AU2006201335B2 - Novel starch from cassava plants - Google Patents

Description

P/00/0 Il Regulation 3.2 AUSTRALIA Patents Act 1990 COMPLETE SPECIFICATION FOR A DIVISIONAL PATENT ORIGINAL TO BE COMPLETED BY APPLICANT Name of Applicants: NATIONAL STARCH AND CHEMICAL INVESTMENT HOLDING CORPORATION and AVEBE Actual Inventors: Richard G.F. VISSER; Christiaan J.J. RAEMAKERS; Evert JACOBSON; and Johanna Elizabeth M. BERGERVOET VAN DEELEN Address for Service: CALLINAN LAWRIE, 711 High Street, Kew, Victoria 3101, Australia Invention Title: NOVEL STARCH FROM CASSAVA PLANTS The following statement is a full description of this invention, including the best method of performing it known to us:- -2 NOVEL STARCH FROM CASSAVA PLANTS This application is related to Continuation-in-Part Application USSN 09/832,626 which itself is a Continuation-in-Part of USSN 09/180,481, the contents of which are incorporated herein by reference. 5 This application is also a divisional patent application of Australian patent application No. 34289/02 (the "parent application"), the specification of which is herein incorporated by reference. BACKGROUND OF THE INVENTION Cassava is a tropical plant grown in many of the equitorial regions of the 10 world for its food value. The plant comprises a tuber in which starch is stored in a granular form. The plant is generally referred to as cassava whilst the starch derived from the tuber is often referred to as tapioca. The starch has good nutritional properties but may also be used for industrial applications. Starch consists of two types of molecules: amylose and amylopectin. 15 Typically, the starch contained in cassava tubers is about 20-30% amylose. SUMMARY OF INVENTION In one aspect, the present invention provides an isolated native amylopectin cassava starch. In another aspect, the present invention provides an altered cassava plant or 20 closely related plant species wherein tubers contain essentially no amylose. In work leading up to the present invention, the inventors have surprisingly developed a cassava plant which produces starch which is primarily amylopectin in content. This is the first time that a cassava plant with this characteristic has been produced. 25 This is advantageous because to obtain amylopectin from existing cassava plants, the starch must be fractionated resulting (in most cases) in a starch which is no longer in a granular state. In some food applications, granular starches are desirous. For example, granular starches are added to meat products to provide more whiteness and opacity. Granular starches also provide a different mouthfeel, texture, 30 and flavor than non-granular starches. The starch preferably contains essentially no amylose. By that it is meant that the starch preferably has an amylopectin content of at least about 90 wt.%, more 29/10/08,15574 - speci.doc.2 -3 preferably at least about 95 wt.%, most preferably at least about 98 wt.%, based on the (dry substance) weight of the starch. As used herein, amylopectin starch or low amylose starch is intended to mean starch obtained from a plant having such an increased amylopectin content. In addition, the starch has a number of distinct 5 properties, such as molecular weight, intrinsic viscosity, particle size, and chain length distribution, which will be further elucidated below. When the amylopectin cassava starch is in an isolated form, i.e., it is removed or extracted from the tuber. Substantially pure starch may be extracted by conventional means. 10 Preferably, there is a method for obtaining starch from the tubers of said cassava plants. The method can be carried out in a manner essentially similar to a method for isolating starch from potato tubers. In one preferred embodiment, the method comprises the steps of washing the tubers, followed by grating and milling them. Subsequently, the starch is separated from fibers and juice in separators, e.g. 15 centrifuges or hydrocyclones. The isolated starch may then be sieved, washed and dried. Washing may be carried out in hydrocyclones. Drying may be carried out in vacuum filters and drying towers. It is preferred that the closely related species include other tuber bearing species and species of the Manihot genus on the basis of morphology, ecology and/or 20 geography. Specific examples of closely related species are Manihot carthaginensis, Manihot aesculifolia, Manihot grahami, Manihotflabellifolia, and Manihot saxicola. It is preferred that the altered cassava plant is obtained by disruption of amylose production. It is possible to disrupt amylose production by transforming a cassava plant with a granular-bound starch synthetase (gbss) gene in an antisense 25 orientation. Preferably the gene is derived from cassava or a closely related species, however, the gene may also be derived from other species such as tuberous species. For example, potato, sweet potato, yam, parsnip, carrot or the like. DETAILED DESCRIPTION OF THE INVENTION The transformed cassava plants may be produced by the methods 30 described in the parent application which include production of friable embryonic callus (FEC). 29/10/08,1t15574 - speci.doc.3 -4 The following part describes how cassava plants genetically were modified with the aim to produce cassava plants with a high amylopectin content in their tuberous roots. Isolation of the Cassava gbss Gene and Construction of a Plant 5 Transformation Vector In a previous study the gbss gene was isolated from cassava using the potato gbss gene (Visser et al., 1989) as probe (Salehuzzaman et al., 1993). The gbss gene was subcloned in anti-sense orientation between the potato gbss promoter (Visser et al., 1991) and the nopaline synthase terminator in pUC 19 resulting in the vector 10 pAG61 (Salehuzzaman et al., 1993). Also, other preferably tuberous root specific promoters such as protein synthesis elongation factor 1-alpha from cassava (Suhandono et al., 2001), cassava gbss or more general promoters such as CaMV can be used to direct expression of genes such as gbss. The complete luciferase gene ( BglII fragment) was isolated from pJIT100 15 (Guerineau et al., 1993) and inserted in the BamHI site of pAG61. This resulted in two different vectors: pGBSSas2 and pGBSSas7 (difference between the two vectors was the orientation of the luciferase and antisense gbss genes to each other). Both constructs were used successfully to produce cassava plants with a high amylopectin content. 20 Plant Material and Tissue Culture Media Used Plants of the genotype TMS60444 were maintained by monthly subculture of one node cuttings on medium supplemented with Murashige and Skoog (1962) salts and vitamins and 40 g/l sucrose (MS4). Friable embryogenic callus (FEC) lines were initiated as follows: 25 -isolation of meristems or immature leaves from donor plants -cultured of meristems/leaves on MS40 supplemented with 6 mg/l NAA and 6 mg/l Picloram -isolation of compact embryogenic tissue and cultured on a medium supplemented with Gresshoff and Doy (1974) salts and vitamins, 60 g/l sucrose and 10 mg/l 30 Picloram (GD6). -isolation of FEC (small clumps of aggregated, spherical units) which were cultured on GD6 medium. FEC was maintained by a 3 weeks subculture on GD6 medium. 29/03/06,at 15S74.speci2,4 -5 Liquid cultures were initiated by transferring 0.5 g of FEC into flask of 200 ml with 50 ml of liquid medium supplemented with Schenk and Hildebrandt (1972) salts and vitamins, 60 g/l sucrose and 10 mg/l Picloram (SH6). The medium was refreshed twice a week and after 2 weeks the content of a each flask was divided 5 over 5 new flasks. The flasks were cultured on a rotary shaker (LAB-line Instruments Inc. Model 3519) at 120 rpm. Coating of DNA on the Particles A method adjusted from Cabe et al. (1988) was used to coat DNA on the particles. Eighty tg of DNA (isolated using WizardTM Maxipreps DNA purification 10 system of Promega from the vectors pGBSSas2 and pGBSSas7) was mixed with 10 mg of gold particles (1.6 ptm, BioRad), 30 ptl 5 M NaCl, 5 pl 2 M tris HCl pH 8.0, 965 ptL H20, 100 pd 25% PEG 1550, 100 ptL 0.1 M spermidine and 50 pd 2.5 M CaCl2. After centrifugation the pellet was resuspended in 10 ml of absolute alcohol and briefly sonificated. Hundred sixty ptL of the suspension was pipetted in the hole 15 of a macrocarrier holder placed up side down on a macrocarrier. After 5 minutes the macro carrier holder was taken away. The macrocarrier covered with a thin layer of gold beads was dried in an oven (10 minutes, 40 OC) and used for bombardment. Bombardment of FEC and Selection of Transgenic Plants FEC cultured in liquid SH6 medium for at least 5 weeks, was sieved (mesh 20 1 mm) and collected. Hundred mg of FEC was spread on GD6 medium and bombarded using the BioRad PDS-100OHe biolistic device (helium pressures 1100 p.s.i., 0.5 cm distance between rupture disc and the macrocarrier and between macrocarrier and the stopper plate, 5.0 cm distance between stopper plate and FEC, 27 inches Hg vacuum). 25 In total 212 and 184 Petri dishes with FEC from TMS60444 were bombarded with construct GBSSas2 or GBSSas7 respectively. After bombardment the bombarded FEC was cultured in plastic pots filled with liquid SH6 medium. Two weeks later the FEC was collected on solid GD6 medium and assayed for luciferase activity. Each luciferase (LUC) spot was subcultured as an individual line. 30 In total 186 luc spots were produced with construct GBSSas2 and 222 with GBSS7. Because it was not possible to locate precisely which FEC unit contained LUC activity and to avoid loss of transgenic tissue, the tissue in a radius of 0.5-1 cm 29/03/06,at 15574.speci2,5 -6 around the LUC spot was transferred in liquid SH6 medium. Two weeks later the FEC was assayed for luciferase activity. Lines without luciferase activity were discarded and lines with 4 or more spots were used for subclump division. The lines with 1-3 spots were transferred again to liquid Sh6 medium and two weeks later the 5 lines in which the number of spots had increased to 4 or more were used for sub clump division; the others were discarded. Forty four lines were obtained from the bombardments with pGBSSas2 and 40 lines from the bombardments with pGBSSas7. The transgenic tissue was isolated and purified via a process called subclump division (Raemakers et al., 2000). 10 Subclump division started with subculturing the tissues around (0.5 to 1 cm diameter) a luciferase positive spot. The tissue was divided as fine as possible on GD6 medium. Two weeks later the Petri dish was covered with small clumps of FEC tissue. Only the LUC positive clumps were subcultured. For this the clumps were divided in subclumps and cultured on GD6 medium. This selection procedure 15 was repeated 1-2 times more before the tissue was cultured for plant regeneration. For this the 84 FEC lines were subcultured every 2-3 weeks for 10-12 weeks on maturation medium (MS4 supplemented with 1 mg/l Picloram). Torpedo shaped somatic embryos were isolated from the FEC and cultured on MS4 supplemented with 0.1 mg/l BAP, which allowed further maturation. Mature somatic embryos 20 were first cultured for two weeks in liquid and hereafter in solid germination medium (MS4+1 mg/l BAP). Plants were rooted on MS4 medium. Plants were obtained from 31 of the 44 GBSSas7 cultured lines and 27 of the 40 GBSSas7 cultured lines. These plants were first grown on Murashige and Skoog medium supplemented with 8% sucrose to allow starch disposition in the stems of the cassava plants 25 (Salehuzzaman et al., 1994). The amylose/amylopectin ratio was visualised by iodine staining of cross sections of the in vitro thickened stems with Lugol's solution (12:KI). The stained stem sections were visualised microscopically. In total 9 lines (3 from GBSSas2 and 6 from GBSSas7) yielded plants with an altered staining pattern, meaning that said plants had a an altered starch composition in the stems. 30 These plants were transferred to the greenhouse to allow formation of tuberous roots. Three months later the roots were peeled and the central cylinder of the storage root was grounded in a laboratory blender in water with a small amount of Na 2

S

2 0 5 . The 29/03106,at 15574.speci2,6 -7 slurry was transferred for starch isolation to a Sanamat. The water-starch granules suspension was transferred to centrifuge tubes and centrifuged. The starch was dried at 20 degrees Celsius for 3 days. The amylopectin/amylose content was determined using the protocol 5 described by Hovenkamp-Hermelink et al. (1988). In total 2 lines (one from GBSSas2 and one from GBSSas7) had yielded plants which in both tests had a high amylopectin content. One year later the same plants were transferred again to the greenhouse. Starch of in total 30 plants of the two lines were analysed via iodine staining and via spectrophotometry. All plants 10 had starch with a high amylopectin content. At the same moment starch was isolated from more than 3000 plants. This starch was analysed in three different laboratories and again it was shown that the starch contained a high percentage amylopectin. Materials & Methods 15 Samples Tapioca starch control sample Amylopectin tapioca starch AFC3KD from Wageningen Potato starch Oostermoer 1998 20 Amylopectin potato starch Oostermoer 1996 Corn starch Meritena A Waxy corn starch Meritena 300 Methods 25 Nitrogen content (Ntotai) ISO 5378 (1978), Starch and derived products - Determination of nitrogen by the Kjeldahl method - Spectrophotometric method. Particle Size Distribution 30 The particle size is measured with a Coulter Multisizer II, calibrated with Coulter calibration standard P.D.V.B. Latex lot F.34, diameter measuring tube 140 pm, number of measured classes 256, measuring range 3,1-107,7 29/03/06,at I 5574.speci2,7 -8 ptm (potato starches) and 2,8-82,0 ptm (other starches). The samples are suspended in isotonic salt solution (DiluidTM azid free, J.T. Baker) and homogenized in a ultrasonic bath (Branson 5510). 5 Differential Scanning Calorimetry Differential scanning calorimetry experiments are performed using a Perkin Elmer DSC-7. At least 10 mg of starch and 40 mg of demineralised water are put in a stainless steel DSC-pan to obtain 80% water content thereby taking the water content of the starch into account. The DSC-pan is 10 hermetically sealed and stored one night at room temperature to equilibrate. Next day, the sample is heated from 5 to 130 'C with a rate of 10 'C/min. Dry Substance Content ISO 1666 (1997), Starch and derived products - Determination of moisture 15 content - Oven drying method. Ash Content ISO 5984 (1978), Animal feeding stuffs - Determination of crude ash content. 20 Intrinsic Viscosity (IV) The intrinsic viscosity is determined in a known manner with a Ubbelohde viscosity meter with 1 M sodium hydroxide as solvent and expressed in g/dl. As described in H.W. Leach in Cereal Chemistry, vol. 40, page 595 (1963). 25 Phosphor Content (P) ISO 3946 (1982), Starch and derived products - Determination of total phosphorous content - Spectrophotometric method. 30 Viscosity Behavior Determined with a Rapid Visco Analyser (RVA) from Newport Scientific. The measurements are carried out in a 6% concentration in demineralized 29/03/06,at 15574. speci2,8 -9 water at 400 rpm. Temperature cycle: 2 min at 45*C, heating to 900 at 140 per min, 5 min at 90* and cooling to 30* at 140 per min. Chain Length Distribution 5 The amylopectin starch is debranched with isoamylase, the resulting linear alfa-1,4 malto-oligosaccharides are measured with High Performance Anion Exchange Chromatography with a Pulsed Amperometric Detection System. Results 10 The results are shown in the following tables. Table 1: Chain length distribution (weight average) Cassava Amylo- Maize Waxy Potato Amylo pectin Maize pectin Cassava Potato DP 6 t/m 9 4.1(5.9) 4.6(5.7) 2.4(3.9) 3.3 (3.7) 2.1(3.7) 2.6(3.9) DP 6 t/m 12 14.7 16.4 11.2 15.3 7.6( 13.5) 9.5 (14.1) (21.7) (20.4) (18.2) (17.0) DP 13 t/m 24 31.9 36.9 33.2 46.2 29.2 33.3 (46.6) (46.0) (54.0) (51.4) (51.7) (49.4) DP 25 t/m 40 14 (20.4) 17.5 13 (21.1) 19.2 12.6 14.9 (21.8) (21.4) (22.3) (22.1) DP >40 7.8(11.4) 9.4(11.7) 4.3(7.0) 8.9(9.9) 7.4(13.1) 9.8(14.5) Recovery(%) 68.5 (100) 80.2 (100) 61.5 (100) 89.9 (100) 56.5 (100) 67.4 (100) 29/03/06,at I 5574.speci2,9 - 10 Table 2: Various parameters Parameter Waxy Amylo- Amylo- Character ACS Maize pectin pectin cassava potato (ACS) (APS) RVA Tg 62.3 oC 61.7 oC 63.3 oC Low Tg for ampec starch Tg - Ttop 10.0 *C 5.9 oC 5.8 OC Fast dissolving Ttop visco 75 RVU 112 RVU 171 RVU Tend visco 48 RVU 87 RVU 77 RVU Brabender Tg 66 oC 60.5 oC 61.5 oC Low Tg for ampec starch Tg - Ttop 6.5 oC 6 oC 7 oC Fast dissolving Ttop visco 685 BU 990 BU 1560 BU T20'90 250 BU 290 BU 440 BU Tend visco 400 BU 495 BU 670 BU Amylose content < 1% <1% Schoch, detection level Visco stability Stable Stable Stable No differences Protein 0.41 mg/g 0.16 mg/g 0.10 mg/g Clear solution Phosphate < 0.05 mg/g 0.06 mg/g 0.81 mg/g Clear solution, viscosity level Chain length 21 27 28 distribution 5 29/03/06,atI5574.speci2, 10 - 11 Table 3: Various parameters dry substance ash Ntotal P IV mg/g as is mg/g ds mg/g ds mg/g ds dl/g ds Tapioca 860 3.5 0.11 0.06 2.4 Amylopectin 863 2.3 0.16 0.06 1.7 tapioca Potato 844 4.7 0.13 0.85 2.4 Amylopectin potato 856 4.7 0.10 0.81 1.8 Maize 879 1.1 0.53 0.18 1.7 waxy maize 881 <1.0 0.41 <0.05 1.6 Table 4: Particle size distribution weight average dio d 50 d 9 o m tm pm pm Tapioca 13.0 7.8 12.6 16.9 Amylopectin 14.0 7.7 13.0 19.2 tapioca Potato 42.9 23.0 42.4 63.3 Amylopectin potato 45.0 23.2 44.7 67.0 Maize 14.0 9.8 14.1 18.4 waxy maize 14.3 9.3 14.0 18.3 5 10 29/03/06,at15574.speci2, II - 12 Table 5: Results of gelatinisation behaviour measured by DSC. Tonset Tpeak Tend LT I H ( 0 C) (OC) (OC) (*C) (J/g) Tapioca 60.2±0.4 65.0 0.6 72.8±0.7 12.6 ±0.3 19.19 1.2 Amylopectin 61.7 ± 0.3 67.6 ± 1.0 75.9 ±0.6 14.2 ±0.4 19.11 ±2.2 tapioca Potato 62.6 ± 0.3 67.3 ± 0.6 75.1 ± 0.9 12.5 ± 0.6 22.12 ± 2.7 Amylopectin potato 63.3 ± 0.3 69.1 ± 0.4 75.2 ± 0.6 11.9 ± 0.5 24.04 ± 1.4 Maize 67.4 ± 0.2 73.2 ± 0.3 79.3 ± 0.9 12.1 ± 0.9 15.92 ± 0.6 waxy maize 62.3 ± 0.3 72.3 ± 0.3 80.4 ± 0.2 18.1 ± 0.4 18.76 ± 0.8 Different properties of amylopectin cassava starch compared to waxy maize starch: 5 e Low Tg e Fast dissolving starch (difference between Tgelatinization and Ttop) " About 20 % higher viscosity e Less protein, low impurity level 10 Properties amylopectin cassava starch compared to amylopectin potato starch * Somewhat lower Tg (< 1 C) " Fast dissolving starch, however the viscosity of APS is higher (delta C = 6 C BU = 1400) * About 30 % lower viscosity level 15 e Small granules * Crystallinity 20 29/03/06,at15574.speci2,12 - 13 Table 6: Genotypes of cassava used for somatic embryogenesis. Indonesia Nigeria TMS90853 M. Col22, Sao Paolo Adira 1 TMS50395 TMS30555 Zimbabwe Thailand 5 Tjurug TMS60444 TMS30211 Line 11 R5 Adira 4 TMS90059 TMS30395 M7 KU50 Mangi 4 TMS30572 TMS30001 Venuzuela R60 Gading TMS4(2)1244 Columbia M.Ven77 R90 Faroka TMS60506 M. Col 1505 Brasil RI 10 Table 7: Influence of light intensity during growth of donor plants in vitro on the number of leaf explants responding with the formation of mature embryos and the number of mature embryos per cultured leaf explant (#ME/CLE). 15 light intensity number of responding production (iEm-s 1 ) explants explantsa (# ME/CLEb) 40 48 18 b 1.7 b 28 48 26 ab 4.9 ab 8 48 31 a 6.6 a 20 a,b means with the same letter are not significantly different by respectively Chi square test (p<0.1) and by LSD test (p<0.1) 25 30 29/03/06,at 15574.speci2,13 - 14 Table 8: Influence of 2,4-D pretreatment on production of primary mature embryos (# mature embryos per cultured leaf explant isolated from in vitro plants), followed by the multiplication of mature embryos by secondary somatic embryogenesis in 11 Nigerian cassava genotypes and in M.Co122. 5 embryogenesis primarya) secondary) 2,4-D pretreatment no yes M.Col22 3.5 9.4 13.5 10 TMS 30555 0 0.7 6.2 TMS 50395 - 0 < 0.1 5.3 TMS 60506 0 < 0.1 0 TMS 90059 0 < 0.1 7.2 TM S 30211 0 0 15 TMS 60444 0 1.1 9.9 TMS 30395 0 0.1 6.7 TMS 90853 < 0.1 0.2 8.2 TMS 4(2)1244 < 0.1 0 5.4 TMS 30001 0 0 20 TMS 30572 0 0 a) average of three experiments (total 48-74 leaf explants), b) average of two experiments (total 24-48 ME explants). 25 BRIEF DESCRIPTION OF THE FIGURES Figure 1: Schematic representation of somatic embryogenesis in cassava, including primary, secondary somatic embryogenesis, selection of friable embryogenic callus, maturation and desiccation followed by germination. gd2= medium supplemented with Gresshoff and Doy salts (1974) and vitamins plus 30 20 g/l sucrose. gd4= medium supplemented with Gresshoff and Doy salts (1974) and vitamins plus 40 g/l sucrose. 29/03/06,at15574.speci2,14 - 15 ms2= medium supplemented with Murashige and Skoog salts and vitamins plus 20 g/l sucrose. pic= 10 mgl/l Picloram, NAA=10 mg/l naphthalene acetic acid, 2,4-D=8 mg/l, 2,4 dichiorophenoxy acetic acid. 5 sh6= medium supplemented with Schenk and Hildebrandt (1972) salts and vitamins plus 60 g/l sucrose. Figure 2: Molecular weight distribution by GPC of the regular and amylopectin tapioca starches. Figure 3: Molecular weight distribution by GPC of the debranched 10 amylopectin present in regular and amylopectin tapioca starches. Figure 4: Molecular weight distribution by GPC of debranched regular and amylopectin tapioca starches. Figure 5: Brabender viscosity of regular and amylopectin tapioca starches. Thus, the present invention also is intended to cover amylopectin cassava 15 (tapioca) starch, both native and derivatized, and compositions containing such starch. The starch is useful in a wide variety of food, pharmaceutical, and industrial applications, either with or without chemical modification. Amylopectin starch, as used herein, is intended to mean a starch or flour which has an amylopectin content substantially higher than that of regular cassava 20 starch, particularly at least about 90%, more particularly at least about 95%, most particularly at least about 98% amylopectin by weight. Amylopectin cassava starch may be obtained by the FEC method, supra. Also included in this invention are amylopectin cassava starches derived from amylopectin cassava plants which may be found in nature, obtained by standard 25 breeding and crossbreeding techniques, or obtained by translocation, inversion, transformation or any other method of gene or chromosome engineering to include variations thereof, whereby the properties of the starch of this invention are obtained. In addition, starch extracted from a plant grown from artificial mutations and variations of the above generic composition which may be produced by known 30 standard methods of mutation breeding is also applicable herein. The substantially pure starch may be extracted from the root of a amylopectin cassava plant. Extraction may be by any method known in the art, including but not 29/03/06,at15574.speci2, 15 - 16 limited to pulverizing the root and separating the starch from the remaining components by water extraction. The amylopectin cassava starches have lower levels of amylose and higher levels of amylopectin. The amylopectin cassava starches also have a higher viscosity 5 than regular cassava starches, particularly at least about 30% greater than, more particularly at least 50% greater than, and most particularly at least about 80% greater than that of regular cassava starches. Particularly suitable amylopectin cassava starches are those which have a peak viscosity of at least about 1200, more particularly at least about 1300 as measure by Rapid Visco Analyzer using the 10 method of Example 3a, infra. The resultant native starch has properties and functionality which are unique and desirable in many applications. Such native starches have the additional benefit of achieving the desired functionality without chemical modification. However, the present starches also may be modified to further enhance their properties and 15 functionality. Any modifications known in the art may be used, including those which are chemical, physical, or enzymatic. Chemical derivitization shall include those to form ethers, esters or half esters such as hydroxyalkyl ethers, acetates, phosphates, succinates, i.e., octenyl succinate, tertiary and quaternary amine ethers, etc., or by any other modification techniques 20 known in the art. Chemical modification of the present starch includes cross-linking. Any cross-linking agent known in the art may be employed for this purpose, including but not limited to epichlorohydrin, linear dicarboxylic acid anhydrides, citric acid acrolein, phosphorus oxychloride, adipic/acetic mixed acid anhydrides, 2'5 trimetaphosphate salts, formaldehyde, cyanuric chloride, diioscyanates, and divinyl sulfones. The present starches may be physically modified, such as by thermal inhibition described in WO 95/04082 (published February 9, 1995) or by shear. The present starches may also be enzymatically modified by one or more 30 enzymes known in the art, including without limitation alpha-amylase, beta-amylase, glucoamylase, maltogenase, isoamylase and pullulanase. 29/03/06,at 15574.speci2,16 - 17 The starches may also be pregelatinized. Exemplary processes for preparing pregelatinized starches are disclosed in U.S. 4,280,851 (Pitchon, et al.), U.S. 4,465,702 (Eastman, et al.), U.S. 5,037,929 (Rajagopalan), U.S. 5,131,953 (Kasica, et al.), and U.S. 5,149,799 (Rubens). Conventional procedures for pregelatinizing 5 starch are well known to those skilled in the art and described in such articles as Chapter XXII- "Production and Use of Pregelatinized Starch", Starch: Chemistry and Technology, Vol. III - Industrial Aspects, R.L. Whistler and E.F. Paschall, Editors, Academic Press, New York 1967. The starches may also be converted to produce, inter alia, fluidity or thin 10 boiling starches prepared by oxidation, enzyme conversion particularly by a-amylase, acid hydrolysis, or heat and or acid dextrinization. The present starches may be purified by any method known in the art to remove off-flavors and colors that are native to the starch or created during starch modification processes. 15 One skilled in the art is capable of using any single or combination of modifications in order to obtain the desired starch properties and functionality. These methods are well known in the art and the resulting starch properties and functionality will vary depending, inter alia, on the type of modification employed, the degree of modification, and the reaction conditions. 20 Functionality obtainable using the amylopectin cassava starches of the present invention includes without limitation encapsulation and emulsification, thickening and viscosifying, gelling, and film-forming. The amylopectin cassava starches of the present invention may be used in a variety of industrial applications, including without limitation paper products, food 25 products, pharmaceutical and nutritional products, personal care products and other industrial products. Paper products is intended to include, without limitation, paper, paperboard, linerboard, corrugating, cardboard, bags, and envelopes. Food products is intended to mean any edible product and includes, without 30 limitation, cereals, breads and bread products, cheese and imitation cheese products, condiments, confectioneries, dressings including pourable dressings and spoonable dressings, pie fillings including fruit and cream fillings, sauces, including white 29/03/06,at15574.speci2,17 - 18 sauces and dairy-based sauces such as cheese sauces, gravies, imitation and lite syrups, puddings, custards, yogurts, sour creams, pastas, beverages including dairy based beverages, glazes, soups and baby food. Pharmaceutical and nutritional products is intended to include pharmaceutical 5 excipients, tablets including effervescent tablets, dusting starches and powders, and prebiotics products. Personal care products is intended to include without limitation deodorants and antiperspirants, hair fixatives including sprays, gels, mousses, lotions and pomades, soaps and cleansers, makeup including eye shadow, powders, foundations, 10 and blushers, shampoos and conditioners, and mouthwashes, breath fresheners and toothpastes. Other industrial products is intended to include without limitation detergents, and biodegradable foamed products including loosefill, sheets and shapes. The amylopectin tapioca starch may generally be used at any desired level, 15 the amount being dependent upon the functionality to be obtained. In general, the amylopectin cassava starch will be used in an amount of from about 1% to about 95%, particularly from about 5% to about 60%, more particularly in an amount of about 10% to about 40% by weight of the product. 20 EXAMPLES The following examples are presented to further illustrate and explain the present invention and should not be taken as limiting in any regard. All percents used are on a weight/weight basis. In the examples below, the tapioca samples used are as follows: 25 Cassava 1 = regular cassava starch grown in Thailand commercially available from National Starch and Chemical Company (Bridgewater, NJ, USA). Cassava 2 = regular cassava starch grown in Indonesia commercially available from Avebe B.A. (Foxhol, The Netherlands). ACS 1-3 = amylopectin cassava starches genetically produced by introducing the 30 GBSS gene in the antisense mode and using FEC from which cassava plants are regenerated. 29/03/06,at15574.speci2,18 - 19 Potato = regular potato starch commercially available from Avebe B.A. (Foxhol, The Netherlands). AMF Potato = low amylose potato starches commercially available from Avebe B.A. (Foxhol, The Netherlands). 5 Corn = regular corn starch commercially available from commercially available from National Starch and Chemical Company (Bridgewater, NJ, USA). Waxy = waxy (low amylose) corn starch commercially available from National Starch and Chemical Company (Bridgewater, NJ, USA). 10 Example 1 - Amylose content a. Amylose content was determined by potentiometric titration. Approximately 0.5 g of a starch sample was heated in 1 Omls of concentrated calcium chloride (about 30% by weight) to 95*C for 30 minutes. The sample was cooled to room temperature, diluted with 5 mls of a 2.5% uranyl acetate solution, mixed well, and 15 centrifuged for 5 minutes at 2000 rpm. The sample was then filtered to give a clear solution. The starch concentration was determined polarimetrically using a 1 cm polarimetric cell. An aliquot of the sample (normally 5 mls) was then directly titrated with a standardized 0.01 N iodine solution while recording the potential 20 using a platinum electrode with a KCl reference electrode. The amount of iodine needed to reach the inflection point was measured directly as bound iodine. The amount of amylose was calculated by assuming 1.0 gram of amylose will bind with 200 miligrams of iodine. The results of the potentiometric titration are showed in Table 9. 25 Table 9 Base starch Amylose Content (%) Cassava 1 20% Cassava 2 17.4% ACS 1 2.0% ACS 2 2.8% ACS 3 2.7% 29/03/06,atI5574.speci2,19 - 20 As can be determined from Table 9, the amylopectin cassava starches contain significantly less amylose than the regular cassava starches. b.The amylose contents were checked by gel permeation chromatograph (GPC). 5 Samples were prepared for analysis by slurrying 4 to 8 mg of starch in 4 g of dimethylsulfoxide (DMSO) containing 5mM sodium nitrate and heating to 1 00 0 C for 2 hours. The sample was filtered if necessary, and injected (30001) into a GPC 150C chromatograph (Waters Corporation, Amherst, MA). The Gel Permeation Chromatograph utilized 4 columns (guard column, 105, 103, 102 micron (nominal) 10 pore size columns, all from Polymer Laboratories, Amherst, MA). The mobile phase was dimethyl sulfoxide containing 5 mM of sodium nitrate. The instrument was operated at a temperature of 80*C and a flow rate of 0.7 ml/minute was used. The columns were calibrated with pullulan standards (Showa Denko K.K., Japan) ranging in molecular weight from 5800 to 850,000. Figure 2 shows the relative molecular 15 weight distribution by GPC of the regular and amylopectin cassava starches. As can be determined from the figure, the three amylopectin cassava starches have significantly more amylopectin as seen by the peak at a relative log (molecular weight) of about 6.83. Further, this is the sole main peak. In contrast, the regular starches each show an additional amylose peak at a relative log (molecular weight of 20 about 6. Example 2 - Structure Debranching was accomplished by the following procedure in this example. 20mg of starch was added to 2ml of 90% DMSO (10% water) and stirred 25 (at 95 0 C) until dissolved. 7.980 ml of mM Acetate Buffer pH 4.8 was added to the vial and stirred. If it appeared that some of the amylose precipitated out of solution, the solution was briefly boiled until clear. Once the sample had been completely dissolved, 20ul of pure isoamylase was added. The vial was incubated in a constant temperature bath for 16 hrs at 38*C. Upon completion, 1 ml of sample was pipetted 30 into a 2ml vial for chain length distribution evaluation. The remainder of the sample was precipitated in 50 ml of acetone. The precipitated material was collected by , filtering using a 0.2 micron nylon filter paper and prepared for GPC. 29/03/06,at 15574speci2,20 -21 Ideally, section b should come before a. The structure of the debranched amylopectin (a) is obtained by taking the overall debranched starch chromatogram (b), and then integrating only the amylopectin peak 5 a.The molecular weight distribution of debranched amylopectin of high amylose and regular tapioca starches were determined using GPC as in Example lb. Figure 3 shows the resultant molecular weight distribution of the debranched amylopectin of cassava starches. As can be seen, the amylopectin structure is substantially the same 10 for the different cassava starches. b.The molecular weight distribution of debranched amylopectin and regular cassava starches were determined as in a, above. Figure 4 shows the resultant molecular weight distribution of the cassava starches. This figure confirms that the 15 amylopectin structure is similar, but present in high quantities by substantially the same (relative) log (molecular weight) peaks at about 3.0 and 4.0. Further, this figure shows no substantial amount of amylose in the amylopectin cassava starches as evidenced by the lack of a (relative) log (molecular weight) peak at about 5.81. 20 Example 3 - Viscosity a. Viscosity was measured using an RVA Series 4 Rapid Visco Analyzer (Newport Scientific, New South Wales, Australia). A slurry containing 5% starch on a dry weight basis was prepared and heated from 50*C to 95'C at a rate of 3.0*C per minute. The sample was then held at 95*C for five (5) minutes. Finally, the sample 25 was cooled to 35*C at 6.0'C per minute. The viscosity measurements were taken at 160 rpm and the results (peak viscosity) are shown in Table 10 below. 30 29/03/06,at I 5574.speci2,21 - 22 Table 10 Base starch Viscosity (RVA Units) Cassava 1 600 Cassava 2 1015 ACS 1 1230 ACS 2 1360 ACS 3 1330 As can be seen from Table 10, the amylopectin cassava starches have significantly higher peak viscosities, about 30% more than that of the Indonesian 5 regular cassava starch and about twice that of the Thai regular cassava starch. b. Viscosity was also measured using a Visco/amylo/graph, Model VA-1A (C.W. Brabender Instrument Co., Hackensack, NJ, USA 07606). A slurry of 5% starch on a dry weight basis was prepared and controlled to pH3 using a citric acid/trisodium citrate buffer solution. The total charge weight of 460 grams was 10 heated from 50 0 C to 92*C at a rate of 1.5'C per minute. The slurry was then held at 92'C for 30 minutes. The hot viscosity was measured while heating the paste in the Visco/amylo/graph and the resulting viscosity profile is shown in Figure 5. This figure confirms that the amylopectin cassava starches have higher peak viscosities than those of the regular cassava starches. 15 Example 4- Gelatinization Temperatures a. Gelatinization temperatures were measured using differential scanning calorimetry. The starch sample is scanned from 5*C to 140*C at a heating rate of 10*C/min with water: starch ratio of 2:1. Duplicate runs are taken and the average is reported. The 20 results are shown in Table 11. 25 29/03/06,at 15574.speci2,22 - 23 Table 11 Base Onset Peak (*C) End (*C) AH (J/g) Cassaval 62.6+0.4 69.0+0.3 81.0+0.1 16.05+1.0 0 Cassava 2 56.7+0.1 63.2+0.2 72.9+0.3 16.01+0.4 3 ACS 1 58.1+0.1 65.7+0.0 74.5+0.0 17.80+0.8 0 ACS 2 58.6+0.0 65.5+0.2 75.6+0.3 18.08+0.3 6 ACS 3 58.6+0.0 65.4±0.0 74.5±0.1 17.71+0.2 0 As shown in Table 11, all three amylopectin cassava starches have a slightly higher onset gelatinization temperature than the Thai regular cassava starch, slightly 5 lower than the Indonesian regular cassava starch. All three amylopectin cassava starches have a higher gelatinization enthalpy than the regular cassava starches. b.Onset gelatinization temperatures were also checked by Brabender using the methodology of Example 3b. As can be seen from Figure 5, the onset gelatinization temperature of the amylopectin cassava starches is lower than the regular Thai 10 cassava. Cited Literature Anonymus, 1985. 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P:205. 10 Wolters, A.M.A., Schoenmakers, H.C.H., van der Meulen-Muiser, J.J.M., van der Knaap, E., Derks, F. H. M., Koornneef, M., and Zelcer, A. 1991. Limited DNA elimination from the irradiated potato parent in fusion products of albino Lycopersicon esculentum and Solanum luberosum. Theor. Apple. Genet. 83:225-232. 15 Wordragen M.F., and Dons, HINL 1992. Agrobacterium tumefaciens mediated transformation of recalcitrant crops. Plant Mol. Biol. Reporter 10:12-36. Woodward, B., and Puonti Kaerlas, 2001. Somatic embryogenesis from floral tissues 20 of cassava (Manihot esculenta Crantz. Euphytica (in press). Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or 25 group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form or suggestion that the prior art forms part 30 of the common general knowledge in Australia. ,It 15574 - speci doc.33

Claims (21)

1. An isolated native amylopectin cassava starch.

2. The starch of claim 1, wherein the starch has an amylopectin content of at least about 90% by weight. 5

3. The starch of claim 1, wherein the starch has an amylopectin content of at least about 95% by weight.

4. The starch of claim 1, wherein the starch has an amylopectin content of at least about 98% by weight.

5. The starch of claim 1, wherein the viscosity of the starch is at least about 10 30% greater than that of regular cassava starch.

6. The starch of claim 1, wherein the viscosity of the starch is at least about 50% greater than that of regular cassava starch.

7. The starch of claim 1, wherein the viscosity of the starch is at least about 80% greater than that of regular cassava starch. 15

8. The starch of claim 1 which has been modified physically, chemically or enzymatically.

9. A composition comprising the starch of claim 1.

10. The composition of claim 9, wherein the composition is selected from the group consisting of paper products, food products, pharmaceutical products, 20 nutritional products, personal care products, detergents, emulsifying agents, encapsulating agents, and biodegradable foamed products.

11. A process of making the composition of claim 9 comprising substituting the starch of claim 1 for the conventionally used starch.

12. A composition comprising the starch of claim 8. 25

13. The composition of claim 11, wherein the composition is selected from the group consisting of paper products, food products, pharmaceutical products, nutritional products, personal care products, detergents, emulsifying agents, encapsulating agents, and biodegradable foamed products.

14. A process of making the composition of claim 1I comprising substituting 30 the starch of claim 8 for the conventionally used starch.

15. An altered cassava plant or closely related plant species wherein tubers contain essentially no amylose. 29/10/08,k 15574 - claims.doc.33 - 34

16. The plant of claim 15 wherein said alteration is a disruption of amylose production.

17. The plant of claim 16 wherein said disruption is caused by transformation of the plant with a granular-bound starch synthetase gene in an antisense orientation. 5

18. The plant of any one of claims 15 to 17 wherein amylopectin content of the tuber is at least about 90%, at least about 95% or at least about 98% by weight.

19. The plant of any one of claims 15 to 17 wherein viscosity of starch in the tuber is about 30% greater, about 50% greater or about 80% greater than that of regular cassava starch. 10

20. An isolated native amylopectin cassava starch substantially as hereinbefore described in any one of the Examples.

21. An altered cassava plant or closely related plant species substantially as hereinbefore described in any one of the Examples. 29/10/08,105574 - claims.doc.34