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MICROBIAL BIOPOLYMER

1. Pullulan

2. Gellan Gum

3. Dextran

4. Xanthan Gum

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Pullulan

Definition

Structure

Microorganism

Properties and quality parameters

Production and Downstream Process of

Pulullan

Pullulan derivatives

 Application

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Pullulan 

Pullulan is a polysaccharide polymer  

consisting of maltotriose units,

also known as α -1,4- ;α -1,6- glucan   Three glucose units in maltotriose are

connected by an α-1,4 glycosidic bond,

whereas consecutive maltotriose unitsare connected to each other by an α-1,6

glycosidic bond.

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Pullulan Structure

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Optimum temperature : 26-28oC, pH 5 -6.5

Aureobasidium pullulan

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Aureobasidium pullulan

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Source of A. pullullans  isolates

Wood

leather

hydrocarbons

synthetic materials

paper (paper, paper pulp) human skin

paint (watercolours)

plants

food products (wheat, oats and bean seeds,flours, fruits and vegetables, fruit juices)

soil

textile (cotton)

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Aureobasidium pullulans  

in leather (flesh)

Aureobasidium pullulans  

in leather (grain)

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Aureobasidium pullulans  

(cotton)

Aureobasidium pul lu lans  

in acidic paper

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Growth curve of A. pullulan

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On Malt-Agar growth medium

MA) pH 6.5) 

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On CYA growth medium (pH 5.5) – 

 Slimycolonies (final pH 6).

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On Czapek growth medium (initial pH

5.5 and final 5.5)

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Properties and quality

parameters 

1. Water soluble, insoluble in organic solvents, non

hygroscopic, aqueous solutions are stable and show a

relatively low viscosity compared to other

polysaccharides.2. It decomposes at 250-280˚C. It forms easily, thermo-

stable, transparent, elastic, antistatic films, with an

extremely low oxygen permeability (compared to celofan,

polypropylene, polyesther, polyvinylchloride, etc)

3. Moldable, and spinnable, good adhesive and binder.

4. Non-toxic, edible, biodegradable and biocompatible.

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Production and Downstream

Process of Pulullan in general

Submerged fermentation using carbohydrate as main media,

nitrogen source, minerals, temperature of 28o C, agitation

of 200 rpm

Downstream processing of fermentation broth includes :

biomass separation precipitation of filtrates by solvent

2- propanol concentration purification of solution

containing the pullulan-type polysaccharide ( partially

purified product or a highly-purified one modern

ultrafiltration techniques are used).

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FERMENTATION STEPS

 A.pululan in complex ni trogen

agar plates with 50 g/L sucrose  

1st Media

 propagation : malt

extract broth

2nd Media

propagation :

complex nitrogen

broth containing 50g/L sucrose.

Fermentation

Centrifugationand separationof biomass

Precipitation ofpullulan by 2 propanolfollowed by furtherpurification

DryingMillingPacking

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Examples of Pullulan Medium

Composition

Complex Nitrogen medium

5 g KH2PO4, 1 g NaCl, 0.6 g (NH4)2SO4, 0.2

g MgSO4·7H2O, 1 g yeast extract, 0.01 gFeSO4, 0.01 g MnSO4 and 0.01 g ZnSO4 in

grams/liter of water

malt extract broth :20 g malt extract, 20 g

glucose and 1 g peptone per liter of water(ATTC Medium 325).

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Factors affecting pullulan

production

Media components

Strain of microorganisms

Characteristic of microorganism : the phylamentous

forms or chlamydospores are less productive thanthe yeast or pigment-free blastospores (Audet et al.,1996).

According to Shabtai and Mukmenev (1995), theyeast-like cells did not produce much pullulan in the

presence of pigment, and the non pigmented swollenblastospores or germinating blastospores help totrigger the elaboration of pullulan

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Commercial pullulan products 

pullulan derivatives

Crosslinked pullulan microparticles

Carboxymethyl pullulan

Sulfopropyl pullulan

Pullulan acetate 

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Crosslinked pullulan

microparticles

Crosslinking reaction between pullulan and

epichlorohydrin followed by separation,washing, drying

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Healing of infected wounds

a b c

Treatment of a leg wound (horse) with crosslinked

 pullulan microparticles: a) infected wound; b) wound

after several applications of crosslinked pullulan

microparticles; c) wound after 2 weeks of treatment.

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Crosslinked pullulan microparticles in dry state

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Application

edible films for food packaging

breath freshener or oral hygiene products

such as Listerine Cool Mint PocketPaks.

 As a food additive, it is known by the E

number  E1204 (e.g in cosmetics (hydrating

creams and gels)

filming agent in pharmaceutics (retard

tablets, capsules and microcapsules)

Entrap colours and flavours

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Innovative use of pullulan film in

sugar confectionary

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Effect of pullulan coating in tablet

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Soft candy manufacturing  Pullulan capsules

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Application (cont.)

Textiles industry (antistatic, in non-

woven)

Paper industry (coating, in composition) Photosensible materials (emulsions)

 Agriculture (low release fertilizers)

Metalurgy (foundery mold or casting)

Mining (floculant, binder).

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Definition

Structure

Microorganism

Production and downstream process

 Application

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Gellan Gum

a water-soluble polysaccharide producedby a bacterium Pseudomonas elodea

used primarily as a gelling agent,alternative to agar  in microbiologicalculture.

MW : 500 000

Forming viscous solution, insoluble inethanol

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Structure

The repeating unit of the polymer is atetrasaccharide which consists of two residuesof D-glucose and one of each residues of L-

rhamnose and D-glucuronic acid. The tetrasacharide repeat has the following

structure:[D-Glc(β1→4)D-GlcA(β1→4)D-Glc(β1→4)L-

Rha(α1→3)]n.  As it is evident from the formula the

tetrasacharide units are connected with eachother using an (α1→3) glycosidic bond. 

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Molecular structure

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Structure (cont.)

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Microorganism

Pseudomonas elodea = Sphingomonas

elodea

The composition and structure of nativegellan gum produced by commercial

fermentation is identical to the naturally

occurring polysaccharide formed by

Sphingomonas elodea on plants of Lily

pad varieties.

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Pseudomonas elodea

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Sphingomonas

Gram-negative, rod shaped, chemoheterotrophic,

strictly aerobic bacteria

containing glycosphingolipids (GSLs) intheir cell envelopes,

typically produce yellow-pigmentedcolonies

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Yellow pigmented colonies of

Sphingomonas

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Properties of gellan gum

ability to suspend while contributing

minimal viscosity via the formation of a

uniquely functioning fluid gel solutionwith a weak gel structure.

setting temperature, degree of structure

and thermal stability.

Water soluble

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Biosynthesis 

1. intracellular formation of the nucleotide-sugar precursors, UDP-Glc, UDP-GlcA, anddTDP-l-Rha.

2. formation of the repeat unit, with sequentialtransfer of the sugar donors to an activatedlipid carrier by committedglycosyltransferases

3. gellan polymerization

4. Export to outer of cells

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Production and downstream

process in general

produced by a pure culture fermentation ofcarbohydrates by Pseudomonas elodea,purified by recovery with alcohol, dried, andmilled.

The direct fermentation of sweet cheese wheydiluted 1:5 with water resulted in production ofapproximately 7 g of EPS (extrapolysaccharides Sellular ) gellan gum per liter

and in a 70% reduction in the initial BOD5  interesting valorisation of this waste of thedairy industry and BOD reduction.

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Factors affecting gellan gum

production

1. Media components

Molasses 112.5 g/L, tryptone 1 g/L,

casamino acid 1 g/L , disodium hydrogenorthophosphate 1 g/L and manganese

chloride 0.947 g/L  produced gellan

gum of 13.81 g/L

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Factors affecting gellan gum

production (cont.)

2. Addition of precursor   The sugarnucleotides providing the activatedprecursors for synthesis oftetrasaccharide i.e UDP-glucose, TDP-rhamnose and UDP-glucuronic acid.

3. Addition of amino acid  triptophan

 enhanced cell growth4. pH  6.5 - 7

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Factors affecting gellan gum

production (cont.)

5.Agitation rate : 250 rpm using a helical

ribbon impeller

6. Higher DOT  (dissolved oxygen tension)levels improve the viscosity and

molecular mass of the polymer

7. Temperature : 30oC

DOWNSTREAM PROCESS

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DOWNSTREAM PROCESS

Fermentationbroth

Heating 90-95oC, 10-15min

Killed bacteria and reduced the viscosity

Dilution

Centrifugation and filtration Separates cells and filtrates

Precipitation by alcoholfollowed by centrifugation

Drying 55oC

Milling and packing ofcrude gellan gum

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Purification of gellan gum

 After alcohol precipitation, the products arewashed repeatedly with acetone and ether,dissolved in deionised water and dialyzedagainst deionised water by using dialysistubing with molecular mass cut-off of 12 000 –14 000.

 After dialysis for 2 –3 days with four or fivechanges of deionised water, the solution was

lyophilized to formulate dry gellan powder gel filtration chromatography (GFC) can also

be used

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Application

 Applications in foods : stabilizer,emulsifier, thickener

 Applications in pharmaceutical industry Solid culture media for growth of

microorganisms and plants

Gel electrophoresis in biological

research Cell immobilization

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DEXTRAN

complex, branched glucan 

(polysaccharide made of many glucose 

molecules) composed of chains ofvarying lengths

Dextran was first discovered by Louis

Pasteur  as a microbial by product in

wine

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Structure

The straight chain consists of α-1,6

glycosidic linkages between glucose

molecules, while branches begin from α-1,4 linkages (and in some cases, α-1,2

and α-1,3 linkages as well).

molecular weights ranging from 10,000

Da to 150,000 Da.

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Molecular Structure

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Microorganisms

Dextran is synthesized from sucrose by

certain lactic-acid bacteria, the best-known

being Leuconostoc mesenteroides andStreptococcus mutans, L. dextranicum , S.

salivarius, S. bovis

Dental plaque   contains dextran

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Leuconostoc mesenteroides Streptococcus mutans  

Slimy dextran produced by Leuconostoc

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y p y

mesenteroides CMG713 on sucrose

containing medium with 0.005 % sodium

azide

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Production of dextran

Medium :  using sucrose as carbon source, examples :

Sucrose 150.0 g., Peptone 2.5 g., Yeast extract 2.5 g.,K2HPO4 , 5.0 g., NaCl 2.5 g., and a water extract ofsugar refining charcoal 2.0 ml.

Sucrose 150.0 g., acid hydrolyzed casein 5.0 g., Yeastextract 1.0 g., K2HPO4 5.0 g., NaCl 2.0 g., and MgSO40.022 g.

(g l –1): sucrose, 150.0; bacto-peptone, 5.0; yeast extract,5.0; K2HPO4, 15.0; MnCl2.H2O, 0.01; NaCl, 0.01; CaCl2,0.05.

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Condition and downstream

process

Condition : 30 oC, 20 hours

Downstream process :

The culture medium after 20 hours was precipitatedusing equal volume of chilled ethanol, shakenvigorously, centrifuged at 10,000 rpm for 15 minutesand the supernatant was decanted. This step wasrepeated twice.

The precipitated dextran was dried under vacuumover calcium chloride at 30ºC. The dextran yield wascalculated on dry weight basis.

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Purification

For removal of impurities, dextran obtainedfrom precipitation was dissolved in distilledwater.

The dextran slurry was again precipitated withequal volume of chilled ethanol. Thisprocedure of re-dissolving, precipitation andwashing was repeated three times to remove

cells debris. Purified dextran was dried under vacuum over

calcium chloride at 30ºC

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Factors affecting dextran

production

1. Incubation time

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Factors affecting dextran

production (cont.)

2. Substrate concentration

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Factors affecting dextran

production (cont.)

3. Temperature

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Factors affecting dextran

production (cont.)

4. pH

Molecular weight of dextran types

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Molecular weight of dextran types  

using glass column chromatography

packed with Sepharose CL6B.

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Remarks :

 Blue Dextran, 2000,000;

 Industrial dextran, 5000,000-40,000,000;

 Dextran from Leuconostoc mesenteroides CMG713, 5,000,000-20,000,000.

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Application medical uses

eye drops as a lubricant

decrease vascular thrombosis.

Dextran in intravenous solution provides anosmotically neutral fluid that once in the body is

digested by cells into glucose and free water.

It also increases blood sugar  levels.

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Other uses

size-exclusion chromatography matrices;

an example is Sephadex.

bead form to aid in bioreactorapplications (immobilization)

stabilizing coating to protect metal

nanoparticles from oxidation make microcarriers for industrial cell

culture

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Application (cont. )

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GUM XANTHAN

ISI KULIAH :

1. Deskripsi Produk ( Sifat fisiko kimia, biosintesa ,

kegunaan dalam industri, dll)

2. Mikrooorganisme

3. Tahapan fermentasi

4. Proses Hilir (recovery)

5. Prospek Pengembangan

di Indonesia

Gum xanthan : eksopolisakarida mikrobial yang diproduksi

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oleh  Xanthomonas campestris dari bahan

berkarbohidrat

Sifat Fisiko Kimia :1. Heteropolisakarida anionik yang bercabang

2. Mengandung D-glukosa (2.8 mol), D-manosa (3.0 mol),

D-glukuronat (2.0 mol), asam asetat dan asam piruvat

3. Rantai utama mirip kerangka selulosa (rantai glukosa

berikatan ß – 1,4)

4. Rantai sisi mengandung dua unit manosa dan satu unit

asam glukuronat

5. Asetat dan piruvat berikatan pada ujung manosa

6. BM : 2-50 x 106  atau 3 x 107 dalton

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Struktur molekul gum xanthan

TURUNAN KIMIA DAN KOPOLIMER CANGKOKAN GUM

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TURUNAN KIMIA DAN KOPOLIMER CANGKOKAN GUM

XANTHAN

1. Gum Karboksimetil xanthan

2. Gum dietilaminotil xanthan

3. Ester propilenglikol xanthan

4. Gum xanthan sulfat5. Ikatan silang aldehid gum xanthan

6. Gum deasetil xanthan

7. Gum xanthan - g – poli akrilamida

Sampai saat ini tidak ada yang diproduksi secara komersil

SIFAT SIFAT GUM XANTHAN

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SIFAT-SIFAT GUM XANTHAN :

1. Viskositas tinggi pada konsentrasi rendah,

2. Kekenyalan semu (pseudoplasticity) tinggi

3. Mudah larut dalam air panas maupun dingin

4. Viskositas larutan gum xanthan stabil terhadap suhu

5. Viskositas larutan gum xanthan stabil terhadap pH

6. Kelarutan dan kestabilan tinggi pada asam

7. Unggul dalam daya suspensi karena yield valuenya tinggi

8. Kesesuaian yang baik dengan berbagai garam

9. Stabilitas pada proses pelelehan dari keadaan bekunya

(freeze thaw)

SIFAT GUM XANTHAN (LANJUTAN)

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  SIFAT GUM XANTHAN (LANJUTAN)

10. Kompatibel terhadap beberapa pelarut seperti methanol,

ethanol, isopropanol dan aseton sampai konsentrasi 50-60%. Konsentrasi pelarut > 60 % akan menyebabkan

gelatinisasi atau pengendapan gum xanthan

11. Tidak terdegradasi oleh enzim protease, selulase

hemiselulase, pektinase dan amilase, tetapi dapatterdegradasi oleh pengoksidasi kuat seperti peroksida,

persulfat dan hipoklorit

12. Sinergi terhadap gum yang lain seperti gum guar, dan

gum dari biji locust (locust bean gum) serta galaktomanan

Viskositas (cp)

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Viskositas (cp)

1000

100

0.5 1.0 1.5 2.0 2.5 3.0 Konsentrasi (%)

Pengaruh konsentrasi gum xanthan thd viskositas

Viskositas (cP) Pengaruh konsentrasi garam thd viskositas lar gum xanthan

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Konsentrasi garam (NaCl) (%)

100

1000

0.01 0.05 0.1 1.0

0.1 % gum xanthan

0.25 % gum xanthan

0.5 % gum xanthan

1.0 % gum xanthan

Viskositas (cP) Pengaruh suhu thd viskositas lar gum xanthan

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Temperatur (oF)

1000

10000

50 100 150 200

0.1 % gum xanthan

0.25 % gum xanthan

0.5 % gum xanthan

1.0 % gum xanthan

Viskositas (cP) Pengaruh pH thd viskositas lar gum xanthan

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 pH

1000

10000

1 3 5 7 9 11 13

0.1 % gum xanthan

0.25 % gum xanthan

0.5 % gum xanthan

1.0 % gum xanthan

Viskositas (cP)

Pengaruh laju geser terhadap viskositas larutan gum xanthan

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Laju geser detik -1

0.1 1 10 100 1000 10 000

1

10

100

1000

10 000

100 000

1 000 000

0.5 %

1 %

2.5 %

Pengaruh laju geser terhadap viskositas larutan gum xanthan

Sifat sinergi gum xanthan denganli k id l i

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Guar gum

Tara gum

polisakarida lainnya

 

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APLIKASI DALAM INDUSTRI :

1. Industri Petroleum :

a. Sebagai cairan pelicin dalam pengeboran sumur minyak

b. Sebagai cairan untuk mengikat dan memisahkan garam-garamdari hasil pengeboran minyak lepas pantai dari cairan petroleumyang diinginkan

c. Sebagai cairan pemecah (fracturing fluid ) berbasis air

d. Untuk mempercepat pengambilan minyak (oil recovery)

Oil drilling

2 Industri Kimia dan lainnya

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2. Industri Kimia dan lainnya

a. Pakan ternak : sebagai cairan pelengkap pakan (Liquid feedsupplements)

b. Flowable pesticides

c. Pewarnaan dan pengecatan tekstil

d. Pelapis keramik

e. Pembersih

f. Pensuspensi cairan tinta, cat dan perekat kertas

3. Industri Pharmasi dan Kosmetika :

a. Stabilizer emulsi cream untuk obat dan kosmetika

b. Pelarut cream dan lotion

c. Pensuspensi pasta gigi atau larutan pencuci gigi

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4. Industri pangan :

a. Pastry fillingb. Penstabil saus dan kaldu

c. Pengemulsi “dressing”  

d. Pengemulsi dan stabilizer “dairy products”  

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BIOSINTESA

 Ada 4 jenis enzim yang terlibat :

1. Enzim untuk metabolisme awal substrat : hexokinase

2. Enzim untuk sintesis dan interkonversi nukleotida gula (UDP

glucose phosphorylase)

3. Enzim untuk pembentukan pengulangan unit polimer(monosakarida) (transferase)

4. Enzim polimerase untuk pembentukan biopolimereksopolisakarida

Tahapan biosintesa :

1. Metabolisme substrat karbohidrat

2. Sintesis dan interkonversi nukleotida gula

3. Pengulangan unit monomer

4. Polimerisasi

MIKROORGANISME :

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MIKROORGANISME :

Bakteri penghasil gum xanthan :Xanthomonas campestris

1. Gram Negatif

2. Membentuk koloni berlendir

berwarna kuning

3. Diisolasi dari daun/tanamanyang berpenyakit,misalnya daunkedelai yang terkena pustule,daun kubis, tebu, dll

TAHAPAN FERMENTASI

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Xanthomonas campestris Pengembanganinokulum

Tangki benih

Fermentasi

Pasteurisasidanpemisahanbiomassa

Pengambilangum denganpelarut

PengeringanPenggilinganPengemasan

Komponen media :

- Karbohidrat sbg sumber C

- Sumber N

- Mineral

- Antifoam

KONTROL PROSES

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Level of control

Metabolic

Environmental Nutrients, pH, P O2, P CO2, shear, temperature, fluid properties

Equipment design

operation Nutrients, acid/base, antifoam addition, air flow, speed, over

pressure, coolant, flow rate and temperature, sparger, and fermenter

impeller geometry

Product biosynthesis

Cell environ

Other processes

Product

Waste (cells, otherproducts, heat)

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SIFAT RHEOLOGI CAIRAN FERMENTASI GUMXANTHAN SANGAT KHAS :

1. Pada penggunaan bioreaktor berpengaduk akantimbul zona stagnasi

2. Masalah ini diatasi dengan penggunaan airliftbioreactor

 Air lift bioreactor

Stirred tank bioreactor

PROSES HILIR :

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Cairan fermentasi

Pasteurisasi Untuk membunuh sel bakteri yang masih tersisa

Pengenceran Untuk memudahkan pemisahan biomassakarena kekentalannya telah diturunkan

Sentrifugasi Untuk memisahkan sel/biomassa

Pengambilan gum dg pelarut Untuk mempresipitasikan gum , bisa dg

etanol, isopropanol, metanol, dll

Pengeringan

Penggilingan

BEBERAPA MERK GUM XANTHAN KOMERSIL :

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1. Keltrol

2. Kelzan

3. Jungbunzlauer

4. Degussa

5. dll

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Prospek Pengembangan di Indonesia :

1. Ketersediaan bahan baku : berbagai jenis sumberkarbon dan nitrogen

2. Ketersediaan sumber isolat

3. Aspek teknis teknologis (penggunaan teknologifermentasi)

4. Aspek pasar yang terbuka luas

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