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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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