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MICROBIAL BIOPOLYMER 1. Pullulan 2. Gellan Gum 3. Dextran 4. Xanthan Gum.

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Presentasi berjudul: "MICROBIAL BIOPOLYMER 1. Pullulan 2. Gellan Gum 3. Dextran 4. Xanthan Gum."— Transcript presentasi:

1 MICROBIAL BIOPOLYMER 1. Pullulan 2. Gellan Gum 3. Dextran 4. 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,polysaccharidepolymermaltotriose also known as α-1,4- ;α-1,6-glucanglucan 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.glucoseglycosidic bond

4 Pullulan Structure


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

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

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.oral hygieneListerinePocketPaks As a food additive, it is known by the E number E1204 (e.g in cosmetics (hydrating creams and gels)food additiveE number 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 Definition Structure Microorganism Production and downstream process Application

34 Gellan Gum a water-soluble polysaccharide produced by a bacterium Pseudomonas elodeapolysaccharidePseudomonas used primarily as a gelling agent, alternative to agar in microbiological culture.agar MW : 500 000 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.glucose rhamnoseglucuronic 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 1. intracellular formation of the nucleotide- sugar precursors, UDP-Glc, UDP-GlcA, and dTDP-l-Rha. 2. formation of the repeat unit, with sequential transfer of the sugar donors to an activated lipid carrier by committed glycosyltransferases 3. gellan polymerization 4. 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 112.5 g/L, tryptone 1 g/L, casamino acid 1 g/L, disodium hydrogen orthophosphate 1 g/L and manganese chloride 0.947 g/L  produced gellan gum of 13.81 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  6.5 - 7

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 Centrifugation and filtration Separates cells and filtrates 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 12 000– 14 000. 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 lengthsglucanpolysaccharideglucose Dextran was first discovered by Louis Pasteur as a microbial by product in wineLouis Pasteur

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). glycosidic 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 Streptococcus mutans Dental plaque  contains dextran

56 Leuconostoc mesenteroides Streptococcus mutans Streptococcus mutans

57 Slimy dextran produced by Leuconostoc mesenteroides CMG713 on sucrose containing medium with 0.005 % sodium azide

58 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 of sugar refining charcoal 2.0 ml. Sucrose 150.0 g., acid hydrolyzed casein 5.0 g., Yeast extract 1.0 g., K2HPO4 5.0 g., NaCl 2.0 g., and MgSO4 0.022 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.

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

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

69 Application (cont. )

70 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

71 Gum xanthan : eksopolisakarida mikrobial yang diproduksi 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 10 6 atau 3 x 10 7 dalton

72 Struktur molekul gum xanthan

73 TURUNAN KIMIA DAN KOPOLIMER CANGKOKAN GUM XANTHAN 1.Gum Karboksimetil xanthan 2.Gum dietilaminotil xanthan 3.Ester propilenglikol xanthan 4.Gum xanthan sulfat 5.Ikatan silang aldehid gum xanthan 6.Gum deasetil xanthan 7.Gum xanthan - g – poli akrilamida Sampai saat ini tidak ada yang diproduksi secara komersil

74 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)

75 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 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 Viskositas (cp) 1000 100 0.5 1.0 1.5 2.0 2.5 3.0 Konsentrasi (%) Pengaruh konsentrasi gum xanthan thd viskositas

77 Konsentrasi garam (NaCl) (%) Viskositas (cP) 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 Pengaruh konsentrasi garam thd viskositas lar gum xanthan

78 Temperatur (oF) Viskositas (cP) 1000 10000 50 100 150 200 0.1 % gum xanthan 0.25 % gum xanthan 0.5 % gum xanthan 1.0 % gum xanthan Pengaruh suhu thd viskositas lar gum xanthan

79 pH Viskositas (cP) 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 Pengaruh pH thd viskositas lar gum xanthan

80 Viskositas (cP) 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

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

82 APLIKASI DALAM INDUSTRI : 1.Industri Petroleum : a.Sebagai cairan pelicin dalam pengeboran sumur minyak b.Sebagai cairan untuk mengikat dan memisahkan garam-garam dari hasil pengeboran minyak lepas pantai dari cairan petroleum yang diinginkan c.Sebagai cairan pemecah (fracturing fluid ) berbasis air d.Untuk mempercepat pengambilan minyak (oil recovery) Oil drilling

83 2. Industri Kimia dan lainnya a.Pakan ternak : sebagai cairan pelengkap pakan (Liquid feed supplements) 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

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

85 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 biopolimer eksopolisakarida Tahapan biosintesa : 1.Metabolisme substrat karbohidrat 2.Sintesis dan interkonversi nukleotida gula 3.Pengulangan unit monomer 4.Polimerisasi

86 MIKROORGANISME : Bakteri penghasil gum xanthan : Xanthomonas campestris 1.Gram Negatif 2.Membentuk koloni berlendir berwarna kuning 3. Diisolasi dari daun/tanaman yang berpenyakit,misalnya daun kedelai yang terkena pustule, daun kubis, tebu, dll

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

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)

89 SIFAT RHEOLOGI CAIRAN FERMENTASI GUM XANTHAN SANGAT KHAS : 1.Pada penggunaan bioreaktor berpengaduk akan timbul zona stagnasi 2.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 Pengenceran Untuk memudahkan pemisahan biomassa karena kekentalannya telah diturunkan Sentrifugasi Untuk memisahkan sel/biomassa Pengambilan gum dg pelarut Untuk mempresipitasikan gum, bisa dg etanol, isopropanol, metanol, dll Pengeringan Penggilingan

91 BEBERAPA MERK GUM XANTHAN KOMERSIL : 1.Keltrol 2.Kelzan 3.Jungbunzlauer 4.Degussa 5.dll

92 Prospek Pengembangan di Indonesia : 1.Ketersediaan bahan baku : berbagai jenis sumber karbon dan nitrogen 2.Ketersediaan sumber isolat 3.Aspek teknis teknologis (penggunaan teknologi fermentasi) 4.Aspek pasar yang terbuka luas


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