Cross-Linked Amylose Bio-Plastic: a Transgenic-Based Compostable Plastic Alternative

International Journal of Molecular Sciences

Article Cross-Linked Amylose Bio-Plastic: A Transgenic-Based Compostable Plastic Alternative

Domenico Sagnelli 1,*, Kourosh Hooshmand 1 ID , Gerdi Christine Kemmer 1, Jacob J. K. Kirkensgaard 2 ID , Kell Mortensen 2 ID , Concetta Valeria L. Giosafatto 3 ID , Mette Holse 4, Kim H. Hebelstrup 5, Jinsong Bao 6, Wolfgang Stelte 7, Anne-Belinda Bjerre 7 and Andreas Blennow 1 1 Department of Plant and Environmental Sciences, University of Copenhagen, 1871 Frederiksberg, Denmark; [email protected] (K.H.); [email protected] (G.C.K.); [email protected] (A.B.) 2 Niels Bohr Institute, University of Copenhagen, 2100 Copenhagen, Denmark; [email protected] (J.J.K.K.); [email protected] (K.M.) 3 Department of Chemical Science, University of Naples, 80126 Napoli, Italy; [email protected] 4 Department of Food Science, University of Copenhagen, 1958 Frederiksberg, Denmark; [email protected] 5 Department of Molecular Biology and Genetics, Aarhus University, 4200 Slagelse, Denmark; [email protected] 6 College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310029, China; [email protected] 7 Center for Bioresources and Bioreﬁnery, Danish Technological Institute, Gregersenvej 7, 2630 Taatsrup, Denmark; [email protected] (W.S.); [email protected] (A.-B.B.) * Correspondence: [email protected]; Tel.: +45-35-333304

Received: 1 September 2017; Accepted: 25 September 2017; Published: 30 September 2017

Abstract: Bio-plastics and bio-materials are composed of natural or biomass derived polymers, offering solutions to solve immediate environmental issues. Polysaccharide-based bio-plastics represent important alternatives to conventional plastic because of their intrinsic biodegradable nature. Amylose-only (AO), an engineered barley starch with 99% amylose, was tested to produce cross-linked all-natural bioplastic using normal barley starch as a control. Glycerol was used as plasticizer and citrate cross-linking was used to improve the mechanical properties of cross-linked AO starch extrudates. Extrusion converted the control starch from A-type to Vh- and B-type crystals, showing a complete melting of the starch crystals in the raw starch granules. The cross-linked AO and control starch specimens displayed an additional wide-angle diffraction reﬂection. Phospholipids complexed with Vh-type single helices constituted an integrated part of the AO starch specimens. Gas permeability tests of selected starch-based prototypes demonstrated properties comparable to that of commercial Mater-Bi© plastic. The cross-linked AO prototypes had composting characteristics not different from the control, indicating that the modiﬁed starch behaves the same as normal starch. The data shows the feasibility of producing all-natural bioplastic using designer starch as raw material.

Keywords: starch; amylose; bioplastic; cross-linker; amylose permeability; cross-link assay; citric acid

1. Introduction In March 2013, the European Commission published a green paper that outlined a strategy for decreasing the impact of plastic on the environment. The primary goal of the new strategy was to promote bio-based and biodegradable plastics [1]. Bio-plastics offer great opportunities for smart and green societal growth [2]. Plastics are materials composed of natural or synthetic high molecular weight polymeric molecules. These can be shaped and used in place of other materials like glass, wood, and metals.

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Their properties are adaptable to many applications using additives or alternative processing technologies. Bio-plastic is a sub-group that contains two qualitative characteristics: it is bio-based and/or biodegradable. The term “bio-based” refers to materials derived from biomass, whereas biodegradable refers to materials that can be assimilated by microorganisms. The main environmental threat of conventional plastics is their low degradability rates and the persistence of macro- and micro-plastics in both soil and water [3]. Micro-plastics are classified as 1–5 mm-sized particles [4]. Over the last years, thermoplastic polymer production has increased, leading to an extensive accumulation of micro-plastics in the environment, and becoming an increasing threat to marine organisms. These particles can carry pesticides. Moreover, their ingestion by sea animals introduces these pollutants to the food chain [5]. As opposed to most conventional plastics, biodegradable plastics are metabolized by environmental microorganisms, and therefore plant-based bio-plastics represent one of the most interesting clean alternative to conventional plastics. Their biochemical structure, favorable physiochemical assets, abundance in nature, and well-established fabrication technologies for plastics support development of novel bio-plastics types [6–9], offering two major, distinct advantages, mainly being that of their renewability and availability. Starch especially, is a raw material suitable for production of new plant-based bio-plastics (e.g., Mater-Bi©, Novamont) due to its abundancy, low cost, and processability with the use of existing technologies. Starch is heterogeneous and its composition typically entails two main polysaccharide types; amylopectin, a branched polymer, and amylose, a chiefly linear polymer. Amylose forms single and double helices that can align in ordered structures. The single helix provides a large hydrophobic cavity that can accommodate hydrophobic molecules, such as lipids [10–15], and lipid content is positively correlated to the amount of amylose in the granule [16]. The natural presence of lipids in the amylose helices may have appealing characteristics for materials science, but their effects on material functionality are not well characterized. Thermoplastic starch (TPS) is an edible and compostable product with functionality that can replace many conventional plastics, thereby assisting the reduction of non-degradable residue release into the environment. The production volume and technical knowledge for different TPS types is steadily increasing, and TPS can now be adapted to a variety of applications. However, it still presents certain limitations due to aging, and brittleness due to recrystallization. As an effect, starch is chemically modified to improve its stability and widen its usability. Etherification, esterification, or oxidation of the available hydroxyl groups on the glucose units are among the most commonly used approaches. For example, hydroxyethylated starch (e.g., vinyl-starch) is grafted with reagents such as 1,3-butadiene and styrene to improve coating properties in paper industries. Cross-linking is a common tactic to improve starch bio-plastic mechanical performances. Improvement of the mechanical properties of starch materials have been achieved with reagents such as sodium trimetaphosphate, phosphorus oxychloride, epichlorohydrin, sodium tripolyphosphate, and 1,2,3,4-diepoxybutane. Most of these compounds are unsafe and potentially toxic, and the use of citric acid in conjunction with sodium hypophosphite is considered to be a cleaner cross-linking technology [17]. Polymer industry and research are increasingly pointing towards more environment-friendly solutions for plastics production. Recent investigations pinpoint opportunities of plant-based biomaterials and composites as food packaging for short- and long shelf-life products [18]. If produced by all-natural plant-based raw materials [9], such products will have a tremendous impact on the economy and environment. An attractive approach is the direct modification of polysaccharides in crops using transgenic techniques. One example is amylose-only (AO) starch, produced by a transgenic barley line synthesizing a starch having 99% amylose. This starch was generated by RNA interference suppressing all three starch branching enzymes in the barley. The AO starch, as shown previously [6], provides a useful raw material for bio-plastics fabrication. In the present study, we extended this initiative using a scale-up extruder and investigating cross-linking with citric acid (CA) with the aim to produce a more Int. J. Mol. Sci. 2017, 18, 2075 3 of 12 robust bio-sustainable, yet compostable bio-plastic. Prototypes were characterized for crystallinity, mechanics, gas permeability, and composting biodegradation.

2. Results and Discussion

2.1. Determination of Lipid Content The presence of lipids complexed to amylose is expected to have a large inﬂuence on starch physics. TheInt. J. major Mol. Sci.internal 2017, 18, 2075 lipids found for the AO and the control starch samples were phospholipids,3 of 12 especially lysophosphatidylcholine (lysoPC), as showed by TLC analysis visualized using primuline 2. Results and Discussion dye (Figure S1). AO showed2.1. Determination a 40-fold of Lipid higher Content free fatty acid content as compared to the control starch (Table1). These data demonstrateThe presence of that lipids the complexed increased to amylose content is ofexpected amylose to have in thea large starch influence granule on starch is positively correlatedphysics. to the increaseThe major of internal lipids. lipids found for the AO and the control starch samples were phospholipids, especially lysophosphatidylcholine (lysoPC), as showed by TLC analysis visualized using primuline dye (Figure S1). Table 1. Free fatty acids (FFA) and phospholipids (PPH) in amylose-only (AO) and control. AO showed a 40-fold higher free fatty acid content as compared to the control starch (Table 1). These data demonstrate that the increased content of amylose in the starch granule is positively correlated to the increaseSample of lipids. FFA (%) PPH (%) Amylose-only 0.04 0.9 Table 1. Free fattyControl acids (FFA) and phospholipids 0.001 (PPH) in amylose-only 0.5 (AO) and control. Sample FFA (%) PPH (%) Amylose-only 0.04 0.9 2.2. Monitoring of Cross-Linking Reaction Control 0.001 0.5

To identify2.2. Monitoring optimal of Cr cross-linkingoss-Linking Reaction conditions, the CA cross-linking reaction temperature was monitored using differential scanning calorimetry (DSC) before the extrusion, with or without sodium To identify optimal cross-linking conditions, the CA cross-linking reaction temperature was hypophosphitemonitored (HP) using catalyst differential included. scanning In calorimetry the absence (DSC) of abefore HP catalyst, the extrusion, no exergonic with or without transition was detected (Figuresodium1 hypophosphiteA) indicating (HP) the catalyst absence included. or presence In the absence of only of a minor HP catalyst, cross-linking. no exergonicThe transition concentration of catalystwas needed detected for the(Figure cross-linking 1A) indicating was the optimized absence or using presence three of CA:HPonly minor ratios cross-linking. (CA:HP; w:wThe ; 1:1; 1:0.5; 1:0.25). Allconcentration three conditions of catalyst showed needed exergonic for the cross- transitionslinking was with optimized increasing using energy three CA:HP release ratios proportional (CA:HP; w:w; 1:1; 1:0.5; 1:0.25). All three conditions showed exergonic transitions with increasing to the amountenergy of release HP catalyst proportional used to (Figure the amount1A). of Subtracting HP catalyst theused control (Figure 1A). (without Subtracting HP) thermogramthe control to the CA/HP thermograms,(without HP) thermogram two HP-dependent to the CA/HP thermogram exergonics, peaks two HP-dependent could be identified exergonic peaks (Figure could1B). be The first occurred atidentified 100–105 (Figure◦C and 1B). the Thesecond first occurred at 120–125 at 100–105◦C. °C The and extrusion the second protocolat 120–125 was°C. The designed extrusion with these catalytic eventsprotocol under was designed consideration. with these In catalytic order toevents avoid under a high consideration. amount In of order residual to avoid catalyst a high in the final amount of residual catalyst in the final extrudate, the ratio CA/HP used for the extrusion was set at extrudate,low the level, ratio 1:0.25. CA/HP used for the extrusion was set at low level, 1:0.25.

(A) (B)

Figure 1. (A) Differential scanning calorimetry (DSC) thermograms of starches showing exergonic Figure 1. (citricA) Differentialacid (CA) cross-linking scanning reactions calorimetry in a sodium (DSC) hypophosphite thermograms (HP)-dependent of starches manner. showing The exergonic citric acid (CA)arrow cross-linkingindicates an endergonic reactions reaction; in a sodium(B) Control hypophosphite data subtracted in (HP)-dependentdicating HP-dependent manner. cross- The arrow indicates anlinking endergonic exotherms reaction; (arrows). (B) Control data subtracted indicating HP-dependent cross-linking exotherms (arrows).

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Int. J. Mol. Sci. 2017, 18, 2075 4 of 12 To conﬁrm the formation of ester bonds, Fourier Transform Infrared Spectroscopy (FTIR) analysis was performedTo confirm with datathe formation being collected of ester from bonds, 4000–550 Fourier cm Transform−1 with a spectralInfrared resolutionSpectroscopy of 8(FTIR) cm−1 and analysis was performed with data being collected from 4000–550 cm−1 with a spectral resolution of 8 attention focused to 1724 cm−1 (Figure S2), previously ascribed to the carboxyl and ester carbonyl cm−1 and attention focused to 1724 cm−1 (Figure S2), previously ascribed to the carboxyl and ester bands [17]. From the loadings and scores of the principal component analysis (PCA) model, which carbonyl bands [17]. From the loadings and scores of the principal component analysis (PCA) model, clearlywhich discriminated clearly discriminated the samples the treatedsamples with treated CA, with it was CA, concluded it was concluded that the that extrusion the extrusion conditions wereconditions appropriate were (Figure appropriate2). (Figure 2).

(A)

(B) (C)

FigureFigure 2. Principal 2. Principal component component analysis analysis (PCA) (PCA) onon MultiplicativeMultiplicative Scatter Scatter Correction Correction pre-processed pre-processed FourierFourier Transform Transform Infrared Infrared Spectroscopy Spectroscopy spectra spectra (1800–1500 (1800–1500 cm−1 )cm obtained−1) obtained from analysisfrom analysis of extrudates. of (A) Scoreextrudates. plot of ( PC1A) Score vs. PC2. plot of Samples PC1 vs. arePC2. grouped Samples according are grouped to bothaccording starch to typeboth andstarch CA type a cross-linking; and CA (B,C)a loading cross-linking; plots ( ofB,C PC1) loading and PC2, plots respectively.of PC1 and PC2, The respective peak arisingly. The from peak carboxylarising from and carboxyl ester carbonyl and −1 absorbanceester carbonyl (1724 cm absorbance−1) is highlighted. (1724 cm ) is highlighted.

2.3. Crystalline Properties of the Extrudates 2.3. Crystalline Properties of the Extrudates The extrudates were characterized for crystallinity by wide angle X-ray scattering (WAXS), Themechanical extrudates analysis, were and characterized gas permeability. for After crystallinity the melting by process wide angleand prior X-ray to any scattering analysis, (WAXS), the mechanicalextrudates analysis, were stored and in gas a controlled permeability. environment After stabilizing the melting the processsemi-crystalline and prior matrix. to any analysis, the extrudatesFor WAXS were analysis, stored in the a extrudates controlled were environment stored at 85% stabilizing relative humidity the semi-crystalline (RH). The effect matrix. of both Forcross-linking WAXS analysis, and glycerol the extrudateswas evident, were withstored both treatments at 85% relative increasing humidity the crystallinity (RH). The of effectthe of both cross-linkingextrudates (Figure and 3). glycerol Previously, was we evident, demonstrated with both thattreatments native AO starch increasing and extrudates the crystallinity fabricated of the from this starch only encompassed Vh- and B-type crystallinity [6]. Cross-linking of both AO and extrudates (Figure3). Previously, we demonstrated that native AO starch and extrudates fabricated control induced the formation of a mixture of Vh, B-, and a further crystal polymorph that may be from this starch only encompassed Vh- and B-type crystallinity [6]. Cross-linking of both AO and associated to A-type [18]. The Vh-type polymorph was substantiated by reflexions at 2θ 7.3, 12.8, control19.6, induced the A-type the at formation 2θ 15, 18.8 ofand a the mixture B-type of at Vh,2θ 5.5, B-, 17.1, and 22.3 a further (Figure crystal3). Both polymorphCA cross-linking that and may be associatedglycerol to facilitated A-type [18 the]. Thegrowth Vh-type of all polymorphcrystalline polymorphs. was substantiated Particularly, by reﬂexions the presence at 2θof 7.3,the 12.8, 19.6, the A-type at 2θ 15, 18.8 and the B-type at 2θ 5.5, 17.1, 22.3 (Figure3). Both CA cross-linking and glycerol facilitated the growth of all crystalline polymorphs. Particularly, the presence of the additives resulted in sharper peaks. The samples without plasticizer and cross-linker were almost Int. J. Mol. Sci. 2017, 18, 2075 5 of 12 Int. J. Mol. Sci. 2017, 18, 2075 5 of 12 additives resulted in sharper peaks. The samples without plasticizer and cross-linker were almost entirely amorphous. An exception was represented by the control starch treated only with CA that entirely amorphous. An exception was represented by the control starch treated only with CA that showed lower crystallinity than AO/CA. showed lower crystallinity than AO/CA.

Figure 3. Wide angle X-ray scattering (WAXS) diffractograms of extruded samples showing the effect Figure 3. Wide angle X-ray scattering (WAXS) diffractograms of extruded samples showing the effect of glycerol and CA cross-linking on crystal formation: (A) Control starch; (B) AO starch. Arrows of glycerol and CA cross-linking on crystal formation: (A) Control starch; (B) AO starch. Arrows highlight the A-type polymorph. highlight the A-type polymorph.

2.4. Mechanical Properties and Glass Transition 2.4. Mechanical Properties and Glass Transition The extrudedextruded specimens, specimens, after after equilibration equilibration at 57% at RH,57% were RH, tested were by tested tensile by tests tensile to characterize tests to characterizetheir stress and their strain stress behavior and strain in behavior standard in conditions. standard conditions. The utilization The utilization of a double of barrela double extruder barrel extruderand controlled and controlled water feeding water allowed feeding the allowed production the production of pure starch of pure extrudates. starch extrudates. The AO-extrudates The AO- extrudateshad superior had performance superior performance compared compared to the control to the starch control extrudates, starch extrudates, with AO with having AO five-fold having five-foldhigher stress higher at breakstressand at break 1.6-fold and higher 1.6-fold strain higher at break strain as at compared break as compared to the control to the starch control specimens starch (Figurespecimens4A–C). (Figure Lack 4A–C). of glycerol Lack of plasticizer glycerol plastici made thezer made specimens the specimens glassy and glassy brittle. and Cross-linkingbrittle. Cross- linkinghad a noteworthy had a noteworthy effect on effect the extrudates, on the extrudates which become, which become more flexible more andflexible cohesive and cohesive even without even withoutglycerol glycerol as a plasticizer as a plasticizer (Figure 4(FigureD). CA 4D). cross-linking CA cross-linking increased increased strain atstrain break at break for both for AOboth and AO andcontrol control starch starch specimens. specimens. The AOThe extrudatesAO extrudates with onlywith cross-linkeronly cross-linker (AOCA) (AOCA) samples samples showed showed a strain a strainat break at break thatwas that 1.6-foldwas 1.6-fold higher, higher, and and a stress a stress at breakat break that that was was two-fold two-fold higher higher than than thethe control (Figure4 4D).D). In In fact, fact, CA CA cross-linking cross-linking resulted resulted in in a bettera better plasticizing plasticizing effect effect than than glycerol, glycerol, as showedas showed by bycomparing comparing the stressthe stress and strainand strain curves curves of AO of with AO onlywith glycerol only glycerol and the and cross-linked the cross-linked samples samples without withoutglycerol. glycerol. The glycerol The plasticized glycerol plasticized samples had sample highers elasticityhad higher but lowerelasticity strength but aslower compared strength to theas comparedcross-linked to ones.the cross-linked (Figure4D,F). ones. The (Figure AO-based 4D,F). specimens The AO-based including specimens glycerol including with CA glycerol crosslinking with (AOglyCA)CA crosslinking extrudates (AOglyCA) had higher extrudates elasticity had but higher a lower elasticity strength but as a comparedlower strength to the as control compared (Figure to4 theE). controlHowever, (Figure for AO 4E). the However, use of only for glycerol AO the and use the of only glycerol/CA glycerol combinationand the glycerol/CA did not differ combination significantly did (Figurenot differ4B). significantly The effect of (Figure the cross-linker 4B). The oneffect the glassof the transition cross-linker temperature on the glass (Tg) transition of AO extrudates temperature was (Tg)monitored of AO byextrudates using dynamic was monitored mechanical by analysis using dynamic (DMA). mechanical The AO-based analysis specimens (DMA). including The AO-based glycerol, ± ◦ AOgly,specimens had including a Tg = 50 glycerol,1 C. Introducing AOgly, had CA a Tg cross-linking = 50 ± 1 °C. to thisIntroducing system (AOglyCA) CA cross-linking decreased to this the ± ◦ ± ◦ Tgsystem to 38 (AOglyCA)3 C. AOCA decreased had a Tgthe that Tg wasto 38 much ± 3 °C. higher AOCA than had all othera Tg that samples was (80much2 higherC), indicating than all other samples (80 ± 2 °C), indicating that CA formed a densely cross-linked network with the AO

Int. J. Mol. Sci. 2017, 18, 2075 6 of 12 Int. J. Mol. Sci. 2017, 18, 2075 6 of 12 thatchains. CA This formed network a densely was cross-linked very sensitive network to glycer withol the as AO demonstrated chains. This by network the very was low very Tg sensitive of the toAOglyCA glycerol specimens. as demonstrated by the very low Tg of the AOglyCA specimens.

Figure 4.4. The stress and strainstrain ofof extrudedextruded samples.samples. ( A) Control starch extrudates;extrudates; (B) AOAO starchstarch extrudates; (C) ComparisonComparison of purepure starchstarch (no(no glycerol)glycerol) extrudates;extrudates; ((DD)) ComparisonComparison ofof extrudatesextrudates cross-linkedcross-linked withwith CA;CA; (E(E)) Comparison Comparison of of extrudates extrudates cross-linked cross-linked and and with with glycerol; glycerol; (F )(F Comparison) Comparison of extrudatesof extrudates with with glycerol. glycerol. Error Error bar bar represent represent the the standard standard deviation. deviation.

2.5. Permeability Tests The permeabilitypermeability tests for cast AO and controlcontrol ﬁlmsfilms showedshowed performancesperformances similar to those ofof commercial plasticsplastics (Mater-Bi(Mater-Bi©©and and Low-Density Low-Density Poly-Ethylene, Poly-Ethylene, LD-PE) LD-PE) (Table (Table2). 2). The The permeability permeability to COto CO2 and2 and O2 Oof2 of the the starches starches analyzed analyzed was was comparable comparable to that to that of the of commercialthe commercial variants. variants. Mater-Bi Mater-Bi was superiorwas superior regarding regarding the water the water vapor vapor permeability permeabili (WVP)ty (WVP) permeability. permeability. Hence, Hence, the gravimetric the gravimetric tests showedtests showed a signiﬁcant a significant effect effect of glycerol of glycerol on water on water vapor vapor permeability. permeability. The The permeability permeability increased increased by increasingby increasing the the amount amount of glycerol of glycerol in the in the formulation formulation (Figure (Figure5). 5). This data supportssupports thethe highhigh potentialpotential behindbehind thethe useuse ofof starchstarch forfor thethe productionproduction ofof plastics.plastics.

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Int. J. Mol. Sci. 2017, 18, 2075 7 of 12 Table 2. Gas permeability of AO-based ﬁlms compared to selected commercially available materials. Table 2. Gas permeability of AO-based films compared to selected commercially available materials.

COCO2 2 OO2 2 WVPWVP PermebilityPermebility 3 2 3 2 3 2 (cm(cm·mm/m3·mm/m·2kPa·kPa 24 24 h) h) (cm(cm3·mm/m·mm/m2·kPa·kPa 24 24 h) h) (cm(cm3·mm/m·mm/m2·kPa· kPa24 h) 24 h) AO/glyAO/gly 4.0 4.0± ±0.2 0.2 0.6 0.6 ±± 0.030.03 0.1 ± 0.1 0.01± 0.01 AO/gly/CAAO/gly/CA 0.4 0.4± ±0.1 0.1 1 1 0.1 ± 0.1 0.03± 0.03 $ $ CT/glyCT/gly NANA $ NANA $ 0.1 0.1 CT/gly/CACT/gly/CA 0.50.5 0.5 0.5 0.02 0.02 ± ± ± Mater-BiMater-Bi (S-301) (S-301) 5.0 5.0 ±0.03 0.03 0.7 0.7 ± 0.0050.005 0.04 0.04 ± 0.0020.002 Mater-Bi ZIO1U/C 5.0 ± 0.02 0.5 ± 0.003 Na Mater-Bi ZIO1U/C 5.0 ± 0.02 0.5 ± 0.003 Na Mater-Bi (Z) Na Na 33 a a LD-PEMater-Bi (Z) Na Na Na Na 33 0.5 a LD-PE Na Na 0.5 a Na: not available a data published in Mariniello et al. 2007 [19], $ the ﬁlm was too fragile for the analysis. water a $ vaporNa: not permeability available (WVP). data published in Mariniello et al. 2007 [19], the film was too fragile for the analysis. water vapor permeability (WVP). 8 Amylose-only 7 Control 6 5 kPa 24h 2 4 3 mm/m 3 2 cm 1 0 30%gly 20%gly 10%gly

Figure 5. Permeability tested for non-crosslinked AO and control starch films as a function of glycerol Figure 5. Permeability tested for non-crosslinked AO and control starch films as a function of content. glycerol content. 2.6. Biodegradation of Grains and Extruded Prototypes 2.6. Biodegradation of Grains and Extruded Prototypes Controlled biodegradation in a composting system of milled AO and control barley grains, and Controlled biodegradation in a composting system of milled AO and control barley grains, and extruded specimens from starch prepared from these, was monitored as CO2 evolution (Figure 6, extrudedTables S1 specimensand S2). The from grain starch and prepared the extruded from sa these,mples was showed monitored significant as CO differences;2 evolution the (Figure milled6, Tablesgrain was S1 and degraded S2). The faster grain andthan the the extruded bio-plastics samples prototypes. showed No significant lag phase differences; in the beginning the milled of grain the wascomposting degraded was faster observed than the for bio-plastics any of the prototypes. samples demo No lagnstrating phase high in the biological beginning activity of the composting in the soil. wasNo significant observed fordifference any of the was samples found between demonstrating the AO high and biologicalthe control activity grain. Likewise, in the soil. the No AO significant and the differencecontrol starch was foundbio-plastic between prototypes the AO andshowed the control no significant grain. Likewise, difference, the AO and and there the controlwas also starch no bio-plasticsignificant prototypesdifference showedbetween nocros significants-linked and difference, non-cross-linked and therewas samples also no(Figure significant 6). The difference slower betweendegradation cross-linked of the extruded and non-cross-linked samples as compared samples (Figure to the6 ).milled The slower grain degradationwas most likely of the due extruded to the samplescompact asstructure compared of to the milledextrudates. grain wasEnzymatic most likely degradation due to the compactof solid structure substrates of theis extrudates.a surface Enzymaticphenomenon, degradation and therefore of solid strongly substrates affected is a surfaceby the accessibility phenomenon, of andthe enzymes therefore to strongly the substrate affected and by the accessibility of the enzymes to the substrate and the availability of moisture. The background CO the availability of moisture. The background CO2 evolutions of organic matter present in the soil were2 evolutionssimilar in both of organic tests. matterThe rate present of degradation in the soil werewas highest similar induring both tests.the first The 20 rate days, of degradation and decreasing was highestsignificantly during afterward. the first 20 After days, 100 and decreasingdays, the degr significantlyadation afterward.had the same After rates 100 days,as the the soil degradation reference hadconcluding the same that rates all astest the specimen soil reference were concludingfully degraded that allat testthat specimenstage. The were results fully demonstrates degraded at that stage.transgenic The resultsthermoplastic demonstrates AO starch that transgenic is not signif thermoplasticicantly different AO starch with isrespect not significantly to composting, different as withcompared respect to toa common composting, thermoplastic as compared starch. to a common thermoplastic starch.

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Figure 6. CO release during the biodegradation test of AO and control grain and bioplastics Figure 6. CO2 release during the biodegradation test of AO and control grain and bioplastics protopypes.protopypes. TheThe datadata hashas beenbeen subtractedsubtracted fromfrom the the soil soil reference reference data. data.

3.3. MaterialsMaterials andand MethodMethod

3.1.3.1. MaterialsMaterials BarleyBarley starchstarch usedused inin thisthis studystudy waswas extractedextracted andand puriﬁedpurified fromfrom twotwo barleybarley lines:lines: aa controlcontrol barleybarley andand amylose-only, amylose-only, a genetically a genetically modiﬁed modified line. The line. AO The line AO was generatedline was bygenerated RNA interference by RNA suppressinginterference allsuppressing three starch all branching three starch enzymes branching in the enzymes Golden Promisein the cultivarGolden backgroundPromise cultivar [20]. Allbackground chemicals [20]. used All were chemicals provided used by Sigma-Aldrichwere provided (St.by Sigma-Aldrich Louis, MO, USA). (St. ForLouis, composting MO, USA). trials, For ◦ 0 topsoilcomposting was collected trials, topsoil from fallowwas collected land located from in fallow the municipality land located of Taastrup,in the municipality Denmark (N of 55Taastrup,39 33”; ◦ 0 EDenmark 12 16 6”). (N 55°39′33″; E 12°16′6″). 3.2. Methods 3.2. Methods 3.2.1. Determination of Lipids 3.2.1. Determination of Lipids The lipid extraction was performed as described by Morrison and coworkers [21]. ButanolThe extractslipid extraction were applied was performed on a thin as described layer chromatography by Morrison and plate coworkers (TLC silica [21]. Butanol gel 60, Merckextracts Darmstadt, were applied Germany) on a thintogether layer chromatogr with appropriateaphy plate standards (TLC silica (PC: gel 60, Phosphatidylcholine, Merck Darmstadt, PG:Germany) Phosphatidylglycerol, together with appropriate PE: Phosphatidylethanolamine, standards (PC: Phosphatidylcholine, PS: Phosphatidylserine, PG: Phosphatidylglycerol, lysoPC: Lysophosphatidylcholine,PE: Phosphatidylethanolamine, and oleic PS: Phosphatidylseri acid (free fatty acid)).ne, lysoPC: The Lysophosphatidylcholine, TLC development was performed and oleic usingacid chloroform/ethanol/water/triethylamine(free fatty acid)). The TLC (30/35/7/35; developmentv/v/v/v ). Thewas TLC platesperformed were assessedusing usingchloroform/ethanol/water/triethylamine a Typhoon Trio variable-mode imager (30/35/7/35; (GE Healthcare, v/v/v/v). Brøndby, The TLC Denmark). plates were Lipid assessed extracts using from a starchTyphoon were Trio analyzed variable-mode using a phosphorousimager (GE Healthcare, assay [22]. Brøndby, Shortly, theDenmark). extracts Lipid together extracts with from a standard starch phosphatewere analyzed solution using (50 a to phosphorou 200 nmol) weres assay digested [22]. byShortly, incubation the extrac in 0.65ts mLtogether 72% perchloricwith a standard acid at 195phosphate◦C until solution colorless. (50 Afterward, to 200 nmol) 3.3 mLwere of digested water, 0.5 by mL incubation of 2.5% ammonium in 0.65 mL molybdate, 72% perchloric and 0.5acid mL at of195 10% °C ascorbicuntil colorless. acid (w/v Afterward,) were added 3.3 mL to the of solution.water, 0.5 The mL color of 2.5% was ammonium developed incubatingmolybdate, the and samples 0.5 mL inof a10% water ascorbic bath at aacid temperature (w/v) were of 80added◦C for to 10 the min. solution. Samples The were color rapidly was cooled developed on ice waterincubating bath andthe thesamples absorbance in a water was measuredbath at a temperature at 812 nm using of 80 a GENESYS°C for 10 min. 10 spectrophotometer Samples were rapidly (Thermo cooled Electron on ice Corporation,water bath and Madison, the absorbance WI, USA). was measured at 812 nm using a GENESYS 10 spectrophotometer (Thermo Electron Corporation, Madison, WI, USA).

Int. J. Mol. Sci. 2017, 18, 2075 9 of 12

Int. J. Mol. Sci. 2017, 18, 2075 9 of 12 3.2.2. Cross-Linking Evaluation by Differential Scanning Calorimetry 3.2.2. Cross-Linking Evaluation by Differential Scanning Calorimetry The cross-linking reaction was performed using CA and HP as a catalyst. During the heating The cross-linking reaction was performed using CA and HP as a catalyst. During the heating process (described below), the CA is converted to an acid anhydride, which reacts with the hydroxyl process (described below), the CA is converted to an acid anhydride, which reacts with the hydroxyl groups of the starch forming ester linkages. The cross-linking temperature was determined by DSC groups of the starch forming ester linkages. The cross-linking temperature was determined by DSC using a DSC 214 polyma (Netzsch, Selb, Germany) instrument. The experiments were carried out on using a DSC 214 polyma (Netzsch, Selb, Germany) instrument. The experiments were carried out on aliquots of 20 ± 1 mg of the polymer, which was placed in stainless steel air tight cells. A single scan aliquots of 20 ± 1 mg of the polymer, which was placed in stainless steel air tight cells. A single scan was performed from 30 to 140 ◦C by an incremental increase of temperature of 3 ◦C·min−1. was performed from 30 to 140 °C by an incremental increase of temperature of 3 °C·min−1. 3.2.3. Melt Processing 3.2.3. Melt Processing The extrusion process was performed on a laboratory scale co-rotating inter-meshing twin-screw extruderThe extrusion (Process 11,process Thermo was Fisherperformed Scientific, on a laborato Karlsruhe,ry scale Germany). co-rotating The inter-meshing barrel diameter twin-screw and its extruderlength-to-diameter (Process 11, ratio Thermo (L/D) wereFisher 11 Scientific, mm and 40:1,Karlsruhe, respectively. Germany). The extruder The barrel barrel diameter was fitted and with its length-to-diametera circular 3 mm die ratio nozzle. (L/D) The were extruder 11 mm was and powered 40:1, respectively. by a 1.5 kW The motor extruder and barrel the screw was speedfitted with was akept circular constant 3 mm at die 350 nozzle. rpm. The The extruder extruder had was seven powered internal by a and1.5 kW one motor external and heating the screw zones speed and was the kepttemperature constant profile at 350 wasrpm. optimized The extruder and had set asseven shown internal in Table and3. one A high external shear heating screw configurationzones and the wastemperature applied. profile The screw was configurationoptimized and was set as designed shown toin produceTable 3. A homogeneity high shear screw in the configuration formulations. wasPermanence applied. ofThe the screw formulation configuration in the heatingwas designed block was to produce permitted homogeneity by an inverted in the screw formulations. positioned Permanenceat the end of of the the sixth formulation heating block in the (115 heating◦C). Theblock raw was material permitted was by metered an inverted into thescrew extruder positioned by a atgravimetric the end of twin-screw the sixth heating feeder block (MT-S, (115 MiniTwin, °C). The Brabenderraw material Technologie, was metered Duisburg, into the Germany) extruder by at a gravimetricspeed of 0.8 twin-screw kg/h. Water, feeder or water (MT-S, plus MiniTwin, cross-linker Brabender was pumped Technologie, into the extruder Duisburg, using Germany) a peristaltic at a pumpspeed (Fillmasterof 0.8 kg/h. TypeWater, 421, or Delta water Scientific plus cross-linke Medical,r Storewas pumped Heddinge, into Denmark) the extruder at a speed using of a 6peristaltic mL/min pumpresulting (Fillmaster in a final Type ratio 421, of 1:0.5 Delta with Scientific the starch Medical, (dry weight, Store Heddinge, d.w.). Glycerol Denmark) was pumped at a speed into of the 6 mL/minextruder resulting using a second in a final peristaltic ratio of pump 1:0.5 (Gilsonwith the Inc., starch Minipulse (dry weight, 3) at a d.w.). speed Glycerol of 3 mL/min was resultingpumped intoin a the final extruder ratio of using 1:0.33 a withsecond the peristaltic starch (d.w.). pump Four (Gilson formulations Inc., Minipulse were 3) prepared at a speed per of starch 3 mL/min type resultingconsisting in of a starchfinal ratio mixed of 1:0.33 with water with the alone, starch water (d.w.). plus Four glycerol, formulations water plus were cross-linker, prepared andper starch water plustype cross-linkerconsisting of plus starch glycerol. mixed Thewith dry water starch:CA alone, ratiowater was plus 1:0.25, glycerol, and water all blends plus were cross-linker, prepared and by watertaking plus into accountcross-linker the dry plus weight glycerol. of starch. The dry starch:CA ratio was 1:0.25, and all blends were preparedThe nomenclatureby taking into ofaccount the samples the dry is weight indicated of starch. accordingly: starch type/plasticizer/cross-linker whereThe the nomenclature starch types were of the AO samples (amylose-only) is indicated or CT accordingly: (control), the starch plasticizer type/plasticizer/cross-linker gly (glycerol) and CA wherecross-linker. the starch For example,types were for AO a plasticized (amylose-only) cross-linked or CT (control), AO the nomenclature the plasticizer is: gly AOglyCA. (glycerol) and CA cross-linker. For example, for a plasticized cross-linked AO the nomenclature is: AOglyCA. Table 3. Screw configuration and temperature profile. The screws were organized to allow longer Tablepermanence 3. Screw of starchconfiguration into the and cross-linking temperature heating profile. blocks. The Thescrews numbers were representsorganized theto allow heating longer and permanencefeeding blocks of configurationstarch into the temperatures. cross-linking heating blocks. The numbers represents the heating and feeding blocks configuration temperatures.

Cross-LinkingCross-Linking Glycerol Glycerol Water/CA Water/CA Starch Starch 135135 °C◦C 125 125 °C ◦C 115 °C 115 ◦C 115 °C 115 ◦ 105C °C 105 ◦C 80 °C 80 ◦C 40 °C 40 ◦C 40 40 °C◦C

3.2.4. Fourier Transform Infrared Spectroscopy 3.2.4. Fourier Transform Infrared Spectroscopy The absorbance measurements were performed on an Arid-Zone MB100 FTIR instrument (ABB The absorbance measurements were performed on an Arid-Zone MB100 FTIR instrument (ABB Bomen, QC, Canada) equipped with an attenuated total reflectance (ATR) device with a triple-bounce Bomen, QC, Canada) equipped with an attenuated total reﬂectance (ATR) device with a triple-bounce diamond crystal. IR spectra were recorded in the range from 4000–550 cm−1 with a spectral resolution diamond crystal. IR spectra were recorded in the range from 4000–550 cm−1 with a spectral resolution of 8 cm−1. The milled sample was squeezed against the crystal surface with a concave needle of 8 cm−1. The milled sample was squeezed against the crystal surface with a concave needle compressor. Each spectrum represents the average of 32 scans ratio against the background (64 scans compressor. Each spectrum represents the average of 32 scans ratio against the background (64 scans measured on the surrounding air) and analysed in triplicate. Data analysis (pre-processing, principal measured on the surrounding air) and analysed in triplicate. Data analysis (pre-processing, principal component analysis—PCA) was performed using LatentiX (v.2.12) in the range 1500–1800 cm−1. component analysis—PCA) was performed using LatentiX (v.2.12) in the range 1500–1800 cm−1.

Int. J. Mol. Sci. 2017, 18, 2075 10 of 12

3.2.5. Wide-Angle X-ray Scattering WAXS of the hydrated samples was performed using the SAXSLab instrument, NBI, University of Copenhagen, equipped with a 100XL + micro-focus sealed X-ray tube (Rigaku) with a 1.54 Å beam. The water content of the samples was adjusted by water phase sorption for 14 days in desiccators at a relative humidity of 85% (saturated KCl) [18].

3.2.6. Mechanical Properties Prior to any measurements, the samples were equilibrated in a desiccator at 57% ± 1 relative humidity (saturated NaBr). The specimens were analyzed using a texture analyzer (TA, Stable Micro Systems, Surrey, UK) equipped with a 300 N tensile load cell. The distance between the clamps was set at 10 mm and the crosshead speed was set at 10 mm min−1. The elongation and the tensile stress at break were measured at 25 ◦C in triplicate.

3.2.7. Dynamic Mechanical Analysis A DMA Q800 (TA Instruments, New Castle, DE, USA) was used in cantilever mode with an amplitude of 0.1% at a frequency of 10 Hz, standard heating rate of 3 ◦C·min−1 and a ramp between −60 to 150 ◦C. The experiments were carried out on prototypes with a length of 4 cm and an average width of 5 ± 0.1 mm.

3.2.8. Permeability to Gases Gas permeability was monitored using thin films prepared by casting. A 2% (w/w) starch suspension was gelatinized using a microwave oven for 3 min in a Duran bottle closed using a membrane screw-cap to avoid over-pressure. After gelatinization, the samples were stirred for 5 min at 300 rpm and poured in the petri dishes coated with teflon, and preheated at 70 ◦C at a surface density of 20 mg/cm2. The suspensions were dried at 70 ◦C for 3 h using maximum oven ventilation and there ◦ after at 50 C for 10 to 12 h without ventilation. CO2,O2, and H2O permeability were determined using the American Society for Testing and Materials (ASTM) Standard Method D 3985 (2010) and F1249 (MultiPerm apparatus-ExtraSolution s.r.l., Pisa, Italy). Duplicate samples were conditioned for 2 days at 50% RH before the measurement. Aluminum masks were used to reduce film test area to 5 cm2. The testing was performed at 25 ◦C and 50% RH. The effect of glycerol plasticizer on the water vapor permeability was performed by a gravimetric test according to ASTM E96 using in-house designed permeability cups in triplicate. The experiments were performed in a desiccator with an RH of 84% using a saturated solution of KCl. The samples were weighed every 30 min the first day (totally 16 measurements) and then every h for 8 h on the following days for at least three days (eight measurements/day).

3.2.9. Biodegradation Test The biodegradation test was slightly modiﬁed from the ASTM standard [23]. Large size impurities such as stones and organic materials i.e. leaves, roots were removed from the soil using a 10-mm metal screen. The soil was subsequently screened to a particle size of <2 mm using a sieving tower with standardized metal screens and a vibrating plate (Retsch, Haan, Germany) (Figures S3–S5). pH was determined in duplicate according to the working document “Determination of pH in soil, sewage sludge and bio-waste” STD5151 [24]. The extruded, bio-plastic prototypes were cut into 5 mm long tube-shaped pieces. Bio-plastic grain samples were separated from ﬁnes using a sieving tower and screens with 2 mm mesh size. 2 g of each material was buried in the soil at 5–10 mm below the surface. For CO2 capture and maintenance of a moist atmosphere, a set of beakers containing 20 ml de-mineralized water and 20 mL of KOH (0.5 N) were placed on the perforated plate inside the desiccators. These were sealed and placed in a dark chamber in a climate controlled room at a ◦ temperature of 20 ± 1 C. The desiccators were ventilated at regular intervals, and the CO2 traps were Int. J. Mol. Sci. 2017, 18, 2075 11 of 12

renewed on a regular basis following the standard procedure. Evolved CO2, absorbed into the KOH solutions was determined by titrating triplicates with HCl (0.5 N) to a phenolphthalein end-point.

4. Conclusions Massive thermoplastic polymer production is leading to an extensive accumulation of micro-plastics in the environment, and is becoming an increasing environmental threat. All-natural, biodegradable bioplastics can form part of this solution, but performance must be improved. We produced and tested extruded and CA cross-linked specimens prepared from AO barley starch. This material had superior mechanical performance as compared to specimens from normal barley starch. Cross-linking produced an additional crystal related to the A-type polymorph. CA cross-linking improved mechanical strength and the cross-linked AO extrudates had superior stress at break and elongation at break. Glycerol plasticizer decreased its performance, and despite its high elasticity, the Tg of the cross-linked AO extrudates was increased indicating the presence of a strongly cross-linked network. AO contained amylose-complexed phospholipids, with free fatty acids constituting an integrated part of the Vh crystals in the specimens. Permeability to CO2,O2, and H2O was comparable to commercial starch-based blends. Biodegradation in composting systems demonstrated that AO designer starch and cross-linked starch degraded at the same rate as normal starch. This new material showed that bioplastic produced using a polysaccharide from a genetically modified plant could be a functional alternative to specific flexible plastics and opens new possibilities for the production of biodegradable materials from e.g., polysaccharides, produced directly in planta, having properties specifically tailored for bioplastics applications.

Supplementary Materials: Supplementary materials can be found at www.mdpi.com/1422-0067/18/10/2075/s1. Acknowledgments: This work was funded by The Danish Council for Independent Research Technology and Production Sciences and Carlsberg Foundation. We acknowledge Thomas Günther-Pomorski for valuable discussions on lipid analysis. Author Contributions: Domenico Sagnelli, Andreas Blennow, and Kell Mortensen conceived and designed the experiments; Domenico Sagnelli performed the experiments for extrusion, DSC, DMA, mechanical properties, and gravimetric permeability; Mette Holse performed the experiments for FTIR; Kourosh Hooshmand and Gerdi Christine Kemmer performed extraction and analysis of starch lipids, Jacob K. Kirkensgaard performed the WAXS experiments; Valeria Giosafatto performed the permeability experiments; Wolfgang Stelte, and Anne-Belinda Bjerre performed the degradation assays; Domenico Sagnelli and Mette Holse analyzed the data; Kim H. Hebelstrup, and Jinsong Bao contributed with grain material; Domenico Sagnelli and Andreas Blennow assembled all data and written contributions from the co-authors, and organized and completed the manuscript. Conﬂicts of Interest: The authors declare no conﬂict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

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