Abstract:
A novel modified thermoplastic starch is manufactured from a native starch using a polysaccharide produced by the fungus species  Ophiostoma ulmi , by growing a culture in a yeast extract medium; adding the native starch; mixing, and harvesting the modified thermoplastic starch. The modified thermoplastic starch may be used in the manufacture of a biodegradable plastic which exhibits low water absorbency and high tensile strength. The plastic may be used to manufacture films or moulding products by casting, extrusion, injection, or compression techniques.

Description:
TECHNICAL FIELD 
       [0001]    The present invention relates to biodegradable plastics. In particular, the present invention relates to modified starch-based biodegradable plastics. 
       BACKGROUND OF THE INVENTION 
       [0002]    An increased emphasis on sustainability, eco-efficiency, and green chemistry has driven a search for renewable and environmentally friendly resources. Starch is a biodegradable polysaccharide, produced in abundance at low cost, which exhibits thermoplastic behaviour. Therefore, it has become one of the most promising candidates for an alternative material to replace traditional plastics in certain market segments such as the food packaging industry. 
         [0003]    Numerous studies have been conducted to optimize the performance of starch-based plastics (Mali, S. et al. (2004),  Food Hydrocolloids,  19 (2005), 157-164); Soest, J. et al. (1997),  Trends in Biotechnology,  15(6), 208-213; Fama, L. et al.,  LWT,  38, 631-639; Lawton, J. W. (1996),  Carbohydrate Polymers,  29 (1996), 203-208). These studies have shown that important properties for evaluation of a packaging material include mechanical properties, gas and water vapour permeability, thermoforming properties, resistance, transparency, and availability (Weber, C. et al. (2001),  Food Additives and Contaminants,  19, Supplement, 172-177). 
         [0004]    However, the design and engineering of a starch-based packaging product that possesses all of these required properties is a significant challenge. Difficulties are encountered with cost, technical hurdles such as brittleness associated with high loads, and poor water and gas barrier properties which must be overcome to commercialize the biomaterial (Lorcks, J. (1997),  Polymer Degradation and Stability,  59 (1998), 245-249). 
         [0005]    Other studies have modified the functional properties of starch to enhance its inherent bonding strength by focusing on incorporating additives such as plasticizers to improve the performance of the material (Poutanen, K. et al. (1996),  TRIP  4-4 (1996), 128-132; Laohakunjit, N. et al. (2003),  Starch,  56 (2004), 348-356). 
         [0006]    It has also been reported that certain fungi have the ability to produce exo-polysaccharides that have great potential for use in cosmetic and food industries because of their bioactive characteristics, rheological behavior, and high stability at high temperature (Selbmann, L. et al. (2003),  Antonie Van Leeuwenhoek,  84 (2003), 135-145). 
       SUMMARY OF THE INVENTION 
       [0007]    According to one embodiment of a method of the present invention, there is provided a method of manufacture of a modified thermoplastic starch from a native starch using a polysaccharide produced by a fungus species, comprising the steps of: growing a culture of the fungus species in a fungal growth medium as a shake culture at an agitation rate sufficient to optimize fungal growth for a time period of between 0.5 and 10 days, preferably between 0.5 and 5 days, until the concentration of spores of the fungus species is between 0.1 and 10 g/L; adding the native starch to the fungus species culture to form a mixture; mixing the mixture at a rate of between 10 and 1000 rpm, preferably at a rate of between 10 and 500 rpm, and a mixing temperature of between 5° C. and 50° C., preferably between 5° C. and 40° C.; and harvesting the modified thermoplastic starch. 
         [0008]    The native starch may be selected from the group comprising native potato starch, native corn starch, and native tapioca starch. The fungus may be  Ophiostoma  sp. or related  Ascomycetes  sp. The  Ophiostoma  sp. may be  Ophiostoma ulmi  sensu lata ( O. ulmi  and  O. novo - ulmi ). 
         [0009]    The fungal growth medium may be a yeast extract medium. The yeast extract medium may comprise DIFCO® yeast extract, KH 2 PO 4 , MgSO 4 , FeCl 3 *6H 2 O, MnCl*4H 2 O, ZnSO 4 *7H 2 O and sucrose, in distilled water. 
         [0010]    According to one embodiment, the harvesting step may comprise extraction of the modified thermoplastic starch and lyophilization. According to an alternative embodiment, the harvesting step may comprise the following steps: centrifuging the spore culture at between 10 and 10000 rpm, preferably at between 200 and 6000 rpm for a centrifugation period of between 0.5 and 60 minutes, preferably between 5 and 40 minutes, at room temperature to obtain a supernatant; decanting the supernatant; lyophilizing the remaining mixture until dry; and removing the dried spores. 
         [0011]    According to another embodiment of a method of the present invention, the step of adding the native starch may be replaced with the following steps: centrifuging the fungus species culture at high speed to obtain a supernatant; and incubating the supernatant with the native starch for between 0.1 and 10 days, preferably between 0.1 and 4 days. 
         [0012]    According to yet another embodiment of a method of the present invention, the fungal growth culture medium may contain native starch, and the following additional steps after growth of the spore cultures may be employed: centrifuging the mixture at high speed to obtain a supernatant; adding ethanol to the supernatant; centrifuging the mixture at high speed to obtain thermoplastic starch as a precipitate; and isolating the thermoplastic starch. 
         [0013]    According to one embodiment, a product of the invention may comprise a modified thermoplastic starch having a tensile strength between 10 and 32 MPa, an elongation at break between 0.5 and 10% and a tensile modulus between 0.3 and 1.5 GPa. Another product of the present invention may comprise a biodegradable plastic. 
         [0014]    According to one embodiment, a use of a product of the invention may comprise use of the biodegradable plastic in the manufacture of films or molding products by casting, extrusion, injection, or compression techniques. 
         [0015]    According to one embodiment, a use of a product of the invention may comprise use of the modified thermoplastic starch in the manufacture of a biodegradable product selected from the group of products comprising a film exhibiting low water absorbance and high tensile strength, a packaging film, a laminate, a sandwiched material, a foamed molded article, an extruded profile, an insulation material, and a filled molded article. 
         [0016]    According to one embodiment, a method of manufacture of a product of the invention may comprise a method of manufacture of a biodegradable plastic containing a modified thermoplastic starch, comprising the steps of: combining the modified thermoplastic starch with glycerol and water in a container; heating the contained mixture in a water bath at about 30° C., preferably at least 70° C., for at least 30 minutes, preferably 1 hour, while maintaining the volume constant, to form a solution; heating the solution at a temperature of at least 30° C., preferably at least 50° C., until a dry plastic is obtained. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0017]    A detailed description of the preferred embodiments is provided by way of example only and with reference to the following drawings, in which: 
           [0018]      FIG. 1  illustrates modified thermoplastic starch production after 4 days, according to one embodiment of the present invention; 
           [0019]      FIG. 2  illustrates water absorbance tests for native starch and modified thermoplastic starch polymer films, according to one embodiment of the present invention; 
           [0020]      FIG. 3  illustrates tensile modulus of native starch and modified thermoplastic starch polymer films, according to one embodiment of the present invention; 
           [0021]      FIG. 4  depicts a Fourier transform infrared (“FT-IR”) spectrum of exo-polysaccharide produced by  O. ulmi  isolate W9, according to one embodiment of the present invention; 
           [0022]      FIG. 5  illustrates FT-IR spectra of unmodified starch, according to one embodiment of the present invention; 
           [0023]      FIG. 6  illustrates detail of FT-IR spectra of unmodified starch and modified thermoplastic starch showing new peaks appearing at 1261.84 and 799.44 cm −1  in the modified starch spectrum, according to one embodiment of the present invention; 
           [0024]      FIG. 7  illustrates detail of FT-IR resonances of unmodified starch and modified thermoplastic starch between 2800 and 3000 cm −1 , related to C—H stretching, according to one embodiment of the present invention; 
           [0025]      FIG. 8  illustrates Raman spectrum of modified thermoplastic starch and unmodified starch in the spectral range 2000-3500 cm −1 , according to one embodiment of the present invention; 
           [0026]      FIGS. 9A and 9B  illustrate Raman mapping of native potato starch, according to one embodiment of the present invention; and 
           [0027]      FIGS. 10A and 110B  illustrate Raman mapping of modified thermoplastic potato starch, according to one embodiment of the present invention. 
       
    
    
       [0028]    In the drawings, one embodiment of the invention is illustrated by way of example. It is to be expressly understood that the description and drawings are only for the purpose of illustration and as an aid to understanding, and are not intended as a definition of the limits of the invention. 
       DETAILED DESCRIPTION OF THE INVENTION 
       [0029]      Ophiostoma ulmi  sensu lata (“ O. ulmi ”) is the causal agent of Dutch Elm disease. This fungus is unique, as its natural habitat resides in xylem fluid. The inventors have been able to demonstrate that isolates of  O. ulmi  are able to produce exo-polysaccharides in a culture medium (.Jeng, R., et al. (2007),  Forest Pathology,  37: 80-95). When starch is used as the substrate in  O. ulmi  culture, a biopolymer is produced that shows characteristics well suited to a bio-packaging material. 
         [0030]    There is disclosed a commercially viable process for large scale production of a biopolymer which can be used as packaging material for a variety of applications. 
         [0031]    The modified thermoplastic starch of the invention is obtained by incubating the spores and/or mycelia of  O. ulmi  in a culture medium containing starch, yeast extract, micro-nutrients and sucrose. The starch may be potato starch, corn starch or tapioca starch. 
         [0032]    Two starch conversion methods are disclosed. According to the first, the ethanol precipitation conversion method,  O. ulmi  is added to a yeast extract medium containing native starch to a desired spore concentration and incubated for a desired period of time. Ethanol precipitation of the incubated mixture and drying of the precipitate produces a novel thermoplastic starch. The rate of native starch conversion can be optimized through selection of spore concentration and incubation time. 
         [0033]    According to one embodiment of an ethanol precipitation starch conversion method of the invention, the conversion medium contains, per litre of distilled water, 2.0 g/L DIFCO® yeast extract, 1.0 g/L KH 2 PO 4 , 0.1 g/L MgSO 4 , 0.48 mg/L FeCl 3 *6H 2 O, 0.36 mg/L MnCl*4H 2 O, 0.44 mg/L ZnSO 4 *7H 2 O and 10 g/L sucrose, (“YE medium”). 25 g/L native starch is added to the YE medium. 200 mL of  O. ulmi  isolate is added to the medium to a concentration of fungal spores of between 3.5 and 4.0 g/L (dry weight). The mixture is incubated on an orbiting shaker at a speed of 150 rpm for between 2 and 5 days at room temperature. Modified thermoplastic starch is obtained by ethanol precipitation using an equal amount of 95% ethanol. The modified thermoplastic starch precipitate is freeze-dried or air-dried. The rate of starch conversion may be optimized by selection of spore concentration and incubation time. 
         [0034]    According to one embodiment of a non-ethanol precipitation starch conversion method from spore-containing culture, according to the invention,  O. ulmi  is grown in a 4 L flask containing 2 L of YE medium. Two isolates (W9 and Q412) of  O. ulmi  are used as a model system, but other isolates would also be acceptable. The spore culture is maintained at room temperature as a shake culture at 150 rpm for 5 days, until the concentration of fungal spores is 3.5 to 4 g/L (dry weight). To initiate starch conversion, 450 g of starch was added to the YE media containing spores. The starch may be steam autoclaved. The mixture is placed on orbiting shaker at speed of 150 rpm at room temperature. Modified thermoplastic starch may be harvested by either of two different methods. 
         [0035]    According to a first harvest method, fungal spores are not removed. Modified thermoplastic starch is harvested through filtration and lyophilized without additional treatment. According to a second harvest method, the mixture is centrifuged at 5000 rpm for 25 minutes at room temperature. The supernatant is discarded and the mixture lyophilized until dry. Dried spores are removed and discarded. This second harvest method produces a modified thermoplastic starch which provides increased clarity and improved mechanical properties in a film. 
         [0036]    According to one embodiment of a non-ethanol precipitation starch conversion method from spore-free culture, according to the invention,  O. ulmi  is grown in a 4 L flask containing 2 L of YE medium. Two isolates (W9 and Q412) of  O. ulmi  are used as a model system, but other isolates would also be acceptable. The spore culture is maintained at room temperature as a shake culture at 150 rpm for 5 days, until the concentration of fungal spores is 3.5 to 4 μL (dry weight). To initiate starch conversion, fungal spores are first removed from the YE medium by high speed centrifugation. The resulting spore-free culture filtrate is mixed with starch and incubated for between 1 and 2 days. Modified thermoplastic starch is obtained by either of the harvest methods previously described. 
         [0037]    According to one embodiment of a non-ethanol precipitation starch conversion method from purified exo-polysaccharide, according to the invention,  O. ulmi  is grown in a 4 L flask containing 2 L of YE medium. Two isolates (W9 and Q412) of  O. ulmi  are used as a model system, but other isolates would also be acceptable. The spore culture is maintained at room temperature as a shake culture at 150 rpm for 5 days, until the concentration of fungal spores is 3.5 to 4 g/L (dry weight). Fungal spores are removed from YE medium by high speed centrifugation. Spore-free culture filtrate is mixed with an equal amount of 95% ethanol. Purified exo-polysaccharide is recovered by centrifugation. Precipitated polysaccharide is re-dissolved with water. To initiate starch conversion, 450 g of starch was added to the YE media containing spores. The starch may be steam autoclaved. The mixture is placed on orbiting shaker at speed of 150 rpm at room temperature. Modified thermoplastic starch may be harvested by either of two different methods. 
         [0038]    The modified thermoplastic starch of the present invention is a novel polymer which appears to result from the interaction between native starch and exo-polysaccharide produced by  O. ulmi . A biodegradable film made by blending the modified thermoplastic starch in a mixture of glycerine and water exhibits low water absorbance and high strength in tensile and modulus tests. 
         [0039]    The film is formulated by combining 8.0 g modified thermoplastic starch with 3.95 g glycerol in a 300 mL beaker, and adding approximately 150 ml water. The suspension is heated in a 90° C. water bath for 1 hour, while maintaining a constant volume by adding water. The solution is poured into a 15 cm diameter Petri-dish. According to the ethanol precipitation method, the dish is left to evaporate at room temperature. According to the non-ethanol methods, the dish is dried in a 50° C. oven. The film is removed from the dish for physical property testing. 
         [0040]    For tensile testing, according to test standard ASTM D638, type I, three “dog bone” shaped specimens are cut from each film. Each specimen has a width of 3.00 mm. Each specimen is measured with a caliper for thickness at a minimum of 5 locations. The smallest measurement is recorded as the thickness of the specimen. Most of the specimens have a thickness of between 0.19 mm and 0.26 mm. 
         [0041]    Tensile tests are done using a Sintech Universal Tensile Test Machine Model #1. The gage length is 25.4 mm. The specimen is fixed into the slit and pulled apart by the machine at a rate of 2.5 mm/min, until specimen failure occurred. The tensile tests are carried out at 23° C. and 50% relative humidity. The atmosphere of the test site may be climate controlled. 
       Experimental Results 
     Experiment 1: Ethanol Precipitated Modified Thermoplastic Starch 
     Starch Conversion 
       [0042]    For ethanol precipitated modified thermoplastic starch, the rate of modified starch conversion using corn starch, potato starch and tapioca starch was measured. Results are shown in  FIG. 1 , which shows that use of tapioca starch produced the highest conversion rate after 4 days conversion, and corn starch the least. Values depicted in  FIG. 1  are mean values with standard deviation as shown, where N=3. By increasing the amount of starch in the medium, a modified starch yield of greater than 85% may be attained. 
       Water Absorption 
       [0043]    Films made of native and modified starches from potato, corn, tapioca, amylopectin, and modified rice starch, were soaked in water. As depicted in  FIG. 2 , after soaking film samples in water, all the unmodified starch films disintegrated within 30 minutes, and continued to absorb water. However, all films made from the modified starch remained intact, even after 24 hours. Furthermore, their water uptake capacities reached a maximum in an hour, and exhibited a plateau thereafter. Values depicted in  FIG. 2  are mean values with standard deviation as shown, for N=1 to 3. 
         [0044]    After modification, biopolymers derived from potato and tapioca starches exhibited a much lower water absorption, which indicated a higher moisture resistance, a favourable property for packaging material applications. 
       Tensile Strength 
       [0045]    Both native starch and modified thermoplastic starch were cast into films, which were dried at room temperature for at least 3 days, then subjected to tensile testing as described above. As depicted in  FIG. 3  and Table I, the experimental results show that the modified starch has improved strength properties and is well suited for use as a packaging material. Tensile modulus values in  FIG. 3  are mean values with standard deviation as shown, for N=5, 4, 6, 6, 10, 4 and 3, respectively. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Tensile Tests of Native and Modified Thermoplastic Starch Films 
               
             
          
           
               
                   
                   
                   
                 95% 
                   
               
               
                   
                   
                   
                 confidence limits 
                 N (number of 
               
               
                   
                 Material 
                 Mean 
                 of the mean 
                 measurements) 
               
               
                   
                   
               
             
          
           
               
                 Peak Stress 
                 Potato Starch 
                 1.60 
                 1.18 
                 2.01 
                 5 
               
               
                 (MPa) 
               
               
                   
                 Potato Polymer 
                 3.58 
                 3.22 
                 3.92 
                 7 
               
               
                   
                 Tapioca Starch 
                 0.37 
                 −0.01 
                 0.75 
                 6 
               
               
                   
                 Tapioca 
                 3.60 
                 3.30 
                 3.89 
                 10 
               
               
                   
                 Polymer 
               
               
                   
                 Rice Polymer 
                 0.43 
                 −0.04 
                 0.89 
                 4 
               
               
                   
                 Corn Polymer 
                 2.52 
                 2.14 
                 2.90 
                 6 
               
               
                   
                 Amylopectin 
                 0.97 
                 0.44 
                 1.51 
                 3 
               
               
                   
                 Polymer 
               
               
                 Elongation at 
                 Potato Starch 
                 40.78 
                 37.05 
                 44.50 
                 3 
               
               
                 break (mm) 
               
               
                   
                 Potato Polymer 
                 10.78 
                 8.34 
                 12.31 
                 7 
               
               
                   
                 Tapioca Starch 
                 48.33 
                 43.76 
                 52.89 
                 2 
               
               
                   
                 Tapioca 
                 10.77 
                 8.73 
                 12.81 
                 10 
               
               
                   
                 Polymer 
               
               
                   
                 Rice Polymer 
                 34.79 
                 30.22 
                 39.35 
                 2 
               
               
                   
                 Corn Polymer 
                 13.36 
                 10.73 
                 16.00 
                 6 
               
               
                   
                 Amylopectin 
                 21.72 
                 17.16 
                 26.28 
                 2 
               
               
                   
                 Polymer 
               
               
                   
               
             
          
         
       
     
         [0046]    Molecular level changes during the modification process were studied by FT-IR. The results are shown in  FIG. 4 . The spectrum of  FIG. 4  represents the native potato starch harvested from the fungal modification of native potato starch. 
         [0047]    The experimental results clearly indicate that isolates of  O. ulmi  can modify native starch into a new polymer which produces a bio-film having low water absorbance and high mechanical strength. Changes in the starch structure may be studied through FT-IR. The pyranose ring is maintained after the modification, but the strength of the hydrogen bonds between molecules is intensified. Peak shifts and ratio changes suggest the fixation of new chemical functional groups or new linkages between starch molecules. Peaks at 798.09 cm −1 , 1257.71 cm −1  and 2860.65 cm −1  are characteristic of the modified thermoplastic starches. 
         [0048]    Based on these results, two possible pathways of the modification are suggested. One pathway may involve the fungus  O. ulmi  producing a polymer which can bond starch molecules together and form new cross-linked structures. The second possible pathway may involve the fungus attaching to one or more functional groups which help strengthen the starch polymer. 
       Non-Ethanol Precipitated Modified Thermoplastic Starch 
       [0049]    Experiments were carried out to determine parameters required for large scale production and improved mechanical strength of bio-films.  O. ulmi  isolates W9 and Q412 were both tested. Results are reported based on tensile testing of bio-film made from modified thermoplastic potato. The method for film casting is as described previously. 
         [0050]    Direct Harvest Method from Spore-containing Culture 
         [0051]    For modified thermoplastic starch film derived by the direct harvest method from spore-containing culture, several experiments were carried out. 
         [0000]    Experiment 2: Non-Ethanol Precipitation with Room Temperature Drying 
         [0052]    In this experiment, the film was dried at room temperature and tensile testing was performed after 5 days. A W9 isolate was used. The results are shown in Table 2. 
         [0000]                                                                                                  TABLE 2                   Tensile testing of modified and unmodified starch films                peak stress   elongation   modulus            Sample   mean   SD   mean   SD   mean   SD                    Unmodified   2.640   0.060   8.960   0.470   0.023   0.001       starch       W day 1   14.310   4.144   2.658   1.372   0.871   0.286       W day 2   9.184   1.446   5.748   1.230   0.369   0.097       W day 3   7.442   1.573   9.596   2.045   0.215   0.109       W day 4   11.617   5.243   0.403   1.139   0.339   0.277       W day 5   6.954   1.627   7.687   1.650   0.210   0.095       W day 6   2.200   0.190   9.080   0.660   0.017   0.001       W day 7   2.050   0.000   9.260   0.000   0.018   0.000       W day 8   2.360   0.040   9.120   0.310   0.027   0.007                    
Experiment 3: Non-Ethanol Precipitation of Q412 Isolate with 50° C. Drying
 
         [0053]    In this experiment, the film was dried at 50° C. for 24 hours. Tensile testing was performed after the film was brought back to room temperature. A Q412 isolate was used, with native starch as a control. Ethanol precipitated modified thermoplastic starch is included as reference. The results are shown in Table 3. 
         [0000]                                                                                                  TABLE 3                   Tensile testing of Q412 isolate with 50° C. Drying                peak stress   elongation   modulus            Sample   mean   SD   mean   SD   mean   SD                    Control   2.28       21.7       0.0353           Q22 hr.   6.32   1.9721   11.05   2.803   0.3768   0.208       Q24 hr.   7.18   0.0987   9.67   1.1372   0.3429   0.059       Q d2   8.51   0.9551   7.33   1.2527   0.4978   0.0882       Q d3   10.6   0.5052   6.73   0.7506   0.6483   0.1071       Q d4   11.08   1.8608   6.23   1.159   0.7694   0.1126       Q d5   10   2.4676   6.93   2.6725   0.5459   0.2076       Q d6   6.95   0.2949   11.37   0.4509   0.2532   0.0451       Q d7   9.12   0.3164   7.47   0.9504   0.4308   0.0998       Q d8   8.92   0.3913   6.27   1.3317   0.4978   0.0929       ETOH   11.49   1.3931   2.23   0.7371   0.785   0.0991                    
Experiment 4: Non-Ethanol Precipitation of W9 Isolate with 50° C. Drying
 
         [0054]    In this experiment, the film was dried at 50° C. for 24 hours. Tensile testing was performed after film was brought back to room temperature. A W9 isolate was used. Day harvested is indicated with ‘d’ in the Sample column. The results are shown in Table 4. 
         [0000]                                                                                                  TABLE 4                   Tensile testing of W9 isolate with 50° C. Drying                peak stress   elongation   modulus            Sample   mean   SD   mean   SD   mean   SD                    W 22 hr.   14.54   0.2307   4.77   0.671   0.9306   0.1201       W 24 hr   8.04   0.2996   7.28   1.0532   0.4637   0.0861       W d2   22.66   1.2061   2.85   0.3514   1.3448   0.1302       W d3   10.42   0.6793   7.16   0.7197   0.6875   0.0639       W d4   17.7   1.0382   3.45   1.002   1.223   0.0157       W d6   11.8   0.2601   6   0.6195   0.6982   0.1686       W d7   10.34   0.2109   5.83   0.7411   0.7453   0.061                    
Centrifugation Method from Spore-containing Culture
 
         [0055]    For modified thermoplastic starch film derived by the centrifugation method from spore-containing culture, several experiments were carried out. 
         [0000]    Experiment 5: Centrifugation from Spore Culture of Modified Thermoplastic Starch 
         [0056]    The film was dried at 50° C. for 24 hours. Tensile testing was performed after film was brought back to room temperature. C represents centrifuged sample, W indicated W9 isolate. The control was native starch. Results are shown in Table 5. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 tensile testing for spore culture of modified thermoplastic starch 
               
             
          
           
               
                   
                 peak stress 
                 elongation 
                 modulus 
               
             
          
           
               
                 Sample 
                 mean 
                 SD 
                 mean 
                 SD 
                 Mean 
                 SD 
               
               
                   
               
             
          
           
               
                 CW d3, 
                 19.85 
                 1.689 
                 2.96 
                 1.013 
                 1.178 
                 0.2765 
               
               
                 Control 
                 8.07 
                 1.274 
                 8.2 
                 3.46 
                 0.4082 
                 0.1368 
               
               
                   
               
             
          
         
       
     
       Experiment 6: Time Interval Testing of Modified Thermoplastic Starch Films 
       [0057]    A series of films made at the same time were subjected to tensile testing at differing time intervals as described in Table 6. C represents a centrifuged sample. Q indicates a Q142 isolate, W indicates a W9 isolate, ‘d’ the day harvested. Native starch was used as a control. Results are shown in Table 6. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Tensile testing with time intervals 
               
             
          
           
               
                   
                 peak stress 
                 elongation 
                 modulus 
                   
               
             
          
           
               
                 Sample 
                 mean 
                 SD 
                 mean 
                 SD 
                 mean 
                 SD 
                 treatment 
               
               
                   
               
             
          
           
               
                 CQ d1 
                 11.81 
                 1.3 
                 6.5 
                 2.893 
                 0.6392 
                 0.1588 
                 50° C. for 24 hr 
               
               
                   
                 25.21 
                 2.6 
                 2.38 
                 1.226 
                 1.088 
                 0.315 
                 50° C. for 48 hrs 
               
               
                   
                 8.53 
                 0.66 
                 10.44 
                 1.543 
                 0.4835 
                 0.2373 
                 To RT after 48 hr 
               
               
                 CQ d2 
                 21.31 
                   
                 3.75 
                   
                 1.276 
                   
                 50° C. for 24 hr 
               
               
                   
                 23.79 
                 2.03 
                 2.083 
                 1.105 
                 1.505 
                 0.042 
                 50° C. for 48 hrs 
               
               
                 CQ d2 
                 9.34 
                 0.61 
                 8.53 
                 1.572 
                 0.6249 
                 0.1785 
                 50° C. for 48 hrs 
               
               
                 CQ d1 
                 22.89 
                 1.7 
                 2.483 
                 1.182 
                 1.087 
                 0.028 
                 50° C. for 48 hrs 
               
               
                   
                 8.37 
                   
                 9.9 
                   
                 0.5516 
                   
                 To RT after 48 hr 
               
               
                 CW d3 
                 19.85 
                 1.689 
                 2.96 
                 1.013 
                 1.178 
                 0.2765 
                 50° C. for 48 hrs 
               
               
                 CW d3 
                 11.55 
                 3.51 
                 6.2 
                 3.203 
                 0.5995 
                 0.0656 
                 50° C. for 48 hrs and 
               
               
                 wash 
                   
                   
                   
                   
                   
                   
                 water wash after 
               
               
                   
                   
                   
                   
                   
                   
                   
                 centrifugation 
               
               
                 CW d1 
                 8.62 
                 1.36 
                 11.1 
                 0.9019 
                 0.5734 
                 0.1771 
                 50° C. for 24 hr 
               
               
                   
                 14.41 
                 3.14 
                 6.673 
                 1.107 
                 0.8362 
                 0.3666 
                 50° C. for 48 hrs 
               
               
                   
                 6.17 
                 1.1 
                 16.1 
                 2.4 
                 0.2438 
                 0.0714 
                 To RT after 48 hr 
               
               
                 Control 
                 5.79 
                 0.58 
                 14.66 
                 2.74 
                 0.3352 
                 0.0325 
                 50° C. for 48 hrs 
               
               
                   
                 3.32 
                 0.03 
                 15.71 
                 1.64 
                 0.1003 
                 0.029 
                 To RT after 48 hr 
               
               
                   
               
             
          
         
       
     
       Experiment 7: Time Interval Testing of Modified Thermoplastic Starch Films 
       [0058]    A series of films made at the same time were subjected to tensile testing at differing time intervals as described in Table 7. C represents a centrifuged sample. W indicates a W9 isolate, ‘d’ the day harvested. Native starch was used as a control. Results are shown in Table 7. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Time interval testing of modified thermoplastic starch films 
               
             
          
           
               
                   
                 peak stress 
                 elongation 
                 modulus 
                   
               
             
          
           
               
                 Sample 
                 mean 
                 SD 
                 mean 
                 SD 
                 mean 
                 SD 
                 treatment 
               
               
                   
               
             
          
           
               
                 CW d1 
                 16.79 
                 2.069 
                 1.12 
                 0.485 
                 0.8174 
                 0.2116 
                 50° C. for 
               
               
                   
                   
                   
                   
                   
                   
                   
                 24 hr 
               
               
                   
                 24.38 
                 3.44 
                 2.95 
                 1.195 
                 1.357 
                 0.161 
                 50° C. for 
               
               
                   
                   
                   
                   
                   
                   
                   
                 48 hrs 
               
               
                 CW d2 
                 15.32 
                 0.87 
                 4.95 
                 1.062 
                 0.948 
                 0.143 
                 50° C. for 
               
               
                   
                   
                   
                   
                   
                   
                   
                 24 hr 
               
               
                   
                 22.69 
                   
                 3.63 
                   
                 1.255 
                   
                 50° C. for 
               
               
                   
                   
                   
                   
                   
                   
                   
                 48 hrs 
               
               
                   
               
             
          
         
       
     
         [0059]    In order to increase the yield of modified thermoplastic starch, 450 g of native potato starch, instead of 225 g, was added to 1 L of YE media. The amount of spores and the procedures for film casting are the same as previously described. The results are as set out in Tables 8, 9 and 10 
         [0000]    Experiment 8: Tensile strength at time intervals for Q412 isolate 
         [0060]    A series of films made at the same time were subjected to tensile testing at differing time intervals as described in Table 8. C represents a centrifuged sample. Q indicates a Q412 isolate. ‘d’ the day harvested. Native starch was used as a control. Results are shown in Table 8. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 Tensile strength at time intervals for Q412 isolate 
               
             
          
           
               
                   
                 peak 
                   
                   
                   
               
               
                   
                 stress 
                 elongation 
                 modulus 
               
             
          
           
               
                 Sample 
                 mean 
                 SD 
                 mean 
                 SD 
                 mean 
                 SD 
                 treatment 
               
               
                   
               
             
          
           
               
                 CQ d1 
                 27.26 
                 0.56 
                 1.939 
                 0.178 
                 1.505 
                 0.086 
                 50° C. for 
               
               
                   
                   
                   
                   
                   
                   
                   
                 24 hr 
               
               
                   
                 25.33 
                 2.59 
                 0.646 
                 0.296 
                 1.604 
                 0.185 
                 50° C. for 
               
               
                   
                   
                   
                   
                   
                   
                   
                 48 hrs 
               
               
                   
                 18.43 
                 2.17 
                 1.34 
                 0.15 
                 1.279 
                 0.088 
                 To RT after 
               
               
                   
                   
                   
                   
                   
                   
                   
                 48 hr 
               
               
                 CQ d2 
                 22.93 
                 1.38 
                 2.73 
                 0.151 
                 1.239 
                 0.0509 
                 50° C. for 
               
               
                   
                   
                   
                   
                   
                   
                   
                 24 hr 
               
               
                   
                 23.59 
                 4.24 
                 2.291 
                 1.142 
                 1.359 
                 0.172 
                 50° C. for 
               
               
                   
                   
                   
                   
                   
                   
                   
                 48 hrs 
               
               
                   
                 13.85 
                 5.02 
                 5.48 
                 3.207 
                 0.8721 
                 0.3481 
                 To RT after 
               
               
                   
                   
                   
                   
                   
                   
                   
                 48 hr 
               
               
                   
               
             
          
         
       
     
       Experiment 9: Tensile Strength at Time Intervals for W9 Isolate 
       [0061]    A series of films made at the same time were subjected to tensile testing at differing time intervals as described in Table 9. C represents a centrifuged sample. W indicates a W9 isolate, ‘d’ the day harvested. Results are shown in Table 9. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                 Tensile strength at time intervals for W9 isolate 
               
             
          
           
               
                   
                 peak 
                   
                   
                   
               
               
                   
                 stress 
                 elongation 
                 modulus 
               
             
          
           
               
                 Sample 
                 mean 
                 SD 
                 mean 
                 SD 
                 mean 
                 SD 
                 treatment 
               
               
                   
               
             
          
           
               
                 CW d1 
                 25.24 
                 3.13 
                 2.8 
                 1.122 
                 1.254 
                 0.203 
                 50° C. for 
               
               
                   
                   
                   
                   
                   
                   
                   
                 24 hr 
               
               
                   
                 25.68 
                 1.35 
                 1.9 
                 0.533 
                 1.376 
                 0.208 
                 50° C. for 
               
               
                   
                   
                   
                   
                   
                   
                   
                 24 hr 
               
               
                 CW d1 
                 25.44 
                 3.31 
                 1.87 
                 0.872 
                 1.292 
                 0.072 
                 50° C. for 
               
               
                   
                   
                   
                   
                   
                   
                   
                 48 hrs 
               
               
                   
                 26.84 
                 2.321 
                 2.03 
                 0.664 
                 1.4813 
                 0.0522 
                 50° C. for 
               
               
                   
                   
                   
                   
                   
                   
                   
                 48 hrs 
               
               
                 CW d1 
                 18.82 
                 3.41 
                 1.84 
                 1.516 
                 1.122 
                 0.1806 
                 To RT after 
               
               
                   
                   
                   
                   
                   
                   
                   
                 48 hr 
               
               
                   
               
             
          
         
       
     
       Experiment 10: Tensile Strength at Time Intervals for W9 Isolate 
       [0062]    A series of films made at the same time were subjected to tensile testing at differing time intervals as described in Table 10. C represents a centrifuged sample. W indicates a W9 isolate, ‘d’ the day harvested. Results are shown in Table 10. 
         [0000]                                                                                                              TABLE 10                   Tensile strength at time intervals for W9 isolate                peak                       stress   elongation   modulus            Sample   mean   SD   mean   SD   mean   SD   treatment                    CW d1   25.68   1.35   1.9   0.533   1.376   0.208   50° C. for                                   24 hr           26.84   2.321   2.03   0.664   1.4813   0.0522   50° C. for                                   48 hrs           18.82   3.41   1.84   1.516   1.122   0.1806   To RT after                                   48 hr                    
Centrifugation Method from Spore-Free Culture
 
         [0063]    For modified thermoplastic starch film derived by the centrifugation method from spore-free culture, several experiments were carried out. 
       Experiment 11: Tensile Strength for Centrifugation Isolation of Modified Thermoplastic Starch 
       [0064]    Films made at the same time were subjected to tensile testing at differing time intervals as described in Table 11. C represents a centrifuged sample. Q indicates a Q412 isolate, ‘d’ the day harvested, −S indicated spores removed before mixing. Results are shown in Table 11. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 11 
               
             
             
               
                   
               
               
                 Tensile strength for centrifugation isolation of 
               
               
                 modified thermoplastic starch 
               
             
          
           
               
                   
                 peak stress 
                 elongation 
                 modulus 
                   
               
             
          
           
               
                 Sample 
                 mean 
                 SD 
                 mean 
                 SD 
                 mean 
                 SD 
                 treatment 
               
               
                   
               
             
          
           
               
                 CW-S d1 
                 27.17 
                 1.01 
                 1.8 
                 0.183 
                 1.524 
                 0.055 
                 50° C. 
               
               
                   
                   
                   
                   
                   
                   
                   
                 for 24 hr 
               
               
                   
                 30.82 
                 1.76 
                 1 
                 0.0617 
                 1.625 
                 0.165 
                 50° C. 
               
               
                   
                   
                   
                   
                   
                   
                   
                 for 48 hrs 
               
               
                 CW-S d2 
                 24.1 
                 2.94 
                 2.4 
                 0.774 
                 1.094 
                 0.134 
                 50° C. 
               
               
                   
                   
                   
                   
                   
                   
                   
                 for 24 hr 
               
               
                   
                 29.72 
                 0.8871 
                 1.75 
                 0.1935 
                 1.454 
                 0.1372 
                 50° C. 
               
               
                   
                   
                   
                   
                   
                   
                   
                 for 48 hrs 
               
               
                 CQ-S d1′ 
                 27.03 
                 0.41 
                 1.8 
                 0.392 
                 1.32 
                 0.124 
                 50° C. 
               
               
                   
                   
                   
                   
                   
                   
                   
                 for 24 hr 
               
               
                   
                 23.2 
                 5.99 
                 1.29 
                 0.8684 
                 1.25 
                 0.063 
                 50° C. 
               
               
                   
                   
                   
                   
                   
                   
                   
                 for 48 hrs 
               
               
                 CQ-Sd2 
                 24.84 
                 1.11 
                 1.92 
                 0.678 
                 1.383 
                 0.189 
                 50° C. 
               
               
                   
                   
                   
                   
                   
                   
                   
                 for 24 hr 
               
               
                   
                 27.99 
                 0.8132 
                 1.6 
                 0.0354 
                 1.396 
                 0.186 
                 50° C. 
               
               
                   
                   
                   
                   
                   
                   
                   
                 for 48 hrs 
               
               
                   
               
             
          
         
       
     
       Experiment 12: Tensile Strength for Filtration Isolation of Modified Thermoplastic Starch 
       [0065]    Instead of centrifuging, the modified thermoplastic starch was obtained by filtration (F) or both filtration followed by water washing (FW). A series of films made at the same time were subjected to tensile testing at differing time intervals. Q indicates isolate Q412; −S indicates spore removed before mixing, and 2 indicates second set. Results are shown in Table 12. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 12 
               
             
             
               
                   
               
               
                 Tensile strength for filtration isolation of modified thermoplastic starch 
               
             
          
           
               
                   
                 peak stress 
                 elongation 
                 modulus 
                   
               
             
          
           
               
                 Sample 
                 mean 
                 SD 
                 mean 
                 SD 
                 mean 
                 SD 
                 treatment 
               
               
                   
               
             
          
           
               
                 Q-SF2 
                 11.88 
                 1.13 
                 9 
                 1.067 
                 0.7305 
                 0.0392 
                 50° C. 
               
               
                   
                   
                   
                   
                   
                   
                   
                 for 24 hr 
               
               
                 Q- 
                 12.49 
                 3.79 
                 8.1 
                 3.661 
                 0.7939 
                 0.193 
                 50° C. 
               
               
                 SFW2 
                   
                   
                   
                   
                   
                   
                 for 24 hr 
               
               
                 Q-SF2 
                 14.3 
                 2.153 
                 8.01 
                 1.11 
                 0.8167 
                 0.0923 
                 50° C. 
               
               
                   
                   
                   
                   
                   
                   
                   
                 for 48 hrs 
               
               
                 Q- 
                 19.84 
                 2.79 
                 3.7 
                 0.794 
                 0.9699 
                 0.883 
                 50° C. 
               
               
                 SFW2 
                   
                   
                   
                   
                   
                   
                 for 48 hrs 
               
               
                 W SF2 
                 11.06 
                 0.99 
                 9.1 
                 0.78 
                 0.7305 
                 0.0392 
                 50° C. 
               
               
                   
                   
                   
                   
                   
                   
                   
                 for 24 hr 
               
               
                 W 
                 11.4 
                 0.3427 
                 8.9 
                 0.4583 
                 0.4811 
                 0.0508 
                 50° C. 
               
               
                 SFW2 
                   
                   
                   
                   
                   
                   
                 for 24 hr 
               
               
                 W SF2 
                 18.82 
                 1.44 
                 5.8 
                 1.334 
                 0.8597 
                 0.1101 
                 50° C. 
               
               
                   
                   
                   
                   
                   
                   
                   
                 for 48 hrs 
               
               
                 W 
                 25.5 
                 4.07 
                 2.91 
                 0.751 
                 1.104 
                 0.1066 
                 50° C. 
               
               
                 SFW2 
                   
                   
                   
                   
                   
                   
                 for 48 hrs 
               
               
                 W-SF 
                 19.01 
                 2.13 
                 3.1 
                 1.353 
                 0.9479 
                 0.0655 
                 50° C. 
               
               
                   
                   
                   
                   
                   
                   
                   
                 for 48 hrs 
               
               
                 W-SFW 
                 19.59 
                 1.44 
                 4.6 
                 0.217 
                 1.049 
                 0.046 
                 50° C. 
               
               
                   
                   
                   
                   
                   
                   
                   
                 for 48 hrs 
               
               
                 W-SF 
                 14.55 
                 0.61 
                 4.3 
                 0.654 
                 0.8751 
                 0.0738 
                 To RT 
               
               
                   
                   
                   
                   
                   
                   
                   
                 after 48 hr 
               
               
                 W-SFW 
                 19.52 
                 2.59 
                 2 
                 1.245 
                 1.025 
                 0.1065 
                 To RT 
               
               
                   
                   
                   
                   
                   
                   
                   
                 after 48 hr 
               
               
                   
               
             
          
         
       
     
       Experiment 13: Tensile Strength for Non-Autoclaved Modified Thermoplastic Starch 
       [0066]    Instead of using autoclaved native starch, the modified thermoplastic starch was obtained by mixing non-autoclaved starch (NAu) with culture filtrate. A series of films made at the same time were subjected to tensile testing at differing time intervals. Q indicates isolate Q412; −S indicates spore removed before mixing. Results are shown in Table 13. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 13 
               
             
             
               
                   
               
               
                 Tensile strength for non-autoclaved modified thermoplastic starch 
               
             
          
           
               
                   
                 peak 
                   
                   
                   
               
               
                   
                 stress 
                 elongation 
                 modulus 
               
             
          
           
               
                 Sample 
                 mean 
                 SD 
                 mean 
                 SD 
                 mean 
                 SD 
                 treatment 
               
               
                   
               
             
          
           
               
                 Q-SNAu 
                 16.84 
                 0.71 
                 5.9 
                 0.583 
                 0.9336 
                 0.0443 
                 50° C. 
               
               
                   
                   
                   
                   
                   
                   
                   
                 for 24 hr 
               
               
                   
                 27.12 
                 1.29 
                 2.2 
                 0.408 
                 1.239 
                 0.187 
                 50° C. 
               
               
                   
                   
                   
                   
                   
                   
                   
                 for 48 hrs 
               
               
                   
               
             
          
         
       
     
         [0067]    These experiments clearly show that modified thermoplastic starch made from the centrifugation method possesses much better mechanical properties for bio-film. These data also show that the films made from a sample having a longer drying time exhibit high peak stress. 
         [0000]    Centrifugation Method from Purified Exo-Polysaccharide 
       Experiment 14: Tensile Strength for Centrifuged Purified Exo-Polysaccharide 
       [0068]    For modified thermoplastic starch film derived by the centrifugation method from purified exo-polysaccharide, tensile testing was carried out. A series of films made at the same time were subjected to tensile testing at differing time intervals. C indicates centrifuged; EPS indicates exo-polysaccharide; and S indicates native starch. Results are shown in Table 14. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 14 
               
             
             
               
                   
               
               
                 Tensile strength for centrifuged purified exo-polysaccharide 
               
             
          
           
               
                   
                 peak 
                   
                   
                   
               
               
                   
                 stress 
                 elongation 
                 modulus 
               
             
          
           
               
                 Sample 
                 mean 
                 SD 
                 mean 
                 SD 
                 mean 
                 SD 
                 treatment 
               
               
                   
               
             
          
           
               
                 EPS + S + 
                 15.63 
                 1.22 
                 2.58 
                 0.6657 
                 0.7301 
                 0.0136 
                 50° C. 
               
               
                 C 
                   
                   
                   
                   
                   
                   
                 for 24 hr 
               
               
                   
                 31.55 
                 1.71 
                 0.8 
                 0.3011 
                 1.411 
                 0.146 
                 50° C. 
               
               
                   
                   
                   
                   
                   
                   
                   
                 for 48 hrs 
               
               
                   
               
             
          
         
       
     
       Structural Analysis 
     Experiment 15: Fourier Transform Infrared Analysis of Modified Thermoplastic Starch 
       [0069]    Table 15 shows results of FT-IR testing, a summary of the frequencies and proposed structural assignments of the most characteristic FT-IR bands of the modified thermoplastic starch spectra. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
             
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 15 
               
             
             
               
                   
               
               
                 Fourier Transform Infrared Analysis of Modified Thermoplastic Starch 
               
             
          
           
               
                   
                 Group 
                 Intensity 
                   
               
             
          
           
               
                 Frequency, cm −1   
                 Vibration 
                 IR 
                 Raman 
                 Description 
               
               
                   
               
               
                 3200-3500 
                 —OH stretch 
                 very strong 
                 very weak 
                 Hydroxyl 
               
               
                 2700-3000 
                 —C—H stretch 
                 strong-medium 
                 medium 
               
               
                 1640-1650 
                 H 2 O 
               
               
                 1300-1400 
                 C—H scissoring 
                 medium 
                 medium-weak 
               
               
                 1300-1350 
                 C—O stretch 
                 strong 
               
             
          
           
               
                  300-1300 
                 Finger print for skeleton 
               
             
          
           
               
                 1100-1300 
                 C—O stretch 
                 strong 
                 medium-weak 
                   
               
               
                 800-900 
                 Skeletal mode 
                   
                 medium 
                 α-(1-4) linkage 
               
               
                 750-800 
                 C—O—C skeletal 
                 medium-weak 
                 medium-weak 
                 β-configuration 
               
               
                 700-750 
                 C—O—C skeletal 
                 medium-weak 
                 medium 
                 α-configuration 
               
               
                 600-650 
                 C—H rocking 
                 very strong- 
                 very weak 
               
               
                   
                   
                 medium 
               
               
                 400-500 
                 Skeletal mode 
                   
                 very strong 
               
               
                   
               
             
          
         
       
     
         [0070]    The FT-IR spectra are shown in  FIGS. 5 ,  6 , and  7 . In  FIG. 5 , new peaks are discernable, and the intensity of the resonances within the spectra, and the resonances at the skeleton mode (400-1500 cm −1 ) are higher compared to resonances due to OH groups in modified starches.  FIG. 6  illustrates detail of FT-IR spectrum of UTTS showing two new peaks appearing at 1261.84 and 799.44 cm −1 .  FIG. 7  illustrates detail of FT-IR resonances between 2800 and 3000, related to C—H stretching. 
         [0071]    In  FIG. 7 , a new peak appears at 2961.40 cm −1  in modified thermoplastic starches. The peak at 2922.80 cm −1  in modified starches may be related to the peak at 2927.19 cm −1  in unmodified starches, the shifted peak may be due to a new interaction within the molecular structure of the modified starch. 
         [0072]    These figures clearly show the presence of three new peaks in the FT-IR spectrum of the modified thermoplastic starch. These peaks are very similar to those detected in ETOH precipitated modified thermoplastic starch. These peaks may be used as bio-makers for the novel modified thermoplastic starch of the invention. 
         [0073]    In  FIGS. 8 ,  9 A,  9 B,  10 A and  10 B, there are depicted Raman spectra for the modified thermoplastic starch.  FIG. 8  illustrates the Raman spectrum of modified thermoplastic starch and native starches, in the spectral range 2000-3500.  FIGS. 9A and 9B  illustrate Raman mapping and Raman spectrum, respectively, of native potato starches.  FIGS. 10A and 10B  illustrate Raman mapping and Raman spectrum, respectively, of modified thermoplastic potato starch. 
         [0074]    The modified thermoplastic starch of the present invention is a new starch-based thermoplastic resulting from the interaction of native starch and exo-polysaccharide produced by isolates of  O. ulmi . Solubility of native starch in the media is not the limiting factor for large scale production of modified thermoplastic starch. Mechanical strength of bio-film may be optimized by regulating the drying temperature and drying duration. Different properties of bio-package material for commercial application can be selected for from modified thermoplastic starch. Although only two isolates of  O. ulmi  are demonstrated here, other isolates of this fungus are able to produce modified thermoplastic starch, as all such isolates have a similar genetic makeup. It will be appreciated by those skilled in the art that other variations of the preferred embodiment may also be practised without departing from the scope of the invention.