspositions ofof thethe DD--mannuronicmannuronic acidacid residuesresidues. . TheThe bacterialbacterial OO--acetylaseacetylase maymay bebe usedused to Oto O--acetylateacetylate thethe algalalgal alginatesalginates, , sosoincreasingincreasing theirtheir waterwater bindingbinding..

StructureStructureofof

AlginatesAlginates

structuralstructural elementselements::homopolysaccharideshomopolysaccharides αα--1,41,4--LL--guluronanguluronan andand ββ--1,41,4--D D mannuronanmannuronanheteropolysaccharideheteropolysaccharideconsistingconsisting ofof alternatingalternating 1,41,4--linkedlinked αα--LL--guluronicguluronic (G) (G) andandββ--DD--mannuronicmannuronic acidacid (M) (M) residuesresidues

–– MM –– GG –– MM –– (M(M –– M)M)nn ––MM –– GG –– (M(M –– G)G)qq –– MM –– GG ––(G(G –– G)G)pp –– GG –– MM –– GG ––

AlginateAlginate

AlginateAlginate

AlginateAlginate

veve vodvoděě rozpustný rozpustný alginalgináátt sodnýsodnýzjemzjemňňovadloovadlo, , emulsifikemulsifikáátortor, jako film, , jako film, žželelíírujrujííccíí lláátkatkanetoxický, lacinýnetoxický, lacinýbiochemicky inertnbiochemicky inertníí, mechanicky stabiln, mechanicky stabilníí

AlginateAlginateAlginateAlginate's 's solubilitysolubility andand waterwater--holding holding capacitycapacity dependdepend on on pHpH((precipitatingprecipitating belowbelow aboutabout pHpH 3.5), 3.5), molecularmolecular weightweight ((lowerlowermolecularmolecular weightweight calciumcalcium alginatealginate chainschains withwith lessless thanthan 500 500 residuesresidues showingshowing increasingincreasing waterwater bindingbinding withwith increasingincreasing sizesize), ), ionicionic strengthstrength ((lowlow ionicionic strengthstrength increasingincreasing thethe extendedextended naturenature ofofthethe chainschains) ) andand thethe naturenature ofof thethe ionsions presentpresent. . GenerallyGenerally alginatesalginatesshow show highhigh waterwater absorptionabsorption andand maymay bebe usedused as as lowlow viscosityviscosityemulsifiersemulsifiers andand shearshear--thinningthinning thickenersthickeners. . TheyThey cancan bebe usedused to to stabilizestabilize phasephase separationseparation in in lowlow fatfat fatfat--substitutessubstitutes e.g.e.g. as as alginatealginate//caseinatecaseinate blendsblends in in starchstarch threethree--phasephase systemssystems. . AlginateAlginate isisusedused in a in a widewide variety variety ofof foodstufffoodstuff such as such as petpet foodfood chunkschunks, , oniononionringsrings, , stuffedstuffed olivesolives, , lowlow fatfat spreadsspreads, , saucessauces andand piepie fillingsfillings. . Propylene Propylene glycolglycol alginatesalginates havehave widespreadwidespread use as use as acidacid--stablestablestabilizersstabilizers for for usesuses such as such as preservingpreserving thethe headhead on on beersbeers..

AlginateAlginateTheThe primaryprimary functionfunction ofof thethe alginatesalginates are as are as thermallythermally stablestable coldcold settingsettinggellinggelling agentsagents in in thethe presence presence ofof calciumcalcium ionsions; ; gellinggelling atat far far lowerlowerconcentrationsconcentrations thanthan gelatingelatin. Such . Such gelsgels cancan bebe heatheat treatedtreated withoutwithout meltingmelting, , althoughalthough theythey maymay eventuallyeventually degradedegrade. . GellingGelling dependsdepends on on thethe ion ion bindingbinding(Mg2+ << Ca2+ < Sr2+ < Ba2+) (Mg2+ << Ca2+ < Sr2+ < Ba2+) withwith thethe controlcontrol ofof thethe dicationdication additionadditionbeingbeing importantimportant for for thethe productionproduction ofof homogeneoushomogeneous gelsgels ((e.g.e.g. by by ionicionicdiffusiondiffusion oror controlledcontrolled acidificationacidification ofof CaCO3). CaCO3). HighHigh GG contentcontent producesproducesstrongstrong brittlebrittle gelsgels withwith goodgood heatheat stability (stability (exceptexcept ifif presentpresent in in lowlowmolecularmolecular weightweight moleculesmolecules) ) butbut proneprone to to waterwater weepageweepage ((syneresissyneresis) on ) on freezefreeze--thawthaw, , whereaswhereas highhigh MM contentcontent producesproduces weakerweaker moremore--elasticelastic gelsgelswithwith goodgood freezefreeze--thawthaw behaviorbehavior andand highhigh MGMGMGMG contentcontent zips zips withwith Ca2+ Ca2+ ionsions to to reducesreduces shearshear. . HoweverHowever, , atat lowlow oror veryvery highhigh Ca2+ Ca2+ concentrationsconcentrationshighhigh MM alginatesalginates produceproduce thethe strongerstronger gelsgels. So . So longlong as as thethe averageaverage chainchainlengthslengths are not are not particularlyparticularly shortshort, , thethe gellinggelling propertiesproperties correlatecorrelate withwithaverageaverage GG blockblock lengthlength (optimum (optimum blockblock sizesize ~12; ~12; seesee alsoalso thethe similaritysimilarity to to pectinpectin gellinggelling) ) andand not not necessarilynecessarily withwith thethe MM//GG ratioratio whichwhich maymay bebeprimarilyprimarily duedue to to alternatingalternating MGMGMGMG chainschains. . TheThe futurefuture prospectsprospects are are excellentexcellent as as recombinantrecombinant epimerasesepimerases withwith differentdifferent specificitiesspecificities maymay bebeusedused to to produceproduce novel novel designerdesigner alginatesalginates..

ApplicationApplication

SodiumSodium alginatealginate isis thethe importantimportant materialmaterial to to produceproduce seaweedseaweed productsproducts such as mansuch as man--mademadegrapegrape andand manmademanmade cherrycherry..b.b.icediced foodfoodSodiumSodium alginatealginate isis as a as a stabilizerstabilizer ofof iceice creamcreambecausebecause ofof itsits densedense organizationorganization isis veryvery equalequalandand thethe slowslow solutionsolution speedspeed. . ItIt isis thethe mainmainmaterialmaterial ofof thethe daintydainty coldcold powderpowder andand jellyjelly......

ApplicationApplication

c.c.cakecake foodfoodAs a As a stabilizerstabilizer ofof cakecake (such as (such as cookiescookies, , breadbread, , finefine drieddried noodlenoodle, , chocolateschocolates etcetc) ) andand a a polishpolish ofof breadbread. . SodiumSodium alginatealginate cancankeepkeep thethe cookiescookies sweetsweet--smellingsmelling andand avoidavoidfallingfalling to to piecespieces, , andand makemake thethe noodlenoodlesmoothsmooth, , reducereduce breakingbreaking ratioratio ofof noodlenoodleandand fragmentationfragmentation ofof breadbread

ApplicationApplicationSodiumSodium alginatealginate isis alsoalso thethe goodgood thickenerthickener ofof jam, jam, chilichilisaucesauce, , jellyjelly, , tomatotomato ketchupketchup, , fishpastefishpaste, , puddingpudding andand salasalaflavoringflavoring..d.d.drinkingdrinking""SodiumSodium alginatealginate isis as as thethe stabilizerstabilizer ofof thethe beerbeer andandclarifierclarifier ofof thethe winewine..e.e.freezingfreezing andand keepingkeeping freshfreshWhenWhen foodfood ((fruitfruit, , fishfish etcetc) ) isis coveredcovered withwith thethe film film ofofsodiumsodium alginatealginate andand keptkept isolatedisolated fromfrom airair, , thethe film film willwillstop stop bacteriabacteria fromfrom invadinginvading, , constraintconstraint waterwaterevaporationevaporation ofof foodfood itselfitself andand prolongedprolonged timetime ofofpreservationpreservation

OriginOrigin andand mainmain sugarsugar moietiesmoieties ofof polysaccharidespolysaccharides

PectinPectin cell cell wallswalls andand middlemiddlelamellalamella ofof higherhigher landland plantsplants: : DD--galacturonicgalacturonic acidacid DD--galacturonicgalacturonicacidacid methylestermethylester

-d-Galactopyranosyluronic acid in 4C1 conformation (above); fragment of galacturonan chain, 40 % methylate

PectinPectin

Schematic representation of pectin backbone, showing the “hairy” regions (rhamnogalacturonan and side-chains) and the “smooth” regions (linear galacturonan

AlginateAlginate cellscells wallswalls ofof brownbrownseaweedsseaweeds exopolysacchridesexopolysacchrides ofofAzetobacterAzetobacter vinelandiivinelandiidd: : mannuronicmannuronic acidacid LL--guluronicguluronicacidacid andand theirtheir acetyl acetyl derivativesderivatives

CarrageenanCarrageenan cell cell wallswalls ofof redredseaweedsseaweeds: : DD--galactosegalactose 3,63,6--anhydroanhydro--DD--galactosegalactose bothbothsugarssugars sulfatedsulfated to to higherhigher ororlowerlower degreedegree

CarageenanCarageenan

33--linkedlinked--ββ--DD--galactopyranosegalactopyranose andand 44--linkedlinked--αα--DD--galactopyranosegalactopyranose unitsunits

CarrageenanCarrageenan

CarrageenanCarrageenan ((E407E407) ) isis a a collectivecollective term term for for polysaccharidespolysaccharides preparedprepared by by alkalinealkalineextractionextraction ((andand modificationmodification) ) fromfrom redredseaweedseaweed ((RhodophycaeRhodophycae), ), mostlymostly ofof genusgenusChondrusChondrus, , EucheumaEucheuma, , GigartinaGigartina andandIridaeaIridaea. . DifferentDifferent seaweedsseaweeds produceproducedifferentdifferent carrageenanscarrageenans..

κκ--carrageenancarrageenan (kappa(kappa--carrageenancarrageenan)) --(1(1--3)3)--ββ--DD--galactopyranosegalactopyranose--44--

sulfatesulfate--(1(1--4)4)--3,63,6--anhydroanhydro--αα--DD--galactopyranosegalactopyranose--(1(1--33))--

ιι--carrageenancarrageenan ((iotaiota--carrageenancarrageenan))--(1(1--3)3)--ββ--DD--galactopyranosegalactopyranose--44--sulfatesulfate--(1(1--4)4)--3,63,6--anhydroanhydro--αα--DD--galactopyranosegalactopyranose--22--sulfatesulfate--(1(1--3)3)--

λλ--carrageenancarrageenan (lambda(lambda--carrageenancarrageenan))--(1(1--3)3)--ββ--DD--galactopyranosegalactopyranose--22--sulfatesulfate--(1(1--4)4)--αα--DD--galactopyranosegalactopyranose--2,62,6--disulfatedisulfate--(1(1--3)3)

GelationGelation ofof carrageenanscarrageenans by by formationformation ofof double double heliceshelices

andand aggregationaggregation ofof heliceshelices

CaragenanCaragenan

CarrageenanCarrageenan

κκ--CarrageenanCarrageenan stabilizesstabilizes milkmilk kk--caseincasein productsproductsduedue to to itsits chargecharge interactioninteraction withwith thethe caseincaseinmicellesmicelles (~200 (~200 nmnm diameterdiameter); ); theirtheir incorporationincorporationintointo thethe network network preventingpreventing wheywhey separationseparation. . Such Such complexescomplexes are are solublesoluble whenwhen bothboth havehavesamesame chargecharge andand are are heldheld togethertogether by by counterionscounterions oror oppositelyoppositely chargedcharged patchespatches. . CarrageenanCarrageenan isis alsoalso usedused as a as a binderbinder in in cookedcookedmeatsmeats, to , to firmfirm sausagessausages andand as a as a thickenerthickener in in toothpastetoothpaste andand puddingspuddings..

CelluloseCellulose

ββ--(1(1--4)4)--DD--glucopyranoseglucopyranose unitsunits in in 4C14C1conformationconformation

CelluloseCellulose

CarboxymethylcelluloseCarboxymethylcellulose

DifferentialDifferential massmass distributiondistribution curvescurvesofof variousvarious cellulosescelluloses

a) Cotton;b) Cottonc) China

grass(ramie)

d) FlaxRamie

e) Balsamf) White fir

DegreeDegree ofof polymerizationpolymerization ofofcellulosescelluloses ofof differentdifferent originorigin

Type Type ofof cellulosecellulose nnCottonCotton, , rawraw 77 000000CottonCotton, , rawraw ((accordingaccording to to RussianRussian workwork)) 1414 000000CottonCotton, , purifiedpurified 11 500500 –– 300300CottonCotton linterslinters 66 500500FlaxFlax 88 000000RamieRamie 66 500500aaCelluloseCellulose ((isolatedisolated fromfrom woodwood fibersfibers)) 11 100100 –– 800800SpruceSpruce, , pulpedpulped 33 300300BeechBeech, , pulpedpulped 33 050050AspenAspen 22 500500FirFir 22 500500BacterialBacterial cellulosecellulose 22 700700AcetobacterAcetobacter cellulosecellulose 600600

ElectronElectronmicrographmicrograph ofof

thethe fibrillarfibrillarnaturenature ofof

cellulosecellulose fibersfibers

TheThe architecturearchitecture ofof elementaryelementaryfibrilsfibrils andand microfibrilsmicrofibrils ofof nativenative

cellulosescelluloses

FringeFringe fibrillarfibrillar model model ofof fiberfiberstructurestructure

Stability Stability ofof somesome polysaccharidespolysaccharides atat variousvarious pHpH.. ResidualResidual viscosityviscosity afterafter1010 min min incubationincubation atat 9090 °°CC

a)a) CarboxymethylcellulosCarboxymethylcellulosee –– methyl methyl cellulosecellulose; ; b)b) LocustLocust beanbean gum; gum; c)c) Agar;Agar;d)d) CarrageenanCarrageenan; ; e)e) PectatePectate;;f)f) PectinPectin

Chitin/ Chitin/ chitosanchitosan

ββ−−(1(1−−4) 2 4) 2 acetamidoacetamido--22--deoxydeoxy ββ−−D D glucopyranosidglucopyranosid

2 amino – 2 deoxy glukosa

HCl

ChitinChitin

DextranDextran