Evaluation of arabinoxylan isolated from sorghum bran, biomass, and bagasse for film formation

Accepted Manuscript Title: Evaluation of Arabinoxylan Isolated from Sorghum Bran, Biomass, and Bagasse for Film Formation Authors: Ryan J. Stoklosa, Renee J. Latona, Laetitia M. Bonnaillie, Madhav P. Yadav PII: DOI: Reference:

S0144-8617(19)30267-X https://doi.org/10.1016/j.carbpol.2019.03.018 CARP 14687

To appear in: Received date: Revised date: Accepted date:

27 September 2018 1 March 2019 1 March 2019

Please cite this article as: Stoklosa RJ, Latona RJ, Bonnaillie LM, Yadav MP, Evaluation of Arabinoxylan Isolated from Sorghum Bran, Biomass, and Bagasse for Film Formation, Carbohydrate Polymers (2019), https://doi.org/10.1016/j.carbpol.2019.03.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Evaluation of Arabinoxylan Isolated from Sorghum Bran, Biomass, and Bagasse for Film Formation Ryan J. Stoklosa1*, Renee J. Latona1, Laetitia M. Bonnaillie2 and Madhav P. Yadav1 1

Sustainable Biofuels and Co-Products Research Unit, Eastern Regional Research Center,

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USDA, ARS, 600 East Mermaid Lane, Wyndmoor, PA, 19038, United States

Dairy and Functional Foods Research Unit, Eastern Regional Research Center, USDA, ARS,

600 East Mermaid Lane, Wyndmoor, PA, 19038, United States †Mention

of trade names or commercial products in this publication is solely for providing

specific information and does not imply recommendation or endorsement by the U.S.

author: Ryan J. Stoklosa, [email protected]; Phone: +1-215-233-6634

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*Corresponding

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Department of Agriculture. ARS is an equal opportunity provider and employer.

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Highlights

Film properties of arabinoxylan isolated from three sorghum fractions were revealed

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Moisture sensitivity at high relative humidity occurred for all arabinoxylan films

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Plasticizer addition decreased tensile strength of sorghum bran arabinoxylan films

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Increasing temperature reduced storage and loss moduli for sorghum bran films

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Greater plasticizer content reduced the onset temperature for material transition

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ABSTRACT Arabinoxylans (AX) are potential agricultural co-products for material applications. Sorghum has seen increased production as a bioenergy crop for biofuel and co-product generation. AX from three sorghum fractions (bran, bagasse, and biomass) were isolated to study film formation. 1

All three AX fractions exhibited high moisture sensitivity. Sorghum biomass AX produced low water vapor permeability compared to sorghum bran or sorghum bagasse AX films. Glycerol addition to sorghum bran AX films reduced tensile strength from 34.8 to 16.0 MPa at 0% and 10% (w/w) glycerol, respectively; reduced the storage and loss moduli during dynamic mechanical analyses at 50% relative humidity (RH) and decreased the rubber-to-plastic material

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transition temperature at 50% RH, from 78.1°C to 38.4°C at 0 and 10% (w/w) glycerol,

respectively. Sorghum bran AX, while sensitive to water absorption at high RH, produced

favorable strength performance compared to AX from other cereal grains indicating potential utilization as a renewable material.

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Keywords: Arabinoxylan, Sorghum, Film formation, Mechanical properties

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1. INTRODUCTION

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Agricultural feedstocks can provide a renewable source of biopolymers for biodegradable material applications. While non-biodegradable materials produced from petroleum still

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dominate many applications, these materials are starting to be phased out by a number of

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countries due to environmental concerns (Siracusa, Rocculi, Romani, & Rosa, 2008). Polysaccharides that originate from plant biomass can be processed to make biodegradable plastics such as polylactic acid or polyhydroxybutyrate that can also reduce the amount of fossil

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derived carbon dioxide (Gross & Kalra, 2002). Governing agencies in many countries are willing to promote these products as long as materials made from biodegradable polymers can provide

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equal or more superior performance compared to non-biodegradable polymers from petroleum sources (Iwata, 2015). Polysaccharides sourced from agricultural feedstocks, mostly cellulose

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and starch, are well known for their use in applications ranging from thickening agents in food (Saha & Bhattacharya, 2010), chemical encapsulation (Glenn et al., 2010), and nanofiber production (Konwarh, Karak, & Misra, 2013). Both cellulose and starch can readily be recovered in polymeric form that is necessary for most applications. Hemicelluloses, which are amorphous polysaccharides present in agricultural feedstocks, are more difficult to obtain in true polymeric

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form as opposed to cellulose and starch but can be utilized as a renewable and biodegradable source of polymers for food and material applications. Hemicelluloses are a diverse and important structural polysaccharide component in both grains and lignified plant cell walls. In general, hemicellulose polysaccharides are β-(1,4)-linked

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pyranosyl units containing a structural conformation that can develop hydrogen bonding with cellulose chains to form cross-linkages that impart hydrophobic properties to the cell wall matrix (Caffall & Mohnen, 2009; Fry, 1986). Individual hemicellulose polysaccharide composition can vary greatly with monocot or dicot grouping. Monocots, which include major cereal grains and

grasses, have different hemicellulose distribution between the grain and lignified plant stalk. The endosperm and bran layer of the cereal grain contain L-arabino-D-xylans (AX) with a

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xylopyranosyl backbone and α-arabinofuranosyl residues, either monosubstituted or disubstituted at the C-2 or C-3 position on the xylopyranosyl monomer (Ebringerová, 2005). In contrast,

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monocot plant stalks typically contain (D-glucurono)-L-arabino-D-xylans (GAX) with the same

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polysaccharide backbone to AX, but possess additional side-chain substitutions with glucuronic

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acid attached at O-2 and O-3 positions and are slightly acetylated (Ebringerová, 2005; Vogel, 2008). Moreover, the arabinosyl unit in GAX within grass cell walls is known to crosslink with hydroxycinnamates (i.e. ferulic and p-coumaric acid) via ester or ether linkages which also have

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the ability to link to lignin (Hartley, Morrison III, Himmelsbach, & Borneman, 1990; Vogel,

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2008). Some gymnosperm species, such as spruce, have also been identified to contain (Larabino)-D-glucurono-D-xylan (AGX) in their plant cell wall (Escalante et al., 2012).

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Alternatively, dicots contain a different hemicellulose profile that is comprised of D-xylo-Dglucan (XG) and, more predominantly, varying ratios of D-galacto-D-mannans (GaM), D-gluco-

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D-mannans (GM), and (D-galacto)-D-gluco-D-mannans (GGM) depending on species type and location in lignified tissue or seed (McCleary, Matheson, & Small, 1976; Timell, 1967). The conformation of xylans in grains or plant cell walls allows them to be extracted by alkaline

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solutions with minimal degradation to the polymer chain (Puls, 1997). The stability of xylans in an alkaline environment provides an option for solubility-based separations where the recovered polysaccharide can be utilized for co-product generation. Direct utilization of hemicellulose can be accomplished for food or materials applications. Xylans as a food additive can increase the total and soluble fiber content of foods 3

for general health benefits by initiating prebiotic effects as a non-digestible oligosaccharide (M. Izydorczyk & Dexter, 2008; Moure, Gullón, Domínguez, & Parajó, 2006; Rosa-Sibakov et al., 2016). For material applications, hemicellulose polysaccharides are known film formers that can be utilized in packaging. The addition of polyols as plasticizers aids in improving the polysaccharide film’s performance properties. AX isolated from oat spelt produced flexible films

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at high plasticizer loadings (40% w/w) with sorbitol providing a better barrier to water vapor

than glycerol and improved film softening (Mikkonen et al., 2009). More recent work has shown improved film formation by utilizing internal plasticization where a xylan polysaccharide

structure was chemically modified by adding hydroxypropyl units as a side chain unit, but the addition of an external plasticizer was still needed to improve the film barrier properties

(Mikkonen et al., 2015). Moreover, AX from corn fiber blended with GGM could produce a

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stable film, but they exhibited sensitivity to high relative humidity (RH) whereas GGM blended

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with konjac GM improved tensile strength properties compared to pure GGM films (Mikkonen et al., 2008). A unique AX aspect is that a lower arabinose content, which indicates less

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substitution on the xylan backbone, induces a loss of plasticizing effect and has been correlated

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to water binding capacity (Sternemalm, Höije, & Gatenholm, 2008). Further improvements in AX hydrophobicity is typically focused on derivatizing hydroxyl group sites along the

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polysaccharide chain (Farhat et al., 2016; Hansen & Plackett, 2008). Prior research on AX

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isolation for food or film applications has focused on the major grains such as rye, wheat, barley, or corn (Gröndahl, Gustafsson, & Gatenholm, 2006; Heikkinen, Mikkonen, Pirkkalainen, et al., 2013; Sárossy, Tenkanen, Pitkänen, Bjerre, & Plackett, 2013; Yadav, Johnston, & Hicks, 2009).

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AX isolated from sorghum has mostly been studied based on food related properties (Nandini & Salimath, 2001); however, AX isolated from sorghum has not been evaluated in depth for film

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formation properties even though sorghum AX has an unusually high degree of substitution with

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arabinofuranosyl units (Pauly & Keegstra, 2008). This research investigated AX isolated from three sorghum sources: sorghum bran,

sorghum biomass, and sweet sorghum bagasse after juice extraction. Each AX fraction was used to make films with and without plasticizer. AX from corn bran was also utilized to make films for property comparisons. The film properties tested included resistance to moisture and mechanical strength. By understanding these baseline properties AX from sorghum can be directed towards more focused material applications 4

2. MATERIALS AND METHODS 2.1 Sorghum Feedstock Sorghum grains were obtained from Bob’s Red Mill Natural Foods, Inc. (Milwaukie, OR, USA)., sorghum bagasse was provided by Delta BioRenewables (Memphis, TN, USA) while

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sorghum biomass was provided by USDA-ARS, SPA, Cropping Systems Research Laboratory (Lubbock, TX, USA). 2.2 Isolation of Arabinoxylans (AX)

Sorghum bran was produced by decortication of sorghum grains (Hums, Moreau, Yadav, Powell, & Simon, 2018) and dried in an oven overnight at 60°C to remove moisture. The

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sorghum bagasse and sorghum biomass were dried in an oven overnight at 60°C to remove

moisture and ground to a 20-mesh particle size using a Wiley mill. The ground materials were

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further dried at 60°C overnight to remove any remaining moisture. The sorghum bran and the

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ground sorghum biomass and bagasse were extracted with hexane to remove oil (Moreau,

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Powell, & Hicks, 1996). The sorghum bran contains a high percent of starch, which was hydrolyzed to maltodextrins by treating the bran with heat stable Termamyl α-amylase from

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Novozymes (Franklinton, NC, USA) at pH 6.8 for 1 hour. A loading of 0.15 g α-amylase per gram of de-oiled sorghum fraction was utilized. The hydrolyzed maltodextrins were removed by

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washing the bran with distilled water.

Arabinoxylans (AX) from de-oiled and de-starched sorghum bran and de-oiled sorghum

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bagasse and sorghum biomass were isolated by an alkaline extraction procedure with some modifications (Yadav & Hicks, 2018). In brief, each sorghum material was boiled at 85oC with

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mechanical stirring in the presence of heat stable Termamyl α-amylase at the same enzyme loading as listed above and held at pH 6.8 for 1 hour to hydrolyze any remaining starch to avoid

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its contamination with AX. The pH of this suspension was raised to 11.5 by adding 50% (w/v) NaOH with boiling and stirring continuing for additional 30 minutes. During the reaction, pH was maintained at 11.5 by adding 50% (w/v) NaOH and the reaction volume was maintained by adding water as needed to compensate water loss due to evaporation. The hot reaction mixture was immediately sheared using a high speed Polytron (PT 10/35 GT) equipped with 12 mm probe (Kinematica Inc., Bohemia, NY, USA) at 10,000 RPM for 30 minutes and cooled to room 5

temperature. The solid residue was separated from the reaction mixture by centrifugation at 14,000g for 10 minutes and discarded. The supernatant was collected in a beaker and its pH was adjusted to 4.0-4.5 by adding concentrated HCl to precipitate acid insoluble Hemicellulose A, which was separated by centrifugation at 10,000g for 30 minutes and discarded. The supernatant was collected in a beaker and two volumes of ethanol were gradually added to it with stirring to

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precipitate the high molecular weight arabinoxylans (also called Hemicellulose B or Bio-fiber gum). The precipitated AX was collected by filtration and dried in the vacuum oven at 50°C overnight. The composition and the functional properties of these AX fractions have been

reported in a previous publication (Qiu, Yadav, & Yin, 2017). Corn bran arabinoxylan (cAX)

was supplied by AgriFiber Solutions LLC (Mundelein, IL, USA). The composition of cAX has

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been reported in a prior study (Yadav & Hicks, 2018).

2.3.1 Film Preparation

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2.3 AX Film Preparation and Analysis

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All AX films were prepared at 20 g/L concentration. In brief, the required volume of water was added to an Erlenmeyer flask and placed on a hot plate with magnetic stirring

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capability. The AX polysaccharide was slowly added to the flask. The solution was heated up to 90°C and held at that temperature for 15 minutes with mixing. The solution was then cooled to

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room temperature followed by centrifugation in an Allegra X-14 (Beckman Coulter, Brea, CA, USA) centrifuge at 3724g to remove a minimal amount of insoluble material. The bulk solution was separated into sample volumes as needed. Next, glycerol (Gly) plasticizer (Sigma-Aldrich,

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St. Louis, MO, USA) was added to each sample at 0%, 5%, or 10% (w/w) loading based on the polymer mass in solution. Films were cast by pouring 30 mL of the AX solution into aluminum pans with 63 mm inner diameter. For tensile strength testing, larger films were prepared at a similar thickness by casting 45 mL of AX solution into aluminum pans with 121 mm inner diameter. Films were dried overnight at 55°C. The films were then stored in a desiccator chamber until testing. 6

2.3.2 AX Composition Analysis The monomeric sugar composition of each AX fraction was determined according to previous methodologies (Sluiter et al., 2006; Stoklosa & Hodge, 2015). In brief, each AX fraction was solubilized by the same process as described in the previous paragraph but at a

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concentration of 10 g/L. Once the solution cooled, a 10 mL aliquot was placed in an Ace Glass Inc. (Vineland, NJ, USA) pressure tube. To each tube 0.378 mL of a 72% (w/w) sulfuric acid solution was mixed into the AX solution to make a 4% (w/w) sulfuric acid solution. Sample tubes were sealed with Teflon screw caps containing an outer O-ring. The samples were

autoclaved at 121°C for one hour for their hydrolysis to monomeric sugars. After autoclaving the samples were cooled to room temperature. The liquid phase was analyzed for sugar monomers

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by HPLC. An aliquot of the hydrolyzed solution was filtered through a 0.2 µm PALL Life

Sciences (Westborough, MA, USA) Acrodisc syringe filter into a HPLC vial. An Agilent (Santa

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Clara, CA, USA) 1260 Infinity II HPLC equipped with a refractive index (RI) detector was

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utilized for analysis. The injected samples were separated by a Bio-Rad (Hercules, CA, USA)

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Aminex HPX-87P column coupled in series with a de-ashing guard column. The mobile phase was ultrapure water from an EMD Millipore (Billerica, MA, USA) Simplicity filtration system. Calibration standards containing glucose, xylose, arabinose, and galactose were analyzed

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simultaneously with the samples. A set of sugar recovery standards for individual sugar

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monomers were prepared and autoclaved with samples to determine sugar degradation during hydrolysis. The composition analysis for each AX fraction was performed in duplicate.

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2.3.3 AX Molecular Weight Analysis The molecular weight for each AX fraction was determined by size exclusion

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chromatography (SEC). The samples were prepared and analyzed by SEC according to previous methods (Yadav, Fishman, Chau, Johnston, & Hicks, 2007).

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2.4 AX Film Moisture Properties 2.4.1 Water Vapor Permeability (WVP) AX films were tested for water vapor permeability (WVP) using Gardco Perm Cups (Paul N. Gardner Co., Inc., Pompano Beach, FL, USA). The cups possessed an open cross section of 56.4 mm diameter with an open area of 25 cm2. Each sample cup was loaded with 4 7

mL water. The depth of the sample cup was 10.0 mm. The volume of water utilized filled the cup approximately halfway which left around a 5.0 mm air gap between the water and film sample. Each film sample was cut to the approximate dimensions of the outside clamp ring and placed over the cup. The film thickness was measured with a B.C. Ames (Framingham, MA, USA) digital micrometer. The sample was held in place with a Teflon and metal gasket, and secured in

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place with a screw cap. The samples were placed in a Caron Environmental Chamber (Marietta, OH, USA) at 22°C and 50% relative humidity (RH). The cups were gravimetrically analyzed at 0, 1, 2, 4, 6, 8, and 24 hours to determine the water loss with respect to time. The water vapor transmission rate (WVTR) was calculated by a linear regression of the slope of the mass loss versus time divided by the test cup open sample area as stated previously (Mikkonen et al., 2009). The sample’s underside water vapor partial pressure was corrected according to a

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previously described method (Gennadios, Weller, & Gooding, 1994). The calculated WVP was

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determined by multiplying the film thickness by the WVTR and dividing by the water vapor partial pressure difference between the two sides of the film. Samples were analyzed in

2.4.2 Vapor Sorption Analysis

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duplicate.

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Additional water absorption properties were measured with a TA Instruments (New Castle, DE, USA) Q5000 SA vapor sorption analyzer. Each film sample was first measured for

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thickness then a hole-puncher was utilized to cut out three circular samples that were evenly spaced in a quartz weigh pan. Prior to placement the weigh pan was tared by the instrument. The

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samples were dried for 600 minutes at 40°C and 0% RH, then analyzed at 25°C with 240-minute step increases in RH starting at 0% RH and ending at 90% RH, with 10% RH intervals. Samples

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were measured for initial rates of moisture-sorption at the beginning of each RH step and watercontent after equilibration.

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2.5 AX Film Mechanical Properties 2.5.1 Tensile Strength Testing Film tensile strength was determined with a MTS (Eden Prairie, MN, USA) Insight 5 Electromechanical instrument. Each film sample was cut into five strips with 10 mm width by 100 mm length. Prior to testing samples were conditioned for at least 40 hours in an 8

environmental chamber at 23°C and 50% RH. After conditioning each strip’s thickness was measured. The testing occurred in a room that was temperature controlled at 25°C and 50% RH. A 100 N load cell was utilized with a 50 mm gauge length and strain rate at 5 mm per minute. For each sample 5-10 replicate specimens were tested.

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2.5.2 Dynamic Mechanical Analysis with Humidity Control (DMA-RH) Film mechanical properties were also determined with a TA Instruments Q800 Dynamic Mechanical Analyzer equipped with a humidity-controlled chamber (DMA-RH) (New Castle,

DE, USA). Samples were cut to 5 mm width and 25 mm and mounted into a clamp with 15 mm gauge length. After mounting, the sample was inspected for proper vertical alignment with no

slack. To determine the measurement parameters, frequency and strain sweeps were conducted

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on film samples held at 25°C and 50% RH. From this analysis, a test frequency of 4 Hz and a

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strain of 0.1% was utilized for all test samples. Test samples were equilibrated at 10°C for 240 minutes at 50% RH. A temperature ramp rate of 0.2°C per minute was utilized to record changes

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in clamp displacement, storage modulus (E’), and loss modulus (E’’) up to 90°C under 50% RH.

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If samples did not break prior to reaching 90°C, the specimen was held at 90°C for 10 minutes before cooling.

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2.7 Data Analysis

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The reported results for tensile strength and WVP testing were analyzed for statistical significance using Microsoft Excel (Redmond, WA, USA). Sample mean differences for AX

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composition and tensile strength measurements were determined by a paired two sample t-test at a probability level of 0.05. WVP results were analyzed by both a two-way ANOVA and a paired

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two sample t-test at a probability level of 0.05. 3. RESULTS AND DISCUSSION

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3.1 AX Composition Arabinoxylans (AX) are the primary hemicellulose component found in cereal bran and

the cell wall surrounding seed endosperm (Ebringerová, Hromadkova, & Heinze, 2005). Film properties such as response to moisture can be intrinsically related to polysaccharide composition (Phan The, Debeaufort, Voilley, & Luu, 2009). The sorghum fraction AX and corn bran

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arabinoxylan (cAX) utilized to prepare films in this study were subjected to an acid hydrolysis procedure to determine the monomeric sugar composition. The sugar monomer mass fraction for each AX fraction is reported in Table 1. Table 1. AX Fraction Monomeric Sugar Composition (Relative Mass %) Glucose

Xylose a

41.31 ± 0.08

Galactose

a,b

Sorghum Bran

7.75 ± 0.08

Sorghum Biomass

5.99 ± 0.87

71.53 ± 0.72a

Sorghum Bagasse

15.93 ± 2.44b

54.8 ± 1.14

cAX

0.58 ± 0.02a,b

62.23 ± 0.08b

Arabinose

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AX Fraction

2.29 ± 0.01

a,b

48.66 ± 0.01a,b,c

6.49 ± 0.10a

16.86 ± 0.05b,e

7.53 ± 0.81

21.75 ± 0.48a,d

6.46 ± 0.11b

30.73 ± 0.01c,d,e

Values in the same column with superscripts are statistically different (p < 0.05)

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All four AX samples a majority monomer composition consisting of xylose and arabinose. The xylose and arabinose in AX polysaccharide are the main sugars that determines polymer

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branching, which can impact both film applications and performance. An AX polysaccharide

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with more branching, that is, a higher arabinose-to-xylose ratio (A/X), should allow greater water

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uptake compared to a less branched polysaccharide (Höije, Sternemalm, Heikkinen, Tenkanen, & Gatenholm, 2008). The composition for the sorghum AX and cAX fractions reported here are higher in both xylose and arabinose monomeric constituents than reported for wheat bran (Maes

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& Delcour, 2002) and rye bran (Falck et al., 2014), while having similar composition to barley

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bran (M. Izydorczyk & Dexter, 2008). The high glucose content of sorghum bagasse AX can be attributed to residual non-structural sugars that remain after sweet sorghum juice removal. The

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predominant content of arabinose and xylose in the sorghum AX fractions and cAX is expected to contribute to the overall mechanical function and moisture barrier properties to the prepared

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films (Stepan, Höije, Schols, De Waard, & Gatenholm, 2012; Ying et al., 2013). 3.2 AX Film Moisture Properties

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3.2.1 Water Vapor Permeability (WVP) and Molecular Weight Films prepared from AX were reported to be hygroscopic, even without plasticizer

addition due to the presence of arabinose side chain units (Sternemalm et al., 2008). The WVP for each film sample was tested to determine sorghum AX susceptibility to moisture permeability. Table 2 shows the corrected WVP and weight average molecular weight for each film sample. There is a clear increase in WVP with the addition of Gly as a plasticizer. This is an 10

expected result since the RH gradient between the two sides of the film was high and Gly acted as a conduit for water transfer between the film underside (exposed to higher RH) and the film topside (exposed to lower RH). A two-way ANOVA found significant differences between the WVP with respect to film type, plasticizer loading, and the interaction between film type and plasticizer loading. A paired two-sample t-test was also utilized to determine statistical

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differences between film type at each plasticizer loading. The statistical differences among film types are indicated by superscripted letters in Table 1. At 0% (w/w) Gly the WVP for the

sorghum bran AX film was found not to be statistically different from the other AX film fraction. Opposite this result, the cAX film was found to be statistically different from both sorghum biomass and bagasse at 0% (w/w) Gly. At 10% (w/w) Gly only the sorghum biomass and

bagasse AX films proved to have statistically different WVP values. Although glycerol addition

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slightly increased the WVP for each film the plasticizer appears to have a greater impact on film

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performance above 50% RH conditions as will be discussed later.

10% (w/w) Gly

Corrected WVP

Corrected WVP

Weight Average

(g*mm*day^-1*m^-2*kPa^-1)

(g*mm*day^-1*m^-2*kPa^-1)

Molecular Weight (kDa)

26.16 ± 6.52

32.19 ± 0.96

620

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43.04 ± 9.35a

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52.13 ± 9.53

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Sorghum Bran

0% (w/w) Gly

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AX Film

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Table 2. Water Vapor Permeability (WVP) and Molecular Weight for AX Film Samples

Sorghum Biomass

19.86 ± 0.25

Sorghum Bagasse

19.74 ± 1.82b

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cAX

32.42 ± 0.51

23.50 ± 4.96

a,b

Values in the same column with superscripts are statistically different (p < 0.05)

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The overall distribution of arabinose groups over the xylan backbone can have an impact

on film morphology as well (Heikkinen, Mikkonen, Pirkkalainen, et al., 2013). In Figure 1A the reported WVP for each film sample is plotted as a function of the A/X ratio. As shown in Figure 1B the A/X ratio is greatest for sorghum bran, followed by cAX, sorghum bagasse, and sorghum biomass possessing the lowest overall A/X ratio. When Gly was added as a plasticizer, the largest increase in WVP came about in films from sorghum bagasse and cAX as opposed to 11

sorghum bran. Although sorghum bran contained a higher A/X ratio, sorghum bagasse and cAX had a higher galactose content as listed in Table 1 when compared to sorghum bran. This higher content of galactose could be an additional contribution to higher WVP since previous film studies on cAX and galactoglucomannan (GGM) showed high sensitivity to relative humidity (Mikkonen et al., 2008). Trends are apparent in Figure 1A for the AX films WVP at both 0%

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(w/w) and 10% (w/w) Gly loadings. Although the WVP increases for AX films at 10% (w/w) Gly no statistical difference was identified by a paired two-sample t-test performed on the

sorghum AX or cAX films. The increase in the WVP for the sorghum bagasse AX film is an

interesting result explained by two properties. First, AX polysaccharides with lower A/X ratios, i.e. more unsubstituted polysaccharide chains, are known to cause more interactions and eventual aggregation between unsubstituted polysaccharides (Andrewartha, Phillips, & Stone, 1979;

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Sternemalm et al., 2008). These unsubstituted interactions have the potential to be disrupted at

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high plasticizer loadings that create more free volume within the AX film (P. Zhang & Whistler, 2004). The sorghum bagasse AX film with 10% (w/w) Gly appears to have reached the critical

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plasticizer threshold that allowed more unbound water to pass through.

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Figure 1. AX Film WVP (A) and Molecular Weight (B) As a Function of the A/X Ratio

Secondly, the composition of the sorghum bagasse AX fraction was not pure polysaccharide. It is reasonable to suspect that sorghum bagasse AX also contains phenolic compounds derived from lignin which can associate with this form and other forms of hemicellulose (Kato, Azuma, & Koshuima, 1987; Takahashi & Koshijima, 1988; Yuan, Sun, Xu, & Sun, 2011). Conversely, sorghum bran and sorghum biomass AX are expected to contain less lignin content. The lignin 13

content of barley husk AX was previously shown to impact both the mechanical and water properties of the AX when made into a film (Höije, Gröndahl, Tømmeraas, & Gatenholm, 2005). While the trends identified in Figure 1A show how A/X ratio impacts WVP, the AX films are expected to show more deviant water properties at RH levels above 50%. The WVP reported in Table 2 are higher than previously reported WVP values for AX films. A different source of

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cAX plasticized with 15% Gly was reported to have a WVP of 15.3 at a RH gradient of 62% (Péroval, Debeaufort, Despré, & Voilley, 2002). Rye bran AX with no plasticization was reported to have a WVP of 7.7 (RH gradient 52%), while wheat endosperm AX without

plasticization had a WVP of 7.2-7.9 (RH gradient 54%) (Mikkonen & Tenkanen, 2012; Sárossy et al., 2013). Further improvements to WVP for hemicellulose films can be accomplished by

direct chemical modification of the polysaccharide known as internal plasticization. Films made

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from birch xylan exhibited improved resistance to WVP when an external plasticizer was

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combined with internal plasticization of the polysaccharide (Mikkonen et al., 2015).

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The molecular weight for each AX fraction was also determined to better understand the

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AX structure. Table 2 reports the weight average molecular weight for the AX fractions while Figure 1B shows an almost logarithmic trend appears as molecular weight is plotted as a function of A/X ratio. As the A/X ratio increases to allow more polysaccharide branching the molecular

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weight for the biopolymer increases accordingly. The AX fractions reported molecular weight

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falls within ranges previously reported for wheat bran AX obtained by graded ethanol precipitation (Schooneveld-Bergmans, Beldman, & Voragen, 1999) and rye AX (Knudsen &

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Lærke, 2010). Higher molecular weight AX with increased branching is known to improve water-holding capacity when made into a hydrogel (M. S. Izydorczyk & Biliaderis, 1992). As

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sorghum bran AX has the most branching, as indicated by both A/X ratio and molecular weight, it is reasonable to suspect this fraction can hold the most water within its structure. This aspect of the sorghum bran AX fraction is shown indirectly by it’s WVP performance. As listed in Table 2

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and shown in Figure 1A the sorghum bran AX film at 10% (w/w) Gly has a lower WVP than compared to sorghum bagasse or cAX. The higher WVP for sorghum bagasse and cAX indicate that more water can pass through the film structure. The lower WVP for sorghum bran not only indicates that ultimately less water passes through the film, but also the potential exists that more water ends up being bound within the film structure. This occurs through water getting into the film pore structure and ending up bound through hydrogen bonding to the AX itself, or the added 14

glycerol disrupting AX interactions in the film to allow greater affinity for the AX to bind water (Ghasemlou, Khodaiyan, & Oromiehie, 2011; Jouki, Khazaei, Ghasemlou, & HadiNezhad, 2013). Improving the hydrophobicity of sorghum bran AX films should be the focus of future research by taking advantage of strategies such as combining external plasticizers with internal plasticization on the AX polysaccharide chain to improve barrier performance (Mikkonen et al.,

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2015), or utilizing fatty acid esters with β-glucan to provide a more uniform film structure (Ali, Bijalwan, Basu, Kesarwani, & Mazumder, 2017). 3.1.2 Vapor Sorption Properties of AX Films

While WVP can indicate how well a film can allow or block water from passing through a film layer, a more in-depth analysis of a film’s response to water can be determined by

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subjecting a film sample to a range of humidity levels. Figure 2 presents the vapor sorption

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properties for AX films showing moisture sensitivity at increasing RH.

Figure 2. Vapor Sorption Isotherms for AX Films at 25°C Showing (A,C) Film Moisture Content and (B,D) Moisture-Sorption Rates 15

Figures 2A and 2C show the moisture-sorption profiles for sorghum bran AX films at different plasticizer loadings and sorghum bagasse, biomass, and cAX plasticized at 10% (w/w) glycerol, respectively. From 20% RH to 50% RH the AX films show a relatively low amount of water absorption, only reaching about a 10% mass increase at 50% RH. However, increasing the RH beyond 50% shows a noticeable increase in water absorption almost approaching an exponential

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increase. At 90% RH the sorghum biomass and sorghum bagasse show the highest amount of water absorbed reaching beyond a 50% mass increase. One trend to note is that with higher

glycerol loading, sorghum bran AX films are more sensitive to moisture absorption as shown in Figure 2A. This moisture sensitivity is even more apparent in Figures 2B and 2D showing initial moisture-sorption rates for each film type. In Figure 2B a much faster rate of water absorption is achieved with a sorghum bran AX film plasticized with 10% (w/w) Gly compared to sorghum

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bran AX film plasticized with 0% (w/w) or 5% (w/w) Gly. In Figure 2D the film moisture

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absorption rates do not drastically increase until 40-50% RH is reached. At that RH level both sorghum bagasse and biomass plasticized with 10% (w/w) Gly have a faster water absorption

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rate than compared to cAX at the same plasticizer loading. The rates of water absorption are

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specifically influenced based on plasticizer amount as confirmed in Figure 2B. Both AX and Gly are highly hydrophilic, but the specific rate increase in water absorption can be attributed to the

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Gly since the diffusion rate of water can be increased through the film network having greater

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polymer chain mobility (Bertuzzi, Vidaurre, Armada, & Gottifredi, 2007). Xylan structure is also known to possess favorable hydrophilic qualities due to a natural tendency toward hexagonal packing structures (Almond & Sheehan, 2003). Moreover, the sorghum bran AX film with 10%

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(w/w) Gly exhibited the fastest moisture absorption rate as shown in Figure 2B. As stated above the sorghum bran AX fraction has the highest A/X ratio at 0.84. Lower A/X ratios can lessen the

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effect of moisture sensitivity at high relative humidity (Stevanic et al., 2011). Interestingly, Figure 2C shows that sorghum biomass and sorghum bagasse films at 10% (w/w) Gly absorbed

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as much water at 80% RH when compared sorghum bran in Figure 2A. This is a unique result as both sorghum biomass and sorghum bagasse AX have a lower A/X ratio than sorghum bran AX as shown in Figure 1. In this case the addition of Gly to AX films prepared from sorghum biomass and bagasse appears to be the primary driver for water absorption at high RH. One way this can occur is through replacing hydrogen bonding between polysaccharide polymers with hydrogen bonding between the polymer and water itself (Gröndahl, Eriksson, & Gatenholm, 16

2004). Overall, each isolated AX fraction showed sensitivity to moisture absorption especially at high (over 50%) RH. However, at or below 50% RH the films exhibit less sensitivity to water. This condition helps to identify a workable range for not only film applications, but also film mechanical property testing.

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3.2 AX Film Mechanical Properties 3.2.1 Tensile Strength

The mechanical properties of films prepared from AX isolated from sorghum and corn brans are displayed in Figure 3. Figure 3A shows that the tensile strength for cAX remains

relatively constant with an increase in Gly content. Alternatively, the sorghum bran AX film

plasticized with 10% (w/w) Gly displays a notable drop in tensile strength. This drop in tensile at

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increased plasticizer loading has been documented previously and can be attributed to both

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plasticizer and additional water taking up more free volume between the polysaccharide matrix

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(Banker, 1966; Mikkonen et al., 2009). Although cAX film did not exhibit the same trend, the difference in film strength might be attributed to the A/X substitution ratio (Heikkinen,

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Mikkonen, Pirkkalainen, et al., 2013). cAX tensile strength was found to have similar values to both oat spelt AX and corn hull AX plasticized with the same loading of glycerol (Mikkonen et

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al., 2009; P. Zhang & Whistler, 2004). Figure 3B shows that increasing Gly loading improves the

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elongation at break for both sorghum bran AX and cAX films. An improvement in film flexibility is usually associated with a higher content of plasticizer as has been shown previously

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where the percent elongation at break doubled with an increase in plasticizer loading to 40% (w/w) Gly for oat spelt AX films (Mikkonen et al., 2009). However, the sorghum bran film sample at 10% (w/w) Gly loading exhibited a large standard deviation for the elongation at

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break. This indicates that the elongation at break for sorghum bran AX at 10% (w/w) Gly could be a similar value to both the 0% (w/w) and 5% (w/w) Gly loadings. The large deviations can

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also be attributed to the overall small percentage values for elongation at break. Although the sorghum bran AX films were strong, they were still very brittle even with

10% (w/w) Gly added as a plasticizer. Figure 3C presents Young’s modulus for sorghum bran AX and cAX films. While Young’s modulus remains consistent for cAX, sorghum bran AX exhibits a sizeable decrease in the Young’s modulus. Figure 3 is an indication that the mechanical properties of the cAX and sorghum bran AX films can be dependent on 17

polysaccharide composition and structure. Polysaccharide film properties are known to be functions not only of overall composition, but also degree of substitution (DS) by side chain moieties (Heikkinen, Mikkonen, Koivisto, et al., 2013; Mikkonen, Pitkänen, et al., 2012). The process of film drying could also impact overall strength properties as xylan chains might orient differently during the final film formation stage depending on the amount of branching in the

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polysaccharide (Heikkinen, Mikkonen, Pirkkalainen, et al., 2013). Sorghum bran AX was the

only film that could be properly tested for mechanical properties as both sorghum bagasse and sorghum biomass AX produced brittle films that could not be properly cut for tensile strength

testing. The sorghum bran AX film at 10% (w/w) Gly was somewhat flexible, while the sorghum bagasse AX film was completely brittle and would easily crack under very slight strain.

Moreover, a difference in film coloration existed and which can be attributed to the AX source in

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the sorghum plant. While AX from bran originates around the cereal grain, the bagasse AX is

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obtained from the lignified stalk portion of the plant. Although some lignin can be found in cereal grain components such as wheat bran (Y. Zhang et al., 2011), the AX isolated from

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sorghum bagasse is expected to be more abundant in lignin and could have an impact on film

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forming performance as documented previously in barley husk AX film formation (Höije et al., 2005). A pared two-sample t-test indicated that only tensile strength at 10% (w/w) Gly and

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and sorghum bran AX films.

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Young’s modulus at 0% (w/w) and 10% (w/w) Gly were significantly different between cAX

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Figure 3. (A) Tensile Strength, (B) Elongation at Break, and (C) Young’s Modulus for Films Prepared from AX With and Without Plasticization; Lower Case Letters Indicate Statistical

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Difference (p < 0.05)

3.2.2 Dynamic Mechanical Analysis with Humidity Control (DMA-RH) of AX Films DMA-RH of AX films was performed utilizing a temperature and humidity-controlled ramp program to determine how the films are impacted under different conditions. Multiple property relationships can be determined from DMA-RH analysis. Figure 4 represents a typical film sample showing film characteristics that were identified. 19

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Figure 4. Typical DMA-RH Curve Plotting Clamp Displacement (Left Y-Axis) and Tan δ

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(Right Y-Axis) as a Function of Temperature at 50% RH for Sorghum Bran AX Film with 5%

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(w/w) Gly

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It is important to note that while each sample was tested at 50% RH, the absolute humidity (H(T)) increased exponentially inside the sample test chamber as temperature increased. By

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combining heat and moisture, the plasticized film sample has increased molecular activity causing swelling which increases mobility of the AX molecules via the absorption of water

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molecules throughout the network (Bonnaillie & Tomasula, 2015). Therefore, the combined effects of T and H(T) triggered different material transitions within the films’ network that can

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be clearly observed in Figure 4. The green curve with filled circles represents the clamp displacement recorded during film analysis. The solid black curve is the parameter tan δ, which

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is a ratio of the dissipated energy to the energy stored per sample deformation cycle. Figure 4 displays how the material transition regions for each film were determined. First, T1 is termed the rubber-to-plastic material transition temperature. It is at this point where a critical amount of

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water is absorbed by the film. Most likely this absorbed water starts to disrupt the network of hydrogen bonding between the polysaccharide and glycerol components which increases the polysaccharide network mobility (Aulin, Gällstedt, & Lindström, 2010). Second, T2 is termed the film displacement melting point temperature. Although the film has not yet broken at this temperature, the film polysaccharide network is compromised enough where a sharp decline in the film strength properties occur (Gröndahl et al., 2004). Lastly, Tm is termed the true melting 20

point of the film. At this temperature some of the main intermolecular bonds within the polysaccharide network break apart and the film becomes liquid-like and ruptures. These identifiable material transition parameters by DMA-RH analysis can be applied to determine the effect of glycerol loading for sorghum bran AX films.

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Figure 5 displays DMA-RH data for sorghum bran AX films at different levels of

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plasticization.

Figure 5. Sorghum Bran AX Films DMA-RH Profiles at 50% RH Showing (A) Clamp Displacement and Tan δ and (B) Storage (E’) and Loss (E’’) Moduli as a Function of Temperature 21

The plateau region on Figure 5A correspond to the rubber material region for the AX films. At this temperature the material softens enough to experience a rapid loss in storage modulus which has been shown to previously occur for other xylan fractions (Gröndahl et al., 2004; Mikkonen et al., 2009). At 0% and 5% (w/w) Gly loading the rubber plateau extends all the way to 60°C before clamp displacement accelerates. Gly loadings above 5% (w/w) have a much shorter

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rubber plateau that only reaches about 20°C. This is a clear indication that a greater amount of

Gly for plasticization can uptake water more favorably at lower temperatures and influence the polysaccharide network mobility (Karbowiak, Debeaufort, Champion, & Voilley, 2006). This

occurs by the plasticizer reducing intermolecular polymer chain interactions which increases the association between water and hydroxyl groups on the polysaccharide (Escalante et al., 2012). Upon reaching the first secondary material transition, T1, a sudden, linear increase occurs in

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clamp displacement and tan δ, which corresponds to the material transitioning to the plastic

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region, with non-reversible stretching of the films. Figure 5A demonstrates clearly that with increasing Gly loading the T1 transition for each film sample moves to a lower onset

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temperature. Figure 6 displays the material transition temperatures as a function of Gly loading.

Figure 6. Material Transition Temperature for Sorghum Bran AX Films as a Function of Gly Loading

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The transition at 0% and 5% (w/w) Gly are both above 70°C, while at 10% (w/w) Gly the transition occurs at 38.4°C. The combined effects of Gly and greater H(T) are the parameters most responsible for this material transition (Bertan, Tanada-Palmu, Siani, & Grosso, 2005). Sorghum bran AX films at Gly loadings lower than 5% (w/w) glycerol can maintain both shape and strength as a rubber-like material beyond ambient temperatures. However, Gly loadings

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above 5% (w/w) produce a transition to a plastic-like material at ambient conditions that can

hinder the films mechanical performance (Lourdin, Bizot, & Colonna, 1997). Similar trends for T2 and Tm are also apparent in Figure 6. Higher Gly loadings allow film shrinkage to occur at

lower temperatures and this eventually leads to a lower temperature for film breakage. Although film plasticization is usually necessary to produce flexible films, a critical plasticization amount

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should exist to impart the necessary flexibility without sacrificing film strength performance.

Next, the peak in clamp displacement in Figure 5A indicates partial melting of the films

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at temperature T2, when the network became sufficiently plasticized by T and H(T) for the

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surface-tension forces to dominate and cause the sample to shrink (Bonnaillie & Tomasula,

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2015). With a higher Gly loading this shrinkage at T2, like the rubber-to-plastic transition at T1, occurs at an earlier temperature for the sorghum bran AX film. This peak was not detected for the film sample with 0% (w/w) Gly indicating that the polysaccharide network had not reached a

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sufficient level of plasticization. Prior research on DMA-RH of AX films is limited as most

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DMA research on polysaccharide films is performed to more or less identify glass transition temperatures of the polymer (Laaksonen, Kuuva, Jouppila, & Roos, 2002). AX from corn cob

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was shown to have a decrease in storage modulus starting at 25% RH (Egüés et al., 2014). Glucuronoxylan from aspen produced a rapid storage modulus loss not until 85% RH

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(Dammström, Salmén, & Gatenholm, 2005). Even with increasing plasticization the sorghum bran AX films did not decrease in storage modulus until after 50°C. While the 50% RH condition was maintained, the absolute humidity within the chamber increased as documented

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previously to higher than 50% RH (Bonnaillie & Tomasula, 2015). The final parameter of interest is the true melting point, Tm. This temperature is best

identified in Figure 5B that shows the storage modulus (E’; filled circles) and loss modulus (E’’; solid line) as a function of temperature. Eventually, the film shrinkage led to film breakage which can be identified at the intersection of E’ and E’’ in Figure 5B. As stated previously at this 23

temperature, Tm, some of the sorghum bran AX film intermolecular bonds break and polysaccharide network mobility becomes more liquid-like and irreversible (Vaikousi, Biliaderis, & Izydorczyk, 2004). Figure 5B also clearly shows that at 0% (w/w) Gly loading the E’ and E’’ curves do not intersect. This further confirms that the film sample with no plasticizer did not break, but also indicates sorghum bran AX films can withstand high temperatures with low or no

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plasticizer added. Once again by increasing Gly loading in the sorghum bran AX film, the Tm

and film breakage occur at lower temperatures. The DMA-RH analysis on the sorghum bran AX films has shown how Gly loading and increasing temperature at constant RH can soften the

polysaccharide film network through combined heat and moisture-sorption (Mikkonen, Heikkilä, Willför, & Tenkanen, 2012). This has the effect of increasing the polysaccharide network mobility and dissolving or breaking chemical bonds within the film network (P. Zhang &

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Whistler, 2004).

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As shown in Figure 5B the glycerol content can also influence the general strength

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properties of the films. Figure 7 presents E’ and E’’ at 50% RH for sorghum bran AX films with

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and without plasticizer at several temperature readings. Figure 7A shows that E’ decreases linearly with temperature and glycerol loading. The increase in the chamber water content by an increase in H(T) can influence the film by decreasing the strength properties (Cho & Rhee,

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2002). Figure 7B shows E’’ which appears to decrease linearly except for a slight spike in E’’ at

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7.5% (w/w) Gly loading. This slight increase in E’’ might be attributed to the age of the film. The film with 7.5% (w/w) Gly loading was about a week and half old when tested on the DMA.

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All other films were 2-3 weeks in age at the time of testing. Film age is known to have an impact on the film physical properties such as strength with oat spelt AX showing an increase in tensile

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strength between films 1 week old and 2 months old (Heikkinen, Mikkonen, Koivisto, et al., 2013). Additionally, replicates for the sorghum bran AX film plasticized with 10% (w/w) Gly were tested. The error bars shown in Figure 4 for sorghum bran AX at 10% (w/w) Gly loading

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represent the standard deviation of the replicates. While some variance exists each replicate, the samples behaved similarly when analyzed by the DMA-RH temperature ramp program. Replicate samples for each film condition were not analyzed due to sample analysis taking around a whole day to complete a sample sequence. Sorghum biomass AX and cAX film samples were analyzed by DMA for comparison and showed a similar decrease (data not shown) in both E’ and E’’ with increase in temperature. 24

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Figure 7. (A) Storage Modulus and (B) Loss Modulus for Sorghum Bran AX Films at 50% RH as a Function of Gly Loading at Different Temperatures

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4. CONCLUSIONS AX isolated from different sorghum fractions were characterized with respect to their film forming properties. AX films from sorghum bran, bagasse, and biomass all showed sensitivity to moisture absorptions and increasing relative humidity (RH). Sorghum biomass AX

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had the lowest WVP while sorghum bagasse AX and cAX had the highest. The weight average molecular weight of the AX fractions was shown to be a function of the A/X ratio. Sorghum bran AX exhibited greater water absorption with increasing amounts of Gly as a plasticizer. This property of the sorghum bran AX correlated to the higher A/X substitution ratio and high

molecular weight allowing for greater water uptake. The AX film mechanical properties were

dependent both on overall polysaccharide composition and Gly loading. Sorghum bagasse and

sorghum biomass AX films were unable to be tested for mechanical strength properties as they

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were too brittle even with plasticization. Tensile strength and Young’s modulus decreased for

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sorghum bran AX films with increasing plasticization while corn bran AX showed relatively

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consistent strength parameters even with plasticization. DMA-RH tests performed on sorghum

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bran AX films exhibited decreases in the film’s storage modulus and loss modulus with increasing temperature and Gly loading. Earlier onset material property transitions at specific temperatures, such as rubber-to-plastic material change, were identified with increasing Gly

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loading since higher Gly content influenced greater water absorption by the material. The

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sorghum AX film moisture-sorption properties indicate that an additional hydrophobic functionality is needed to improve moisture barrier properties. However, films prepared from

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sorghum bran AX produced similar mechanical properties when compared to AX fractions from other cereal grains. Although the sorghum AX fractions from bagasse or biomass might be better

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utilized with higher Gly loadings to improve film brittleness, the goal of utilizing less plasticizer (e.g. around 10% w/w) to decrease processing costs should be encouraged especially if acceptable strength properties can be achieved at lower plasticizer loadings as shown by the

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sorghum bran AX film strength characteristics. Additionally, internal plasticization by chemical modification or derivatization with hydrophobic components to the polysaccharide structure should be investigated to determine an improvement in film performance; primarily for improved resistance to water permeability. Future applications of sorghum bran AX should be dedicated to packaging applications where strength is needed. Additional future research should focus on in depth structural characterization of each sorghum AX fraction to determine degrees of 26

substitution for certain chemical moieties to better determine underlying conditions for optimal film performance. ACKNOWLEDGEMENTS The authors would like to thank Stefanie Simon for preparing sorghum bran, bagasse, and

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biomass AX fractions; John Mulherin for set up and running film vapor sorption analysis; and

Dr. Cheng-Kung Liu and Nick Latona for coordinating MTS usage and testing samples on the

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instrument, respectively.

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