This work was supported by the Swedish governmental strategic research program Trees and Crops for the Future (TC4F), VINNOVA, and the EC FP7 project ICON. Hannah Rasel is gratefully acknowledged for performing most of the experiments in the previous paper (Reference 5).

NOTE: Crambe seeds (cultivar Galactica) were supplied by the Plant Research International, Wageningen, Netherlands. Oil was extracted from the seeds by the method of Appelqvist13. Both the crambe meal and the wheat gluten were stored at -18 &#176;C until further use.
1. Dough Preparation
<ol>
	<li>Sieving crambe
	<ol>
		<li>Sieve the crambe meal with a round, fine-mesh stainless steel kitchen sieve (pore size: ~1.5 mm, 14 mesh), to remove large fiber fractions and uncrushed seeds. Store the sieved meal at -18 &#176;C to prevent material ageing.</li>
	</ol>
	</li>
	<li>Milling crambe
	<ol>
		<li>To reduce the particle size and make the material more homogeneous, mill the sieved crambe meal in a rotary ball mill.</li>
		<li>Mill 250 g of crambe meal each time in a 7 L jar with 21-25 mm diameter ceramic balls using a 53 rpm jar revolution rate and a milling time of 24 hr.</li>
	</ol>
	</li>
	<li>Conditioning
	<ol>
		<li>Before further processing, condition all the ball-milled crambe meal and wheat gluten powder in open jars for a minimum of 48 hr at 23 &#176;C and a relative humidity of 50% in a climate-controlled room.</li>
	</ol>
	</li>
	<li>Mixing the components
	<ol>
		<li>Grind the urea powder (stored in a closed beaker at ambient conditions) to fine particles with a mortar and pestle.</li>
		<li>Blend urea and glycerol (25.5 g of glycerol and 15 g of urea per 100 g of the final mixture).
		<ol>
			<li>Heat the glycerol to 65 &#176;C in a glass flask in an oil bath and add the urea powder slowly.</li>
			<li>Stir the mixture with a magnetic stirrer at 65 &#176;C until the urea powder is completely dissolved.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Mixing the crambe and wheat gluten
	<ol>
		<li>Blend the crambe meal powder and wheat gluten powder in a kitchen mixing machine for 5 min. For example, for a 60/40 (w/w) crambe/wheat gluten ratio, use 35.7 g of crambe meal and 23.8 g of wheat gluten per 100 g of the final mixture.</li>
	</ol>
	</li>
	<li>Mixing glycerol/urea with crambe/wheat gluten
	<ol>
		<li>Slowly add the glycerol/urea mixture to the crambe/wheat gluten blend in the kitchen mixing machine while stirring the mixture. Continue mixing for approximately 2 min, until a homogeneous dough is obtained. Prepare 500 g of the mixture each time.</li>
		<li>For the material with 60/40 (w/w) crambe and wheat gluten, use the following relative contents of the respective constituents: 35.7 g of crambe meal, 23.8 g of wheat gluten, 25.5 g of glycerol, and 15 g of urea (per 100 g). For the other two material combinations (i.e., 70/30 and 80/20), change only the crambe and wheat gluten contents. Keep the glycerol and urea contents the same as in the 60/40 combination.</li>
	</ol>
	</li>
</ol>
2. Film Extrusion
<ol>
	<li>Low-temperature profile
	<ol>
		<li>Perform the film extrusion in a twin-screw extruder. Set zones 1-10 (each 80 mm-long) along the extruder barrel at a low temperature profile (subsequently called &#34;low-T profile&#34;), as follows: 75-75-75-80-80-80-80-85-85-85 &#176;C. This prevents the wheat gluten from crosslinking in the barrel.</li>
		<li>Use a flat sheet die (45 mm x 0.7 mm) to extrude the films.</li>
		<li>Choose a screw speed between 30 rpm and 200 rpm and record the die pressure.</li>
		<li>Feed the dough manually through the hopper with the help of a wooden pusher to support the material flow towards the screws.</li>
		<li>At the die, pick up the extrudate with a conveyor belt operating at a speed of 2.0 m/min. Place air ventilation cooling (fans) along the belt.</li>
		<li>Run different die temperatures (105 &#176;C-(105 &#176;C), 110 &#176;C-(110 &#176;C), 125 &#176;C-(115 &#176;C), 130 &#176;C-(120 &#176;C), and 140 &#176;C-(125 &#176;C)) to select the conditions that give the smoothest extrudate with a minimum amount of voids. 
		NOTE: The values in the brackets correspond to the temperature in zone 11, next to the die. It is adjusted to reach the target temperature in the die.</li>
		<li>After extrusion, store the extrudates in sealed polyethylene bags until further processing or analysis in order to prevent ageing and atmospheric water absorption.</li>
	</ol>
	</li>
	<li>Film extrusion using the high-temperature profile
	<ol>
		<li>Extrude films as described in section 2.1, but use a high-temperature profile (subsequently called &#34;high-T profile&#34;), as follows: 85-85-85-100-100-100-110-110-120-120-120 &#176;C for zones 1 to 11 of the extruder.</li>
		<li>Use die temperatures of 125 &#176;C and 130 &#176;C.</li>
	</ol>
	</li>
	<li>Film extrusion after pelletization
	<ol>
		<li>In order to get pellets, extrude the material as continuous strands in the extruder using a two-strand die.
		<ol>
			<li>Use the low-T profile for the extruder barrel, as described above, and a 60-rpm screw rotational speed.</li>
			<li>Use different die-(zone 11) temperatures (130 &#176;C-(125&#176;C), 125 &#176;C-(115 &#176;C), 105 &#176;C-(100 &#176;C), and 85 &#176;C-(85 &#176;C)) to obtain the strands with the smoothest surfaces.</li>
		</ol>
		</li>
		<li>Pelletization
		<ol>
			<li>After passing the conveyor belt (the belt situated after the extruder that helps to feed material out of the extruder), feed the strands into a pelletizer operated with a cutting speed of 7 m/min.</li>
		</ol>
		</li>
		<li>Film extrusion from the pellets
		<ol>
			<li>Feed the pellets manually into the extruder and extrude films with the low-T profile inside the barrel and with a 125 &#176;C-(115 &#176;C) flat-sheet die temperature. Use a screw rotational speed of 30 rpm.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Film extrusion using volumetric feeding
	<ol>
		<li>In order to simulate automatic feeding (commonly used in industry), use the pellets previously extruded at 85 &#176;C (steps 2.3.1-2.3.2.1).</li>
		<li>Connect the feeder to the extruder and choose the volumetric feeder mode of the hopper.</li>
		<li>Use a feeding volume of 35 kg/h and hopper and extruder screw speeds of 16 and 120 rpm, respectively.</li>
		<li>Extrude with the low temperature profile of the barrel and use a die-(zone 11) temperature of 125 &#176;C-(115 &#176;C).</li>
	</ol>
	</li>
</ol>
3. Post-extrusion Process (Compression Molding)
<ol>
	<li>Pressing with the frame
	<ol>
		<li>For the first setup, cut two extrudates into pieces of 4.4 cm x 7.0 cm and 2.6 cm x 7.0 cm. 
		NOTE: This is needed because the frame is broader than the extrudates.</li>
		<li>Place them next to each other in an aluminum rectangular frame (70 x 70 x 0.5 mm3).</li>
		<li>Sandwich the frame between two aluminum plates using poly(ethylene terephthalate) (PET) films on both sides to prevent adhesion, and then place them into the press.</li>
		<li>Set the pressure gauge on the press to 200 or 400 bar.</li>
		<li>For each molding pressure, press the films with a plate temperature of 110, 120, and 130 &#176;C for 10 and 20 min.</li>
		<li>As a reference to the pre-extruded samples, press films from unextruded material. Center 7.2 g of fresh material (from section 1.6) in the aluminum frame.</li>
		<li>Press with the same parameter settings as for the pre-extruded films above (steps 3.1.4- 3.1.5).</li>
	</ol>
	</li>
	<li>Pressing without a frame
	<ol>
		<li>Cut out and sandwich rectangular samples (4.4 cm x 4.4 cm) between two aluminum plates using PET films on both sides to prevent adhesion.</li>
		<li>Place them into the press. Set the pressure gauge to 50 bar, 75 bar, or 100 bar.</li>
		<li>For each molding pressure, press films for 5 or 10 min using 110 &#176;C, 120 &#176;C and 130 &#176;C plate temperatures.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Aeschelmann, F., Carus, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bio-based building blocks and polymers in the world. Capacities, production and applications: Status quo and trends towards 2020. , (2015).</li><li>T&#252;re, H., G&#228;llstedt, M., Kuktaite, R., Johansson, E., Hedenqvist, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+network+structure+and+properties+of+wheat+gluten+extrudates+using+a+novel+solvent-free+approach+with+urea+as+a+combined+denaturant+and+plasticizer.">Protein network structure and properties of wheat gluten extrudates using a novel solvent-free approach with urea as a combined denaturant and plasticizer.</a> Soft Matter. 7, 9416-9423 (2011).</li><li>Belyea, R. L., Steevens, B. J., Restrepo, R. J., Clubb, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+in+Composition+of+By-Product+Feeds.">Variation in Composition of By-Product Feeds.</a> J. Dairy. Sci. 72 (9), 2339-2345 (1989).</li><li>G&#243;mez-Estaca, J., Gavara, R., Catal&#225;, R., Hern&#225;ndez-Mu&#241;oz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+proteins+for+producing+food+packaging+materials:+A+review.">The potential of proteins for producing food packaging materials: A review.</a> Packag. Technol. Sci. , (2016).</li><li>Lazzeri, L., Leoni, O., Conte, L. S., Palmieri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+technological+characteristics+and+potential+uses+of+Crambe+abyssinica+products.">Some technological characteristics and potential uses of Crambe abyssinica products.</a> Ind. Crops and Prod. 3, 103-112 (1994).</li><li>Lalas, S., Gortzi, O., Athanasiadis, V., Dourtoglou, E., Dourtoglou, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Full+Characterisation+of+Crambe+abyssinica+Hochst+Seed+Oil.">Full Characterisation of Crambe abyssinica Hochst Seed Oil.</a> J. Am. Oil Chem. Soc. 89, 2253-2258 (2012).</li><li>Carlson, K. D., Tookey, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crambe+Meal+as+a+Protein+Source+for+Feeds.">Crambe Meal as a Protein Source for Feeds.</a> J. Am. Oil Chem.Soc. 60, 1979-1985 (1983).</li><li>Massoura, E., Vereijken, J. M., Kolster, P., Derksen, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteins+from+Crambe+abyssinica+oilseed.+II.+Biochemical+and+functional+properties.">Proteins from Crambe abyssinica oilseed. II. Biochemical and functional properties.</a> J. Am. Oil Chem. Soc. 75, 323-335 (1988).</li><li>Quispea, C. A. G., Coronadoc, C. J. R., Carvalho, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycerol:+Production,+consumption,+prices,+characterization+and+new+trends+in+combustion.">Glycerol: Production, consumption, prices, characterization and new trends in combustion.</a> Renew. Sust. Energ. Rev. 27, 475-493 (2013).</li><li>Kuktaite, R., Plivelic, T. S., T&#252;re, H., Hedenqvist, M. S., G&#228;llstedt, M., Marttila, S., Johansson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+hierarchical+protein+polymer+structure:+urea+and+temperature+effects+on+wheat+gluten+films.">Changes in the hierarchical protein polymer structure: urea and temperature effects on wheat gluten films.</a> RSC Advances. 2, 11908-11914 (2012).</li><li>Bennion, B. J., Daggett, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+for+the+chemical+denaturation+of+proteins+by+urea.">The molecular basis for the chemical denaturation of proteins by urea.</a> Proc.Natl.Acad.Sci. 100, 5142-5147 (2003).</li><li>Rasel, H., Johansson, T., G&#228;llstedt, M., Newson, W., Johansson, E., Hedenqvist, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+bioplastics+based+on+agricultural+side-stream+products:+Film+extrusion+of+Crambe+abyssinica/wheat+gluten+blends+for+packaging+purposes.">Development of bioplastics based on agricultural side-stream products: Film extrusion of Crambe abyssinica/wheat gluten blends for packaging purposes.</a> J. Appl. Polym. Sci. 133, 42442 (2016).</li><li>Appelqvist, L. -. &. #. 1. 9. 7. ;. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Further+studies+on+a+multisequential+method+for+determination+of+oil+content+in+oilseeds.">Further studies on a multisequential method for determination of oil content in oilseeds.</a> J. Am. Oil Chem. Soc. 44, 209-214 (1967).</li><li>Verbeek, C. J. R., van den Berg, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrusion+Processing+and+Properties+of+Protein-Based+Thermoplastics.">Extrusion Processing and Properties of Protein-Based Thermoplastics.</a> Macromol. Mater. Eng. 295, 10-21 (2010).</li><li>Ralston, B. E., Osswald, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viscosity+of+Soy+Protein+Plastics+Determined+by+Screw-Driven+Capillary+Rheometry.">Viscosity of Soy Protein Plastics Determined by Screw-Driven Capillary Rheometry.</a> J Polym. Environ. 16, 169-176 (2008).</li><li>Nur Hanani, Z. A., Beatty, E., Roos, Y. H., Morris, M. A., Kerry, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manufacture+and+characterization+of+gelatin+films+derived+from+beef,+pork+and+fish+sources+using+twin+screw+extrusion.">Manufacture and characterization of gelatin films derived from beef, pork and fish sources using twin screw extrusion.</a> J. Food Eng. 113, 606-614 (2012).</li></ol>

The methods and results were previously presented as an article by Rasel et al.5.

The reason the die pressure was of such high importance was probably due to the fact that the material needed a certain pressure to avoid bubble formation. However, the different components could phase separate if the pressure was too high. When extruding at too low a temperature, the cohesion was poor, possibly due to a low degree of crosslinking, while too high a temperature resulted in the release of gas (probably moisture together with urea and protein degradation products).
The two step extrusion (i.e., where strands were first extruded, pelletized, and then extruded again) resulted in a more homogeneous extrudate because of the more extensive blending that the first extrusion step provided.
The poor dough cohesion when decreasing the urea concentration from 15 to 10 wt% was probably due to a lower crosslink density. In analogy to this, a lower glycerol concentration, and thus a lower ability to dissolve the urea, resulted in poorer films unless a higher die pressure was applied.
Increasing the crambe meal concentration, and thus decreasing the wheat gluten concentration, resulted in a lower degree of aggregation/network formation. This lowered the viscosity of the material in the extrudate, resulting in the need to increase the die temperature to 130 &#176;C to raise the viscosity and generate homogeneous films.
It is difficult, if not impossible, to extrude plasticized crambe into films of sufficient quality for any use. We show here that this can be overcome by blending crambe with a more readily extrudable protein like wheat gluten. For the best quality, the extrudates need to be compression-molded in a separate step after extrusion.
It is shown here that extrusion works on a smaller scale, and upscaling is likely to be more demanding. Extrusion, along with injection molding, are the most important commercial methods for producing plastics. In order to replace existing conventional plastics, it is necessary that the protein material can be produced with the same techniques14-16. We show here that it is possible to extrude crambe oilseed meal with the help of wheat gluten.
Possible applications include packaging and applications for various extruded profiles (e.g., rods and cylinders). We consider the most critical step during the preparation of the samples to be the extrusion step. The final quality of the films depended strongly upon the extrusion parameters and the properties of the material prior to extrusion.

When upgrading a material from a low value to a higher value, two main issues need to be considered: the type of potential end-product(s) and the required properties. This study focuses on the extrusion of protein-based plastics for potential use in packaging for two reasons. The present variety of packages is extensive, but the request for renewable and biodegradable low-cost packaging material has increased rapidly in the last decade. This trend seems to be continuing, as most brand owners and legislators are searching for options to create plastics from petroleum1. The required material properties for packaging are, in many cases, more demanding than for other plastic products. However, if a successful material is obtained, the potential market is very large.
Packaging material needs to fulfil a number of criteria to be suitable. The exact criteria differ depending on the type of package, filling/sealing systems, transport, storage, content, appearance, product design, etc. All these parameters should be considered by a packaging developer, but all cannot be of the highest priority at once when initiating the development of a new and unexplored material. The properties in focus for this study were the mechanical and barrier performances.
Extrusion is the processing method of choice for two reasons: extrusion is a common and efficient method for making packaging plastics, and it generally does not involve a solvent, as in solution casting. Thus, no drying step is needed at the end of the process2.
Wheat gluten is also a side stream material coming from a starch product3. It has shown potential as a packaging plastic in a number of studies. Despite this, some challenges remain4. Crambe abyssinica is an interesting oilseed plant in that it is not a food resource and can be grown in many different agronomic conditions5,6. As with wheat gluten, crambe protein is a byproduct, in this case, from oil production. It is obtained as a defatted crambe meal, with protein as the largest component. It also contains a sizeable amount of nitrogen-free extracts, such as carbohydrates and fiber7,8. The meal has relatively poor cohesive properties and needs to be blended with a material of higher cohesion. In this study, wheat gluten is used as a supportive additive to the crambe meal. To improve the toughness/extensibility of the protein material, a plasticizer is commonly used as an additive as well. In this study, glycerol is used, which is a side product of the plant oil industry (e.g., rape seed methyl ester fuels) and is readily available at a low cost9. Urea, also renewable, is used as a denaturant in order to give the extrudate the proper cohesion2,10,11. It may also work as a plasticizer.
Renewable materials, especially those that are used directly from nature, without purification, modification, or chemical synthesis, are, in most cases, not suitable for high-temperature treatment. The challenge is to find suitable processing parameters and compositions that result in an extrudate with properties that allow it to compete with products from petroleum.
This study focuses on the characterization of the mechanical and barrier properties of a new bio-based material produced from crambe meal processed with different additives and at different conditions12. The full details of the mechanical and oxygen barrier features are found in Rasel et al.12.

<table><tbody><tr><td>Crambe meal</td><td>Plant Research International</td><td></td><td>Defatted crambe meal, Residual from oil extraction of cultivar Galactica seeds</td></tr><tr><td>Wheat gluten</td><td>Lantm&amp;auml;nnen Reppe AB</td><td></td><td>It contains 77% (w/w) gluten, 8.1% (w/w) starch and 1.34% (w/w) fat.</td></tr><tr><td>Glycerol</td><td>Karlshamn Tefac AB</td><td>99.5% purity</td><td></td></tr><tr><td>Urea</td><td>Sigma Aldrich</td><td>purity &amp;ge; 99.5%</td><td></td></tr><tr><td>The dough</td><td></td><td></td><td>(per 100 g) prepared with 35.7 g crambe meal, 23.8 g wheat gluten, 25.5 g glycerol and 15 g urea, hence with a liquid (glycerol/urea) to solid (crambe/wheat gluten) ratio of 0.342.</td></tr><tr><td>Round, fine meshed stainless steel kitchen sieve (pore size: ~1.5&amp;nbsp;mm, 14 mesh)</td><td></td><td></td><td>Sieve the crambe meal</td></tr><tr><td>Rotary ball mill</td><td>Pascal Engineering</td><td></td><td>Milling crambe/The volume of the mill house is 7 L and it contained 215 ceramic balls, each with a diameter of 25&amp;nbsp;mm.</td></tr><tr><td>Mortar and pestle</td><td></td><td></td><td>Grinding urea</td></tr><tr><td>Kitchen machine Cloer 660</td><td>Cloer</td><td></td><td>Blending crambe and wheat gluten</td></tr><tr><td>Twin-screw extruder Type LTE20-48</td><td>Labtech Engineering LTD</td><td></td><td>Compounding and film extrusion</td></tr><tr><td>Flat sheet die</td><td></td><td></td><td>Produce extruded flat films with a cross-section of 45 mm x 0.7 mm</td></tr><tr><td>Air Cooling Conveyor Unit type LAC-2.6</td><td>Labtech Engineering LTD</td><td></td><td>Used in the extrusion</td></tr><tr><td>Pelletizer Type LZ-120</td><td>Labtech Engineering LTD</td><td></td><td>Making pellets</td></tr><tr><td>Polystat 200T Hot Press&amp;nbsp;</td><td>Servitec Machine GmbH</td><td></td><td>Hot press to press extrudates</td></tr></tbody></table>

film extrusion of crambe abyssinica/wheat gluten blends

Crambe abyssinica is a plant with potential for use in industrial (non-food) plant oil production. The side stream from this oil production is a high-protein crambe meal that has limited value, as it is not fit for food or feed use. However, it contains proteins that could potentially make it a suitable raw material for higher-value products. The purpose of this study was to find methods of making this side stream into extruded films, showing that products with a higher value can be produced. The study mainly considered the development of material compositions and methods of preparing and extruding the material. Wheat gluten was added as a supportive protein matrix material, together with glycerol as a plasticizer and urea as a denaturant. The extrudate was evaluated with respect to mechanical (tensile testing) and oxygen barrier properties, and the extrudate structure was revealed visually and by scanning electron microscopy. A denser, more homogeneous material had a lower oxygen transmission rate, higher strength, and higher extensibility. The most homogeneous films were made at an extruder die temperature of 125-130 &#176;C. It is shown here that a film can be extruded with promising mechanical and oxygen barrier properties, the latter especially after a final compression molding step.

The blended materials (60 wt% crambe meal and 40 wt% wheat gluten) resulted in a tough dough after the initial mixing procedure. The material was rested for a few minutes before the first extrusion. However, the dough had a too high a viscosity to be able to be fed into the extruder hopper in a regular manner. Therefore, it was fed piece-by-piece, directly into the screw. The screws had a constant speed, and the resulting film extrudate was continuous and had a visually smooth surface. An example of an extruded film is shown in Figure 1.
The die pressure and temperature were found to be the two most important processing parameters to control in order to obtain homogeneous and smooth film extrudates. Too low a die temperature, typically below 110 &#176;C, did not result in continuous film extrudates, whereas a temperature above 130 &#176;C resulted in the formation of bubbles in the material. The most suitable die temperature to obtain homogeneous and smooth films was found to be around 125 &#176;C.
To get the most homogeneous extrudates, a two-step process was found to be advantageous, where, in the first step, strands were extruded at a lower temperature (typically 85 &#176;C) and pelletized. The pellets were then fed to the hopper for the second extrusion step.
When the urea content was decreased from 15 to 10 wt%12, the cohesion of the dough decreased substantially, resulting in a powder-like material; no continuous film could be extruded12.
When the glycerol concentration was decreased (with a retained 15 wt% urea), the dough was found to be more brittle, and the urea did not fully dissolve in the glycerol. Also, a considerably higher die pressure was required to get homogeneous films. However, these films were found to be smoother and more homogeneous than those with a higher glycerol content.
When increasing the crambe meal powder concentration and decreasing the wheat gluten concentration, the extruded films appeared darker, but also smoother and more homogeneous5. The feeding rate could also be increased12. The drawback was that the films were only partially continuous, and film breakages appeared a few meters apart. However, when increasing the die temperature to approximately 130 &#176;C, continuous films could be prepared, although with some discolorations12.
Compression molding without a frame yielded thin (thickness: 0.1-0.2 mm) films that were very flexible and translucent (Figure 2).
Depending on how the extrudates were made and what they contained, the stiffness ranged from 4.9-5.6 MPa and the strength from 0.3-0.7 MPa, whereas the extensibility ranged from 7 to 16%12. The corresponding values for the extrudates after compression molding were 6.4-15.0 MPa, 0.3-1.1 MPa, and 8-19%5. Details of the mechanical measurements are given in reference12. 64 mm-long dumbbell specimens were tensile-tested according to ASTM D882-02 at 23 &#177; 1 &#176;C and 50 &#177; 1% RH, with a crosshead speed of 10 mm/min. Figure 3 shows the importance of extruding crambe with the addition of wheat gluten. The strength, and especially the extensibility, decreased with decreasing wheat gluten content. The oxygen permeability ranged from 17 to 39 cm3 mm/(day m2 atm), depending on the composition and whether a compression molding step (with a frame) was used or not.
<img alt="Figure 1" src="/files/ftp_upload/54770/54770fig1.jpg" /> 
Figure 1: Extruded material. Extruded film using a die temperature of 130 &#176;C. It contains 35.7 wt% crambe, 23.8 wt% wheat gluten, 25.5 wt% glycerol, and 15 wt% urea. The width of the film is 44 mm. <a href="http://cloudflare.jove.com/files/ftp_upload/54770/54770fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54770/54770fig2.jpg" /> 
Figure 2: Compression-molded films. Extruded material compression-molded, without a frame, into thin, translucent films using a 130 &#176;C press temperature at 75 bar for 10 min. The flat and wrinkled films are from the same material. The width of the left film is ~17 mm. <a href="http://cloudflare.jove.com/files/ftp_upload/54770/54770fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54770/54770fig3.jpg" /> 
Figure 3: Mechanical properties as a function of crambe content. Maximum stress (filled circles) and strain at maximum stress (open circles) as a function of crambe content in the crambe/wheat gluten mixture. The error bars represent the standard deviations. Maximum stress was obtained from the maximum force per initial sample cross-section of the narrow part of the dumbbell specimen (narrow section: 16 mm long and 4 mm wide). The low-temperature profile was used with an initial die temperature of 125 &#176;C and a zone 11 temperature of 115 &#176;C. The screw speed was 30 rpm, and the extrusion was performed without prior pelletization. Data obtained from reference12. <a href="http://cloudflare.jove.com/files/ftp_upload/54770/54770fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Film Extrusion of Crambe abyssinica/Wheat Gluten Blends at JoVE.com

Film Extrusion of Crambe abyssinica/Wheat Gluten Blends

The side stream from plant oil production of Crambe abyssinica has limited value. The purpose of this study was to find methods for extruding materials based on this side stream, demonstrating that products with a higher value can be produced. The extrudates were found to have promising properties.

Film Extrusion of Crambe abyssinica/Wheat Gluten Blends

Crambe abyssinica is a plant with potential for use in industrial (non-food) plant oil production. The side stream from this oil production is a ...

film-extrusion-of-crambe-abyssinicawheat-gluten-blends

Research

JoVE Journal

Bioengineering

9.9K Views.  SIG Combibloc. The side stream from plant oil production of Crambe abyssinica has limited value. The purpose of this study was to find methods for extruding materials based on this side stream, demonstrating that products with a higher value can be produced. The extrudates were found to have promising properties.

Video: Film Extrusion of Crambe abyssinica/Wheat Gluten Blends

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper and food industries. A deeper understanding of CAZymes is important from both fundamental biology and industrial standpoints. Vast numbers of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used for screening the activities of enzymes, enzyme cocktails, and broths.

A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

Biochemistry

Method to Produce Durable Pellets at Lower Energy Consumption Using High Moisture Corn Stover and a Corn Starch Binder in a Flat Die Pellet Mill

A major challenge in the production of pellets is the high cost associated with drying biomass from 30 to 10% (w.b.) moisture content. At Idaho National Laboratory, a high-moisture pelleting process was developed to reduce the drying cost. In this process the biomass pellets are produced at higher feedstock moisture contents than conventional methods, and the high moisture pellets produced are further dried in energy efficient dryers. This process helps to reduce the feedstock moisture content by about 5-10% during pelleting, which is mainly due to frictional heat developed in the die. The objective of this research was to explore how binder addition influences the pellet quality and energy consumption of the high-moisture pelleting process in a flat die pellet mill. In the present study, raw corn stover was pelleted at moistures of 33, 36, and 39% (w.b.) by addition of 0, 2, and 4% pure corn starch. The partially dried pellets produced were further dried in a laboratory oven at 70 &#176;C for 3-4 hr to lower the pellet moisture to less than 9% (w.b.). The high moisture and dried pellets were evaluated for their physical properties, such as bulk density and durability. The results indicated that increasing the binder percentage to 4% improved pellet durability and reduced the specific energy consumption by 20-40% compared to pellets with no binder. At higher binder addition (4%), the reduction in feedstock moisture during pelleting was &#60;4%, whereas the reduction was about 7-8% without the binder. With 4% binder and 33% (w.b.) feedstock moisture content, the bulk density and durability values observed of the dried pellets were &#62;510 kg/m3 and &#62;98%, respectively, and the percent fine particles generated was&#160;reduced to &#60;3%.

In this study, a protocol was developed to produce good quality pellets using a flat die pellet mill at reduced specific energy consumption testing high-moisture corn stover and a starch based binder. The results indicated that adding a corn starch binder improved the pellet durability, reduced percent fines and decreased specific energy consumption.

A major challenge in the production of pellets is the high cost associated with drying biomass from 30 to 10% (w.b.) moisture content. At Idaho National ...

Environment

Composition and Properties of Aquafaba: Water Recovered from Commercially Canned Chickpeas

Chickpea and other pulses are commonly sold as canned products packed in a thick solution or a brine. This solution has recently been shown to produce stable foams and emulsions, and can act as a thickener. Recently interest in this product has been enhanced through the internet where it is proposed that this solution, now called aquafaba by a growing community, can be used a replacement for egg and milk protein. As aquafaba is both new and being developed by an internet based community little is known of its composition or properties. Aquafaba was recovered from 10 commercial canned chickpea products and correlations among aquafaba composition, density, viscosity and foaming properties were investigated. Proton NMR was used to characterize aquafaba composition before and after ultrafiltration through membranes with different molecular weight cut offs (MWCOs of 3, 10, or 50 kDa). A protocol for electrophoresis, and peptide mass fingerprinting is also presented. Those methods provided valuable information regarding components responsible for aquafaba functional properties. This information will allow the development of practices to produce standard commercial aquafaba products and may help consumers select products of superior or consistent utility.

Aquafaba is a viscous juice from canned chickpea that, when stirred vigorously, produces a relatively stable white froth or foam. The primary research goal is to identify the components of aquafaba that contribute viscosifying/thickening properties using nuclear magnetic resonance (NMR), ultrafiltration, electrophoresis, and peptide mass fingerprinting.

Chickpea and other pulses are commonly sold as canned products packed in a thick solution or a brine. This solution has recently been shown to produce ...

A Protocol for the Production of Gliadin-cyanoacrylate Nanoparticles for Hydrophilic Coating

This article presents a protocol for the production of protein-based nanoparticles that changes the hydrophobic surface to hydrophilic by a simple spray coating. These nanoparticles are produced by the polymerization reaction of alkyl cyanoacrylate on the surface of cereal protein (gliadin) molecules. Alkyl cyanoacrylate is a monomer that instantly polymerizes at RT when it is applied to the surface of materials. Its polymerization reaction is initiated by the trace amounts of weakly basic or nucleophilic species on the surface, including moisture. Once polymerized, the polymerized alkyl cyanoacrylates show a strong affinity with the object materials because nitrile groups are in the backbone of poly (alkyl cyanoacrylate). Proteins also work as initiator for this polymerization because they contain amine groups that can initiate the polymerization of cyanoacrylate. If aggregated protein is used as an initiator, protein aggregate is surrounded by the hydrophobic poly(alkyl cyanoacrylate) chains after the polymerization reaction of alkyl cyanoacrylate. By controlling the experimental condition, particles in the nanometer range are produced. The produced nanoparticles readily adsorb to the surface of most materials including glass, metals, plastics, wood, leather, and fabrics. When the surface of a material is sprayed with the produced nanoparticle suspension and rinsed with water, the micellar structure of nanoparticle changes its conformation, and the hydrophilic proteins are exposed to the air. As a result, the nanoparticle-coated surface changes to hydrophilic.

This article presents a protocol for the production of protein-based nanoparticles that changes the hydrophobic surface to hydrophilic. The produced nanoparticle is an assembly of gliadin-cyanoacrylate diblock copolymers. Spray coating with the produced nanoparticle changes the surface of target material to a hydrophilic surface.

This article presents a protocol for the production of protein-based nanoparticles that changes the hydrophobic surface to hydrophilic by a simple spray ...

Chemistry

Ammonia Fiber Expansion (AFEX) Pretreatment of Lignocellulosic Biomass

Lignocellulosic materials are plant-derived feedstocks, such as crop residues (e.g., corn stover, rice straw, and sugar cane bagasse) and purpose-grown energy crops (e.g., miscanthus, and switchgrass) that are available in large quantities to produce biofuels, biochemicals, and animal feed. Plant polysaccharides (i.e., cellulose, hemicellulose, and pectin) embedded within cell walls are highly recalcitrant towards conversion into useful products. Ammonia fiber expansion (AFEX) is a thermochemical pretreatment that increases accessibility of polysaccharides to enzymes for hydrolysis into fermentable sugars. These released sugars can be converted into fuels and chemicals in a biorefinery. Here, we describe a laboratory-scale batch AFEX process to produce pretreated biomass on the gram-scale without any ammonia recycling. The laboratory-scale process can be used to identify optimal pretreatment conditions (e.g., ammonia loading, water loading, biomass loading, temperature, pressure, residence time, etc.) and generates sufficient quantities of pretreated samples for detailed physicochemical characterization and enzymatic/microbial analysis. The yield of fermentable sugars from enzymatic hydrolysis of corn stover pretreated using the laboratory-scale AFEX process is comparable to pilot-scale AFEX process under similar pretreatment conditions. This paper is intended to provide a detailed standard operating procedure for the safe and consistent operation of laboratory-scale reactors for performing AFEX pretreatment of lignocellulosic biomass.

Ammonia Fiber Expansion AFEX Pretreatment of Lignocellulosic Biomass

Ammonia fiber expansion (AFEX) is a thermochemical pretreatment technology that can convert lignocellulosic biomass (e.g., corn stover, rice straw, and sugarcane bagasse) into a highly digestible feedstock for both biofuels and animal feed applications. Here, we describe a laboratory-scale method for conducting AFEX pretreatment on lignocellulosic biomass.

Lignocellulosic materials are plant-derived feedstocks, such as crop residues (e.g., corn stover, rice straw, and sugar cane bagasse) and purpose-grown ...

Manufacturing Of Robust Natural Fiber Preforms Utilizing Bacterial Cellulose as Binder

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose and short sisal fibers are dispersed into a water suspension containing bacterial cellulose. The fiber and nanocellulose suspension is then filtered (using vacuum or gravity) and the wet filter cake pressed to squeeze out any excess water, followed by a drying step. This will result in the hornification of the bacterial cellulose network, holding the loose natural fibers together.
Our method is specially suited for the manufacturing of rigid and robust preforms of hydrophilic fibers. The porous and hydrophilic nature of such fibers results in significant water uptake, drawing in the bacterial cellulose dispersed in the suspension. The bacterial cellulose will then be filtered against the surface of these fibers, forming a bacterial cellulose coating. When the loose fiber-bacterial cellulose suspension is filtered and dried, the adjacent bacterial cellulose forms a network and hornified to hold the otherwise loose fibers together.
The introduction of bacterial cellulose into the preform resulted in a significant increase of the mechanical properties of the fiber preforms. This can be attributed to the high stiffness and strength of the bacterial cellulose network. With this preform, renewable high performance hierarchical composites can also be manufactured by using conventional composite production methods, such as resin film infusion (RFI) or resin transfer molding (RTM). Here, we also describe the manufacturing of renewable hierarchical composites using double bag vacuum assisted resin infusion.

We present a novel method of manufacturing rigid and robust short natural fiber preforms using a papermaking process. Bacterial cellulose acts simultaneously as the binder for the loose fibers and provides rigidity to the fiber preforms. These preforms can be infused with a resin to produce truly green hierarchical composites.

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose ...

Molecular Entanglement and Electrospinnability of Biopolymers

Electrospinning is a fascinating technique to fabricate micro- to nano-scale fibers from a wide variety of materials. For biopolymers, molecular entanglement of the constituent polymers in the spinning dope was found to be an essential prerequisite for successful electrospinning. Rheology is a powerful tool to probe the molecular conformation and interaction of biopolymers. In this report, we demonstrate the protocol for utilizing rheology to evaluate the electrospinnability of two biopolymers, starch and pullulan, from their dimethyl sulfoxide (DMSO)/water dispersions. Well-formed starch and pullulan fibers with average diameters in the submicron to micron range were obtained. Electrospinnability was evaluated by visual and microscopic observation of the fibers formed. By correlating the rheological properties of the dispersions to their electrospinnability, we demonstrate that molecular conformation, molecular entanglement, and shear viscosity all affect electrospinning. Rheology is not only useful in solvent system selection and process optimization, but also in understanding the mechanism of fiber formation on a molecular level.

Electrospinning is a fascinating technique used to fabricate micro- to nano-scale fibers from a wide variety of materials. Molecular entanglement of the constituent polymers in the spinning dope is essential for successful electrospinning. We present a protocol for utilizing rheology to evaluate the electrospinnability of two biopolymers, starch and pullulan.

Electrospinning is a fascinating technique to fabricate micro- to nano-scale fibers from a wide variety of materials. For biopolymers, molecular ...

Twin-Screw Extrusion Process to Produce Renewable Fiberboards

A versatile twin-screw extrusion process to provide an efficient thermo-mechano-chemical pre-treatment on lignocellulosic biomass before using it as source of mechanical reinforcement in fully bio-based fiberboards was developed. Various lignocellulosic crop by-products have already been successfully pre-treated through this process, e.g., cereal straws (especially rice), coriander straw, shives from oleaginous flax straw, and bark of both amaranth and sunflower stems.
The extrusion process results in a marked increase in the average fiber aspect ratio, leading to improved mechanical properties of fiberboards. The twin-screw extruder can also be fitted with a filtration module at the end of the barrel. The continuous extraction of various chemicals (e.g., free sugars, hemicelluloses, volatiles from essential oil fractions, etc.) from the lignocellulosic substrate, and the fiber refining can, therefore, be performed simultaneously.
The extruder can also be used for its mixing ability: a natural binder (e.g., Organosolv lignins, protein-based oilcakes, starch, etc.) can be added to the refined fibers at the end of the screw profile. The obtained premix is ready to be molded through hot pressing, with the natural binder contributing to fiberboard cohesion. Such a combined process in a single extruder pass improves the production time, production cost, and may lead to reduction in plant production size. Because all the operations are performed in a single step, fiber morphology is better preserved, thanks to a reduced residence time of the material inside the extruder, resulting in enhanced material performances. Such one-step extrusion operation may be at the origin of a valuable industrial process intensification.
Compared to commercial wood-based materials, these fully bio-based fiberboards do not emit any formaldehyde, and they could find various applications, e.g., intermediate containers, furniture, domestic flooring, shelving, general construction, etc.

A versatile twin-screw extrusion process to provide an efficient thermo-mechano-chemical pretreatment on lignocellulosic biomass was developed, which leads to an increased average fiber aspect ratio. A natural binder can also be added continuously after fiber refining, leading to bio-based fiberboards with improved mechanical properties after hot pressing of the obtained extruded material.

A versatile twin-screw extrusion process to provide an efficient thermo-mechano-chemical pre-treatment on lignocellulosic biomass before using it as ...

Engineering

Design of an Open-Source, Low-Cost Bioink and Food Melt Extrusion 3D Printer

Three-dimensional (3D) printing is an increasingly popular manufacturing technique that allows highly complex objects to be fabricated with no retooling costs. This increasing popularity is partly driven by falling barriers to entry such as system set-up costs and ease of operation. The following protocol presents the design and construction of an Additive Manufacturing Melt Extrusion (ADDME) 3D printer for the fabrication of custom parts and components. ADDME has been designed with a combination of 3D-printed, laser-cut, and online-sourced components. The protocol is arranged into easy-to-follow sections, with detailed diagrams and parts lists under the headings of framing, y-axis and bed, x-axis, extrusion, electronics, and software. The performance of ADDME is evaluated through extrusion testing and 3D printing of complex objects using viscous cream, chocolate, and Pluronic F-127 (a model for bioinks). The results indicate that ADDME is a capable platform for the fabrication of materials and constructs for use in a wide range of industries. The combination of detailed diagrams and video content facilitates access to low-cost, easy-to-operate equipment for individuals interested in 3D printing of complex objects from a wide range of materials.

The aim of this work is to design and construct a reservoir-based melt extrusion three-dimensional printer made from open-source and low-cost components for applications in the biomedical and food printing industries.

Three-dimensional (3D) printing is an increasingly popular manufacturing technique that allows highly complex objects to be fabricated with no retooling ...

Harnessing Gracilaria gracilis: Bioactive Potential for Sustainable Innovation

Valorization of the Red Seaweed Gracilaria gracilis Through a Biorefinery Approach

The interest in seaweeds as an abundant feedstock to obtain valuable and multitarget bioactive ingredients is continuously growing. In this work, we explore the potential of Gracilaria gracilis, an edible red seaweed cultivated worldwide for its commercial interest as a source of agar and other ingredients for cosmetic, pharmacological, food, and feed applications.
G. gracilis growth conditions were optimized through vegetative propagation and sporulation while manipulating the physicochemical conditions to achieve a large biomass stock. Green extraction methodologies with ethanol and water were performed over the seaweed biomass. The bioactive potential of extracts was assessed through a set of in vitro assays concerning their cytotoxicity, antioxidant, and antimicrobial properties. Additionally, dried seaweed biomass was incorporated into pasta formulations to increase food&#39;s nutritional value. Pigments extracted from G. gracilis have also been incorporated into yogurt as a natural colorant, and their stability was evaluated. Both products were submitted to the appreciation of a semi-trained sensorial panel aiming to achieve the best final formulation before reaching the market.
Results support the versatility of G. gracilis whether it is applied as a whole biomass, extracts and/or pigments. Through implementing several optimized protocols, this work allows the development of products with the potential to profit the food, cosmetic, and aquaculture markets, promoting environmental sustainability and a blue circular economy.
Moreover, and in line with a biorefinery approach, the residual seaweed biomass will be used as biostimulant for plant growth or converted to carbon materials to be used in water purification of the in-house aquaculture systems of MARE-Polytechnic of Leiria, Portugal.

Author Spotlight: Exploring Seaweed's Bioactive Compounds for Sustainable Innovations in Industries 

Here, we describe several protocols aiming at an integrated valorization of Gracilaria gracilis: wild species harvesting, in-house growth, and extraction of bioactive ingredients. The extracts&#39; antioxidant, antimicrobial, and cytotoxic effects are evaluated, along with the nutritional and stability assessment of food enriched with whole seaweed biomass and pigments.

The interest in seaweeds as an abundant feedstock to obtain valuable and multitarget bioactive ingredients is continuously growing. In this work, we ...

Film Extrusion of Crambe abyssinica/Wheat Gluten Blends

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Rozdziały w tym wideo

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Method to Produce Durable Pellets at Lower Energy Consumption Using High Moisture Corn Stover and a Corn Starch Binder in a Flat Die Pellet Mill

Composition and Properties of Aquafaba: Water Recovered from Commercially Canned Chickpeas

A Protocol for the Production of Gliadin-cyanoacrylate Nanoparticles for Hydrophilic Coating

Ammonia Fiber Expansion (AFEX) Pretreatment of Lignocellulosic Biomass

Manufacturing Of Robust Natural Fiber Preforms Utilizing Bacterial Cellulose as Binder

Molecular Entanglement and Electrospinnability of Biopolymers

Twin-Screw Extrusion Process to Produce Renewable Fiberboards

Design of an Open-Source, Low-Cost Bioink and Food Melt Extrusion 3D Printer

Valorization of the Red Seaweed Gracilaria gracilis Through a Biorefinery Approach