Biodegradable polysaccharide films for packaging

Abstract

Biodegradable polysaccharide films for packaging are disclosed. One general aspect includes a biodegradable polysaccharide film exhibiting properties comparable to polyethylene and is biodegradable. The biodegradable polysaccharide film includes a blend of pullulan and chitosan. The biodegradable polysaccharide film also includes a crosslinking agent selected from glutaraldehyde or sodium tripolyphosphate. Another general aspect includes a method of making the biodegradable polysaccharide film.

Description

Attorney Docket: 222204-2980 BIODEGRADABLE POLYSACCHARIDE FILMS FOR PACKAGING CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 586,837, titled “Biodegradable Polysaccharide Films for Packaging,” filed September 29, 2023, the entire contents of which are hereby incorporated by reference herein. BACKGROUND

[0002] Plastics have become an integral part of modern life, particularly in the medical, food packaging, and industrial sectors, due to their versatility, durability, and cost- effectiveness. However, the overwhelming reliance on petroleum-based plastics, such as high-density polyethylene (HDPE), polypropylene (PP), and polyethylene terephthalate (PET), has led to significant environmental challenges. These plastics are non- biodegradable and persist in the environment for hundreds of years, contributing to pollution and ecological damage.

[0003] The global demand for plastics continues to rise, with the medical plastics market alone valued at $22.26 billion as of 2020, growing at an annual rate of 6.1%. This market includes products such as medical devices, packaging, and disposables that are often made from non-biodegradable materials. The accumulation of plastic waste, particularly from single-use applications, has prompted researchers and industries to explore alternatives that can mitigate the environmental impact associated with plastic disposal. BRIEF SUMMARY OF THE INVENTION

[0004] This Summary introduces a selection of concepts in a simplified form that are further described below in the Detailed Description. As such, this Summary is not intended to identify essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0005] The present disclosure is directed to biodegradable polysaccharide films for packaging. One general aspect includes a biodegradable polysaccharide film exhibitingAttorney Docket: 222204-2980 properties comparable to polyethylene and is biodegradable. The biodegradable polysaccharide film includes a blend of pullulan and chitosan. The biodegradable polysaccharide film also includes a crosslinking agent selected from glutaraldehyde or sodium tripolyphosphate (TPP).

[0006] Implementations may include one or more of the following features. The biodegradable polysaccharide film where the crosslinking agent is glutaraldehyde, applied in vapor phase for 3 to 6 hours. The crosslinking agent is sodium TPP added during a film fabrication process. The biodegradable polysaccharide film has a thickness between 0.1 millimeter (mm) and 0.5 mm. The film is used as a packaging material for medical devices. The pullulan and the chitosan are blended in an acidic solution of acetic acid with a concentration of 1-3%. The biodegradable polysaccharide film may include glycerol as a plasticizer in a concentration of 1-10% by weight. The biodegradable polysaccharide film exhibits antibacterial properties. The biodegradable polysaccharide film degrades in soil within 60 days. The biodegradable polysaccharide film swells by less than 50% in saline water over a period of 7 days.

[0007] Another general aspect includes a method of forming a biodegradable polysaccharide film. The method includes blending pullulan, chitosan, glycerol, and an acetic acid solution to form a polysaccharide solution. The method also includes degassing the polysaccharide solution to remove air bubbles. The method also includes casting the degassed polysaccharide solution into a film. The method also includes curing the film to solidify the film. The method also includes crosslinking the film with using either glutaraldehyde vapor or sodium TPP during the blending step to form a crosslinked biodegradable polysaccharide film.

[0008] Implementations may include one or more of the following features. The method where the acetic acid solution has a concentration of 1-3%. The glycerol is added in a concentration of 1-10% by weight. The casting step produces a film thickness between 0.1 mm and 0.5 mm. The degassing is performed using a vacuum chamber to remove air bubbles from the polysaccharide solution. The crosslinking with sodium TPP is performed to enhance biodegradability and mechanical strength of the crosslinked biodegradable polysaccharide film. The method may include fabricating a package using the crosslinked biodegradable polysaccharide film. The package is a food package. The package is aAttorney Docket: 222204-2980 medical package. The method may include incorporating the crosslinked biodegradable polysaccharide film into a biodegradable application. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Many aspects of the present disclosure can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale, with emphasis instead being placed upon clearly illustrating the principles of the disclosure. Moreover, in the drawings, like reference numerals designate corresponding parts throughout the several views.

[0010] FIG. 1 depicts a block flow diagram of a process used to manufacture a biodegradable polysaccharide film according to an example implementation.

[0011] FIG. 2 depicts an antibacterial leakage test setup used to evaluate the antibacterial properties of the biodegradable polysaccharide film according to an example implementation.

[0012] FIG.3 depicts a thermogravimetric analysis (TGA) graph illustrating the weight loss behavior of several fabricated film samples over a temperature range according to an example implementation.

[0013] FIG. 4 depicts a TGA graph for the raw materials of pullulan and chitosan, according to an example implementation.

[0014] FIG. 5 depicts a stress-strain graph for different film samples according to an example implementation.

[0015] FIG. 6 depicts graphs showing the mean penetration of Staphylococcus aureus bacteria through different film samples during antibacterial testing according to an example implementation.Attorney Docket: 222204-2980

[0016] FIGS. 7A-7C depict swelling percentage graphs showing how film samples absorb water over time in different conditions (deionized water and saline water) according to an example implementation.

[0017] FIGS. 8A-8C depict weight loss plots over 27 days, illustrating the biodegradability of the films under soil conditions according to an example implementation.

[0018] FIG. 9 depicts a method for producing a biodegradable polysaccharide film according to an example implementation. DETAILED DESCRIPTION

[0019] The accumulation of plastic waste, particularly from single-use packaging, poses a significant environmental challenge due to the non-biodegradable nature of most petroleum-based plastics. One area of significant interest is the development of biopolymers—materials derived from renewable sources that exhibit biodegradable properties. Polysaccharides, such as pullulan and chitosan, have garnered attention due to their natural abundance, biocompatibility, and potential for forming biodegradable materials. These biopolymers can be sourced from plants, algae, and microorganisms, making them sustainable alternatives to petroleum-based plastics.

[0020] While biopolymers like pullulan and chitosan have shown promise in terms of biodegradability and biocompatibility, their mechanical properties often fall short when compared to conventional plastics. As a result, research efforts have focused on enhancing the performance of biopolymer films through techniques such as crosslinking, which can improve their thermal stability, mechanical strength, and resistance to environmental factors. Crosslinking involves creating bonds between polymer chains, which increases the overall stability and durability of the material.

[0021] The biodegradable polysaccharide film disclosed herein addresses this problem by combining the natural biodegradability of pullulan and chitosan with enhanced mechanical strength and thermal stability achieved through crosslinking. The biodegradable polysaccharide film uses a 50:50 blend of pullulan and chitosan, which is plasticized with glycerol and fabricated into a film using a casting process. The biodegradable polysaccharide film is then subjected to crosslinking. For example, one ofAttorney Docket: 222204-2980 two crosslinking methods can be relied upon, such as vapor-phase glutaraldehyde crosslinking or ionic crosslinking with sodium TPP. Other crosslinkers and crosslinking methods can be relied upon.

[0022] The crosslinking process significantly improves the structural integrity of the biodegradable polysaccharide film, allowing the biodegradable polysaccharide film to maintain its performance over a wider range of conditions compared to unmodified biopolymer films. Specifically, glutaraldehyde crosslinking creates covalent bonds between polymer chains, resulting in films with increased stiffness, tensile strength, and resistance to thermal degradation. The sodium TPP crosslinking, while less rigid, enhances the biodegradability of the film and retains favorable mechanical properties, making it a suitable choice for applications requiring non-toxic materials.

[0023] The biodegradable polysaccharide films described herein help to solve the problem of balancing biodegradability with the necessary physical performance for packaging applications. The biodegradable polysaccharide films have been tested for mechanical strength, thermal stability, antibacterial resistance, swelling behavior in different water environments, and soil degradation. The films demonstrate suitability as viable alternatives to petroleum-based plastics. Unlike existing solutions, which often compromise either biodegradability or performance, the disclosed biodegradable polysaccharide films offer a comprehensive solution that provides both environmental sustainability and practical utility. The ability to tune the properties of the films through different crosslinking methods further enhances the adaptability of the films for various applications, including food and medical packaging. The polysaccharide films not only degrade in soil or dissolve in water over time, they also exhibit shelf-stability at ambient temperature, pressure, and humidity. Film blends with or without crosslinking have remained stable for over one year. No loss of mechanical properties or change in color have been detected after being stored.

[0024] Referring now to FIG. 1, shown is a block flow diagram of a process 100 used to manufacture a biodegradable polysaccharide film, with each block representing a distinct step in the production according to an example implementation. The process 100 is illustrated as a representative example and is not exhaustively illustrated. A number of additional steps or actions may be taken between or among the blocks shown in FIG. 1.Attorney Docket: 222204-2980 One or more of the steps shown in FIG. 1 can be omitted in some cases, as not all of the steps are needed, required, or essential in every circumstance. One or more of the steps shown in FIG. 1 can also be reordered or rearranged in some cases.

[0025] The process 100 starts with blending the core ingredients, followed by degassing to remove trapped air bubbles, casting the solution into a thin film, curing the film to solidify it, and finally crosslinking the film to enhance its properties. Two different crosslinking methods are depicted—glutaraldehyde vapor crosslinking and sodium TPP crosslinking—each providing unique advantages depending on the desired film characteristics. The use of other crosslinking approaches to obtain other characteristics is within the scope of the embodiments.

[0026] The process 100 begins with blending block 102. A number of constituent parts, including one or more polysaccharides and biopolymers, are blended to form a solution at the blending block 102. As one example, a combination of pullulan 104, chitosan 106, glycerol 108, and acetic acid 110 are combined to form an initial polysaccharide solution 112. The use of other polysaccharides and biopolymers, other than pullulan and chitosan, are within the scope of the embodiments.

[0027] Pullulan 104 is a naturally occurring polysaccharide produced by the fungus Aureobasidium pullulans. Pullulan 104 is composed of repeating units of maltotriose, which consist of three glucose molecules linked by Į-1,4 glycosidic bonds. Pullulan 104 is known for its high solubility in water, flexibility, and film-forming properties, making it a suitable material for use in biodegradable films. In the context of the embodiments described herein, pullulan 104 acts as a component in the formation of the polysaccharide solution, providing flexibility, biocompatibility, and aiding in the development of the biodegradable structure of the resulting films. Pullulan 104 also has excellent oxygen barrier properties that also make it suitable for use in packaging applications.

[0028] Chitosan 106 is a biopolymer derived from chitin, which is found in the exoskeletons of crustaceans like shrimp, crabs, and lobsters, as well as in the cell walls of fungi. Chitosan 106 is composed of repeating units of D-glucosamine and N-acetyl-D- glucosamine linked by ȕ-1,4-glycosidic bonds. Chitosan is known for its biocompatibility, biodegradability, and antimicrobial properties, making it a valuable material for biomedical, environmental, and packaging applications.Attorney Docket: 222204-2980

[0029] In the context of the embodiments, chitosan 106 is blended with pullulan 104 to form a biodegradable film. Chitosan 106 contributes mechanical strength, antibacterial properties, and enhances the overall structural integrity of the polysaccharide film. The cationic nature of chitosan also allows for effective crosslinking with agents like glutaraldehyde or sodium tripolyphosphate, improving the mechanical and thermal performance of the resulting films. Additionally, chitosan’s antimicrobial activity is particularly beneficial for packaging applications that require bacterial resistance.

[0030] Glycerol 108, also known as glycerin, is a simple polyol compound with the chemical formula C3H8O3. Glycerol 108 is a colorless, odorless, and viscous liquid that is widely used in various industries due to its humectant (moisture-retaining), plasticizing, and solvent properties. Glycerol is commonly sourced from plant and animal fats or as a byproduct of biodiesel production.

[0031] In the context of the embodiments, glycerol 108 is used as a plasticizer in the biodegradable polysaccharide films. The glycerol 108 can be relied upon to increase the flexibility and reduce the brittleness of the resulting films, by introducing intermolecular interactions that reduce rigidity. Glycerol can also help to prevent the resulting films from becoming too rigid or brittle, making the films more suitable for practical applications such as packaging. The addition of glycerol also enhances the processability and durability of the resulting films, ensuring that the films remain flexible without significantly compromising other mechanical properties.

[0032] Acetic acid 110, with the chemical formula CH3COOH, is a colorless liquid organic compound with a strong, pungent odor commonly associated with vinegar. Acetic acid is a weak acid, and it is widely used in chemical processes, food preservation, and manufacturing.

[0033] In the context of the biodegradable polysaccharide film production described herein, the acetic acid 110 is relied upon as a solvent to dissolve the chitosan 106. Chitosan is typically insoluble in water and most organic solvents, but it dissolves in acidic solutions such as acetic acid. In one example, the pullulan 104 and the chitosan 106 can be added to and blended in an acidic solution of acetic acid and distilled water, with a concentration of 1-3% of the acetic acid in the acidic solution. The acetic acid 110 provides an acidic environment that enables the blending of the chitosan 102 and pullulan 104 to form aAttorney Docket: 222204-2980 homogeneous polysaccharide solution 112. This acidic solution facilitates the integration of the film components during the blending stage, ensuring that the final film is uniform and well-formed.

[0034] Pullulan 104 and chitosan 106, the two primary biopolymers, are blended in equal parts in one example, although it is not necessary that the pullulan 104 and chitosan 106 are blended in equal parts in all cases. Pullulan 104 provides flexibility and biocompatibility, while chitosan 106 contributes mechanical strength and antibacterial properties. Both biopolymers are dissolved in acetic acid (e.g., a 2wt% acetic acid solution), which helps solubilize the chitosan 106. Glycerol 108 is added as a plasticizer, which imparts flexibility to the final film, preventing it from becoming brittle. The mixing may occur in a stainless steel mixing vessel equipped with a mechanical stirrer, and other suitable equipment can be used. The stirrer can operate at an adjustable speed, typically between 200 and 500 rpm, ensuring thorough blending. The output of the blending block 102 step is a homogeneous polysaccharide solution 112 with all components fully mixed and dissolved.

[0035] After blending, the polysaccharide solution 112 moves to a degassing process depicted at degassing block 114. The degassing process removes any air bubbles trapped during blending. The air bubbles could create voids or defects in the resulting films if not removed, compromising the structural integrity of the films. The polysaccharide solution 112 may be placed in a vacuum degassing chamber for a suitable period of time, where the pressure is reduced to around 100-200 mbar, allowing the air bubbles to escape. Alternatively, the polysaccharide solution 112 can be left to rest for 30 minutes to an hour in an open container to naturally release the bubbles. In some cases, ultrasonic degassing equipment may be used to accelerate the process. The output of the degassing block 114 is a degassed polysaccharide solution 116 free of air bubbles.

[0036] Once degassing is complete, the degassed polysaccharide solution 116 is transferred to a casting process depicted at casting block 118. During the casting process, the degassed polysaccharide solution 116 is spread onto a flat surface to form a thin layer of wet polysaccharide film 120. For instance, the degassed polysaccharide solution 116 can be poured onto a Teflon®-coated casting tray to prevent sticking. A casting knife (e.g., doctor blade) can be used to evenly spread the degassed polysaccharide solution 116 acrossAttorney Docket: 222204-2980 the tray, and the thickness of the wet polysaccharide film 120 can be precisely controlled by the casting knife using a micrometer or related tool in some cases. The thickness of the wet polysaccharide film 120, typically between 1 and 2 mm, influences the mechanical properties of the final film. The output of the casting block 118 is the wet polysaccharide film 120, which has an even thickness but has not yet been solidified. Other approaches to casting are within the scope of the embodiments, including approaches suitable for higher- volume manufacturing.

[0037] The wet polysaccharide film 120 is then subjected to a curing process depicted at curing block 121. During the curing process, the wet polysaccharide film 120 is dried to remove excess moisture, solidifying the film into its final form. The film can be placed, still on a flat surface such as a Teflon®-coated sheet, into a forced-air convection oven in one example, which may be preheated to an appropriate temperature to cure the wet polysaccharide film 120 (e.g., 50°C for a suitable time). Forced air circulates evenly, ensuring uniform drying. The wet polysaccharide film 120 remains in the oven for a time sufficient to fully solidify (e.g., approximately 3 hours). Alternatively, a vacuum oven can be used to provide a more controlled drying environment, although the curing time and temperature may need adjustment. The output of the curing block 121 is a cured polysaccharide film 122, which is now solid and ready for crosslinking.

[0038] The cured polysaccharide film 122 can be crosslinked in a variety of different ways depending on the desired properties of the resulting film, and two main approaches are described herein. The crosslinking process strengthens the cured polysaccharide film 122 by forming bonds between the polymer chains, either through covalent or ionic interactions.

[0039] The first example crosslinking method is represented by glutaraldehyde vapor crosslinking block 124. In this method, the cured polysaccharide film 122 is placed in a vapor-phase crosslinking chamber, where it is exposed to glutaraldehyde vapor 126. The glutaraldehyde vapor 126 reacts with the amine groups in the chitosan 106, forming covalent bonds that enhance the mechanical strength and thermal stability of the cured polysaccharide film 122. For instance, the cured polysaccharide film 122 can be suspended above a dish containing a 10% glutaraldehyde solution, and the vapor reacts with the cured polysaccharide film 122 over a time period of approximately 3 to 6 hours, depending onAttorney Docket: 222204-2980 the desired degree of crosslinking. As an alternative to the vapor-phase method, a fine mist of glutaraldehyde solution can be sprayed onto the cured polysaccharide film 122. The output of the glutaraldehyde vapor crosslinking block 124 is a biodegradable glutaraldehyde crosslinked polysaccharide film 128, which exhibits improved strength and thermal resistance.

[0040] The second crosslinking method is represented by sodium TPP crosslinking block 130. In this method, sodium TPP 132 is introduced into the degassed polysaccharide solution 116 before the casting process at casting block 118, allowing the sodium TPP 132 to interact with the chitosan 106 during the curing stage to form ionic crosslinks. Sodium TPP crosslinking enhances the biodegradability and provides structural integrity to the resulting films. The sodium TPP 132 crosslinks the chitosan 106 molecules ionically, improving the mechanical properties of the film. As an alternative to sodium TPP 132, other ionic crosslinkers such as genipin or sodium citrate can be used to achieve similar results. The output from this method is a biodegradable sodium TPP crosslinked polysaccharide film 134, which offers enhanced biodegradability along with good mechanical performance.

[0041] The final output from the process 100 is a fully formed, biodegradable polysaccharide film, either the biodegradable glutaraldehyde crosslinked polysaccharide film 128 or the biodegradable sodium TPP crosslinked polysaccharide film 134, that can be tailored for various applications depending on the selected crosslinking method. For example, although not separately illustrated in FIG. 1, the process can also include fabricating a package using the biodegradable polysaccharide films. Example packages including those for food, medical-related devices and components, consumer products, and other products in a range of different fields. The films are not limited to use for packaging, however, as the films can be relied upon for a range of different applications. These films can be used in packaging materials for medical, food, or other industries where biodegradability and sustainability are desirable. Additionally, the films can be subjected to further testing for mechanical properties, thermal stability, and antibacterial effectiveness.

[0042] Turning to other aspects, FIG. 2 shows an antibacterial leakage test setup 200, including filter paper 202, a test film 204, and a plastic cover sheet 206. The antibacterialAttorney Docket: 222204-2980 leakage test setup 200 is used to evaluate the integrity and antibacterial properties of polysaccharide films, such as the biodegradable glutaraldehyde crosslinked polysaccharide film 128, the biodegradable sodium TPP crosslinked polysaccharide film 134, or a non- crosslinked film as the test film 204. More particularly, the test evaluates the ability of the test film 204, both crosslinked and non-crosslinked, to prevent the penetration of bacteria, specifically Staphylococcus aureus, through the film material. FIG. 2 specifically illustrates the arrangement of the test components inside a sterile Petri dish.

[0043] The antibacterial leakage test setup 200 can be implemented to follow one or more standards, such as the International Organization for Standardization (ISO) 22196 standard titled “Measurement of antibacterial activity on plastics and other non-porous surfaces.” The ISO 22196 standard specifies a method for evaluating the antibacterial activity of plastics and other non-porous surfaces. The test involves applying a bacterial inoculum onto a test surface, incubating the test surface for a defined period, and then assessing the number of surviving bacteria to determine the effectiveness of the material in inhibiting bacterial growth. This standard is used to measure how well a material prevents the growth and proliferation of bacteria such as Staphylococcus aureus or Escherichia coli. The results are reported as a measure of antibacterial efficacy, which is particularly important for materials used in medical devices, food packaging, and other applications where bacterial contamination needs to be controlled.

[0044] The filter paper 202 is positioned at the base of the antibacterial leakage test setup 200. The filter paper 202 serves as a collection medium to detect any bacteria that might penetrate through the test film 204 during the test. The filter paper 202 can be sterilized, using an ultraviolet (UV) light or other suitable sterilization method. On top of the filter paper 202, the test film 204 (also sterilized), which is crosslinked with glutaraldehyde or sodium TPP or non-crosslinked, is placed using sterile forceps to avoid contamination.

[0045] An inoculum containing Staphylococcus aureus is mixed with Polyoxyethylene (80) sorbitan monooleate (e.g., Tween-80®) and is distributed evenly over the surface of the test film 204. The plastic cover sheet 206 (also sterilized) is then placed on top of the test film 204. The plastic cover sheet 206 spreads the inoculum evenly across the surfaceAttorney Docket: 222204-2980 of the test film 204, ensuring proper contact between the bacterial solution and the test film 204 for accurate testing of bacterial penetration.

[0046] The antibacterial leakage test setup 200 is left a sufficient time (e.g., approximately 3 hours) for the inoculum to interact with the test film 204 and for any potential bacterial leakage to occur. After this incubation period, the plastic cover sheet 206 and the test film 204 are carefully removed, and the filter paper 202 is transferred to a conical tube containing phosphate-buffered saline (PBS) with Polyoxyethylene (80) sorbitan monooleate (e.g., Tween-80®). The conical tube is then vortexed for a time (e.g., five seconds) to dislodge any bacteria that may have penetrated the test film 204 and adhered to the filter paper 202.

[0047] The wash fluid collected from the conical tube is transferred to tubes for further analysis. If no significant bacterial leakage is observed, the wash fluid is spread on bacterial culture plates to confirm the absence of bacterial penetration. In cases where bacterial leakage is detected, smaller volumes of the wash fluid are diluted and plated for bacterial colony counting.

[0048] After an overnight incubation (e.g., at 37°C), bacterial colonies are manually counted or analyzed using an automated colony counter. The concentration of Staphylococcus aureus colonies present in the wash fluid is used to determine the level of bacterial penetration through the test film 204.

[0049] Turning to FIG. 3, shown is a thermogravimetric analysis (TGA) graph 300 with TGA curves of several fabricated film samples, with a temperature range from room temperature up to 500 °C, illustrating how weight loss occurs over this range. The different lines in FIG.3 represent the four types of films tested: a 0-hour crosslinked (CL) film (302), a 3-hour CL film (304), a 6-hour CL film (306), and a sodium TPP crosslinked film (308). The TGA graph 300 highlights the initial weight loss that occurs due to moisture content being boiled off up to approximately 100 °C – 110 °C. This initial loss of mass can be interpreted as the moisture content loss. The weight percentage of the samples was evaluated at 110 °C, where all water would have evaporated. As shown by Table 1, all film samples experienced weight loss of at least 10%, indicating that all films retained some moisture. This should be accounted for when considering other forms of degradation, as water loss could lead to variations in the overall mass reduction process.Attorney Docket: 222204-2980Attorney Docket: 222204-2980Table 1

[0050] Following this, the weight percentage gradually decreases, indicating the decomposition of the polymer matrix of the films. The slopes and weight loss patterns shown in FIG. 3 aid in understanding the degradation behavior of the various crosslinked films. After the water evaporated, the first site of degradation was determined by analyzing the change in slope in the curves, and the average temperature values are provided in Table 2. The region / temperature at which this occurs can be associated with the decomposition of the plasticizer glycerol. Several studies performing TGA on pure / reagent-grade glycerol have demonstrated that initial mass loss and decomposition take place at temperatures between 125 °C and 150 °C. While there is no significant difference observed between the four averages in Table 2, the glutaraldehyde crosslinked films appear to show a slight delay in the onset of decomposition, which suggests that the crosslinking improved thermal stability to some extent. In contrast, the sodium TPP crosslinked films did not exhibit the same degree of thermal stability enhancement.Table 2

[0051] The final area of focus is a highlighted region 310 on the TGA graph 300 of FIG. 3. This extended phase of weight loss is most likely attributed to the degradation of both polysaccharides, pullulan, and chitosan. To gain a further understanding of how the two polysaccharides behave individually, TGA was performed on the raw materials forAttorney Docket: 222204-2980 reference, as shown in raw TGA graph 400 in FIG.4. The initial expectation was that there would be two distinct steps in sequence of mass loss—one for each polymer. Chitosan (402) is expected to begin to degrade sooner, at approximately 250 °C, while pullulan (404) degradation starts at around 295 °C. However, the overall mass loss for both polymers thermally overlaps significantly. Consequently, instead of observing two distinct and short declines in the curve, there is one larger region of descent.

[0052] The mechanical properties of the fabricated films were assessed using tensile testing to compare them with current plastic materials. Tensile testing involves applying a controlled force to a sample and measuring how it responds to that force by stretching until it breaks. The stress-strain behavior of each film type was recorded and graphed, providing insight into the material’s mechanical characteristics.

[0053] Turning to FIG.5, a stress-strain graph 500 is shown to display the mechanical properties of the different fabricated film samples under tensile testing. In the stress-strain graph 500, stress is plotted on the y-axis, measured in megapascals (MPa), and strain is plotted on the x-axis, represented as a percentage of elongation. The curves represent the tensile behavior of various film samples as a controlled rate of force is applied until the material reaches its breaking point.

[0054] In the stress-strain graph 500, curves representing a standard barrier film (502), a 0-hour crosslinked (CL) film (504), a 3-hour CL film (506), a 6-hour CL film (508), a sodium TPP crosslinked film (510), and, which is non-crosslinked, are shown. The shape of these curves indicates the material’s elastic region, the ultimate tensile strength, and the point at which the material fails.

[0055] As shown in Table 3, the elastic modulus values for each film were calculated based on the slope of the initial linear region of the stress-strain curves. It was found that crosslinking with both 6 hours of glutaraldehyde exposure and sodium TPP crosslinking enhanced the modulus of the polysaccharide films compared to the non-crosslinked film. This indicates that crosslinked films exhibit higher stiffness and greater resistance to elastic deformation, which can be attributed to efficient modification from the two crosslinking agents.

[0056] The non-crosslinked fabricated biopolymer films also demonstrated a higher elastic modulus compared to the standard barrier film. This suggests that the biopolymerAttorney Docket: 222204-2980 films could potentially perform at the same level as currently used plastics, if not better. While there is a noticeable dip in the modulus for the films crosslinked with 3 hours of glutaraldehyde, which showed the lowest value of 0.601 ± 0.364 MPa, statistical analysis revealed no significant difference between the elastic moduli across all five types of films.

[0057] However, the variability observed in the elastic modulus values, highlighted by the standard deviations in Table 3, suggests a wide range of measurements for each sample type. This variability indicates that a larger sample size may be necessary to reduce the range of distribution and provide a clearer understanding of the relationship between crosslinking and the elastic modulus of the pullulan-chitosan films.

[0058] Turning to FIG. 6, shown are graphs 600A, 600B with the calculated mean penetration of Staphylococcus aureus bacteria through different fabricated film samples, displaying the quantitative results of bacterial penetration tests. The graphs 600A, 600B help visualize the magnitude of bacterial colonies that successfully penetrated the films, offering a comparison between the various film types tested. This can be used to understand the balance between biodegradability and bacterial resistance, indicating how the film can prevent harmful bacteria from penetrating while still degrading over time. The films tested were non-crosslinked and crosslinked samples with both 3-hour and 6-hour exposure to glutaraldehyde.

[0059] In the antibacterial leakage testing setup 200 (shown in FIG. 2), a goal was to evaluate the effectiveness of the films as a barrier against bacterial penetration while maintaining their biodegradable properties. Staphylococcus aureus was chosen due to its relevance as a common bacterium found in the human respiratory tract and its widespread use in antimicrobial testing, including in the ISO 22196 standard.Attorney Docket: 222204-2980

[0060] Table 4 presents the quantitative data showing the bacterial colony counts from the plates for each film type. The results were determined based on a mock control experiment using 2 μL of inoculum to evaluate the bacterial concentration. It was calculated that for every 2 μL of inoculum, there were 50 colonies, which translates to 1 colony per 0.04 μL. Using this baseline, the penetration of bacteria through the different films was calculated and subsequently graphed as shown in FIG. 6.Table 4

[0061] The films tested include non-crosslinked samples, and samples crosslinked with glutaraldehyde for 3 hours and 6 hours. The results in FIG. 6 highlight the wide variation in bacterial penetration between the different film types, demonstrating the effectiveness of the crosslinked films in minimizing bacterial penetration compared to the non- crosslinked samples.

[0062] Swelling studies were conducted to evaluate the film samples’ ability to absorb water, as well as to assess any changes in the polymeric network and decomposition over time. Two water conditions were used: deionized (DI) water and laboratory-grade simulated sea salt (SS) water. These two conditions allow for a comparison of the film’s behavior in pure water and a more complex environment containing salts, which mimics the real-world conditions that the films might encounter during use or disposal.

[0063] Each film sample was subjected to a four-week swelling test. The initial weight of each sample was recorded before being submerged in water. At specific time points, the wet weight of the films was measured, followed by drying the films in a vacuum ovenAttorney Docket: 222204-2980 to determine their dry weight. Swelling was calculated as a percentage using the following formula: 100where Wwetis the wet weight and Wdriedis the dried weight of the film.

[0064] The average swelling percentages for each film type in both DI water and SS water were calculated and recorded in Table 5. The values were plotted across the four- week timeline, as shown in FIGs. 7A-7C.Table 5

[0065] FIGs. 7A-7C depict swelling percentage graphs 700A, 700B, 700C, respectively, and each of these graphs contains two lines: solid lines represent the swelling behavior in DI water, and dashed lines show the swelling behavior in SS water. The swelling percentage is calculated by measuring the increase in weight of the films after being immersed in water and compared over time. Error bars are included to account for standard deviations. The results indicate that all films experienced a higher swelling rate in DI water compared to SS water, which can be attributed to the different ionic content in the solutions.Attorney Docket: 222204-2980

[0066] In FIG. 7A, shown is a swelling percentage graph 700A of the swelling percentage of the 0-hour crosslinked (CL) film. The swelling percentage graph 700A shows an initial high swelling percentage in both DI water and SS water at the four-day time point, particularly in DI water, where the swelling percentage was significantly higher. This can be attributed to the hydrophilic nature of the non-crosslinked films. However, over time, the swelling percentage drastically declined due to the breakdown of the polymer chains, rendering the film incapable of absorbing water to the same degree. In SS water, the presence of salts further inhibited the swelling by creating more rigid interactions between the salt ions and the chitosan’s cationic backbone, reducing the available space for water absorption. Statistical analysis revealed significant differences between the water conditions, particularly at the four-day and fourteen-day time points.

[0067] In FIG. 7B, shown is a swelling percentage graph 700B for the sodium TPP crosslinked film. The initial swelling percentages were lower than the non-crosslinked films, as expected, due to the crosslinked polymeric network inhibiting water absorption. However, unlike the non-crosslinked films, the sodium TPP crosslinked films displayed an increase in swelling over time in both water conditions, with a slight preference for swelling in DI water. This phenomenon indicates that the crosslinking agent provided a level of structural integrity that prevented the polymer chains from breaking down as quickly, allowing the films to continue absorbing water while maintaining their form. At the fourteen-day time point, a significant difference in swelling was observed between the sodium TPP CL and the other film types, further confirming the impact of crosslinking on the swelling behavior.

[0068] In FIG.7C, shown is a swelling percentage graph 700C for the 6-hour CL film. These films also displayed lower swelling percentages initially compared to the non- crosslinked films, but their ability to absorb water increased slightly over time. Interestingly, the films showed greater variability in the later time points, particularly after the fourteenth day, which can be attributed to the embrittlement of the films. Qualitative observations during the experiment noted that many 6 hr CL samples turned yellow-brown and became increasingly brittle, often breaking into smaller pieces during handling. This fragility, caused by glutaraldehyde crosslinking, created challenges in measuring full samples, leading to a higher variability in the swelling data.Attorney Docket: 222204-2980

[0069] In general, the crosslinked films (both sodium TPP CL and 6 hr CL) exhibited more stable swelling behavior compared to the non-crosslinked films. The presence of salt in the SS water environment consistently reduced swelling in all film types, as the salt ions restricted water uptake by creating rigidity in the polymer matrix. Statistical analysis, including a two-way ANOVA test, revealed significant differences between water types and crosslinking conditions, particularly at earlier time points.

[0070] FIGS. 8A-8C illustrate the measured weight of the various film samples over a 27-day period, plotted against time in days. These figures aid in understanding the biodegradability of the films under soil conditions, which is a unique attribute of the disclosed, differentiating them from traditional plastics.

[0071] In particular, FIG.8A shows the degradation behavior of the 0-hour crosslinked (0 hr CL) film samples 802-812. The weight of each sample decreases gradually over the course of the 27-day study. Six samples were used (802, 804, 806, 808, 810, 812), and the data points clearly depict a consistent loss of mass over time. The slope and intercept data, as calculated and shown in Table 6, confirm that the non- crosslinked films degrade at a slower rate compared to crosslinked films, with a projected full degradation occurring after 54.4 ± 14.6 days, as outlined in Table 7.Attorney Docket: 222204-2980Table 6Table 7

[0072] FIG. 8B presents the data for sodium TPP crosslinked (TPP CL) film samples 814-824. The sodium TPP crosslinked films, like the non-crosslinked films, also show a decrease in mass over time, but the rate of degradation is slower. The use of the sodiumAttorney Docket: 222204-2980 TPP crosslinker resulted in an extended degradation period compared to non-crosslinked films, with a projected full degradation occurring after 60.6 ± 13.5 days. The curves in this figure show slightly less steep declines compared to the 0-hour samples, indicating improved structural integrity due to the crosslinking.

[0073] FIG. 8C illustrates the degradation behavior of 6-hour glutaraldehyde crosslinked (6 hr CL) film samples 826-832. The weight of these samples decreases at the fastest rate among the three types of films, indicating that glutaraldehyde crosslinking may result in faster biodegradation in soil. The slope of the curves is steeper than those of the other films, with a projected full degradation occurring after 45.2 ± 2.06 days, as outlined in Table 7. The data suggest that while glutaraldehyde crosslinking strengthens the films initially, it also promotes faster breakdown under soil conditions, potentially making these films suitable for applications where quicker degradation is desired.

[0074] Overall, the results plotted in FIGS. 8A-8C demonstrate the impact of crosslinking and film composition on the biodegradability of the films. The choice of crosslinking agent—whether glutaraldehyde or sodium TPP—allows for the customization of the film’s degradation rate, depending on the application’s requirements. Whether faster or slower degradation is needed can be controlled by selecting the appropriate crosslinker and modifying the exposure conditions.

[0075] Turning to FIG. 9, shown is a method 900 for producing a biodegradable polysaccharide film according to an example implementation. It should be understood that the operations of the methods disclosed herein are not necessarily presented in any particular order and that performance of some or all of the operations in an alternative order(s) is possible and is contemplated. The operations have been presented in the demonstrated order for ease of description and illustration. Operations may be added, omitted, and / or performed simultaneously, without departing from the scope of the appended claims.

[0076] The method 900 will be described with additional reference to FIG. 1. The method begins at block 902, where the pullulan 104, chitosan 106, glycerol 108, and acetic acid 110, are blended to form a homogeneous polysaccharide solution 112. The blending process ensures the uniform dissolution and mixing of all ingredients, resulting in aAttorney Docket: 222204-2980 smooth, particle-free solution. Following this, the polysaccharide solution 112 undergoes a degassing step at block 904.

[0077] At block 904, the polysaccharide solution 112 is degassed to remove any trapped air bubbles that could lead to defects in the final film. Degassing can be carried out by allowing the polysaccharide solution 112 to rest or by placing it in a vacuum chamber where the pressure is reduced to 100-200 mbar to facilitate the release of air bubbles. After degassing, the degassed polysaccharide solution 116 is ready for casting, as shown at block 906.

[0078] At block 906, the degassed polysaccharide solution 116 is poured onto a flat casting surface, such as a Teflon®-coated tray. A casting knife, or doctor blade, is used to evenly spread the solution into a thin layer, with the thickness controlled by adjusting the casting knife height. Alternatively, for smaller-scale production, a spin coater may be used to ensure even distribution of the degassed polysaccharide solution 116 across the surface. The wet polysaccharide film 120 is then prepared for curing at block 908.

[0079] At block 908, the wet polysaccharide film 120 undergoes curing, such as in a preheated oven at 50°C for approximately 3 hours. The curing process evaporates any remaining moisture from wet polysaccharide film 120, solidifying the structure and creating a cured polysaccharide film 122 that is durable and can be further processed. Once curing is complete, the cured polysaccharide film 122 may undergo one of two crosslinking methods to modify its mechanical properties, represented by blocks 910 and 912.

[0080] At block 910, glutaraldehyde vapor crosslinking is performed. The cured polysaccharide film 122 is placed in a vapor-phase crosslinking chamber, where it is exposed to glutaraldehyde vapor 126 for 3 to 6 hours. The glutaraldehyde vapor 126 reacts with the amine groups in chitosan 106, forming covalent crosslinks that enhance the film’s mechanical strength and thermal stability. Alternatively, at block 912, the film can be crosslinked using sodium TPP 132.

[0081] At block 912, the sodium TPP crosslinking method involves mixing sodium TPP 132 directly into the degassed polysaccharide solution 116 before casting. The sodium TPP 132 creates ionic bonds between chitosan 106 molecules, improving the film’s structural integrity and biodegradability. The crosslinking method selected, eitherAttorney Docket: 222204-2980 glutaraldehyde vapor 126 or sodium TPP 132, allows for customization of the film’s properties based on the intended application.

[0082] At block 914, the completed biodegradable polysaccharide film 128, 134 emerges as the final product. The crosslinking method used allows for variations in the mechanical properties and degradation rate, making the film suitable for a range of applications, including medical or food packaging, where eco-friendly materials are required.

[0083] A novel pullulan-chitosan composite film has been described, offering a biodegradable alternative to conventional plastics. This film is created using two distinct crosslinking methods—glutaraldehyde and sodium TPP—which enhance the mechanical, thermal, antibacterial, water swelling, and soil degradation properties of the material. The formulation and process allow for reproducible film production, with over 200 samples successfully created and tested during experimentation. These films demonstrate comparable, and in some cases superior, performance to current plastic films, making them a viable solution for packaging and other applications where environmental sustainability is critical.

[0084] This biodegradable film addresses the urgent need to reduce plastic waste and its environmental impact. By combining pullulan and chitosan, two naturally occurring polysaccharides, the film leverages renewable resources to provide a sustainable solution. Its ability to be crosslinked enables customization for various applications, whether short- term products requiring quick degradation or long-lasting materials with enhanced durability. The scalability of the production process further ensures that this film can be manufactured at an industrial level, making it practical and economically feasible for use in industries such as food packaging, medical supplies, and other environmentally focused sectors.

[0085] The features, structures, or characteristics described above may be combined in one or more implementations in any suitable manner, and the features discussed in the various implementations are interchangeable, if possible. In the following description, numerous specific details are provided in order to fully understand the embodiments of the present disclosure. However, a person skilled in the art will appreciate that the technical solution of the present disclosure may be practiced without one or more of the specificAttorney Docket: 222204-2980 details, or other methods, components, materials, and the like may be employed. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the present disclosure.

[0086] In this specification, the terms such as “a,” “an,” “the,” and “said” are used to indicate the presence of one or more elements and components. The terms “comprise,” “include,” “have,” “contain,” and their variants are used to be open ended, and are meant to include additional elements, components, etc., in addition to the listed elements, components, etc. unless otherwise specified in the appended claims.

[0087] The terms “first,” “second,” etc. are used only as labels, rather than a limitation for a number of the objects. It is understood that if multiple components are shown, the components may be referred to as a “first” component, a “second” component, and so forth, to the extent applicable.

[0088] The above-described implementations of the present disclosure are merely possible examples set forth for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the above-described implementations without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure and protected by the following claims.

Claims

Attorney Docket: 222204-2980 CLAIMS What is claimed is:

1. A biodegradable polysaccharide film, comprising: a blend of pullulan and chitosan; and a crosslinking agent selected from glutaraldehyde or sodium tripolyphosphate.

2. The biodegradable polysaccharide film of claim 1, wherein the crosslinking agent is glutaraldehyde applied in vapor phase for 3 to 6 hours.

3. The biodegradable polysaccharide film of claim 1, wherein the crosslinking agent is sodium tripolyphosphate added during a film fabrication process.

4. The biodegradable polysaccharide film of any of claims 1-3, wherein the biodegradable polysaccharide film has a thickness between 0.1 millimeter (mm) and 0.5 mm.

5. The biodegradable polysaccharide film of any of claims 1-4, wherein the film is used as a packaging material for medical devices.

6. The biodegradable polysaccharide film of any of claims 1-5, wherein the pullulan and the chitosan are blended in an acidic solution of acetic acid with a concentration of 1-3% of the acetic acid in the acidic solution.

7. The biodegradable polysaccharide film of any of claims 1-6, further comprising glycerol as a plasticizer in a concentration of 1-10% by weight of the biodegradable polysaccharide film.

8. The biodegradable polysaccharide film of any of claims 1-7, wherein the biodegradable polysaccharide film exhibits antibacterial properties.Attorney Docket: 222204-2980 9. The biodegradable polysaccharide film of any of claims 1-8, wherein the biodegradable polysaccharide film degrades in soil within 60 days.

10. The biodegradable polysaccharide film of any of claims 1-9, wherein the biodegradable polysaccharide film swells by less than 50% in saline water over a period of 7 days.

11. A method, comprising: blending pullulan, chitosan, glycerol, and an acetic acid solution to form a polysaccharide solution; degassing the polysaccharide solution to remove air bubbles; casting the degassed polysaccharide solution into a film; curing the film to solidify the film; and crosslinking the film with using either glutaraldehyde vapor or sodium tripolyphosphate during the blending step to form a biodegradable polysaccharide film.

12. The method of claim 11, wherein the acetic acid solution has a concentration of 1-3% of the acetic acid in the acidic solution.

13. The method of any of claims 11 or 12, wherein the glycerol is added in a concentration of 1-10% by weight of the biodegradable polysaccharide film.

14. The method of any of claims 11-13, wherein the casting produces a film thickness between 0.1 millimeter (mm) and 0.5 mm.

15. The method of any of claims 11-14, wherein the degassing is performed using a vacuum chamber to remove air bubbles from the polysaccharide solution.

16. The method of any of claims 11-15, wherein the crosslinking is performed with the TPP to enhance biodegradability and mechanical strength of the biodegradable polysaccharide film.Attorney Docket: 222204-2980 17. The method of any of claims 11-16, further comprising fabricating a package using the biodegradable polysaccharide film.

18. The method of claim 17, wherein the package is a food package.

19. The method of claim 17, wherein the package is a medical package.

20. The method of any of claims 11-19, further comprising incorporating the biodegradable polysaccharide film into a biodegradable application.

21. A method, comprising: blending pullulan and chitosan to form a polysaccharide solution; casting the polysaccharide solution into a film; curing the film to solidify the film; and crosslinking the film to form a crosslinked biodegradable polysaccharide film.

22. The method of claim 21, wherein: the blending comprises blending the pullulan, the chitosan, glycerol, and an acetic acid solution to form the polysaccharide solution; and the acetic acid solution has a concentration of 1-3% of the acetic acid in the acidic solution.

23. The method of any of claims 21 or 22, wherein the casting produces a film thickness between 0.1 mm and 0.5 mm.

24. The method of any of claims 21-23, wherein the degassing is performed using a vacuum chamber to remove air bubbles from the polysaccharide solution.

25. The method of any of claims 21-24, wherein the crosslinking enhances biodegradability and mechanical strength of the biodegradable polysaccharide film.Attorney Docket: 222204-2980 26. The method of any of claims 21-25, further comprising fabricating a package using the biodegradable polysaccharide film.

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