**Biopolymers from Natural Resources**

Editors

**Rafael Antonio Balart Gimeno Marina Patricia Arrieta Dillon Daniel Garc´ıa Garc´ıa Lu´ıs Jes ´us Quiles Carrillo Vicent Fombuena Borr´as**

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This is a reprint of articles from the Special Issue published online in the open access journal *Polymers* (ISSN 2073-4360) (available at: https://www.mdpi.com/journal/polymers/special issues/ Biopolymers Natural Resources).

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## **Contents**

Reprinted from: *Polymers* **2021**, *13*, 384, doi:10.3390/polym13030384 ................ **107**

Thermomechanical Behavior Reprinted from: *Polymers* **2020**, *12*, 2248, doi:10.3390/polym12102248 ................ **259**

Reprinted from: *Polymers* **2020**, *12*, 1097, doi:10.3390/polym12051097 ................ **385**

## **About the Editors**

**Rafael Antonio Balart Gimeno** (h-index 41, Scopus) is a faculty staff member in the Department of Mechanical and Materials Engineering, in the area of Materials Science and Metallurgical Engineering. He has been a Full Professor at Universitat Politecnica de Val ` encia since 2015. ` His research activity has been developed in the Institute of Materials Technology at UPV in the "RESEARCH GROUP ON POLYMERS AND GREEN COMPOSITES - GiPCEco", a group that he has led since its formation. This group focuses its activity on the following research lines, all of them with a marked environmental concern: (1) the potential of polymers of renewable origin and their transfer to industrial sectors; (2) minimizing the impact of additives and active ingredients of natural origin; (3) the development of materials under the "bio-refinery" concept; (4) the use of waste from the agro-food industry and agro-forestry sector through their incorporation into polymer and composite formulations; and (5) circular economy.

**Marina Patricia Arrieta Dillon** (h-index 30, Scopus) works at the Department of Industrial and Environmental Chemical Engineering at the School of Technical Industrial Engineering of the Technical University of Madrid, (ETSII-UPM) Madrid, Spain. She holds a Biochemistry BS from the National University of Cordoba (Argentina), a MS in Food Technology from the Catholic ´ University of Cordoba (Argentina), a MS in Polymer Science and Technology from UNED (Spain), and an international PhD in Science, Technology and Food Management from the Polytechnic University of Valencia (Spain), awarded with the Extraordinary PhD Thesis Award. She has been an AECID and Santiago Grisol´ıa predoctoral fellow, as well as a Juan de la Cierva (formacion and ´ Incorporacion) and UCM postdoctoral fellow. Dr. Arrieta has experience on the synthesis, processing ´ (melt-blending, extrusion, injection moulding, electrospinning, etc.), recycling, and characterization of multifunctional sustainable polymers and their nanocomposites with active and multifunctional properties for sustainable food packaging or agricultural applications.

**Daniel Garc´ıa Garc´ıa** (h-index 19, Scopus) is an Associate Professor of Materials Science and Engineering at the Department of Mechanical and Materials Engineering (UPV Campus d'Alcoi). He is a Technical Engineer in Industrial Design and Materials Engineering. He studied a Master's degree in Engineering, Processing and Characterisation of Materials and obtained an international Ph.D. in Engineering and Industrial Production with a national FPU grant at the UPV. He was awarded with the National Award for End of Degree in University Education by the Spanish Government and with the Extraordinary Doctoral Thesis Award from the UPV. He joined the Technological Institute of Materials (ITM) in 2009 and specializes on the development and formulation of sustainable polymers. His areas of interest include the extraction of active compounds from agroforestry residues for use as additives in biopolymers and the development and characterization of bionanocomposites.

**Lu´ıs Jes ´us Quiles Carrillo** (h-index 18, Scopus) works at the Department of Mechanical and Materials Engineering of the Escuela Politecnica Superior de Alcoy (EPSA) of the Universitat ´ Politecnica de Val ` encia, (UPV) Alcoy, Spain. He holds a degree in Mechanical Engineering, a Master's ` degree in Engineering, Processing and Characterisation of Materials, and obtained an international PhD in engineering and industrial production with a national FPU grant at the UPV. He was awarded the Best Doctoral Thesis in Polymers Award by the Royal Spanish Society of Chemistry and Physics (RSEQ and RSEF), granted by the group specialised in polymers (GEP).

His research work has focused on the search for new formulations of polymers with high environmental performance from waste and natural compounds. He has extensive experience in the characterisation of polymeric and composite materials, the reuse of waste and by-products for the creation of highly eco-efficient packaging, and works extensively with techniques such as electrospinning and polymer injection moulding.

**Vicent Fombuena Borr´as** (h-index 17, Scopus) works at the Department of Nuclear and Chemical Engineering of the Escuela Politecnica Superior de Alcoy (EPSA) of the Universitat ´ Politecnica de Val ` encia, (UPV) Alcoy, Spain. He holds degrees in Technical Engineering in Industrial ` Chemical Engineering and Materials Engineering. He joined the Technological Institute of Materials (ITM) in 2008 as a research technician and obtained an international PhD in Engineering and Industrial Production in 2012. His research work has focused on the implementation of a circular economy model in the polymer industry. He has focused his efforts on obtaining multiple active compounds from seeds and agroforestry residues to be applied in thermoplastic and thermosetting polymers. He has extensive experience in techniques of the chemical, thermal and mechanical characterization of materials.

## **Preface to "Biopolymers from Natural Resources"**

The increase in the production and application of biobased polymers has been posed as an extremely promising method of sustainable development goals, offering the option of replacing traditional petroleum polymers in several industrial sectors. In this context, there is great interest in biobased building blocks, including: the revalorization of agri-food wastes; biopolymers extracted directly from biomass, such as polysaccharides and proteins, as well as those produced by yeast biomass or by bacterial fermentation; medical devices; food packaging; agricultural films; membrane process applications, and so on. Despite the successful development of biobased polymers to an industrial scale, huge interest in their optimization still exists in order to extend their industrial exploitation. Therefore, significant attention has been given to the improvement of these sustainable plastics' overall performance, with the hope of further expanding their advantageous properties. As a versatile product, biobased polymers can be modified through chemical synthesis, copolymerization, or surface modification, as well as by the use of different additives, i.e., micro and nanoparticles, plasticizers, and active agents, among others.

This work covers all aspects related to recent, original, and cutting-edge research works focused on enhancing the performance of biopolymers from natural resources, and emphasising their potential use in industrial sectors, in line with the worldwide trend of building a circular economy. Each chapter is concerned with a given polymer's sustainable origin, in addition to their prospective obtainment, production, design, and processing at an industrial level, as well as possible improvements for specific industrial applications.

We acknowledge each author for their substantial contribution to the research field of biopolymers from natural resources.

### **Rafael Antonio Balart Gimeno, Marina Patricia Arrieta Dillon, Daniel Garc´ıa Garc´ıa, Lu´ıs Jes ´us Quiles Carrillo, and Vicent Fombuena Borr´as** *Editors*

## *Editorial* **Biopolymers from Natural Resources**

**Rafael Balart 1,\*, Daniel Garcia-Garcia 1,\*, Vicent Fombuena 1,\*, Luis Quiles-Carrillo 1,\* and Marina P. Arrieta 2,3,\***

During the last decades, the increasing ecology in the reduction of environmental impact caused by traditional plastics is contributing to the growth of more sustainable plastics with the aim to reduce the consumption of non-renewable resources for their production. Thus, in the last few years, the increased bio-based polymer production and application have positioned biopolymers as one of the most promising ways to meet the sustainable development goal of replacing traditional petroleum polymers with more sustainable materials in several industrial sectors.

In this context, the revalorization of agro-food wastes, biopolymers extracted directly from biomass such as polysaccharides, proteins and lipids, as well as those produced by yeast biomass, algae or by bacterial fermentation, have attracted a significant amount of interest, especially for medical devices, food packaging, agricultural films, membrane process applications, sustainable clothes and so on—interest which, despite successful recent developments in bio-based polymers up to the industrial scale, extends to their optimization for industrial exploitation. Therefore, significant attention has been paid to the improvement of those sustainable plastics directly derived from biomass overall performance to provide more than one advantageous property attributed to their versatility to be modified through chemical synthesis, copolymerization, and surface modification, as well as through the use of different additives (i.e., micro- and nanoparticles, plasticizers, and active agents, among others). Moreover, nowadays, the starting monomers or building blocks to obtain traditional plastics can be obtained from biomass instead of petrochemical resources, known as drop-in bioplastics. Drop-in bioplastics present the same advantages of traditional plastics in terms of performance and are more sustainable, while at the same time they can be directly transformed into the desired products by means of the same technology already available at the plastic industrial sector. Figure 1 summarizes several biobased polymers classified on the basis of their origin and obtainment process.

The present Special Issue gathers a series of twenty-four articles focused on the manufacturing and characterization of biopolymers and building blocks extracted from natural resources, as well as their potential scalability to the industrial level for several industrial applications. The Special Issue paid special focus on the improvement of the overall performance of biobased polymers by chemical modification process, the incorporation of sustainable additives such as biobased oligomers, by blending with another biobased or biodegrable polymeric matrix, as well as by the development of composites and nanocomposites through the incorporation of naturally occurring particles and nanoparticles.

**Citation:** Balart, R.; Garcia-Garcia, D.; Fombuena, V.; Quiles-Carrillo, L.; Arrieta, M.P. Biopolymers from Natural Resources. *Polymers* **2021**, *13*, 2532. https://doi.org/10.3390/ polym13152532

Received: 30 June 2021 Accepted: 20 July 2021 Published: 30 July 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

1

**Figure 1.** Classification of biobased polymers.

Among other naturally derived biopolymers, polysaccharides are nontoxic and biodegradable, which increases their potential application in several fields. Polysaccharides, with cellulose as the most important representative since it is the most abundant polymer in nature and is found all around the world, are widely investigated. Although the most-used biopolysaccharides are obtained from plant origin (e.g., cellulose), they can be obtained from animal origin (e.g., chitin/chitosan) and also from microbial origin (e.g., bacterial cellulose). In this sense, the production of biobased polymers by using microorganisms has gained considerably attention during the last decade as a sustainable production method. In this regard, Irbe et al. [1] developed novel blends based on biopolysaccharides derived from biomass, softwood cellulose fibers such as softwood Kraft (KF) or hemp fibers (HF) and fungal fibers (hyphae). Regarding the fungal fibers, they concluded that among several fungal fibers, the fibers from screened basidiomycota fungi *Ganoderma applanatum* (Ga), *Fomes fomentarius* (Ff), *Agaricus bisporus* (Ab), and *Trametes versicolor* (Tv) were good for blending with softwood cellulose fibers. Thus, the fungal fibers were blended with KF and HF fiber pulp in different mass ratios (i.e., 50:50 and 33:33:33). The materials were characterized in terms of mechanical properties, air permeability and virus filtration efficiency. The overall performance of the hyphal polysaccharide/cellulose blends was highly affected by the microstructural features of each cellulosic material, while the ability of functional groups of hyphal polysaccharides to establish hydrogen bonding interactions with cellulose fibers was one of the key elements of network formation which determined the final blend properties and the potential applications. In this context, highly fibrillated hemp fibers showed the highest mechanical strength, while the blends containing Kraft fibers increased air permeability and showed a high virus reduction capacity. Thus, depending on the intended use the different microstructure of Hemp fibers or Kraft fibers will allow obtaining materials with interesting properties. For instance, materials with a low air permeability are interesting for food packaging applications where high gas barrier properties are of fundamental importance. Meanwhile, materials with a higher air permeability and virus filtration efficiency have potential for being used as gas permeable membranes, for example, as biobased filter layers in face masks. Despite its outstanding characteristics, cellulose in its native form is hard and provides some limitations for the plastic industry in terms of cellulose processing. Nevertheless, cellulose derivatives can overcome such problems. Thus, cellulose chemical modifications have been widely

studied and nowadays several cellulose derivatives can be found commercially such as carboxymethyl cellulose (CMC), methyl cellulose (MC), hydroxyethyl cellulose (HEC), hydroxypropyl methylcellulose (HPMC), and cellulose acetate (CA).

Among others, CMC possesses good film-forming abilities because of the presence of a hydrophilic carboxyl group (–CH2COONa), which allows its dissolution in water. In this context, Rachtanapun et al. [2] revalorized coconut juice, a waste product from the coconut milk industry, into bacterial cellulose using *Acetobacter xylinum* to further obtain carboxymethyl cellulose (CMC) through chemical synthesis with different degrees of substitution. With this propose in mind, CMC was obtained from bacterial cellulose in the presence of monochloroacetic acid (MCA) via a carboxymethylation reaction and by simply varying the sodium hydroxide content (NaOH from 20% to 60%), the degree of substitution was controlled. The degree of substitution significantly increased with NaOH concentration up to 30% and then progressively decreased with further NaOH concentrations. As CMC is a water soluble cellulose derivative, the higher the degree of substitution, the higher the water vapor permeability. The flexibility of the material also increased with the degree of substitution. Thus, the developed method to modify cellulose from nata de coco allows tuning of the water permeability and the mechanical properties of the final material according to the need of the intended application by simple varying the NaOH content. CMC with different degrees of substitutions has been also obtained by Kluklin et al. [3] from the *Asparagus officinalis* stalk end, a typical herbal medicine used in Asia, by simply varying the NaOH content (from 20% to 60%) too, and it was also found that there is a higher degree of substitution for the NaOH 30% (*w*/*v*). CMC has been also used in the development of solid polymer electrolyte (SPE) film for sustainable energy storage systems due to its solubility in water and its naturally high degree of an amorphous phase that allows easier transport of conducting ions like lithium (Li+) and proton (H+). In this context, Sohaimy et al. [4] tuck this advantageous properties of CMC to develop CMC-based films for electrochemical applications. However, the inherent solid properties of the polymeric film impede ionic mobility leading to a low ionic conductivity, especially at room temperature. Thus, ammonium carbonate (AC), with two groups of ammonium ions, was used as a dopant in amounts from 1 wt.% to 11 wt.% with the main objective of injecting more H+ into the system and the CMC-AC thin electrolyte film was obtained by a solvent casting method. Both CMC and AC salts dissolved completely and had a low degree of crystallinity (Xc) as it was demonstrated by X-ray diffraction (XRD). Although, the ionic conductivity for a biopolymer electrolyte intended for commercial energy storage technology needs to be at least a minimum value of ~10−4 Scm−1, they obtained promising results since the highest ionic conductivity obtained for CMC doped with 7 wt.% was 7.71 × 10−6 Scm−1, resulting in higher results than other diammonium salts tested (i.e., ammonium adipate and ammonium sulfate), showing that further enhancement is still needed to increase the ionic conductivity for suitable electrochemical applications (~10−4 Scm−1) and the CMC-AC films show promising prospects for electrochemical applications.

Panraksa et al. [5] developed orodispersible films (ODF) based on hydroxypropyl methylcellulose (HPMC: E5 and E15) by extrusion-3D printer technology to enhance the solubility and dissolution of poorly water-soluble drugs (i.e., phenytoin). ODFs are required to rapidly disintegrate in the buccal cavity (i.e., within a minute), without requiring water, thus HPMC, which is a well-known hydrophilic and film-forming polymer, is an ideal candidate for this propose. HPMC E5 and E15 were used as the film-forming polymers, while propylene glycol and glycerin were used as plasticizers. They optimized the processing conditions and the results showed that the phenytoin-loaded HPMC E15 (at a concentration of 10% *w/v* of HPMC E15) was the most suitable formulation of HPMC, since it exhibited a good physical appearance, good mechanical strength, low Young's modulus and high elongation at break, rapid in vitro disintegration time (within 5 s) as well as a rapid drug release (up to 80% within 10 min), showing the improvement of

solubility and dissolution rate of phenytoin at the same time as allowing ease of handling and application.

Another widely used cellulose derivative is cellulose acetate (CA). For instance, it has been used by Arrieta et al. [6] as a carrier of *Cucumis metuliferus* fruit extract to provide to corona-treated low-density polyethylene (LDPE) films with antioxidant activity by coating a CA-based polymeric solution, containing different amounts of *Cucumis metuliferus* fruit extract (1, 3 and 5 wt.%), onto the LDPE film. The materials showed antioxidant activity (between 0.3 and 0.6 mg Trolox/dm2) after performing migration studies in a fatty food simulant and also showed their ability to reduce the browning effect of fresh-cut apples in direct contact. Additionally, the coating not only provided an antioxidant active functionality, but also improved the oxygen barrier performance to the final bilayer material, showing their use in active food packaging applications.

Another interesting approach to obtain a solution of insoluble cellulose is regenerated cellulose (RC). RC refers to the chemical dissolution of insoluble natural cellulose followed by the recovery of the material from the solution by means of different methods, such as cellulose carbamate processes, lyocell processes, and viscose processes. In this context, Husseinsyah et al. [7] used an ionic liquid (1-butyl-3-methylimidazolium chloride) as a novel method to obtain RC from empty fruit bunches (EFB) to develop a self-reinforced composite for biodegradable packaging applications. They studied the possibility of improving the interphase bonding between the cellulosic matrix and the filler in the selfreinforced composite by means of EFB chemical treatment with a methyl methacrylate (MMA) chemical treatment. They observed that the substitution of the –OH group of the EFB cellulose with the ester group of the MMA allowed the dissolution process of the EFB during the regeneration process owing to weakening of the hydrogen bonding of the cellulose. This allowed us to obtain a more crystalline and homogeneous EFB RC biocomposite film, with an improved thermal stability and mechanical performance. Another very important cellulosic material is cotton, which is widely distributed worldwide. It has a higher yield compared to other commercial crops, and thus several products are obtained from cotton seed, such as oil. In this context, Chen et al. [8] used bio-based cottonseed oil (CO) and two of its derivatives, fatty acid of cottonseed oil (COF) and sodium soap of cottonseed oil (COS), to tune the density and mechanical strength of polysulfide polymers by means of a free radical addition mechanism. COF reacts with sulfur to generate serials of polysulfide-derived polymers with a lesser viscosity and tensile strength. Whereas COS was not involved in the reaction with sulfur, as a consequence of the high melting point of sodium linoleate and sodium oleate. Nevertheless, COS it was able to increase the density and tensile strength of polysulfide-derived polymers when it was used as a filler. Moreover, the developed polysulfide-based polymers showed good reprocessability and recyclability, showing their potential as bio-based functional supplementary additives.

Ammar et al. [9] developed a novel adsorbent material for cationic dyes based on a polyelectrolyte multi-layered (PEM) system cross-linked to a cellulosic-based material. The PEM was formed by an alternation of layers of two polysaccharides, sodium alginate polyanion and reticulated citric acid with k-carrageenan, thus providing many carboxylate and sulfonate groups grafted on the cellulosic surface. The developed system provides outstanding adsorption capacities for cationic dyes (e.g., with a capacity above 522.4 mg/g for methylene blue).

The processing and revalorization of lignocellulosic materials obtained from agro-food wastes, as well as the extraction through sustainable approaches, have gained considerable interest. Gómez-Patiño et al. [10] extracted cellulosic cutins from of *S. aculeatissimum* and *S. myriacanthum* fruit peels, using enzymatic (i.e., *A. niger pectinase*, *A. niger cellulose* and *A. niger hemicellulose*) and chemical (trifluoroacetic acid hydrolysis) methods. They selected these fruits since they are not for human or animal consumption and are thus interesting as a potential raw material to produce sustainable materials. The hydrolyzed cutins showed mainly 10,16-dihydroxyhexadecanoic acid (10,16-DHPA) monomers composition and the obtained films showed a good homogeneity.

Lignocellulosic derivatives have also gain considerable interest as micro and nanofillers. In this context, Adamcyk et al. [11] studied the extraction of lignin from wheat straw, spruce, and beech using ethanol organosolv pretreatment at temperatures from 160–220 ◦C, and further precipitated by solvent-shifting obtaining lignin micro- and nanoparticles (with mean hydrodynamic diameters from 67.8 nm to 1156.4 nm). They observed that higher pretreatment temperatures increased the delignification of the raw materials but also favor depolymerization and structural alteration of the extracted lignin, increasing the particle sizes as well as agglomeration at pretreatment temperatures of over 200 ◦C. The obtained particles were then purified by dialysis and the showed interesting antioxidant activity (i.e., from 19.1 and 50.4 mg Lignin/mg ascorbic acid equivalents).

On the other side, lignocellulosic nanoparticles have been obtained from yerba mate (*Ilex paraguariensis*) waste, a typical infusion widely consumed in Latin America (i.e., Argentina, Brazil, Uruguay and Paraguay), by means of an aqueous extraction procedure (with mean average size of 495 nm). The obtained nanoparticles were used by Beltrán et al. [12] to reinforce mechanically recycled bioplastic (i.e., polylactic acid). Poly(lactic acid) (PLA) is chemically synthesized starting from simple sugars obtained from biomass fermented to lactic acid. It is one of the most promising biopolyesters for massive industrial applications due to its availability in the market at a competitive cost, and it can be recycled following the mechanical recycling process used for other traditional plastics (e.g., polyethylene terephthalate, PET). Low amounts of yerba mate nanoparticles (i.e., 1 wt.%) were able to increase the intrinsic viscosity and to improve the oxygen barrier properties of mechanically recycled PLA, showing the interest of such nanocomposites in the food packaging field. Ramos et al. [13] also studied PLA-based nanocomposites for food packaging applications. In this case, PLA was loaded with commercial montmorillonite (D43B) at two different concentrations (2.5 and 5 wt.%) and was further incorporated with thymol as an antioxidant and antimicrobial agent for active food packaging applications. The results showed that the addition of 2.5 wt.% D43B and 8 wt.% thymol leads to a material with a good balance of properties, with antibacterial activity against *Escherichia coli* and *Staphylococcus aureus.*

Another interesting source for sustainable additives are seeds fruits, peel and/or nut's shells, from which can be obtained flours and natural particles and/or nanoparticles, as well as vegetable oils. In this regard, Jorda-Reolid et al. [14] obtained micronized Argan shell (MAS) particles from residues of the *Argania spinosa* plant, a tropical plant whose fruit is used in Morocco to prepare oil. The authors used MAS particles (with mean average long of 70 μm and mean average wide of 45 μm) as reinforcing fillers of high-density polyethylene obtained from sugarcane (Bio-HDPE). Meanwhile, to improve the low compatibility between MAS and Bio-HDPE, they studied two compatibilizing agents, polyethylene-grafted maleic anhydride (PE-g-MA) and maleinized linseed oil (MLO); as well as halloysite nanotubes (HNTs), a second reinforcing filler. The obtained materials showed improved stiffness, high ductility, and good thermal stability, as well as a visual appearance very similar to that of reddish-color woods, showing their interest as wood plastic composites with the additional advantage of being chipper rather than neat Bio-HDPE. González Martínez et al. [15] optimized the carbonation reaction of epoxidized linseed oil (ELO) with carbon dioxide (CO2) in the presence of tetrabutylammonium bromide (TBAB) as catalysts, to obtain carbonated vegetable oils (CVOs), which have potential applications as interesting starting materials for the formation of polymers (e.g., monomers, additives, lubricants and plasticizers), particularly in the synthesis of nonisocyanate polyurethanes (NIPUs). They were able to obtain large conversion (96%), carbonation (95%), and selectivity (99%) at low temperature (90 ◦C), and thus the high carbonate content obtained for carbonated ELO showed their promising performance for the synthesis of NIPU with the required properties and more sustainable characteristics. On the other hand, Dominici et al. [16] studied the possibility of obtaining bioplastics

from wheat flour particles as a novel source and an energetically and economically cheap alternative to other thermoplastics. The refined flours, with different contents of bran, were first plasticized with glycerol and then the authors attempted to find how different contents of grinded bran could affect the deformability of the flour by blending with low melting polymeric fractions (such as poly(ε-caprolactone) (PCL), and polybutyleneadipate-*co*-terephthalate (PBAT)). They studied different processing parameters and they were able to obtain a novel thermoplastic wheat flour (TPWF), which showed an improved performance when glycerol was partially replaced by water and when it was blended with PCL, as well as adding citric acid as a compatibilizer.

Ferrándiz et al. [17] studied the potential use of soy protein (SP) fibers functionalized with an undisclosed antimicrobial agent as well chitin (with inherent antimicrobial properties) fibers as sustainable active textiles to replace synthetic polymers widely used in this sector. The thermal resistance of both weft-knitted fabrics was similar to that of cotton, whereas their air permeability was higher, particularly in the case of chitin due to its higher fineness, which makes these natural fibers very promising for summer clothes.

After cellulose, starch is the second most abundant polymer in nature and, thus, has been widely studied for several sustainable industrial applications. Zhang et al. [18] developed potato starch-based foams by means of a microwave treatment. They blended potato starch with chitosan as a reinforcing phase and it was found that starch-based foams with a larger proportion of starch showed a small pore size and a low density with higher compressive strength ascribed to the good compatibility between both polymeric matrices due to the formation of hydrogen bond interactions between amino groups of chitosan and hydroxyl groups of starch. Those interactions were also responsible to maintained intact the morphological structure of the foam in water for 10 days, while the starch-based foams completely degraded in water after 30 days. Thus, the developed starch/chitosan foams resulted in interesting results for their use as active materials in biomedical applications as well as in drug and food packaging.

For several industrial applications, starch is particularly used in its thermoplastic form, known as thermoplastic starch (TPS). To obtain TPS, native starch requires disruption of the granule organization by means of a combination of high water content and heat, leading to the starch granule swelling and consequently starch gelatinization. In this regard, Crucean et al. [19] studied the gelatinization process of three native starches: (i) wheat, (ii) potato, and (iii) waxy corn starch, as well as starch from a wheat flour in mixtures of water and choline chloride (an ionic compound, which has a "structure making" effect). They demonstrated that choline chloride/water system exhibits an allotropic change at low water concentrations and solubilization for water contents greater than 30%. They observed a stabilization, or a better organization, of the ordered regions of the starch in the presence of choline chloride as it was demonstrated by an X-ray diffraction analysis in a heating cell that the crystalline rearrangement of the structure of the starch grain takes place simultaneously with the solubilization phenomenon of amylose.

Osman et al. [20] studied thermoplastic starch (TPS)-based biocomposites using dolomite (DOL) as a filler in its pristine form (DOL(P)), as well as dolomites, after a simple and scalable sonication process (DOL(U)). TPS-DOL biocomposites intended for packaging applications were prepared at different loadings (i.e., 1, 2, 3, 4 and 5 wt.%). The TPS-based biocomposites with a high dolomite loading (i.e., 4 and 5 wt.%) showed better mechanical performance, showing greater tensile and tear properties, particularly in the case of biocomposites loaded with a sonicated process assisted by dolomite due to a reduction in particle size allowing their better dispersion within the TPS matrix. Thus, the high abundancy and low cost of dolomite, in combination with the simple, scalable and environmental friendly method of sonication, showed their interest to be of use as reinforcing fillers for TPS to produce a sustainable biocomposite for packaging applications. Although biomass-derived biopolymers are mainly extracted from plants, they can also be obtained from animal resources. For instance, gelatin has been traditionally used in the development of soft capsules (softgels) due to its biodegradable nature, as well as its ability to form

thermo-responsive hydrogels, inspiring interest in the biomedical sector and food industry. In recent years, new materials have been explored in the food industry to partially or completely replace gelatin with other non-animal natural hydrocolloids. In this context, Otálora et al. [21] developed gelatin/cactus mucilage softgel capsules to encapsulate oil extracted from sacha inchi (*Plukenetia volubilis L.*) seeds, a rich source of polyunsaturated fatty acids (PUFAs) that are beneficial to human health. They submitted the softgel capsules to an in vitro digestion process to simulate gastric conditions and the protective capacity of the gelatin/cactus mucilage-based softgel against digestive processes was evaluated. Although the study revealed a reduction in the content of polyunsaturated fatty acids (PUFAs) after the digestion process and, thus, a reduction of the nutritional value, they concluded that gelatin/cactus mucilage microcapsules can act as interesting bioactive delivery systems for acidic food (e.g.,: fruit juices or dairy drinks) before being subjected to digestive processes.

As has already been mentioned, the biobased polymer obtainment, by means of microorganisms, has gained a lot of interest. In this context, the family of polyhydroxyalkanoates (PHAs) are polyesters biologically synthesized by controlled bacterial fermentation in response to nutrient limitation as an intracellular storage of food and energy. The homopolymer, poly(hydroxybutyrate) (PHB), is the simplest and most common representative of the PHA family, but PHAs also comprise many copolyesters, polyhydroxybutyrate-*co*hydroxyalkanoates, that have gained high industrial interest in the bioplastic sector, such as polyhydroxybutyrate-*co*-hydroxyvalerates (PHBV), or polyhydroxybutyrate-*co*-hydroxyhexanoate (PHBH), polyhydroxybutyrate-*co*-hydroxyoctonoate (PHBO), and polyhydroxybutyrate-*co*-hydroxyoctadecanoate (PHBOd). For instance, Ivorra-Martinez et al. [22] developed PHBH/PCL blends with different compositions ranging from 0 to 40 of PCL wt.% by means of extrusion process followed by injection molding, with the main objective of reducing the inherent embrittlement of PHBH produced due to the aging process (i.e., secondary crystallization). Although they mainly found a lack of miscibility between both polymeric matrices, as revealed by the thermogravimetric analysis, they concluded that the addition of high amounts of PCL (i.e., 40 wt.%) contributed to an increase in ductility and to a decrease in the typical brittleness of PHA. PCL considerably improved the toughness, as well as the impact resistance, of neat PHBH. Therefore, PHBH/PCL blends showed their suitability for the packaging industry. Then, Ivorra-Martinez et al. [23] developed Wood Plastic Composites (WPCs) based on PHBH loaded with lignocellulosic particles of almond shell flour (ASF), a by-product from the agro-food industry, by means of extrusion process followed by injection molding. Since the addition of ASF (with a maximum particle size of 150 μm) leads to an embrittlement and reduced toughness of the WPC, they used oligomeric lactic acid (OLA) to provide improved properties to the final PHBH-ASF/OLA material. Interestingly, a remarkable increase in impact strength with 20 phr OLA addition was achieved. In fact, OLA provides PHBH polymer chains of a high mobility due to a plasticizing effect, leading to an improvement in toughness, even on composites with 30 wt.% ASF. Additionally, OLA decreased the water absorption capacity of WPC, thus broadening potential industrial applications of PHBH-ASF/OLA in high humidity environments.

As it was previously mentioned, nowadays many traditional plastics, which were obtained for decades through the classic petrochemical production routes, have recently found "green" routes for their production (e.g., Bio-PE, Bio-PET, etc.). In this regard, polyamide 6 (PA6) can be replaced with a biobased variant that exhibits a high renewable content and with very similar properties known as PA610. In this context, Marset et al. [24] used PA610 partially biobased polyamide (Bio-PA) to develop nanocomposites reinforced with 10%, 20%, and 30% halloysite nanotubes (HNTs) as a natural flame retardancy filler by means of an extrusion process, followed by injection molding. The results showed that HNTs promote a significant reduction in the optical density and in the number of toxic gases (i.e., CO2) emitted during combustion, especially when PA610 was loaded with 30% HNTs. Thus, the nanocomposites showed good flame retardancy properties.

In summary, the Special Issue Biopolymers from Natural resources published in *Polymers*, compiles the recent research works in biopolymers obtained from natural resources and the strategies to improve their overall performance for sustainable industrial applications. From the above, improvements of biobased polymers was successfully achieved by means of copolymerization, blending, the use of compatibilizers, or plasticizers (vegetable oils, oligomers, etc.), as well as with naturally occurring or naturally derived particles on the micro- and nano-scale. Special interest has been shown to the revalorization of agro-food wastes for the extraction/production of additives or polymers with an interest in the industrial sector. The resulting, more sustainable materials can find potential applications in several industrial sectors, such as in the controlled release of active compounds for active food packaging, bioactive delivery systems, or biomedical devices (e.g., soft capsules, etc.), textiles (e.g., sustainable clothes) or as wood plastic composites (e.g., furniture and outdoor applications).

Future research efforts on biobased polymer extraction and production methods, as well as the optimization of their final production into co-polymers, blends, composites, and nanocomposites, are still needed to properly transfer the proposed developments, as well as future ones from the laboratory scale level to the industrial sector to reach more environmentally friendly materials within a circular economy approach.

**Author Contributions:** All the guest editors read the twenty-four articles of the Special Issue and wrote this editorial letter. All authors have then reviewed the final version of the manuscript and agreed its publication. All authors have read and agreed to the published version of the manuscript.

**Acknowledgments:** The guest editors thank all the authors for submitting their work to this Special Issue and for its successful completion. We also acknowledge all the reviewers participating in the peer-review process of the submitted manuscripts for enhancing their quality and impact. We are also grateful to Chris Chen and the editorial assistants of *Polymers* who made the entire Special Issue creation a smooth and efficient process.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

## *Article* **Influence of the Presence of Choline Chloride on the Classical Mechanism of "Gelatinization" of Starch**

**Doina Crucean 1,2,3, Bruno Pontoire 2,3, Gervaise Debucquet 4, Alain Le-Bail 1,3 and Patricia Le-Bail 2,3,\***

**Abstract:** The aim of this research is to contribute to a better understanding the destructuration of three native starches and a wheat flour in mixtures of water and choline chloride. Model systems have thus been defined to allow a better approach to hydrothermic transformations related to the interactions between choline chloride and starch. We have observed that choline chloride has an impact on the gelatinization of starch which corresponds to the stabilizing salts phenomenon. The depolymerization and dissolution of the starch have also been demonstrated and can there dominate the gelatinization. However, the results obtained in X-ray diffraction by heating cell have shown that the exotherm which appeared was not only related to the depolymerization of the starch, but that a stage of crystalline rearrangement of the starch coexisted with this phenomenon.

**Keywords:** allotropic transition; choline chloride; plasticizer; starch dissolution

### **1. Introduction**

Choline chloride, because of its unique nutrient functionality and its ability to enhance flavor, provides a new option as a partial substitute for sodium chloride in reformulations of processed foods to reduce their sodium content. To date, very few studies have explored the potential of choline chloride to act as a substitute for salt. Locke and Fielding were the first to identify the properties of choline chloride to improve the salty taste of food products [1–3].

Starch is a natural polymer with particular properties unlike those of traditional polymers. As a heterogeneous material, it has macromolecular structures bound in a granular superstructure.

When native starch granules are heated in water (excess of water conditions, around 60% water wb), their semicrystalline nature and three-dimensional architecture are gradually disrupted, resulting in a phase transition from the ordered granular structure to a disordered state in water, which is known as "gelatinization" [4,5]. Gelatinization is an irreversible process that includes several steps, such as granule swelling, native crystalline melting (loss of birefringence), and leaching of fractions of polymers or of polymers in particular amylose chains resulting in a molecular solubilization [6]. The gelatinization process is essential in the processing of foods. For improving the properties of starch and adding new functionalities, it is common to carry out the hydrothermal transformation of starch in environments containing substances other than water. Various plasticizers and additives for starch processing have been used, including polyols (glycerol, glycol, sorbitol, etc.) and nitrogen-containing compounds (urea, ammonium derived, and amines) [7,8]. An alternative class of materials known as ionic liquids, now commonly defined as salts, that

**Citation:** Crucean, D.; Pontoire, B.; Debucquet, G.; Le-Bail, A.; Le-Bail, P. Influence of the Presence of Choline Chloride on the Classical Mechanism of "Gelatinization" of Starch. *Polymers* **2021**, *13*, 1509. https:// doi.org/10.3390/polym13091509

Academic Editors: Rafael Antonio Balart Gimeno, Daniel García García, Vicent Fombuena Borrás, Luís Jesús Quiles Carrillo and Marina Patricia Arrieta Dillon

Received: 21 March 2021 Accepted: 28 April 2021 Published: 7 May 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

melt below 100 ◦C has recently attracted much interest for the processing of biopolymers such as starch.

Choline chloride solubilized in water gives an ionic liquid. Several studies have shown the effects of ionic liquids (solvents and plasticizers) based on the effects of choline chloride on starch [9–14]. A study conducted by Decaen et al. highlights the plasticization of starch by ionic liquids based on an examination of choline [15]. The destructuration of native maize starch in mixtures of water and ionic liquids containing choline cations was studied in dynamic heating conditions, combining calorimetry, rheology, microscopy, and chromatographic techniques by Sciarini et al. [16].

This work presents the mechanism of choline chloride penetration into the starch grain and its impact on gelatinization and the structural evolution of starch during heating kinetics.

#### **2. Materials and Methods**

#### *2.1. Materials*

Wheat flour (type 65) was supplied by Evelia, La Varenne, France. Wheat starch was purchased from Loryma GmbH (Zwingenberg, Germany), and waxy corn starch was purchased from Roquette Italia SpA (Cassano Spinola, Italy). Potato starch and choline chloride (C5H14ClNO) were supplied by Sigma-Aldrich (France).

### *2.2. Sample Preparations*

The measurements were carried out on wheat flour (Fw) and wheat, potato, and waxy corn starch (Sw, Sp, and Swc, respectively) solubilized in an aqueous solution containing different concentrations of choline chloride.

The model systems studied being composed of three ingredients (flour, chlorine choline, and water) and were presented under the form of Equation (1) with Fx, Ccy, and Wz being the flour (or starch), choline chloride, and water content, respectively.

$$\text{F}\_{\text{x}}\text{C}\text{c}\_{\text{Y}}\text{W}\_{\text{x}}\tag{1}$$

A mixing design has been used (1). The factors are the concentrations of each component of the mixture. Responses are expressed as a function of these concentrations with the sum of the mass fraction of each component being equal to 100 (x+y+z= 100). For each analysis, 5 g of mixture was prepared.

The mode of preparation (quoted ILS = "Ionic Liquid + Starch" for the rest of the paper) consisted in adding the choline chloride under the form of ionic liquid [15] (previous dissolution of Cc and water for one hour before starting the experiments); in that case, the script that was used was "(Cc + W)", and the ionic liquid was then added to the flour. These systems were written with the following script: Fx[CcyWz].

All analyses were repeated in triplicate. A statistical analysis was performed with variance analysis (*p* < 0.005) to detect significant differences.

#### *2.3. Methods*

#### 2.3.1. Differential Scanning Calorimetry Measurements

Samples weighing 500 mg were placed in stainless steel pans, and a reference cell was prepared by adding an amount of water equal to the volume of water in the sample cells. The cells were then sealed with a heat-resistant seal. The cell containing the sample was agitated (50 rpm at room temperature) for one hour and then placed in the oven of the appliance. The pans were heated at a rate of 1 ◦C/min from 10 to 120 ◦C by using the SETARAM microcalorimeter (μDSC) III (France). Scans were run on the following starch suspensions: SP20[CcxWy], SW20[CcxWy], SWC20[CcxWy], and FW20[CcxWy]. All measurements were made at least in triplicate.

Thermal transitions were defined as TGe (peak temperature gelatinization), and ΔHGe denotes the transition enthalpies for different endotherms. The enthalpies were determined by endotherm integration, and all traces were normalized to 1 mg of dried sample. The partial enthalpies were calculated from the onset of the endotherm to the end (every 1 ◦C).

#### 2.3.2. X-ray Diffraction

The samples containing flour and starch suspended in [Cc56W24] were prepared as follows: SWC20[Cc56W24], SP20[Cc56W24], SW20[Cc56W24], and FW20[Cc56W24]. The appropriate amounts of choline chloride, water (when required), and starch were weighed. The same formulations without choline chloride were studied: SWC45W55, SP45W55, SW45W55, and FW45W55, corresponding to the same F/W ratio as the previous mixtures.

The samples were examined by wide-angle (WAX) X-ray diffraction. The measurements were performed using a D8 Discover spectrometer from Bruker-AXS (Karlsruhe, Germany). Cu Kα1 radiation, produced in a sealed tube at 40 kV and 40 mA, was selected and parallelized using a double Gobël mirror parallel optics system and collimated to produce a 500 μm beam diameter with sample alignment by a microscopic video and laser. The data were monitored by a VANTEC 500 2D detector (Bruker, Karlsruhe, Germany) for 10 min and normalized.

The sample was placed in a capillary with a diameter of 1.5 mm, and a second capillary was introduced into the first to avoid loss of water during the rise in temperature; the two capillaries were sealed. The as-prepared capillary was placed in a heating stage HFS91 (Linkam, Tadworth, UK). The detector was positioned at a focusing distance of 8.6 cm from the sample surface. It was in direct beam position [17]. The heating kinetic applied to the sample was 1 ◦C/min from 20 to 120 ◦C. Every 10 ◦C, a plateau of 10 min was introduced to enable the acquisition of the diffraction spectrum.

### **3. Results and Discussion**

### *3.1. Gelatinization of Different Starches*

The thermograms of wheat flour (FW), wheat starch (SW), potato starch (SP), and waxy-corn starch (SWC) in excess water (W = 80%) should serve as a reference and allow comparison with the F20[CcxWy] systems, i.e., when choline chloride is added to the aqueous phase. All the samples exhibited an endotherm on their thermograms, which corresponded to the gelatinization of the starch at the following temperatures: FW 61.7 ± 0.2 ◦C, SW 58.1 ± 0.1 ◦C, SWC 70.8 ± 0.1 ◦C, and SP 65.7 ± 0.1 ◦C (Table 1). The gelatinization temperatures of the different samples studied increased in the following order: TGe (SW) < TGe (FW)<TGe (SP)<TGe (SWC), whereas the gelatinization enthalpies increased in the following order: ΔHGe (FW) < ΔHGe (SW) < ΔHGe (SWC) < ΔHGe (SP).

**Table 1.** Enthalpies (ΔHGe, J/g) and gelatinization temperatures (TGe, ◦C) of different flour/starch and plasticizer model systems. Enthalpies were calculated on flour or starch total dry basis.

The thermograms of the FW and SW samples showed a second endotherm characterized by the fusion of the amylose-endogenous lipid complexes formed during gelatinization [18,19]. The melting temperatures of the amylose-lipid complexes for SW and FW were 97 ± 1 ◦C and 92.3 ± 0.2 ◦C, respectively.

#### *3.2. Influence of Choline Chloride on Starch Destructuration*

Since choline chloride behaves as an ionic liquid when it is in the presence of water, its behavior during the heating of a starch granule suspension may strongly influence gelatinization. A study comparing the previously discussed aqueous-phase suspensions containing solubilized choline chloride was carried out.

Micro-DSC was used to study the physicochemical characteristics of the following suspensions: SWC20[CcxWy], SP20[CcxWy], SW20[CcxWy], and FW20[CcxWy].

The thermograms of the suspensions are shown in Figure 1, and their enthalpies ΔHGe and gelatinization temperatures TGe are shown in Table 1.

**Figure 1.** μDSC heating curves, 10–120 ◦C at 1 ◦C/min, of: (**a**) waxy-corn starch, (**b**) potato starch, (**c**) wheat starch, and (**d**) flour at different water and chlorine chloride contents.

At low concentrations of Cc, the starch undergoes a typical gelatinization, represented by an endothermic transition. At the same concentration, the endothermic transition attributed to the fusion of endogenous amylose-lipid complexes is also observed for FW and SW. When the concentration of Cc increases from 0 to 40% for SWC, SW, and FW and from 0 to 48% for SP, the peak of gelatinization moves to a higher temperature and then decreases for all three starches and flour. The observed gelatinization temperature is always higher than that observed in pure water. However, beyond 40% for SWC, SP, and FW and beyond 48% for SW, the gelatinization of the starch decreases with an increase in the B4 concentration. The same trend is observed for the enthalpies of gelatinization.

The increase in gelatinization temperature caused by Cc and the subsequent decrease with an increasing Cc level agree with the results of Chiotelli et al. for wheat and potato starches, Chungcharoen and Lund for rice starch, Jane for corn starch, and those of Ahmad and William and Ghani et al. for sargo starch [20–24].

The shift of the gelatinization temperature toward higher temperatures as well as the increase in the total enthalpy of gelatinization in the presence of Cc may be due to the reduction of the water activity in the starch/plasticizer solution, which causes an increase in the energy required for chemical and physical reactions involving water [25] Jane [22] claimed that the effect of salts on starch gelatinization follows the "Hofmeister" series, the increase in the gelatinization temperature follows the charge density order of the ions (LiCl > NaCl > KCl > RbCl), that is, the order of the "structure making" effect. However, at concentrations greater than 4 M, these salts decrease the onset temperature of gelatinization. Choline reacts as an "ion structure maker".

For high Cc concentrations, exothermic transitions were observed. The exotherms started at higher temperatures as the water content decreased, and the corresponding heat released (ΔH) increased as well. There was a critical concentration, which depends on the type of starch used, for which both exothermic and endothermic transitions took place. For this concentration, both phenomena seem to happen at very close temperatures, at which they overlapped, resulting in a neutralization with no visible thermal effect. The same behavior was found by Sciarini et al. and Mateyawa et al. working with choline acetate and EMIMAc and by Koganti et al. using N-methyl morpholine N-oxide (NMMO) [12,16,26]. These authors attributed the exothermic transition to starch dissolution in these solvents. For low water contents (16%), the thermograms of SP and SWC show wide endotherms at low temperatures (19.7 ◦C). This phenomenon was not visible for SW and FW. A DSC study was then carried out on the SWC20[CcxWy] systems (with x included between 56 and 70 and y between 24 and 10) to understand this phenomenon (Figure 2).

The thermogram of pure Cc (data not shown) shows an endotherm near 73.6 ± 0.1 ◦C. With the addition of water, this endotherm moves to lower temperatures and undergoes a strong shift from the baseline until disappearing. In the presence of waxy maize starch (20%), the concentration of Cc was increased in steps of 1% (from 56 to 70%), and water was decreased likewise (from 24 to 10%), which produced an endotherm at low temperatures for the sample with 17% water.

During these investigations, the Cc was solubilized in water to form the plasticizer, and then the starch was added to the plasticizer. When the amount of water was sufficient, complete solubilization of the Cc was achieved. When the water concentration was less than 17%, the solubilization of Cc was partial, and we observed a solubilization endotherm that broadened with a decrease in the water content. For the 14% water sample, there was enough unsolubilized choline chloride for the allotropic change in choline chloride at 68 ◦C to be observed.

It was assumed that the solution (water + Cc) became more viscous with increasing Cc concentrations, resulting in more difficulty for the Cc solution to penetrate inside the starch granule; in turn, a shift to higher gelatinization temperatures was observed.

**Figure 2.** Microcalorimeter heating curves, 10–120 ◦C at 1 ◦C/min, of waxy-corn starch at different water, and choline chloride contents.

The effect of chlorine choline (Cc) on the gelatinization of starch is very different from that of NaCl observed in the literature. Indeed, if the gelatinization temperature follows the same trend when adding Cc or NaCl, the enthalpy of gelatinization shows the opposite behavior. The decrease in the total enthalpy of starch gelatinization at high NaCl concentrations suggests a destabilization of the ordered regions of the starch in the presence of NaCl. As explained by Chiotelli et al., sodium chloride may be hindered by polymer–polymer interactions in favor of water–polymer interactions, resulting in a lower enthalpy for fusion of organized regions. The addition of Cc causes a reorganization of the internal structure of the starch grain [20].

#### *3.3. Penetration of the Plasticizer into the Starch Grain*

To understand the penetration kinetics of the ionic liquid (Cc + water) in the starch granule, the cumulative enthalpy curves were calculated and reported as a function of temperature.

The curves of the partial enthalpy of gelatinization for all the plasticizer contents studied are shown in Figure 3. This study presents the kinetics of the loss of ordered structure in starch for temperatures between 40 and 110 ◦C. An offset of the melting toward higher temperatures was observed when the Cc content increased; this was observed for the three starches and the flour.

The shapes of the curves shown in Figure 3 are rather different for SP and SW: the melting of the potato starch ordered zones was more abrupt than it was for the wheat starch and the wheat flour. These observations are in good agreement with the results of Waigh et al. on the two-stage destructuration of SW (crystallinity loss and helices destruction) [27]. Fannon, Hauber, and Bemiller showed that the SP granules appear smooth on the surface by scanning electron microscopy, whereas cereal grains such as SW have pores of about 100 nm at their surfaces, which are generated at the time of granule biosynthesis and cause a greater hydration sensitivity [28]. These observations may partly explain the differences in the plasticizer penetration into the granules for wheat starch and potato starch.

**Figure 3.** Curves of the partial enthalpy of gelatinization for all the plasticizer contents studied. (**a**) [SWCxCcy]Wz systems; (**b**) [SPxCcy]Wz systems; (**c**) [SWxCcy]Wz systems; (**d**) [FWxCcy]Wz systems.

For potato starch, the total gelatinization enthalpy (ΔH) in the presence of Cc remained nearly constant at low concentrations of Cc but decreased slightly for higher concentrations. For wheat starch, the ΔH increased significantly as the concentration of Cc increased, to a greater extent than that for the potato starch. This considerable increase in the total enthalpy of gelatinization of wheat starch at high Cc concentrations suggests a better organization of the ordered regions of starch in the presence of Cc; on the other hand, when the concentration reached 40% for the Cc, the total enthalpy of the starch due to the fusion decreased perceptibly. The effect of high Cc concentrations on the gelatinization process

was different for potato starch compared to waxy maize and wheat starch and wheat flour; it is assumed that this was due to the differences in the polymorphic structures.

The effect of Cc on the gelatinization of SP is more pronounced in the first step of the gelatinization process (retardation of the loss of crystalline order), whereas for SW, FW, and SWC, Cc also affects the second stage (delay in the loss of molecular order) and the overall enthalpy of the transition. These results are again opposite to those found in the literature for potato starch and wheat starch in the presence of high salt concentrations (NaCl) [20].

#### *3.4. Impact of the Formulation on Starch Gelatinization*

As previously described, choline chloride in the absence of water is not considered as an ionic liquid. Therefore, it seemed interesting to observe the action of Cc on starch when Cc is no longer acting as a plasticizer, but when it enters into the formulation as dry matter, Cc is placed directly in competition with water in front of starch. The relevant suspensions were formulated as follows: the starch (or flour) was mixed with the choline chloride (dry powder), and the water was added to the mixture. The obtained mixes were quoted using the following script: [FxCcy]Wz. These mixtures were studied by μDSC.

The temperatures and enthalpies of starch gelatinization did not vary with the formulation. However, there was a marked increase in the solubilization/rearrangement exotherm when the choline chloride was used as an ionic liquid (data not shown). This observation is explained by the fact that the solubilization of Cc before deposition on the starch allowed better penetration into the grain.

Another comparative study was carried out on the FW20Cc62W18 system, with three different formulations (Figure 4):

**Figure 4.** Comparative study of the thermograms of the systems [FW20Cc64]W16, FW20[Cc64W16], and [FW20W16]Cc64.

The results clearly showed that when the water (in small quantities) is premixed with the flour, it becomes inaccessible to the choline chloride, causing gelatinization at 90 ◦C corresponding to a hydration of approximately 50%, which agrees with the starting hydration (following formula).

There is also a broad endotherm of allotropic change in Cc, suggesting that choline chloride has access to only a very small amount of water.

For the other two formulations, very similar results were obtained: namely, no change in the gelatinization temperature, a slightly more prominent exotherm for formulation 2, and a small endotherm associated with the allotropic change in Cc.

In this part, we have shown that the order of incorporation of water into the system can influence the gelatinization of the studied starches. Indeed, if the water is added to the starch first, it binds enough to the starch to become unavailable for Cc. On the other hand, if the water is first added to the choline chloride, the latter is solubilized before entering as an ionic liquid in the starch.

### *3.5. Evolution of the Structure of Different Starches in the Presence of Choline Chloride during Heating Kinetics*

To understand the phenomena underlying the exothermic transitions observed with the microcalorimeter, measurements by an XRD heating cell were made.

Two types of systems were compared with and without Cc. The starch/flour and water concentrations were the same in both systems. The X-ray diffraction spectra of each of the suspensions of waxy corn starch and potato starch are collated in Figure 5.

SWC, SW, and FW possess A-type polymorphic structures, and SP possesses a B-type polymorphic structure. In the diffraction spectra of the SW20W24 (not shown), FW20W24 (not shown), and SWC20W24 systems, characteristic peaks of the A-type structure were identified. Spectra of the SP20W24 system showed characteristic peaks of the B-type structure.

A notable difference is seen in the evolution of the spectra during heating between the samples without Cc (Figure 5a,c) and those containing Cc (Figure 5b,d). Indeed, the peaks are more well defined in the systems without Cc, as with the addition of Cc at 20 ◦C. This confirms the previous conclusion: Cc causes a loss of crystalline order for all types of starch at room temperature. For the SP45W55 system, the type-B structure disappears at a temperature of 90 ◦C. However, for type-A systems, the structure collapses near 90 ◦C; this is consistent with Waigh's observations [29]. When Cc was added to the systems, a collapse of structure A or B was observed at much higher temperatures, which agrees with the results obtained by DSC.

For the SP20[Cc56W24] system, an increase in the peak intensity occurred at 5.6◦ in 2Θ at a temperature of 60 ◦C, followed by the onset of collapse at 100 ◦C and the total disappearance of the structure at 110 ◦C. This phenomenon corresponds to the appearance of the exotherm on the thermogram from the DSC study.

For systems SWC20[Cc56W24] and SW20[Cc56W24], the transition to an increase in crystallinity before melting is much less visible, and it is even absent for the FW20[Cc56W24] system. This explains the absence of the exotherm for the FW20[Cc56W24] system. In the literature, the exotherm is related to the solubilization of the starch in the plasticizer. However, during the X-ray diffraction study, we showed that the exotherm also corresponded to the increase in the intensity of the diffraction peaks and therefore to the increase in the crystallinity of the starch before its solubilization and then its melting.

**Figure 5.** Evolution of the X-ray diffraction spectra during heating associated with the thermogram: waxy maize starch without Cc: SWC45W55 (**a**) and with Cc: SWC20[Cc56W24] (**b**); potato starch without Cc: SP45W55 (**c**) and with Cc: SP20[Cc56W24] (**d**).

To complete this work, a μDSC study was conducted on the SP20[Cc56W24] system at low rates (0.1 ◦C/min). Two peaks were clearly observed (Figure 6), indicating that more than one phenomenon is responsible for the exothermic transition observed.

**Figure 6.** μDSC heating curves, 20–120 ◦C at 0.1 ◦C/min, for SP20[Cc56W24].

#### **4. Conclusions**

The starch/water system shows a gelatinization accompanied or unaccompanied by a fusion, according to the water content of the matrix. The choline chloride/water system possesses the characteristics of an ionic liquid and exhibits an allotropic change at low water concentrations and solubilization for water contents greater than 30%.

Choline chloride is an ionic compound, which, like NaCl, has a "structure making" effect: that is, it increases the viscosity of the aqueous solution while decreasing the water fraction with high mobility. As a result, the gelatinization temperatures are displaced to higher temperatures. In contrast to the addition of NaCl, the addition of choline chloride entails a significant increase in the gelatinization enthalpy, which suggests stabilization or better organization of the ordered regions of the starch in the presence of choline chloride.

An X-ray diffraction study in a heating cell demonstrated the crystalline rearrangement of the structure of the starch grain, which takes place simultaneously with the solubilization phenomenon of amylose.

**Author Contributions:** Conceptualization, A.L.-B. and P.L.-B.; methodology, D.C. and B.P.; formal analysis, D.C.; data curation, D.C., P.L.-B.; writing—original draft preparation, D.C., A.L.-B. et P.L.-B.; writing—review and editing, D.C., G.D. and P.L.-B.; supervision, P.L.-B.; project administration and funding acquisition, A.L.-B. All authors have read and agreed to the published version of the manuscript.

**Funding:** Co fundings by French Ministry of Agriculture (ONIRIS-ID for FOODS programme), by ONIRIS-GEPEA and by INRA-BIA.

**Institutional Review Board Statement:** Nothing to report.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Nothing to report.

**Acknowledgments:** This project was supported by the French Ministry of Agriculture (ONIRIS-ID for FOODS programme), by ONIRIS-GEPEA and by INRA-BIA co fundings.

**Conflicts of Interest:** I certify that there is no conflict of interest.

### **References**

## *Article* **Characterization of Novel Biopolymer Blend Mycocel from Plant Cellulose and Fungal Fibers**

**Ilze Irbe 1,\*, Inese Filipova 1, Marite Skute 1, Anna Zajakina 2,\*, Karina Spunde 2 and Talis Juhna 3**

**Abstract:** In this study unique blended biopolymer mycocel from naturally derived biomass was developed. Softwood Kraft (KF) or hemp (HF) cellulose fibers were mixed with fungal fibers (FF) in different ratios and the obtained materials were characterized regarding microstructure, air permeability, mechanical properties, and virus filtration efficiency. The fibers from screened Basidiomycota fungi *Ganoderma applanatum* (Ga), *Fomes fomentarius* (Ff), *Agaricus bisporus* (Ab), and *Trametes versicolor* (Tv) were applicable for blending with cellulose fibers. Fungi with trimitic hyphal system (Ga, Ff) in combinations with KF formed a microporous membrane with increased air permeability (>8820 mL/min) and limited mechanical strength (tensile index 9–14 Nm/g). HF combination with trimitic fungal hyphae formed a dense fibrillary net with low air permeability (77–115 mL/min) and higher strength 31–36 Nm/g. The hyphal bundles of monomitic fibers of Tv mycelium and Ab stipes made a tight structure with KF with increased strength (26–43 Nm/g) and limited air permeability (14–1630 mL/min). The blends KF FF (Ga) and KF FF (Tv) revealed relatively high virus filtration capacity: the log10 virus titer reduction values (LRV) corresponded to 4.54 LRV and 2.12 LRV, respectively. Mycocel biopolymers are biodegradable and have potential to be used in water microfiltration, food packaging, and virus filtration membranes.

**Keywords:** air permeability; fungal fibers; hemp fibers; microstructure; mechanical properties; mycocel; softwood fibers; virus membrane filtration

### **1. Introduction**

The importance of biobased polymers is well known, and much research and development activities concern the use of biobased polymers in science, engineering, and industry. Biopolymers from renewable resources are used in multiple fields, namely health, food, energy, and the environment, due to their intrinsic features, versatility, biocompatibility, and degradability. Besides, the widespread use of biopolymers also addresses concerns about environmental sustainability [1]. As long as biopolymers stand as a sustainable, biodegradable compound, new biopolymer products and materials filled, blended, or reinforced with natural fibers, will predominate with the promise of sustainability benefits [2].

Generally, biobased polymers are classified into three classes: (1) naturally derived biomass polymers such as cellulose, cellulose acetate, starches, chitin, modified starch, etc.; (2) bio-engineered polymers bio-synthesized by using microorganisms and plants such as poly(hydroxy alkanoates (PHAs), poly(glutamic acid), etc.; (3) synthetic polymers produced from naturally derived molecules or by the breakdown of naturally derived macromolecules through the combination of chemical and biochemical processes such as polylactide (PLA), poly(butylene succinate) (PBS), bio-polyolefins, bio-poly(ethylene terephtalic acid) (bio-PET) [3]. The first and second class polymers are biodegradable, they

**Citation:** Irbe, I.; Filipova, I.; Skute, M.; Zajakina, A.; Spunde, K.; Juhna, T. Characterization of Novel Biopolymer Blend Mycocel from Plant Cellulose and Fungal Fibers. *Polymers* **2021**, *13*, 1086. https:// doi.org/10.3390/polym13071086

Academic Editor: Rafael Antonio Balart Gimeno

Received: 3 March 2021 Accepted: 25 March 2021 Published: 30 March 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

allow for more efficient production, which can produce desired functionalities and physical properties, but chemical structure designs have limited flexibility. The third class polymers such as bio-polyolefins and bio-PET are not biodegradable and the only contribution for reducing environmental impact comes from reducing the carbon footprint. The origin of a polymer does not determine its biodegradability; this condition depends on the chemical structure of the polymer [3].

The naturally derived biopolymers, among others, include polysaccharides of plant and fungal origin. Polysaccharides are nontoxic and biodegradable, which increases their potential application in biopolymers. The most used biopolysaccharides are obtained from plant origin (e.g., cellulose), microbial origin (e.g., bacterial cellulose), and animal origin (e.g., chitin/chitosan). Moreover, these natural derivatives present a considerable number of reactive functional groups (e.g., hydroxyl, carboxyl, and amino groups), which significantly increase their applicability through chemical modification or physical blend [1]. Cellulose is the most abundant polysaccharide of natural origin in the world, and is mostly produced by plants. The cellulose chains structure leads to areas of high crystallinity within the polymer and to high stability structures, which as a consequence promote considerable strength, remarkable inertness, and insolubility in water and common organic solvents [4]. In its turn, fungal cell walls share a common chemical structure composed of homo- and heteropolysaccharides, protein, protein–polysaccharide complexes, lipids, melanin, and polysaccharide chains of chitin. Chitin is a biopolymer of N-acetylglucosamine with some glucosamine, which is the main component of the cell walls of fungi and is considered the second most abundant natural polymer after cellulose [5].

Current research in membrane science is now focusing more on biopolymers from natural raw materials with a well-defined structure to develop new membrane materials. In fact, the combination of polysaccharides and proteins is a method frequently used to design blended materials with improved performance regarding swelling, mechanical resistance, and biocompatibility, among other features. The attention has been focused also on membranes based on chitosan blended with other biomacromolecules such as alginate, cellulose, collagen, gelatin, keratin, sericin, and soy protein [6]. Chitin-related materials from fungal sources with focus on nanocomposites and nanopapers have been suggested as greener alternative to synthetic polymers [7]. Fungal chitin–glucan nanopapers have manufactured from chitin nanofibrils, which is a native composite material (chitin–glucan) combining the strength of chitin and the toughness of glucan. These nanopapers showed distinct physico-chemical surface properties, being more hydrophobic than crustacean chitin [8]. Nanopapers from chitin nanofibrils have exhibited tunable mechanical and surface properties with potential use in coatings, membranes, packaging [9] and ultrafiltration of organic solvents and water [10]. Fungal mycelium–nanocellulose has been produced by the agitated liquid culture of a white-rot fungus with nanocellulose as part of the culture media. The obtained biomaterials are suggested for diverse applications, including packaging, filtration, and hygiene products [11].

The literature survey demonstrates that fungal chitin nanofibrils have been used to manufacture nanopapers. Additionally, fungal mycelium has been combined with nanocellulose to obtain a blended biomaterial. However, there are no studies on blended biopolymers from plant cellulose and fungal hyphae. The aim of this study was to develop a novel blended biopolymer from naturally derived biomass that is made of softwood cellulose fibers and fungal fibers (hyphae) and to test it for air permeability, mechanical properties, and virus filtration efficiency. More detailed investigation of specific properties of biomaterial blend containing fungal fibers from basidiomycete *Ganoderma applanatum* is published in our previous paper [12]. The cellulose fibers from softwood and hemp shives are easily available natural resource. The basidiomycetes selected for this study have shown a high importance in medical and nutritional applications. For example, *Ganoderma applanatum* has been reported for its phytochemical properties for potential application in nanotechological engineering for clinical use [13]. *Fomes fomentarius* has been used in a traditional medicine for centuries and is still economically important as

a source of medicinal and neutraceutical products [14]. *Trametes versicolor* is known for its general health-promoting effects and is widely employed in traditional medicine [15]. *Agaricus bisporus* represents the leading position among edible cultivated mushrooms [16].

### **2. Materials and Methods**

#### *2.1. Synthesis of Biopolymers*

The screening of several basidiomycetes was carried out to select the fungal candidates forming homogenous hyphal biomass convenient for biopolymer material development. The fungal biomass was obtained from (1) fruiting bodies of the forest growing species *Ganoderma applanatum* (Pers.) Pat., *Fomes fomentarius* (L.) Fr., *Lentinus lepideus* (Fr.) Fr., *Polyporus squamosus* (Huds.) Fr., *Fomitopsis betulina* (Bull.) B.K. Cui, (2) commercially cultivated mushroom stipes of *Agaricus bisporus* (J.E. Lange) Imbach and (3) mycelium of pure culture strain *Trametes versicolor* CTB 863 A.

Fungal biomass was kept in 4% NaOH solution for 24 h at a room temperature in order to extract proteins and alkali soluble polysaccharides. Then the samples were washed in tap water and mechanically disintegrated using Blendtec 725 (Orem, UT, USA) at 360 W for 30 s. Obtained fungal fibers (FF) were dried at room temperature and kept in a dry state until used.

Bleached softwood Kraft fibers (KF) were provided by Metsä Fibre (Äänekoski, Finland) as pressed sheets and used without specific pre-treatment.

Hemp fibers (HF) were obtained from industrial hemp *Cannabis sativa* (USO-31). After the decortication fibers were treated in 4% NaOH solution at 165 ◦C for 75 min, then washed with tap water to neutral and refined using Blendtec 725 (Orem, UT, USA) at 179 W for 7 min at 1.5% consistency, dried at room temperature and kept in dry state until used.

For biopolymer blend development, a certain amount (g) of FF in combinations with KF and HF fiber pulp in different mass ratios (50:50 and 33:33:33) was placed in a glass baker and soaked in 1–2 L of distilled water for 8 h, then disintegrated using 75,000 revolutions in the disintegrator (Frank PTI, Laakirchen, Austria). Material sheets were produced according to ISO 5269-2:2004 with a Rapid Köthen paper machine (Frank PTI, Laakirchen, Austria). Grammage (weight per unit area in g/m2) of samples was calculated by dividing the mass with area according to ISO 536:2019. At least five parallel samples at grammage 50 ± 15 g/m2 of each composition were prepared.

#### *2.2. Micromorphology*

The micromorphology was examined by a light microscope (LM) Leica DMLB (Leica Microsystems GmbH, Wetzlar, Germany) at a magnification of 200×. Lactophenol blue solution (Fluka) was used to observe cyanophilic reaction of fungal hyphae. The images were captured by a video camera Leica DFC490 using calibrated image analysis software Image-Pro plus 6.3 (Media Cybernetics, Inc., Rockville, MD, USA).

For scanning electron microscopy (SEM), the surface of samples was coated with gold plasma using a K550X sputter coater (Emitech, Ashford, UK) and examined with Vega TC (Tescan, Brno-Kohoutovice, Czech Republic) with accelerating voltage of 15 kV, software <IP_ADDRESS>.

#### *2.3. Air Permeability*

Air permeability was tested by Bendsen method according to ISO 5636-3:2013 using an air permeability tester 266 (Lorentzen and Wettre, Stockholm, Sweden). Samples were clamped between a metal ring and a rubber gasket and the air flow rate was measured through sample area of 10 cm2 under 1.47 kPa air pressure for 5 s. Commercially available disposable face masks consisting of three layers made of polypropylene spunbondmeltblown-spunbond nonwoven fabrics were tested for air permeability as comparison for developed biopolymer samples.

### *2.4. Mechanical Properties*

For evaluation of tensile properties samples with a width of 1 cm were prepared with a strip cutter (Frank-PTI, Laakirchen, Austria) and tested according to ISO 1924-1:1992 using a tensile tester vertical F81838 (Frank-PTI, Laakirchen, Austria). Tensile index (Nm/g) was calculated by dividing measured maximum tensile strength (N/m) by grammage (g/m2) of test sample. Breaking length (km) and stretch (%) was used as calculated by software of equipment. Burst index (kPa m2/g) was calculated dividing the measured burst strength (kPa) by grammage (g/m2), where burst strength was measured as hydrostatic pressure necessary to cause rupture in a circular area of a 4 cm diameter of sample according to ISO 2758:2014 using burst tester (Frank-PTI, Laakirchen, Austria).

#### *2.5. Virus Preparation and Membrane Filtration Procedure*

The recombinant Semliki forest virus (SFV) pSFVenh/Luc, encoding firefly luciferase gene, was produced as previously described [17,18]. The virus-containing cell medium was harvested and concentrated by ultracentrifugation through two sucrose cushions, as previously described [19]. The virus was rapidly frozen and subsequently used as a virus stock. The virus titre expressed in infectious units per ml (i.u./mL) was quantified by immunostaining with rabbit polyclonal antibodies specific to the nsp1 subunit of SFV replicase, as previously described [20].

The ability of developed biopolymer materials to retain/remove virus particles was tested using centrifugal filtration test. EMA/CPMP/BWP/268 guidelines were considered for development of virus filtration procedures and calculation of virus reduction values. Two-layer cellulose hygienic paper (AB Grigeo, Vilnius, Lithuania), three-layer cotton mask with antibacterial *Silverplus* coating (SIA P.E.M.T., Riga, Latvia) and a standard surgical mask type II EN 14,683 (Matopat, Toru ´n, Poland) were used as reference materials.

A 1.5 × 1.5 cm sample was cut out from each material, folded in a form of a conical funnel, placed into truncated 1 mL plastic pipette tip (for better fixation), and subsequently placed into an Eppendorf 1.5 mL tube as presented in Figure 1.

**Figure 1.** Schematic representation of the virus filtration test (technical details are provided in text). (1) 1.5 cm × 1.5 cm filter sample is cut out from each material, folded into the form of a conical funnel, and placed into a 1.5 mL tube; (2) 50 μL of recombinant Semliki forest virus (SFV)-enh/Luc virus solution (107 i.u./mL) is added into the cone; (3) the tube is centrifuged to allow the virus to pass through the material; (4) the filtrate (indicated by arrow) is collected; (5) the filtrated is diluted and used for cell infection in a 24-well cell culture plate; (6) after overnight incubation of the plate the cell lysates are prepared and the virus infection is measured by detection of the luciferase activity in infected cells (luminometry). The cell infection with the standard dilutions of the virus is used to generate a standard curve and to calculate the amount of virus in the filtrate.

50 μL of recombinant SFVenh/Luc virus solution with a titer of 107 infectious units per milliliter (i.u./mL) were poured into each cone containing the test material. The tubes were then centrifuged for 20 s at 500× *g* (3500 rpm) by FVL-2400N Combi-Spin, Mini-Centrifuge/Vortex (Biosan, Riga, Latvia) to allow the liquid to pass (filtrate) through the material under low pressure conditions. The filtrated virus samples were collected (at least 20 μL each) and used for BHK-21 cell infection in 24-well plate as previously described [18]. Briefly, 10 μL of the virus sample in duplicate was mixed with 190 μL PBS (containing M2+ and Ca2+) and incubated with BHK-21 cells for 1 h at 37 ◦C, 5% CO2, then 800 μL of BHK medium (1% fetal bovine serum) was added. In parallel, standard dilutions of the SFVenh/Luc virus in a range 1 × 103–5 × 105 i.u./mL per well were generated and used for BHK-21 cell infection in duplicate in the 24-well plate. The cells were incubated overnight at 37 ◦C, 5% CO2 to allow complete cell infection to complete and expression of firefly luciferase gene.

To quantify the virus titer in filtered samples the relative luminescence units (RLU) were measured in cell lysates and the respective values were plotted on the standard curve with serial SFVenh/Luc virus dilutions. The RLUs were measured by the Luciferase assay (Promega, Madison, WI, USA), as recommended by manufacturer. Briefly, the cell medium was removed, and the cells (24-well) were lysed in 100 μL of the Cell Culture Lysis buffer (Promega, Madison, WI, USA), centrifuged at 600 cfr for 5 min, and 1 μL of the cell lysate was used immediately to measure the luciferase enzymatic activity by luminometer Luminoskan Ascent (Thermo Scientific, Loughborough, UK). The cell infection was done in duplicate in each independent experiment. Two independent experiments were performed with each sample (repeats). The virus titer standard curve was generated in each experiment and the negative control signal (RLU of uninfected cells) was subtracted from all values. The log10 reduction value (LRV) represents the difference between loaded and eluted virus infectious units per ml, and respectively was calculated according to the following equation:

LRV = [log10 (virus titer before filtration) − log10 (virus titer after filtration)]. (1)

Statistical analysis of obtained results was performed using an Excel 2016 MSO data statistical analysis tool.

### **3. Results and Discussion**

The fruiting bodies of *L. lepideus*, *P. squamosus*, and *F. betulina* formed gelatinous biomass in NaOH solution which was unusable for further experiments. FF were successfully separated from the biomass of fungi *G. applanatum*, *F. fomentarius*, *A. bisporus*, and *T. versicolor* and integrated in biopolymer compositions with softwood and hemp cellulose fibers.

#### *3.1. Morphological Characterization*

The microscopic structure of mycocel biopolymer blends and macrostructure of developed materials is shown in Figure 2.

Kraft fibers, hemp fibers, and fungal fibers were most likely physically bound together making a net of the biopolymer material. The fungal fibers separately or in bundles were randomly distributed within the net of cellulose fibers. The structure of materials was affected by individual properties of fibers. The size of Kraft fibers was average 2 mm in length and 30 μm in width. Hemp fibers were 1 mm long and 10–20 μm wide with a net of microfibers (2–5 μm). Fungal fibers from fruiting bodies of poroid species (*G. applanatum*, *F. fomentarius*) (Figure 2a,b) were 2–7 μm across. The fibers of *T. versicolor* mycelium were 2–3 μm wide (Figure 2c). The stipes of mushroom *A. bisporus* were composed of swollen hyphae, ascending 7–20 μm across (Figure 2d).

**Figure 2.** *Cont*.

(**e**)

**Figure 2.** Microstructure (left column) and macrostructure (right column) of mycocel biopolymer compositions made of Kraft fibers (KF), hemp fibers (HF), and fungal fibers (FF): (**a**) KF FF (Ff); (**b**) KF FF (Ga); (**c**) KF FF (Tv); (**d**) KF FF (Ab); (**e**) KF HF FF (Ga). Microimages show flat hemp and softwood fibers (10–30 μm) (**a**–**e**) and narrow fungal fibers (2–7 μm) of polypores (**a**–**c**,**e**) and swollen hyphae (7–20 μm) of agaric (**d**). LM, 200×. Bar = 100 μm.

The hyphae of basidiomycetes (primarily poroid species) are divided in three main types: generative, binding, and skeletal hyphae with key differences in cell wall thickness, internal structure, and branching characteristics. Monomitic species comprise only generative hyphae, dimitic species comprise two hyphal types (usually generative and skeletal) and trimitic species contain all three hyphal types [21].

The fungal fruiting bodies under this study, namely, *G. applanatum* and *F. fomentarius*, had trimitic hyphal system. Depending on the species, the generative hyphae were 2–5 μm, skeletal hyphae 3–7 μm and binding hyphae 2–4 μm across (Figure 2a,b). In the case of poroid species *T. versicolor*, the mycelium of pure culture strain consisted of thin-walled generative hyphae, while hyphae of agaric *A. bisporus* were flat and swollen. The fibers of two later species formed dense hyphal bundles among the cellulose fibers. These can be observed in Figure 2c,d where cyanophilic reaction turned hyaline hyphae of Tv and Ab blue after staining. The trimitic species Ga and Ff presented brown pigmented hyphal network (Figure 2a,b,e). The pigmented layer within the hyphal cell wall is likely crosslinked to polysaccharides and has a mesh-like structure with pores, through which small and large molecules can penetrate into cells. Pigment melanin is believed to enhance the strength of the cell wall and has antioxidant properties and propensity to bind to a variety of substances [22].

SEM micrographs (Figure 3) support the findings of LM and display a detailed ultrastructure of the raw materials and mycocel biopolymer blends. Fiber orientation appeared randomly distributed, entangled fibers were physically bound together with H bonds, forming non-oriented, multi-layered net. Kraft fibers (Figure 3a) were flattened and made a microporous network. Similarly, the fungal fibers Ga (Figure 3c) formed a network of microporous structures. Hemp material (Figure 3b) displayed a dense structure composed of flat fibers and thin microfibers.

The microstructure of mycocel biopolymers was affected by individual properties of raw materials. The structure of KF FF (Ga) (Figure 3d) was determined by individual properties of Kraft fibers and fungal fibers. Distribution FF among the flattened KF favoured formation a loose microporous structure of the material. The structure of biopolymer HF FF (Ga) was affected by a dense network of variable diameter hemp fibers (Figure 3e). The FF were incorporated in a tight HF network of microfibers forming a compact structure with decreased porosity. The surface of KF HF FF (Ga) material (Figure 3f) displayed dense areas of hemp fibers and Kraft fibers with few areas of loose fungal fibers. The porosity and the tortuosity fractal dimension are two critical parameters to determine the permeability. It is reported [23] that increase in the tortuosity fractal dimension leads to decrease in the dimensionless permeability and absolute permeability; an increase in the porosity increases

the dimensionless permeability; increase in the fiber diameter yields an increase in the absolute permeability.

**Figure 3.** Ultrastructure of raw materials (left column) from (**a**) Kraft fibers (KF), (**b**) hemp fibers (HF), and (**c**) fungal fibers (FF Ga), and mycocel biopolymer compositions (right column): (**d**) KF FF (Ga); (**e**) HF FF (Ga); (**f**) KF HF FF (Ga). SEM, 1000×. Bar = 100 μm.

### *3.2. Air Permeability*

The air permeability results were supported by microscopy findings. Mycocel biopolymer compositions had significant variations in air permeability properties starting from almost non-air permeable ones to materials with air permeability above measuring limit of device, which was 8820 mL/min (Table 1). The fungal type of hyphae had a significant effect on results. Biopolymer blends having *F. fomentarius* (Ff) or *G. applanatum* (Ga) trimitic hyphal system in combination with Kraft fibers (KF FF (Ff) and KF FF (Ga)) had the highest

air permeability >8820 mL/min. Biopolymers consisting of cellulose fibers (KF and HF) and *A. bisporus* (Ab) hyphae had the lowest air permeability 14.1 mL/min, <1 mL/min and 7.5 mL/min for KF FF (Ab), HF FF (Ab), and KF HF FF (Ab), respectively.

**Table 1.** Mechanical and air permeability properties of mycocel biopolymer materials (KF = Kraft fibers; HF = hemp fibers; FF = fungal fibers; Ga = *G. applanatum*; Ab = *A. bisporus*; Tv = *T. versicolor*; Ff = *F. fomentarius*). The marked (\*) samples have been described previously [12].

It should be noted that materials with HF in their composition had the lowest air permeability numbers because of high Shopper Riegler freeness of hemp cellulose fiber (91.5 ◦SR) [12], which led to high bonding and tight networks of fibers (Figure 3b). The bonding of cellulose fibers is based primarily on hydrogen bonding between hydroxyl functional groups during close contacting of fibers [24]. The same bonding theory can be attributed to investigated mycocel biopolymer materials, since cellulose and chitin the main constituent of hyphae—are biopolymers and have similar polysaccharide chain structure with the main difference being the replacement of one of the three hydroxyl groups with acetyl amine group in chitin monomeric unit [25]. The ability of functional groups of hyphal polysaccharides to make hydrogen bonding with cellulose fibers is one of the key elements of network formation in investigated mycocel materials. Furthermore, better bonding leads to a more compact packing of fibers and lower free volumes associated with lower air permeability [26], which can be seen in the cases of biopolymer blends with Tv mycelial and Ab stipe fibers. The higher numbers of air permeability showed lower ability of Ga and Ff hyphae to get involved in the network of cellulose fibers through hydrogen bonding. It can be explained by presence of non-polysaccharidic substances, such as pigments, which were indicated by the dark color of fruiting bodies and isolated hyphae from Ga and Ff.

Mycocel blends with higher air permeability have potential for using as gas permeable membranes, for example, as biobased filter layer in face masks. Disposal medical face masks, used as reference material, correspondingly showed high air permeability above 8820 mL/min, therefore only KF FF (Ga) and KF FF (Ff) are appropriate for this application. Blends with very low air permeability, such as those containing Ab hyphae can be used in applications, where high air or gas barrier properties are important, for example, food packaging materials [27].

### *3.3. Mechanical Properties*

Comparison of cellulose and fungal fiber biopolymers showed significant differences among different fungal species and hyphal types regarding their effects on mechanical properties of investigated materials (Table 1). It was possible to produce a pure fiber material from *G. applanatum* (Ga) and *A. bisporus* (Ab) biomass, however FF (Ga) material had a rather low tensile index 8.2 Nm/g, but FF (Ab) material was too fragile for handling and it was not possible to measure mechanical properties. When compare two-component blends of Kraft fibers and fungal fibers, the best results were obtained in the case of KF FF

(Ab) and KF FF (Tv), 32.5 and 26.5 Nm/g, respectively, which is by 92% and 57% more than tensile index measured for material containing only KF. However, addition of *G. applanatum* (Ga) or *F. fomentarius* (Ff) fibers to cellulose fibers decreased mechanical strength by 18% for KF FF (Ga) and by 48% for KF FF (Ff) if compare with pure KF. This can be explained by specific character of Ab and Tv hyphae which formed a dense microstructure with cellulose fibers increasing the mechanical strength of material. It is reported [28] that generative hyphae alone (monomitic hyphal system), which are hollow and contain cytoplasm, are suggested to provide limited mechanical performance, with binding hyphae (dimitic and trimitic hyphal systems) responsible for material strength. Contrary, our results showed that the trimitic hyphal system of fungi Ga and Ff did not favor an improvement of mechanical properties of mycocel compositions. Lower mechanical performance of Ga and Ff hyphae containing materials can be explained also by the aspects mentioned in description of air permeability properties and are related to lower ability for bonding with cellulose caused by presence of non-polysaccharidic substances and lower amount of hydrogen bonds formed. It is known that fiber to fiber bonding or bonding strength is directly related to mechanical strength of cellulose fiber-based materials and can be evaluated using results of mechanical tests [29].

Tensile index was almost threefold higher in wet stage when compare KF and KF FF (Ab) showing the ability of Ab hyphae to significantly improve the strength of fiber material in wet conditions. Two-component blends consisting of hemp fibers and fungal fibers HF FF were produced using hemp cellulose and Ga or Ab fibers. It must be noted that highly fibrillated hemp fibers with freeness 91.5 ◦SR used in the research, had the most significant effect on the mechanical strength of all investigated materials. Furthermore, pure HF material had the highest tensile index among all the tested materials by reaching 60.4 Nm/g. Addition of hyphae significantly decreased tensile index reaching 30.8 Nm/g in the case of HF FF (Ga) and 32.5 Nm/g in the case of HF FF (Ab). When compare threecomponent blends KF HF FF, here also Ab hyphae showed the highest effect on mechanical properties of composites by KF HF FF (Ab) reaching tensile index 46 Nm/g, while KF HF FF (Ga) reached 35.9 Nm/g. Overall, *A. bisporus* (Ab) and *T. versicolor* (Tv) fibers showed higher potential as component of biopolymer for improving the mechanical properties of cellulose fiber-based materials, whether they consist of wood or hemp cellulose fibers. Biopolymers containing Ga hyphae demonstrated the highest flexibility measured as percentage elongation or stretch. HF FF (Ga) and KF HF FF (Ga) samples reached 3.4% and 3.9% stretch, while other composites had numbers equal or below 2.0%.

Mechanical performance of materials was evaluated also by measuring burst index, which is the hydrostatic pressure necessary to cause rupture in a circular area of a given diameter and tells how much pressure material can tolerate before rupture. Three component blends showed the highest burst index 2.8 kPa m2/g and 2.4 kPa m2/g for KF FF FF (Ga) and KF HF FF (Ab) respectively, indicating the prevalence of fiber diversity presented in mechanical blend. If two-component blends are compared, Tv and Ab containing KF FF materials have higher results than Ga and Ff containing ones. Breaking length is used to characterize inherent strength of material and is defined as the length of imaginary material strip, if suspended vertically from one end, would break by its own weight. Numeric values of breaking length of biopolymer blends were in direct correlation with tensile strength values showing the predominance of Ab and Tv containing materials and lower results in Ga and Ff containing materials. Mechanical properties of disposal medical face masks layers were not possible to measure according to the standard and device used for biopolymer blends testing, however apparently they are less strong than materials of biopolymer blends.

### *3.4. Virus Filtration Properties*

In order to evaluate the filtration properties of the experimental biomaterials, the recombinant Semliki forest virus (SFV) was applied, which belongs to the Togaviridae family of enveloped RNA viruses (60–70 nm in diameter) structurally similar to human pathogenic viruses such as influenza and coronaviruses. The test system was based on a safe replication deficient SFV vector (pSFVenh/Luc), allowing to perform one round cell infection to be performed with precise quantification of the virus filtration rates using rapid measurement of luciferase activity in infected cells.

The efficiency of the SFV/enhLuc virus filtration through the tested materials are summarized in Table 2. The efficient virus retention properties were observed both for raw materials (Kraft fibers, hemp fibers, and fungal fibers Ga) and mycocel blends KF FF (Ga) and KF FF (Tv), which revealed very low virus permeability rates (<2%). Cellulose hygiene paper also showed the ability to retain the virus, albeit with lower efficiency (the permeability <30%) comparing to cellulose containing biopolymers. The properties of cellulose materials are determined by production technology and additives which can result in lower mechanical strength, density, and virus filtering efficiency as observed in the case of hygiene paper. Regarding the reference materials, i.e., face masks, the highest virus permeability was observed for the tested surgical mask (>92%) and hydrophobic outer layer of the cotton mask (>70%), whereas the *Silverplus* layer retained the virus significantly (the permeability <1%).

**Table 2.** SFV1enh/Luc recombinant virus titer change after pressure filtration through experimental materials (KF = Kraft fibers; HF = hemp fibers; FF = fungal fibers; Ga = *G. applanatum*; Tv = *T. versicolor*).

According to EMA guidelines (CPMP/BWP/268/95, European Medicines Agency) the log10 reduction value (LRV) is an important parameter to quantify the virus reduction capacity. Only the LRV > 4 is considered as a "very high" virus reduction potential. In this study, the biopolymer KF FF (Ga) demonstrated sufficiently high virus reduction capacity (4.54 LRV). Remarkably, the silver containing cotton layer of the commercial mask also showed efficient virus removal properties (3.6 LRV), which can be related to the viricidal capacity of the immobilized silver nanoparticles [30].

Due to safety aspects, handling of human viruses is a labor-intensive and timeconsuming process. In this study, we have established a protocol for a relatively simple virus filtration method, which is based on replication deficient SFV vector encoding firefly luciferase used for rapid virus quantification. The proposed method can be efficiently applied for primary screening of virus filtration properties of the membranes.

The mechanisms underlying the membrane filtration of viruses include size exclusion and/or adsorptive interactions (e.g., hydrophobic/hydrophilic and electrostatic interactions) between virus envelope and membrane compounds [31]. The diameter of the virus particles is much smaller than the pore size of the tested membranes. Therefore, the adsorptive interactions can be considered as the main mechanism of filtration in the tested system. A surgical mask, which is made of hydrophobic polypropylene layers, did not adsorb aqueous virus containing solution, resulting in low virus retention values (Table 2). Practically, the surgical masks serve as a barrier to aqueous aerosol and are designed to block direct fluid entry into the wearer's respiratory tract and mostly act as a repellent of the water-based liquids [32]. In contrast to surgical mask and tested cotton mask, the row fiber materials and mycocel blends exhibited hygroscopic properties and showed the ability to binding, or adsorption of SFV particles. A high virus filtration capacity of mycocel biopolymer blends might be attributed to the structural properties of the fungal cell wall as an insoluble polysaccharide-based sorbent. The fungal polysaccharides possess direct virus inactivation properties as shown for several highly pathogenic human viruses, including human immunodeficiency virus, herpes simplex virus, etc. [33]. The application of mycocel-based filters alone or in combination with polypropylene-based layers can represent an advanced individual respiratory protective device against airborne pathogens. Furthermore, chitin, one of the main polymers of fungal cell walls, is widely used for controlled drug delivery systems, protein and enzyme carriers, and packaging materials, based on its natural antimicrobial activity. Chitin and its derivatives (e.g., chitosan) have many useful properties that make them suitable for a wide variety of biomedical applications. Their products are known to be antibacterial, antifungal, antiviral, nontoxic, and nonallergic [34]. Therefore, the proposed mycocel-based biopolymers are promising multifunctional materials for biomedical and bioengineering applications.

### **4. Conclusions**

A novel mycocel biopolymer from naturally derived biomass of plant cellulose fibers and fungal fibers (hyphae) was developed and characterized regarding its air permeability, mechanical properties, and virus filtration efficiency.

Air permeability and mechanical properties of mycocel biopolymer blends were affected by microstructural features of raw materials. Highly fibrillated hemp fibers had the most significant effect on the mechanical strength while Kraft fibers revealed increased air permeability. The ability of functional groups of hyphal polysaccharides to make hydrogen bonding with cellulose fibers was one of the key elements of network formation which determined biopolymer properties. The loose fiber net of trimitic fungal species *G. applanatum* and *F. fomentarius* with incorporated Kraft fibers formed a microporous structure of mycocel blends with improved air permeability (>8820 m/min) and limited mechanical properties in comparison with individual raw fibers. *A. bisporus* and *T. versicolor* fibers showed higher potential as components of biopolymer for improving the mechanical properties of cellulose fiber-based materials, whether they consist of wood or hemp cellulose fibers.

Virus testing provided promising results regarding virus filtration efficiency of biopolymer blends. Mycocel biopolymer KF FF (Ga) demonstrated sufficiently high virus reduction capacity (4.54 LRV) than surgical mask and outer and inner layers of commercial face mask. Feasibly, the adsorptive interactions can be considered as the main mechanism of filtration in tested system.

The natural, biodegradable mycocel blends have a potential for use in biomaterial membranes depending on the target application. Blends with higher air permeability and virus filtration efficiency have potential for being used as gas permeable membranes, for example, as biobased filter layer in face masks. Blends with low air permeability can be used in areas where high air or gas barrier properties are important, for example, food packaging materials. The water microfiltration and ultrafiltration also are considered for future application.

**Author Contributions:** Conceptualization, I.I. and I.F.; methodology, M.S., K.S., A.Z.; validation, I.I., I.F. and A.Z.; formal analysis, K.S., A.Z.; writing—original draft preparation, I.I.; writing—review and editing, I.F., A.Z. and T.J.; project administration, I.F. and T.J.; funding acquisition, T.J. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research is funded by the Ministry of Education and Science, Republic of Latvia, Project "Integration of reliable technologies for protection against Covid-19 in healthcare and high-risk areas", Project No. VPP-COVID-2020/1-0004. The Article Processing Charge was kindly provided by the Latvian State Institute of Wood Chemistry Base financing source.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

## *Article* **Upgrading Argan Shell Wastes in Wood Plastic Composites with Biobased Polyethylene Matrix and Different Compatibilizers**

**Maria Jorda-Reolid 1, Jaume Gomez-Caturla 2, Juan Ivorra-Martinez 2,\*, Pablo Marcelo Stefani 3, Sandra Rojas-Lema 1 and Luis Quiles-Carrillo 1,\***

**Abstract:** The present study reports on the development of wood plastic composites (WPC) based on micronized argan shell (MAS) as a filler and high-density polyethylene obtained from sugarcane (Bio-HDPE), following the principles proposed by the circular economy in which the aim is to achieve zero waste by the introduction of residues of argan as a filler. The blends were prepared by extrusion and injection molding processes. In order to improve compatibility between the argan particles and the green polyolefin, different compatibilizers and additional filler were used, namely polyethylene grafted maleic anhydride (PE-g-MA 3 wt.-%), maleinized linseed oil (MLO 7.5 phr), halloysite nanotubes (HNTs 7.5 phr), and a combination of MLO and HNTs (3.75 phr each). The mechanical, morphological, thermal, thermomechanical, colorimetric, and wettability properties of each blend were analyzed. The results show that MAS acts as a reinforcing filler, increasing the stiffness of the Bio-HDPE, and that HNTs further increases this reinforcing effect. MLO and PE-g-MA, altogether with HNTs, improve the compatibility between MAS and Bio-HDPE, particularly due to bonds formed between oxygen-based groups present in each compound. Thermal stability was also improved provided by the addition of MAS and HNTs. All in all, reddish-like brown wood plastic composites with improved stiffness, good thermal stability, enhanced compatibility, and good wettability properties were obtained.

**Keywords:** argan shell particles; wood plastic composite; polyethylene; mechanical properties; compatibilization

### **1. Introduction**

In the last decades, an increasing concern about environmental issues related to the great use of petrochemical-derived plastics has been rising, as well as the necessity to reduce the carbon footprint associated with the production processes of those polymers. This trend is driving the industry toward the use of polymeric materials derived from natural resources, with the objective of moving away from petroleum. Moreover, this fact has propitiated the use of biodegradable polymers, some of which are capable of decomposing in composting conditions. At the same time, they exhibit very similar properties to those of their petrochemical counterparts [1]. One of the main drawbacks of plastics derived from natural sources is that they are more expensive than those from petrochemical origin. This leads to a direct rejection by industry. For this reason, incorporation of fillers and creation of new more economic materials is gaining increasing relevance.

**Citation:** Jorda-Reolid, M.; Gomez-Caturla, J.; Ivorra-Martinez, J.; Stefani, P.M.; Rojas-Lema, S.; Quiles-Carrillo, L. Upgrading Argan Shell Wastes in Wood Plastic Composites with Biobased Polyethylene Matrix and Different Compatibilizers. *Polymers* **2021**, *13*, 922. https://doi.org/10.3390/ polym13060922

Academic Editor: Gianluca Tondi

Received: 27 February 2021 Accepted: 15 March 2021 Published: 17 March 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Among those materials, "Wood plastic composites" (WPC) are becoming increasingly popular. This technology implies the addition of natural organic fillers obtained from wastes into polymer matrices, with the objective of decreasing their processing cost. At the same time, those fillers enhance their environmental value and vary their mechanical, physical and chemical properties [2]. Initially, only wood flour or sawdust were used as bio-fillers [3–6], but over time fillers from agroforestry waste or food industry have started to be used, such as orange peel [7,8], almond shell [9], pomegranate peel [10], or argan seed shell [11]. Those fillers are introduced in the structure of polymers like poly (lactic acid) (PLA), high density polyethylene (HDPE) or polyester [12–14]. Nowadays, WPC can be employed as a substitute of wood due to its appearance for the fabrication of indoor and outdoor furniture, decks pergolas, and so on [15,16].

In this sense, high density bio-polyethylene (Bio-HDPE) is one of the most interesting polymer matrices, as it combines its natural origin with the ease of processability of polyolefins. This polymer is produced by conventional polymerization of ethylene obtained from catalytic dehydration of bio-ethanol [17], which is extracted from natural sources, such as sugarcane [18]. Bio-HDPE possesses the same physical properties than its fossilderived counterpart; particularly, good mechanical resistance and high ductility [19]. Injection-molded Bio-HDPE pieces have great application in packaging [20]. However, one of the main problems that limits the use of bio-fillers as reinforcing agents is their hydrophilic nature, which causes a poor interfacial adhesion with a hydrophobic (non-polar) matrix, such as Bio-HDPE. This provokes a deterioration in the mechanical properties of the material and a bad compatibility between filler and matrix [21,22].

Several studies have been carried out to improve the compatibilization between those fillers and polymeric matrices. On the one hand, some of the methods that have been applied to improve this compatibility include the modification of the surface of the filler particles from the waste, such as mercerization, benzoylation, acetylation, silanation, or graft copolymerization [23,24]. On the other hand, compatibilizing agents have also been used in order to improve the adhesion and affinity between filler and matrix. Additionally, they have also been utilized to increase the dispersion of the reinforcing particles within the matrix and give additional properties to the blend [25–27]. In this context, polyethylene-grafted maleic anhydride (PE-g-MA) is one of the most efficient compatibilizers. It acts as a chemical connection between ligno-cellulosic particles and polymeric chains, owing to its double functionality. First, the polyethylene fraction of PE-g-MA interacts with polymeric chains due to its chemical affinity, while the anhydride groups can react with the hydroxyl groups from the ligno-cellulosic structure in the organic particles by esterification. Thus, leading to an increase in the matrix–filler interaction and a positive effect in the dispersion of the particles and the stiffness of the blend [28–30]. However, ductile properties tend not to get better, because of the fragility given by those fillers when they form stress-concentrator aggregates in the matrix.

In recent years, vegetable oils have generated a great deal of interest as compatibilizing additives. Modified vegetable oils have been used as plasticizers, stabilizers, crosslinkers, compatibilizers, and so on. The use of epoxidized vegetable oils (EVOS) should be remarked, such as epoxidized soybean oil (ESBO) [31,32], epoxidized linseed oil (ELO) [33,34], or epoxidized palm oil (EPO) [35]. All of them have been tested in polymeric blends and composites, achieving positive compatibilization/plasticization effects, owing to the reactivity of the oxirane group. Maleinization is an interesting alternative chemical modification for vegetable oils. Maleinized linseed oil (MLO) has been widely used in WPC, for example, in a PBS matrix with almond shell flour [36]. Furthermore, the excellent ductile properties that this oil is capable of giving to composites have also been reported [13]. In addition, inorganic particles can also be introduced as reinforcing agents in polymeric matrices, such as carbon nanotubes (CNTs), nanoclays, or metallic oxide nanoparticles [37–39]. In recent years, halloysite nanotubes (HNTs) have gained great importance in this sense. They are cheaper and more abundant than, for example, carbon nanotubes (CNTs). In this sense, halloysite is a natural mineral clay with nanotube

morphology, similar to kaolinite (Al2[Si2O5(OH)4].nH2O), where n is 2 for hydrated HNTs, and 0 for dehydrated HNTs [40]. Frost et al. [41] reported two types of hydroxyl groups, inner and outer hydroxyl groups, placed between the nanotubes layers and in the surface of HNTs [41]. Because of their multilayer structure, most of the hydroxyl groups are in the inner side, while a few of them are in the surface of HNTs. As a result of this, HNTs dispersion in polymer matrices is simpler than other inorganic nanoparticles [40]. Numerous studies have reported the ability of HNTs to improve thermal stability, fire resistance, mechanical properties, and crystalline behavior of polymers such as polypropylene [42– 46], low density polyethylene (LLDPE) [47], polyamide 6 (PA6) [48], epoxy resin [49], polylactide (PLA) [50,51], and so on. These are the reasons for utilizing HNTs in this work. As it is the case with organic particles, the challenge here is to achieve a good compatibility and adhesion between HNTs and the polymer matrix, especially in non-polar matrices, such as polyolefins.

The objective of this work is the reuse of micronized argan shell (MAS) as a reinforcing organic filler in a Bio-HDPE matrix derived from sugarcane, with the objective of developing a wood plastic composite with enhanced properties compared to the original matrix following the trends proposed by the circular economy, which seeks to reuse or recycling the waste and to reintroduce them into the industry [52]. Argan (*Argania spinosa*) is a tropical plant that belongs to the *Sapotaceae* family, whose fruit is used in morocco to prepare oil [53]. This oil has great application in cosmetics owing to its high E vitamin content [53], while fruit residues and seeds are generally used to feed cattle [54]. The main drawback of micronized argan shell (MAS) is its low compatibility with Bio-HDPE. In particular, the challenge of this investigation is to improve the affinity between both elements through the use of compatibilizing agents. Several formulations have been developed to meet this end, using the compatibilizers PE-g-MA and MLO; and halloysite nanotubes (HNTs) as additional reinforcing fillers; altogether with Bio-HDPE as the polymer matrix and argan shell particles as the main reinforcing filler; these elements have been used both individually and in combination. Additionally, MLO and HNTs concentrations have been varied (7.5 parts per hundred resin (phr) when used individually and 3.75 phr when used in combination). In order to evaluate the effects of these compatibilizers and fillers over each blend, mechanical, morphological, thermal, thermomechanical, colorimetric, and wettability properties are presented.

### **2. Materials and Methods**

### *2.1. Materials*

Bio-HDPE, SHA7260 grade, was supplied in pellets form by FKuR Kunststoff GmbH (Willich, Germany) and manufactured by Braskem (São Paulo, Brazil). This green polyethylene has a density of 0.955 g cm−3 and a melt flow index (MFI) of 20 g/10 min, measured with a load of 2.16 kg and a temperature of 190 ◦C. Micronized argan shell (MAS) was supplied by MICRONIZADOS VEGETALES S.L. (Benamejí (Córdoba), Spain) company. Halloysite nanotubes were supplied by Sigma Aldrich (Madrid, Spain) with CAS number 1332-58-7.

Interlocking is one of the most important mechanisms in polymer composites. This mechanism depends on the filler shape. Figure 1a shows the morphology of the argan particles observed by SEM. Most of the particles exhibit a rough surface, which can be ascribed to the milling process due to the high hardness of this filler. A closer observation of the particles shows some porosity on their surface, which can produce a good interaction with the polymer matrix acting as mechanical anchoring points. A similar structure was observed by Laaziz et al. [55] when studying argan nut shell particles with different treatments. Additionally, Figure 1b and c show histograms for the length and diameter of the particles, respectively, determined from the SEM image. Particles were average 70 μm long and 45 μm wide. These parameters are also important to consider when talking about mechanical properties. Too large particles could lead to a great mechanical impairment, increasing the heterogeneity of the blends. Crespo et al. [56] observed this effect for almond shell particles superior to 150 μm diameter.

**Figure 1.** (**a**) Scanning electron microscope image (SEM) of micronized argan shell (MAS). Image was taken with a magnification of 100× and a scale marker of 100 μm; (**b**) histogram of the argan particles length; (**c**) histogram of the argan particles diameter.

In relation to the additives, polyethylene-grafted maleic anhydride (PE-g-MA) with CAS Number 9006-26-2 and MFI values of 5 g/10 min (190 ◦C/2.16 kg), was obtained from Sigma-Aldrich S.A. (Madrid, Spain). This PE-based copolymer was selected due to its functionality. The maleinized linseed oil—MLO, VEOMER LIN was supplied by Vandeputte (Mouscron, Belgium). This modified vegetable oil is characterized by a viscosity of 10 dPa s at 20 ◦C and an acid value comprised in the 105–130 mg KOH g−1 range.

### *2.2. Preparation of Bio-HDPE Blends*

Bio-HDPE, PE-g-MA, MAS y HNTs were initially dried at 40 ◦C for 48 h in a dehumidifying dryer MDEO (Barcelona, Spain) to remove any residual moisture prior to processing to avoid the possibility of hydrolysis due to the moisture. Then, the corresponding wt.% of each component (see Table 1) were mixed and pre-homogenized in a zipper bag. The corresponding formulations were compounded in a twin-screw co-rotating extruder from Construcciones Mecánicas Dupra, S.L. (Alicante, Spain). This extruder has a 25 mm diameter with a length-to-diameter ratio (L/D) of 24 to allow a correct blending process. The extrusion process was carried out at a rate of 22 rpm, using the following temperature profile (from the hopper to the die): 140–145–150–155 ◦C. The compounded materials were pelletized using an air-knife unit. In all cases, residence time was approximately 1 min. Table 1 shows the compositions of the formulations developed in this work.

To transform the pellets into standard samples, a Meteor 270/75 injector from Mateu & Solé (Barcelona, Spain) was used. The temperature profile in the injection molding unit was 135 ◦C (hopper), 140 ◦C, 150 ◦C, and 160 ◦C (injection nozzle). A clamping force of 75 tons was applied while the cavity filling and cooling times were set to 1 and 10 s, respectively. Standard samples for mechanical and thermal characterization with an average thickness of 4 mm were obtained.

**Table 1.** Summary of compositions according to the weight content (wt.%) of Bio-HDPE/MAS and different compatibilizers and additives.

### *2.3. Characterization of Bio-HDPE Blends*

2.3.1. Mechanical Characterization

Mechanical properties were obtained with different tests like tensile test, shore hardness, and Charpy impact test. Tensile properties of Bio-HDPE/PE-g-MA/MAS blends were obtained in a universal testing machine ELIB 50 from S.A.E. Ibertest (Madrid, Spain) as recommended by ISO 527-1:2012 with dog bone samples 1B specification. A 5-kN load cell was used and the cross-head speed was set to 5 mm/min. Shore hardness was measured in a 676-D durometer from J. Bot Instruments (Barcelona, Spain), using the D-scale, on rectangular samples with dimensions 80 × 10 × 4 mm3, according to ISO 868:2003. The impact strength was also studied on injection-molded rectangular samples with dimensions of 80 × 10 × 4 mm3 in a Charpy pendulum (1-J) from Metrotec S.A. (San Sebastián, Spain) on notched samples (0.25 mm radius V-notch), following the specifications of ISO 179-1:2010. All mechanical tests were performed at room temperature, and at least six samples of each material were tested and the corresponding values were averaged.

### 2.3.2. Morphology Characterization

The morphology of fractured samples from Charpy tests, obtained from the impact tests, were studied by field emission scanning electron microscopy (FESEM) in a ZEISS ULTRA 55 microscope from Oxford Instruments (Abingdon, UK). Before placing the samples in the vacuum chamber, they were sputtered with a gold-palladium alloy in an EMITECH sputter coating SC7620 model from Quorum Technologies, Ltd. (East Sussex, UK). The FESEM was operated at an acceleration voltage of 2 kV.

#### 2.3.3. Thermal Analysis

The most relevant thermal transitions of Bio-HDPE/PE-g-MA/MAS blends were obtained by differential scanning calorimetry (DSC) in a Mettler-Toledo 821 calorimeter (Schwerzenbach, Switzerland). Samples with an average weight of 6–7 mg, were subjected to a thermal program divided into three stages: a first heating from 25 ◦C to 160 ◦C followed by a cooling to 0 ◦C, and a second heating to 250 ◦C. Both heating and cooling rates were set to 10 ◦C/min. All tests were run in nitrogen atmosphere with a flow rate of 66 mL/min using standard sealed aluminum crucibles with a capacity of 40 μL.

The thermal degradation of the Bio-HDPE/PE-g-MA/MAS blends was assessed by thermogravimetric analysis (TGA). TGA tests were performed in a LINSEIS TGA 1000 (Selb, Germany). Samples with a weight of 15–17 mg were placed in 70 μL alumina crucibles and subjected to a dynamic heating program from 40 ◦C to 700 ◦C at a heating rate of 10 ◦C/min in air atmosphere. The first derivative thermogravimetric (DTG) curves were also determined. All tests were carried out at least three times in order to obtain reliable results.

### 2.3.4. Thermomechanical Properties

In order to obtain the thermomechanical properties of the Bio-HDPE composites a dynamical mechanical thermal analysis (DMTA) was carried out in a DMA1 dynamic analyzer from Mettler-Toledo (Schwerzenbach, Switzerland), working in single cantilever flexural conditions. Rectangular samples with dimensions 20 × 6 × 2.7 mm3 were subjected to a dynamic temperature sweep from −150 ◦C to 120 ◦C at a constant heating rate of 2 ◦C/min. The selected frequency was 1 Hz and the maximum flexural deformation or cantilever deflection was set to 10 μm.

#### 2.3.5. Color and Wetting Characterization

The colorimetric modifications of the samples were measured with a Konica CM-3600d Colorflex-DIFF2, from Hunter Associates Laboratory, Inc. (Reston, VA, USA) was used. Color coordinates (L\*a\*b\*) were measured according to the following criteria: L\* = 0, darkness; L\* = 100, lightness; a\* represents the green (a\* < 0) to red (a\* > 0); b\* stands for the blue (b\* < 0) to yellow (b\* > 0) coordinate.

Hydrophilicity/ hydrophobicity of each blend was assessed by contact angle measurements through time were carried out with an EasyDrop Standard goniometer model FM140 (KRÜSS GmbH, Hamburg, Deutschland) which is equipped with a video capture kit and analysis software (Drop Shape Analysis SW21; DSA1). Double distilled water was used as test liquid, a drop of it was put in each sample and contact angle measurements were taken at 0, 5, 10, 15, 20, and 30 min after the administration of the water. For color and wetting measurement, traction samples were used to facilitate the measures.

### 2.3.6. Water Absorption Test