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Pullulan Gellan Gum Dextran Xanthan Gum

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Presentasi berjudul: "Pullulan Gellan Gum Dextran Xanthan Gum"— Transcript presentasi:

1 Pullulan Gellan Gum Dextran Xanthan Gum
MICROBIAL BIOPOLYMER Pullulan Gellan Gum Dextran Xanthan Gum

2 Pullulan Definition Structure Microorganism
Properties and quality parameters Production and Downstream Process of Pulullan Pullulan derivatives Application

3 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 units are connected to each other by an α-1,6 glycosidic bond.

4 Pullulan Structure

5 Microorganism

6 Aureobasidium pullulan
Optimum temperature : oC, pH Aureobasidium pullulan

7 Aureobasidium pullulan

8 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)

9 Aureobasidium pullulans in leather (flesh)
Aureobasidium pullulans in leather (grain)

10 Aureobasidium pullulans (cotton)
Aureobasidium pullulans in acidic paper

11 Growth curve of A. pullulan

12 On Malt-Agar growth medium (MA) (pH 6.5)

13 On CYA growth medium (pH 5.5) – Slimy colonies (final pH 6).

14 On Czapek growth medium (initial pH 5.5 and final 5.5)

15 Properties and quality parameters of pulullan
Water soluble, insoluble in organic solvents, non hygroscopic, aqueous solutions are stable and show a relatively low viscosity compared to other polysaccharides. It decomposes at ˚C. It forms easily, thermo-stable, transparent, elastic, antistatic films, with an extremely low oxygen permeability (compared to celofan, polypropylene, polyesther, polyvinylchloride, etc) Moldable, and spinnable, good adhesive and binder. Non-toxic, edible, biodegradable and biocompatible.

16 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).

17 FERMENTATION STEPS 2nd Media propagation : complex nitrogen broth containing 50 g/L sucrose. 1st Media propagation : malt extract broth A.pululan in complex nitrogen agar plates with 50 g/L sucrose Fermentation Centrifugation and separation of biomass Precipitation of pullulan by 2 propanol followed by further purification Drying Packing Milling

18 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 g FeSO4, 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).

19 Factors affecting pullulan production
Media components Strain of microorganisms Characteristic of microorganism : the phylamentous forms or chlamydospores are less productive than the yeast or pigment-free blastospores (Audet et al., 1996). According to Shabtai and Mukmenev (1995), the yeast-like cells did not produce much pullulan in the presence of pigment, and the non pigmented swollen blastospores or germinating blastospores help to trigger the elaboration of pullulan





24 Commercial pullulan products pullulan derivatives
Crosslinked pullulan microparticles Carboxymethyl pullulan Sulfopropyl pullulan Pullulan acetate

25 Crosslinked pullulan microparticles
Crosslinking reaction between pullulan and epichlorohydrin followed by separation, washing, drying

26 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.

27 Crosslinked pullulan microparticles in dry state

28 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

29 Innovative use of pullulan film in sugar confectionary

30 Effect of pullulan coating in tablet

31 Soft candy manufacturing
Pullulan capsules

32 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).

33 GELLAN GUM Definition Structure Microorganism
Production and downstream process Application

34 Gellan Gum a water-soluble polysaccharide produced by a bacterium Pseudomonas elodea used primarily as a gelling agent, alternative to agar in microbiological culture. MW : Forming viscous solution, insoluble in ethanol

35 Structure The repeating unit of the polymer is a tetrasaccharide which consists of two residues of 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 each other using an (α1→3) glycosidic bond.

36 Molecular structure

37 Structure (cont.)

38 Microorganism Pseudomonas elodea = Sphingomonas elodea
The composition and structure of native gellan gum produced by commercial fermentation is identical to the naturally occurring polysaccharide formed by Sphingomonas elodea on plants of Lily pad varieties.

39 Pseudomonas elodea

40 Sphingomonas Gram-negative, rod shaped, chemo heterotrophic,
strictly aerobic bacteria containing glycosphingolipids (GSLs) in their cell envelopes, typically produce yellow-pigmented colonies

41 Yellow pigmented colonies of Sphingomonas

42 Properties of gellan gum
ability to suspend while contributing minimal viscosity via the formation of a uniquely functioning fluid gel solution with a weak gel structure. setting temperature, degree of structure and thermal stability. Water soluble

43 Biosynthesis intracellular formation of the nucleotide-sugar precursors, UDP-Glc, UDP-GlcA, and dTDP-l-Rha. formation of the repeat unit, with sequential transfer of the sugar donors to an activated lipid carrier by committed glycosyltransferases gellan polymerization Export to outer of cells

44 Production and downstream process in general
produced by a pure culture fermentation of carbohydrates by Pseudomonas elodea, purified by recovery with alcohol, dried, and milled. The direct fermentation of sweet cheese whey diluted 1:5 with water resulted in production of approximately 7 g of EPS (extra polysaccharides Sellular ) gellan gum per liter and in a 70% reduction in the initial BOD5  interesting valorisation of this waste of the dairy industry and BOD reduction. 

45 Factors affecting gellan gum production
1. Media components Molasses g/L, tryptone 1 g/L, casamino acid 1 g/L , disodium hydrogen orthophosphate 1 g/L and manganese chloride g/L  produced gellan gum of g/L

46 Factors affecting gellan gum production (cont.)
2. Addition of precursor  The sugar nucleotides providing the activated precursors for synthesis of tetrasaccharide i.e UDP-glucose, TDP-rhamnose and UDP-glucuronic acid. 3. Addition of amino acid  triptophan  enhanced cell growth 4. pH 

47 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

48 DOWNSTREAM PROCESS Fermentation broth Heating 90-95oC, 10-15 min
Killed bacteria and reduced the viscosity Dilution Separates cells and filtrates Centrifugation and filtration Precipitation by alcohol followed by centrifugation Drying 55oC Milling and packing of crude gellan gum

49 Purification of gellan gum
After alcohol precipitation, the products are washed repeatedly with acetone and ether, dissolved in deionised water and dialyzed against deionised water by using dialysis tubing with molecular mass cut-off of – After dialysis for 2–3 days with four or five changes of deionised water, the solution was lyophilized to formulate dry gellan powder gel filtration chromatography (GFC) can also be used

50 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


52 DEXTRAN complex, branched glucan (polysaccharide made of many glucose molecules) composed of chains of varying lengths Dextran was first discovered by Louis Pasteur as a microbial by product in wine

53 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.

54 Molecular Structure

55 Microorganisms Dextran is synthesized from sucrose by certain lactic-acid bacteria, the best-known being Leuconostoc mesenteroides and Streptococcus mutans, L. dextranicum , S. salivarius, S. bovis Dental plaque  contains dextran

56 Leuconostoc mesenteroides Streptococcus mutans

57 Slimy dextran produced by Leuconostoc mesenteroides CMG713 on sucrose containing medium with % sodium azide

58 Production of dextran Medium : using sucrose as carbon source, examples : Sucrose g., Peptone 2.5 g., Yeast extract 2.5 g., K2HPO4 , 5.0 g., NaCl 2.5 g., and a water extract of sugar refining charcoal 2.0 ml. Sucrose g., acid hydrolyzed casein 5.0 g., Yeast extract 1.0 g., K2HPO4 5.0 g., NaCl 2.0 g., and MgSO 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.

59 Condition and downstream process
Condition : 30 oC, 20 hours Downstream process : The culture medium after 20 hours was precipitated using equal volume of chilled ethanol, shaken vigorously, centrifuged at 10,000 rpm for 15 minutes and the supernatant was decanted. This step was repeated twice. The precipitated dextran was dried under vacuum over calcium chloride at 30ºC. The dextran yield was calculated on dry weight basis.

60 Purification For removal of impurities, dextran obtained from precipitation was dissolved in distilled water. The dextran slurry was again precipitated with equal volume of chilled ethanol. This procedure of re-dissolving, precipitation and washing was repeated three times to remove cells debris. Purified dextran was dried under vacuum over calcium chloride at 30ºC

61 Factors affecting dextran production
1. Incubation time

62 Factors affecting dextran production (cont.)
2. Substrate concentration

63 Factors affecting dextran production (cont.)
3. Temperature

64 Factors affecting dextran production (cont.)
4. pH

65 Molecular weight of dextran types  using glass column chromatography packed with Sepharose CL6B.

66 Remarks : Blue Dextran, 2000,000;
Industrial dextran, 5000,000-40,000,000; Dextran from Leuconostoc mesenteroides CMG713, 5,000,000-20,000,000.

67 Application medical uses
eye drops as a lubricant decrease vascular thrombosis. Dextran in intravenous solution provides an osmotically neutral fluid that once in the body is digested by cells into glucose and free water. It also increases blood sugar levels.

68 Other uses size-exclusion chromatography matrices; an example is Sephadex. bead form to aid in bioreactor applications (immobilization) stabilizing coating to protect metal nanoparticles from oxidation make microcarriers for industrial cell culture

69 Application (cont. )

Deskripsi Produk ( Sifat fisiko kimia, biosintesa , kegunaan dalam industri, dll) Mikrooorganisme Tahapan fermentasi Proses Hilir (recovery) Prospek Pengembangan di Indonesia

71 Gum xanthan : eksopolisakarida mikrobial yang diproduksi oleh Xanthomonas campestris dari bahan berkarbohidrat Sifat Fisiko Kimia : Heteropolisakarida anionik yang bercabang Mengandung D-glukosa (2.8 mol), D-manosa (3.0 mol), D-glukuronat (2.0 mol), asam asetat dan asam piruvat Rantai utama mirip kerangka selulosa (rantai glukosa berikatan ß – 1,4) Rantai sisi mengandung dua unit manosa dan satu unit asam glukuronat Asetat dan piruvat berikatan pada ujung manosa BM : x atau 3 x 107 dalton

72 Struktur molekul gum xanthan

Gum Karboksimetil xanthan Gum dietilaminotil xanthan Ester propilenglikol xanthan Gum xanthan sulfat Ikatan silang aldehid gum xanthan Gum deasetil xanthan Gum xanthan - g – poli akrilamida Sampai saat ini tidak ada yang diproduksi secara komersil

Viskositas tinggi pada konsentrasi rendah, Kekenyalan semu (pseudoplasticity) tinggi Mudah larut dalam air panas maupun dingin Viskositas larutan gum xanthan stabil terhadap suhu Viskositas larutan gum xanthan stabil terhadap pH Kelarutan dan kestabilan tinggi pada asam Unggul dalam daya suspensi karena yield valuenya tinggi Kesesuaian yang baik dengan berbagai garam Stabilitas pada proses pelelehan dari keadaan bekunya (freeze thaw)

10. Kompatibel terhadap beberapa pelarut seperti methanol, ethanol, isopropanol dan aseton sampai konsentrasi %. Konsentrasi pelarut > 60 % akan menyebabkan gelatinisasi atau pengendapan gum xanthan 11. Tidak terdegradasi oleh enzim protease, selulase hemiselulase, pektinase dan amilase, tetapi dapat terdegradasi 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

76 Pengaruh konsentrasi gum xanthan thd viskositas
Viskositas (cp) 1000 100 Konsentrasi (%) Pengaruh konsentrasi gum xanthan thd viskositas

77 Pengaruh konsentrasi garam thd viskositas lar gum xanthan
Viskositas (cP) 1.0 % gum xanthan 1000 0.5 % gum xanthan 0.25 % gum xanthan 100 0.1 % gum xanthan Konsentrasi garam (NaCl) (%)

78 Pengaruh suhu thd viskositas lar gum xanthan
Viskositas (cP) 1.0 % gum xanthan 10000 0.5 % gum xanthan 0.25 % gum xanthan 1000 0.1 % gum xanthan Temperatur (oF)

79 Pengaruh pH thd viskositas lar gum xanthan
Viskositas (cP) 1.0 % gum xanthan 10000 0.5 % gum xanthan 0.25 % gum xanthan 1000 0.1 % gum xanthan pH

80 Pengaruh laju geser terhadap viskositas larutan gum xanthan
Viskositas (cP) Pengaruh laju geser terhadap viskositas larutan gum xanthan 10 000 1 % 2.5 % 1000 0.5 % 100 10 1 Laju geser detik -1

81 Sifat sinergi gum xanthan dengan polisakarida lainnya
Guar gum Tara gum

Industri Petroleum : Sebagai cairan pelicin dalam pengeboran sumur minyak Sebagai cairan untuk mengikat dan memisahkan garam-garam dari hasil pengeboran minyak lepas pantai dari cairan petroleum yang diinginkan Sebagai cairan pemecah (fracturing fluid ) berbasis air Untuk mempercepat pengambilan minyak (oil recovery) Oil drilling

83 2. Industri Kimia dan lainnya
Pakan ternak : sebagai cairan pelengkap pakan (Liquid feed supplements) Flowable pesticides Pewarnaan dan pengecatan tekstil Pelapis keramik Pembersih Pensuspensi cairan tinta, cat dan perekat kertas 3. Industri Pharmasi dan Kosmetika : Stabilizer emulsi cream untuk obat dan kosmetika Pelarut cream dan lotion Pensuspensi pasta gigi atau larutan pencuci gigi

84 4. Industri pangan : Pastry filling Penstabil saus dan kaldu
Pengemulsi “dressing” Pengemulsi dan stabilizer “dairy products”

85 BIOSINTESA Tahapan biosintesa : Metabolisme substrat karbohidrat Sintesis dan interkonversi nukleotida gula Pengulangan unit monomer Polimerisasi Ada 4 jenis enzim yang terlibat : Enzim untuk metabolisme awal substrat : hexokinase Enzim untuk sintesis dan interkonversi nukleotida gula (UDP glucose phosphorylase) Enzim untuk pembentukan pengulangan unit polimer (monosakarida) (transferase) Enzim polimerase untuk pembentukan biopolimer eksopolisakarida

86 MIKROORGANISME : Bakteri penghasil gum xanthan : Xanthomonas campestris Gram Negatif Membentuk koloni berlendir berwarna kuning Diisolasi dari daun/tanaman yang berpenyakit,misalnya daun kedelai yang terkena pustule, daun kubis, tebu, dll

87 TAHAPAN FERMENTASI Komponen media : Karbohidrat sbg sumber C Sumber N
Pengembangan inokulum Xanthomonas campestris Tangki benih Fermentasi Komponen media : Karbohidrat sbg sumber C Sumber N Mineral Antifoam Pasteurisasi dan pemisahan biomassa Pengambilan gum dengan pelarut Pengeringan Pengemasan Penggilingan

88 KONTROL PROSES 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, other products, heat)

Pada penggunaan bioreaktor berpengaduk akan timbul zona stagnasi Masalah ini diatasi dengan penggunaan airlift bioreactor Air lift bioreactor Stirred tank bioreactor

90 PROSES HILIR : Cairan fermentasi Pasteurisasi
Untuk membunuh sel bakteri yang masih tersisa Untuk memudahkan pemisahan biomassa karena kekentalannya telah diturunkan Pengenceran Untuk memisahkan sel/biomassa Sentrifugasi Pengambilan gum dg pelarut Untuk mempresipitasikan gum , bisa dg etanol, isopropanol, metanol, dll Pengeringan Penggilingan

Keltrol Kelzan Jungbunzlauer Degussa dll

92 Prospek Pengembangan di Indonesia :
Ketersediaan bahan baku : berbagai jenis sumber karbon dan nitrogen Ketersediaan sumber isolat Aspek teknis teknologis (penggunaan teknologi fermentasi) Aspek pasar yang terbuka luas

93 Terima kasih

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