EP4587511A1 - Bio-nanocomposite material - Google Patents

Abstract

The invention concerns a bio-nanocomposite material comprising: - at least a protein extract; - at least an aliphatic hydrocarbon compound selected from: iii) C₂-C₂₁ containing at least an oxygenated functional group selected from the group consisting of hydroxyl, epoxide, ether, acid, ester, carbonate; ii) polyethylene glycol (PEG) with a molecular weight in the range from 0.2 to 35 kDa; and iii) polytetrahydrofuran with a molecular weight in the range from 0.25 to 2.9 kDa; and - at least an adduct of an allotrope of sp² carbon with at least a pyrrolic derivative of Formula (i), wherein - R₁-R₄ are independently selected from the group consisting of hydrogen, C₁-C₃ alkyl, linear or branched C₂-C₁₀ alkenyl or alkynyl, aryl, linear or branched C₁-C₁₀ alkyl-aryl, linear or branched C₂-C₁₀ alken-aryl, linear or branched C₂-C₁₀ alkyn-aryl and heteroaryl; - W is selected from the group consisting of hydrogen, linear or branched C₁- C₁₀ alkyl, linear or branched C₂-C₁₀ alkenyl or alkynyl, aryl, linear or branched C₁-C₁₀ alkyl-aryl, linear or branched C₂-C₁₀ alken-aryl, linear or branched C₂-C₁₀ alkyn-aryl, heteroaryl, carboxyl and a group of Formula, wherein - M, J and Q are independently selected from the group consisting of hydrogen, hydroxyl, amine, linear or branched C₁-C₁₀ alkyl, linear or branched C₂-C₁₀ alkenyl or alkynyl and a group selected from: wherein: - R₅-R₁₉ are independently selected from the group consisting of hydrogen, linear or branched C₁-C₁₀ alkyl, linear or branched C₂-C₁₀ alkenyl or alkynyl, aryl, linear or branched C₁-C₁₀ alkyl-aryl, linear or branched C₂- C₁₀ alken-aryl, linear or branched C₂-C₁₀ alkyn-aryl, heteroaryl or carboxyl; - R₁₄-R₁₆ are selected from -OCH₃ and -OCH₂-CH₃; - R₁₈ is selected from -CH₂-SH and -CH₂-CH₂-S-CH₃; - T is oxygen or sulfur, with the proviso that when T is sulfur m' is an integer from 1 to 4 and when T is oxygen m' is an integer from 1 to 2; - I', p', q', r' are independently integer from 0 to 12; and R₂₀ is selected from the group consisting of hydrogen, alkyl, aryl, benzyl, ami_{ne, alkyl-amine, aryl-amine, benzyl-amine and amino-aryl; and} R₂₁-R₂₅ are independently selected from the group consisting of hydrogen, linear or branched C₁-C₁₀ alkyl, linear or branched C₂-C₁₀ alkenyl or alkynyl and 1-(4-aminocyclohexyl) methylene, wherein said at least a protein extract is extracted from the Black Soldier Fly (BSF) Hermetia illucens, and wherein said allotrope of sp² carbon has a surface area higher than 60 m²/g, measured by BET in accordance with the standard ASTM D3037.

Description

BIO-NANOCOMPOSITE MATERIAL

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FIELD OF THE INVENTION

The present invention concerns the development of an environmentally sustainable and biodegradable bio-nanocomposite material with high properties of resistance to mechanical stresses, flexibility and elasticity and high electrical conductivity, resistance to moisture, and thus with broad flexibility of use.

STATE OF THE ART

The field of printed electronics aims to revolutionise the way electronic circuits and components are manufactured and used, using innovative printing technologies. This is because printed electronics exploit additive manufacturing methods and low- temperature processes. In this respect, this field has already had enormous success in the realisation of printed circuit boards and “components” such as transistors, solar cells and light-emitting diodes on flexible plastic substrates. In the future, for electronic devices in the medical and flexible robotic field, materials with excellent flexibility and extensibility but which are at the same time compatible with the body and non-toxic will be increasingly required in order to meet the extreme mechanical demands they will be subjected to. For certain applications relating to the human body, stresses of up to 50% in elongation must be tolerated. This means that the constituent materials, such as substrates, conductors, dielectrics, semiconductors, sensors and encapsulants, must be carefully designed to function under harsh conditions.

Currently, most of the functional materials used in printed electronics consist of relatively rigid composites, such as those found in conductive wiring (e.g. silver pastes), dielectrics (e.g. clay composites), or sensors (e.g. force-sensitive resistors), or sintered metal layers or conjugated (doped) polymers.

It is therefore essential to use conductive functional materials to be used in printed electronics that are both flexible and elastic. In order to achieve the desired electronic and mechanical properties, the simplest approach is to create a composite, which can represent a stretchable conductive material consisting of a conductive filler, also known as a “filler”, and a polymeric phase. However, the polymers used as a matrix of the composite material are typically derived from petroleum and their main polymer chains are based on C-C or Si-O-Si bonds and, as a result, they are not biodegradable and it is widely recognised that an increasing amount of polymeric waste is being accumulated in the environment. Therefore, preference has been given to the use of more sustainable polymers, derived from natural sources and having functional groups in the main polymeric chain such that they can be more easily attacked by the microorganisms and thus be at least compostable or even biodegradable. In fact, most of the polymers of natural origin that have functional groups in the main chain are actually compostable rather than biodegradable, and even when they are biodegradable, they need more time to be degraded in the environment than in laboratories.

These technical problems appear really relevant, as recycling is hardly considered as an end-of-life option for the polymeric materials of natural origin, which are potentially compostable or even biodegradable.

It is therefore preferable to use polymers of natural origin, as a matrix of the composite materials, whose degradability can be controlled and ideally triggered, and which can make a positive contribution to solving, or at least reducing, the polymeric waste problem in order to contribute to the sustainable technological development and improve the sustainability of the technologies adapted to meet the present and future needs of the population.

However, it is well known that it is not easy to have flexibility and elasticity with polymers of natural origin, which are typically provided with oxygenated and even nitrogenated functional groups that give rise to strong supra-molecular interactions that prevent easy mobility of the polymeric chains, such mobility being a prerequisite for having elasticity. It would be highly desirable to have matrices based on polymers of natural origin that are still flexible and elastic.

Moreover, a further issue to be taken into consideration when selecting and processing the polymer of natural origin, to be used as a matrix of the composite material, is the need for its chemical compatibilization with ingredients such as the fillers, used to provide mechanical reinforcement and/or electrical conductivity.

In particular, the allotropes of sp² carbon (graphite, carbon black, carbon nanotubes) are very attractive fillers for the preparation of composite materials thanks to their optimal intrinsic mechanical and electrical properties. However, their use in the conductive inks for printed electronics is limited, in case a matrix of natural polar origin is used, primarily because of the need for their chemical modification to increase their polarity. Indeed, the allotropes of sp² carbon and a polar polymeric matrix of natural origin typically have significantly different solubility parameters. Thus, supra-molecular interactions (Van der Waals forces) are established in the composite material between the nanoparticles of the allotropes of sp² carbon, which lead to the formation of aggregates that are difficult to separate and thus to obtaining an inhomogeneous distribution and poor dispersion in the polymeric matrix. Therefore, one possibility that has emerged is to modify the solubility parameter of the nanometer-sized allotropes of sp² carbon in order to promote their compatibility with the polymeric matrix. In the case of graphene and of the graphene-correlated materials (few layer graphene, nanographites), this is typically achieved through oxidation to graphene oxide, realising reactions that require drastic and even dangerous conditions and chemicals that can be toxic. In addition, the graphene oxide obtained loses the electrical characteristics of the starting material.

Finally, the printed electronics devices, in their various applications, are exposed to different environments and, among them, to humid environments.

Therefore, of high importance would be the eventual ability of these electronic devices to withstand humid environments with different water concentrations, maintaining the elastic and conductive properties even after exposure to a humid environment.

In addition to the environmental problems linked to the plastics outlined above, there are those related to the organic waste, more precisely food waste, green waste, i.e. , the so-called organic fraction of municipal solid waste (OFMSW).

The annual municipal solid waste (MSW) production is estimated to reach 2.5 billion tonnes. More than one third is represented by food waste, without considering green waste from gardens and parks, and is the so-called organic fraction of municipal solid waste (OFMSW) (FAO, Global Food Losses and Food Waste - Magnitude, Causes and Prevention. Food and Agriculture Organization of the United Nations, Rome, 1-37, 2011, Wilson D.C., Rodic L, Modak P., Soos R., Carpintero Rogero A., Velis C., Iyer M., Simonett 0. Global perspectives on waste management. Report. United Nations Environment Programme, 1-346).

This amount is expected to increase by 70% by 2050 in both high-income and low- income countries (https://www.epa.gov/sustainability. Accessed 27.02.2020).

OFMSW is currently treated by means of known composting and/or anaerobic digestion systems, which however require the use and the optimisation of specific parameters and plants, being thus time-consuming, expensive methods and which require specialised personnel.

An efficient and promising alternative for the treatment and the valorisation of organic waste is the insect-mediated bioconversion.

However, the insect-derived protein films show significant drawbacks, first of all, they are water-soluble. Therefore, they can only be used under controlled conditions. In addition, they are susceptible to the attack by microorganisms. Finally, no remarkable mechanical properties have been documented. For example, the elasticity and the resilience of the film are not documented.

Furthermore, the insect-derived protein films have no functionality, i.e. , they are not electrically conductive.

It would therefore be highly desirable to prepare functional products, in particular functional films from proteins, with adjustable and adaptable properties such as electrical and thermal conductivity and with high resistance to mechanical stresses, i.e., a material that is flexible and features elastic properties.

Therefore, in light of the above considerations and problems, the lacks of solutions in the known art, and the disadvantages of those available listed above, the need is still felt today to provide a functional material for printed electronics that is not only electrically conductive, with good mechanical properties such as extensibility and flexibility and able to withstand humid environments, but above all deriving from natural sources, non-toxic, compostable or even biodegradable, and having the possibility of being variously supplemented to be able to impart it different properties, including for example thermal and electrical conductivity.

SUMMARY OF THE INVENTION

This aim is achieved by the present invention, which proposes to use a bio- nanocomposite material comprising a matrix formed by proteins extracted from the Black Soldier Fly (known in the sector by the abbreviation BSF, which will be used hereafter) in a mixture with an aliphatic hydrocarbon compound selected from: i) C2- C21 containing at least one oxygenated functional group, ii) polyethylene glycol (PEG) having a molecular weight in the range from 0.2 to 35 kDa and iii) polytetrahydrofuran (poly-THF) having a molecular weight in the range from 0.25 to 2.9 kDa, within which a conductive material is dispersed consisting of an adduct of an allotrope of sp² carbon with a pyrrole derivative, wherein the allotrope of sp² carbon, having a surface area higher than 60 m²/g, ensures the development of a network formed by the particles of the allotrope of sp² carbon, and thus ensures the electrical conductivity of the bio-nanocomposite material, while the pyrrole derivative ensures a homogeneous dispersion through stable interactions with the protein matrix. Moreover, the adduct between the two when inserted into the protein matrix decreases its hydrophilicity, making the bio-nanocomposite more resistant to moisture.

It is thus possible to obtain a biodegradable, compostable, moisture-resistant material, which at the same time has high electrical conductivity and optimal mechanical properties of flexibility, starting from natural sources that valorise waste, which otherwise cannot be used.

Thus, in a first aspect, the invention concerns a bio-nanocomposite material comprising:

- at least a protein extract;

- at least an aliphatic hydrocarbon compound selected from: i) C2-C21 containing at least an oxygenated functional group selected from the group consisting of hydroxyl, epoxide, ether, acid, ester, carbonate; ii) polyethylene glycol (PEG) with a molecular weight in the range from 0.2 to 35 kDa; and iii) polytetrahydrofuran with a molecular weight in the range from 0.25 to 2.9 kDa; and

- at least an adduct of an allotrope of sp² carbon with at least a pyrrol ic derivative of Formula (i) wherein

- R1-R4 are independently selected from the group consisting of hydrogen, C1-C3 alkyl, linear or branched C2-C10 alkenyl or alkynyl, aryl, linear or branched C1-C10 alkyl-aryl, linear or branched C2-C10 alken- aryl, linear or branched C2-C10 alkyn-aryl, and heteroaryl;

- W is selected from the group consisting of hydrogen, linear or branched C1-C10 alkyl, linear or branched C2-C10 alkenyl or alkynyl, aryl, linear or branched C1-C10 alkyl-aryl, linear or branched C2-C10 alken-aryl, linear or branched C2-C10 alkyn-aryl, heteroaryl, carboxyl, and a group of Formula wherein

- M, J and Q are independently selected from the group consisting of hydrogen, hydroxyl, amine, linear or branched C1-C10 alkyl, or linear or branched C2-C10 alkenyl or alkynyl, and a group selected from: wherein:

- R5-R19 are independently selected from the group consisting of hydrogen, linear or branched C1-C10 alkyl, linear or branched C2-C10 alkenyl or alkynyl, aryl, linear or branched C1-C10 alkyl-aryl, linear or branched C2-C10 alken- aryl, linear or branched C2-C10 alkyn-aryl, heteroaryl or carboxyl; - R-I4-R-I6 are selected from -OCH3 and -OCH2-CH3;

- Ris is selected from -CH2-SH and -CH2-CH2-S-CH3;

- T is oxygen or sulfur, with the proviso that when T is sulfur m' is an integer from 1 to 4 and when T is oxygen m' is an integer from 1 to 2;

- I’, p’, q’, r’ are independently integer from 0 to 12; and

R20 is selected from the group consisting of hydrogen, alkyl, aryl, benzyl, amine, alkyl-amine, aryl-amine, benzyl-amine and amino-aryl; and

R21-R25 are independently selected from the group consisting of hydrogen, linear or branched C1-C10 alkyl, linear or branched C2-C10 alkenyl or alkynyl, and 1 -(4- aminocyclohexyl) methylene, wherein said at least a protein extract is extracted from the Black Soldier Fly (BSF) Hermetia illucens, and wherein said allotrope of sp² carbon has a surface area higher than 60 m²/g, measured by BET in accordance with the standard ASTM D3037.

The bio-nanocomposite material described above is a viable alternative to the petroleum-derived polymers of synthetic origin, as it ensures the same, if not better, electrical and mechanical properties, while being sustainable, biodegradable and compostable.

In a second aspect thereof, the invention concerns a bio-nanocomposite film obtained from the aforementioned bio-nanocomposite material of the invention.

Said film ensures flexibility of use in different environments, including biological ones, deriving precisely from the components that form the bio-nanocomposite material of the invention.

Said bio-nanocomposite material on the one hand ensures important properties, and on the other hand has been synthesised by means of a specific process that was simple, fast and without the use of toxic solvents and difficult and drastic reaction conditions requiring a large amount of energy.

Thus, in a further aspect thereof, the present invention concerns the synthesis of a bio-nanocomposite material comprising the steps of: a) providing larvae and pupae of Black Soldier Fly (BSF) Hermetia illucens, fed with the organic fraction of urban solid waste (OFMSW); b) extracting proteins from larvae and pupae, thus obtaining at least a protein extract; c) mixing said at least a protein extract of step b) with at least an adduct of an allotrope of sp² carbon with at least a pyrrole derivative, thus obtaining a uniform mixture; d) adding to the mixture of step c) at least an aliphatic hydrocarbon compound selected from: i) C2-C21 containing at least an oxygenated functional group selected from the group consisting of hydroxyl, epoxide, ether, acid, ester, carbonate; ii) polyethylene glycol (PEG) having a molecular weight in the range from 0.2 to 35 kDa; and iii) polytetrahydrofuran (poly-THF) having a molecular weight in the range from 0.25 to 2.9 kDa, thus obtaining a bio-nanocomposite material; wherein in phase b) the protein extract is a mixture of muscle proteins with a molecular weight higher than 5 kDa, obtained from the organic material produced by grinding the larvae and/or pupae of the Black Soldier Fly insect (BSF) Hermetia illucens dried and frozen, degreasing in soxhlet with organic solvents, filtering of the degreased fraction resolubilized at a pH in the range from 9 to 11 and precipitating the protein content at a pH in the range from 3 to 5.

In a last aspect thereof, the present invention concerns a method for obtaining a bio-nanocomposite film, comprising the steps of: e) providing the bio-nanocomposite material of the invention; f) pouring the bio-nanocomposite material from step e) into a mould; g) drying the bio-nanocomposite material of step f) in the mould at room temperature.

BRIEF DESCRIPTION OF THE DRAWINGS

Further features and advantages of the invention will be more apparent after reading the following description provided by way of a non-limiting example, with the aid of the figures illustrated in the accompanying tables, wherein:

Figure 1 : SDS-PAGE analysis of the protein extract according to Example 1 .3

Figure 2: thermal characterisation by TGA/DTA on the protein film according to Example 2.4.

Figure 3: ATR-FTIR infrared spectroscopy on the protein film obtained according to Example 2.5.

Figure 4: figure of a prototype of bio-nanocomposite film of the invention obtained according to Example 2;

Figure 5: Device for electrical resistance measurements;

Figure 6: Geometry of the copper electrode device used to measure the resistance of the sheet of the coating layers;

Figure 7: graph of the conductivity of the bio-nanocomposite films of the invention as a function of the CBC/SP adduct content, expressed in php with respect to the BSF protein;

Figure 8A: graph of the conductivity of the bio-nanocomposite films of the invention as a function of the presence or not of the CBC/SP adduct;

Figure 8B: Photo of a film with CBC (without SP) as filler: the local defects are highlighted by arrows;

Figure 9A: Rectangular specimen obtained from the bio-nanocomposite film of the invention, dimensions: 40x7 mm;

Figure 9B: Cylindrical punches with different radii of curvature: from left to right 10, 8, 7, 5.5, 4, 3 mm.

Figure 10: diagram of mechanical compression and tensile tests, according to Example 3.2;

Figure 11A-D: Experimental configuration for the tests of electrical conductivity of the bio-nanocomposite films of the invention with respect to the radius of curvature, according to Example 3.2;

Figure 12A-B: Mechanisms involved in the compression and traction mode of the bio-nanocomposite film of the invention, according to Example 3.2;

Figure 13A-C: graphs of normalised resistivity of the bio-nanocomposite films of the invention as a function of radius of curvature, in a film containing the CBC/SP adduct in a concentration respectively of: A) 9 Php, B) 10 Php and C) 15 Php. Both modes are displayed, i.e. , compression (solid line) and traction (dashed line).

Figure 14: graph of the water absorption profiles in 4 bio-nanocomposite films having CBC/SP adduct concentrations of 2, 8, 10 and 20 php with respect to the BSF polymeric matrix.

Figure 15A-C: contact angles measured on 3 bio-nanocomposite films of the invention, having respectively A) 2 php, B) 10 php and C) 20 php.

DETAILED DESCRIPTION OF THE INVENTION

In this description and in the claims, the following terms:

- “nanocomposite material” means a mixture of two or more materials that are insoluble in each other. For the purposes of the present invention, composite material means a material of organic origin in combination with a material of inorganic origin. Said material is defined as nano-composite because at least one of its components has nanometric dimensions, i.e. , one of its components has one or more dimensions of the order of 100 nm or less (Vocabulary - Nanoparticles PAS 71 :2005 BSI);

- “bio-nanocomposite” means a nanocomposite material, as defined above, in which the organic component is derived from a natural source with biodegradable or compostable characteristics.

Said bio-nanocomposite, as defined herein, is thus distinguished from the class of bio-polymers which, while remaining biodegradable, are not derived from a natural renewable source but from petroleum; and from the class of the bio-polymers which, despite deriving from natural renewable sources, are not biodegradable.

In fact, in the case of the present invention, the bio-polymeric matrix of the material of the invention is entirely protein-based, thus deriving from a natural source and at the same time being biodegradable.

- “php” means the concentration measured in parts of adduct per 100 parts of protein;

- “percolation threshold” means the threshold above which there is a marked increase in electrical conductivity, due to the continuity of the conductive filler or charge within the protein matrix being reached. The value of the percolation threshold is therefore linked to this increase in continuity and thus also to the increase in the conductive charge within the protein matrix;

- the definition “proteins extracted from the BSF fly” means proteins extracted from the larvae and/or pupae of said fly;

- all the numerical values reported for the measurements of the electrical properties thereof are reported below, taking into account an error, due to the measurement, of 0.5%. This percentage takes into account the evaluation of the double experimental error, i.e. the instrumental (0.05%) and the experimental setup error (0.5% of variability in the measurements during each individual measurement of the same specimen);

- the numerical values of molecular weight of polyethylene glycol (PEG) and polytetrahydrofuran (poly-THF) were obtained by size exclusion chromatography (SEC);

- “room temperature” or its abbreviation “RT” means a temperature in the range 20- 30 °C;

- “water”, “H2O”, “dhhO” means deionised water.

The inventors have surprisingly found that the use of materials coming from renewable sources, i.e. proteins extracted from BSF larvae that develop on food waste, green waste, i.e. from the so-called organic fraction of municipal solid waste (OFMSW), makes it possible to replace the known polyurethane-based or petroleum-derived polymeric matrices, and to obtain a bio-nanocomposite material with strong electrical conductivity and resistivity properties, and at the same time to employ renewable sources with very low or even no impact on the environment, neither from the point of view of the use of agricultural land, nor through the use of excessive amounts of water, also favouring the valorisation of OFMSW, normally treated as unusable waste.

In the first aspect thereof, the invention concerns a bio-nanocomposite material as described above.

The aforementioned bio-nanocomposite material thus combines an inorganic-based component, i.e. at least one sp² carbon adduct in combination with at least one pyrrole derivative, which guarantees its electrical conductivity characteristics, and an organic protein matrix, i.e. said at least one protein extract obtained from the BSF, which not only makes the material more sustainable as it is synthesised starting from raw materials of natural origin, but, precisely because of its protein nature, can be easily recycled and composted by breaking down the polypeptides of the protein into the various amino acids and subsequent reuse.

The bio-nanocomposite material of the invention comprises at least one adduct of an allotrope of sp² carbon with at least one pyrrole derivative.

Preferably, the at least one allotrope of sp² carbon is selected from the group consisting of carbon black, fullerene, single-walled or multi-walled carbon nanotubes, graphene and graphite with a number of stacked graphene layers up to 1000, preferably up to 100, more preferably up to 50, even more preferably up to 15.

More preferably, said allotrope of sp² carbon is carbon black.

Advantageously, the at least one allotrope of sp² carbon is used in the bio- nanocomposite material of the invention, on the one hand to reinforce the biomaterial in the absence of a synthetic polymer as a matrix, and on the other hand to ensure a high and constant thermal and electrical conductivity, which makes the bio- nanocomposite material of the invention suitable for use as a conductive substrate in a variety of applications. Due to their properties, the allotropes of carbon are used both in polymeric, plastic or elastomeric matrices and in liquid media that will then form coating layers. In fact, they favour the mechanical reinforcement, the thermal and electrical conductivity of the materials in which they are incorporated.

When the at least one allotrope of sp² carbon is carbon black, it, in addition to providing mechanical reinforcement, electrical conductivity, ensures a further protection from the ultraviolet radiations and the colouring of the final product.

In a preferred and advantageous embodiment, said at least one allotrope of sp² carbon has a surface area, measured by BET in accordance with the standard ASTM D3037, greater than 100 m²/g, more preferably greater than 200 m²/g, even more preferably equal to 780 m²/g.

For example, an allotrope of sp² carbon with these characteristics is the carbon black from the company Imerys.

Advantageously, the nanometric size of the at least one allotrope of sp² carbon ensures a large surface area exposed to the protein matrix and therefore a large interfacial surface area, which positively influence and increase the electrical conductivity at the same concentration.

The bio-nanocomposite material comprises at least one adduct of an allotrope of sp² carbon with at least one pyrrole derivative.

Preferably, said at least one pyrrole derivative is selected from the group consisting of 2-(2,5-dimethyl-1 H-pyrrol-1 -yl)-ethan-1 -ol (ethanolpyrrole, EP), 2-(2,5-dimethyl- 1 H-pyrrol-1 -yl)-propan-1 ,3-diol (serinol pyrrole), 3-(2,5-dimethyl-1 H-pyrrol-1 -yl)- propan-1 ,2-diol (iso-serinol pyrrole), 2,5-dimethyl-1 -(3-(triethoxysilyl)propyl)-1 H- pyrrole, a-hydro-co-hydroxypoly(oxy-1 ,4-butanedyl) or (poly(1 ,4-butanediol)), 0,0’- bis(2-aminopropyl) polypropylene glycol-b-polyethylene glycol-b-polypropylene glycol) or (O-(2-aminopropyl)-O'-(2-methoxyethyl)polypropylene glycol, a-hydro-co- hydroxypoly (oxy-1 ,4-butanedyl) or (poly( 1 ,4-butanediol)).

Preferably said at least one pyrrole derivative is 2-(2,5-dimethyl-1 H-pyrrol-1 -yl)- propan-1 ,3-diol, commonly referred to as serinol pyrrole.

The pyrrole derivatives, and in particular the serinol pyrrole and the substituted derivatives thereof, were selected because they were found to be able to give rise to reactions with various chemical species and polar interactions. In particular, it was found that the aromatic ring is able to interact with other aromatic molecules, and with hydrocarbon substances, while the hydroxyl groups of the pyrrole can interact with the polar molecules and also generate a wide variety of modifications in the original molecule through reactions with functional groups such as isocyanates, acids, anhydrides, halides and others.

In particular, the serinol pyrrole was found to be highly soluble in various solvents and, thanks to its chemical nature as outlined above, it ensures compatibility with various polymeric and non-polymeric matrices.

As will be evident from the experimental part, the serinol pyrrole has demonstrated that it can be used to functionalise the at least one allotrope of sp² carbon to form a stable adduct and to improve the electrical and mechanical properties of the bio- nanocomposite material into which it is inserted.

Indeed, it is precisely the ability of the serinol pyrrole to form at least one adduct with an allotrope of sp² carbon that allows said resulting adduct to be uniformed within the matrix formed by the at least one protein extracted from BSF of the bio- nanocomposite material of the invention and thus to have uniform and homogeneous electrical properties.

Said at least an adduct of an sp² carbon allotrope with at least a pyrrole derivative is in an amount higher than 10 php, parts by weight of adduct per 100 parts by weight of protein, preferably it is in an amount in the range from 10 to 30 php, parts by weight of adduct per 100 parts by weight of protein, more preferably it is 15 php parts by weight of adduct per 100 parts by weight of protein. Advantageously, when said at least one adduct of an allotrope of sp² carbon with at least a pyrrole derivative has a value of the surface area higher than 200 m²/g, said at least one adduct is added in an amount equal to or higher than 30 php, preferably equal to or higher than 20 php, more preferably equal to or higher than 10 php, even more preferably equal to or higher than 5 php, parts by weight of adduct per 100 parts by weight of protein.

This allows the conductive adduct particles to distribute themselves within the at least one protein, which acts as a non-conductive protein matrix, in a uniform manner but concentrated enough to have a continuous contact between the particles, thus ensuring a uniform and efficient conductive path thereof, which then results in high electrical conductivity.

Preferably, said at least an adduct of an allotrope of sp² carbon with at least one pyrrole derivative is a carbon black adduct with serinol pyrrole (CBC/SP).

The bio-nanocomposite material comprises at least one aliphatic hydrocarbon compound selected from: i) C2-C21 containing at least an oxygenated functional group selected from the group consisting of hydroxyl, epoxide, ether, acid, ester, carbonate; ii) polyethylene glycol (PEG) having a molecular weight in the range from 0.2 to 35 kDa; and iii) polytetrahydrofuran (poly-THF) having a molecular weight in the range from 0.25 to 2.9 kDa, the purpose of which is on the one hand to act as a plasticiser, i.e. to incorporate itself stably within the protein matrix so as to favour the flowing, thanks to the supramolecular interactions (hydrogen bridges), of the protein chains present in the at least one protein extract obtained from BSF, which constitutes the protein matrix, and on the other hand, being a reactive and hydrophilic compound, to have affinity with both the protein matrix and the adduct.

In fact, said at least one aliphatic hydrocarbon compound, as defined above, allows to make a material, which would not otherwise be flexible because of the interactions between the polymer chains, plastic and resistant to breakage even after drying, by means of the interposition of said compound between the polymer chains.

Preferably, said at least an aliphatic hydrocarbon compound is i) a C2-C21 aliphatic hydrocarbon compound containing at least an oxygenated functional group selected from the group consisting of hydroxyl, epoxide, ether, acid, ester, carbonate.

More preferably, said at least one C2-C21 aliphatic hydrocarbon compound contains at least two oxygenated functional groups.

Still preferably, said at least one C2-C21 aliphatic hydrocarbon compound containing at least one oxygenated functional group contains a hydroxyl group as an oxygenated functional group.

More preferably said at least one C2-C21 aliphatic hydrocarbon compound containing at least one oxygenated functional group contains at least two oxygenated functional groups, even more preferably said at least two oxygenated functional groups are hydroxyl.

In a preferred and advantageous embodiment, said at least one aliphatic hydrocarbon compound C2-C21 containing at least one oxygenated functional group is selected from the group consisting of: glycerol, isopropanol, cardanol, 2-amino- 1 ,3-propanediol (serinol), isoserinol, serinol pyrrole, epoxidized castor oil, epoxidized soybean oil, polyethylene glycol, poly(tetrahydrofuran), aldaric acid, glucaric acid, triglycerides where the fatty acids are preferably lauric, myristic, palmitic, oleic, linoleic, linolenic, stearic, adipic acid ester, sebacic acid ester, phthalic acid ester, ethylene carbonate, propylene carbonate, glycerol carbonate, and their mixtures.

Even more preferably, said at least one C2-C21 aliphatic hydrocarbon compound is glycerol.

The use of a protein extract from BSF in the invention guarantees not only the natural raw material of the bio-nanocomposite, which replaces the known polymers obtained synthetically from petroleum, but also enables an efficient reuse and an efficient waste valorisation.

The larvae and/or the pupae of this insect are in fact able to feed and grow on a variety of organic waste, such as the waste generated by the agri-food industry and by-products of processing chains, plant tissue waste and dairy waste, and provides an alternative to valorise the OFMSW and reduce the volume thereof, turning it into protein-rich biomass.

From the larvae and/or from the pupae of BSF, if reared on selected and safe organic substrates as indicated by the current regulations, it is possible to obtain proteins and lipids that can be used in animal feed.

In addition, the fact of having a protein matrix entails as a further advantage the fact of being able to reuse, by breaking down the proteins in the matrix into their individual amino acids and reassembling at a later stage the polypeptides, so that the material can be completely reused.

The bio-nanocomposite material described above is a viable alternative to the known polymers, as it ensures the same, if not better, electrical and mechanical properties, being also sustainable and recyclable.

In the second aspect thereof, the invention concerns a film obtained from the aforementioned bio-nanocomposite material of the invention.

Typically, the films obtained with the method of the invention have a conductivity in the range from 1 x1 O’² to 5 Q’¹ ■ rrr¹.

When produced under the preferred conditions of the invention, the films obtained with the method of the invention have a conductivity between 0.8 and 3 Q’¹*m’¹.

Said film has mechanical properties, such as for example flexibility, such that when subjected to conductivity tests at different curvature angles, by means of metal cylinders of different radii, it retains the original conductivity of the film up to 100%. Thus, advantageously, the inventors observed that said film has a retention capacity of the electrical conductivity, when subjected to mechanical stress, in the range from 90 to 100%.

When produced under the preferred conditions of the invention, the films obtained with the method of the invention show electrical conductivity retention capacity when subjected to mechanical stress comprised in the range from 98% to 100%, more preferably it is 100%.

Furthermore, in view of a medical application, and for most of the applications in which the film comes into contact with water, it is important that the film is resistant to moisture and can therefore be used in direct contact with water.

Advantageously, the inventors observed that the film of the invention has a swelling capacity, expressed as a percentage water absorption property, comprised in the range from 50 to 300% w/w.

When produced under the preferred conditions of the invention, the films obtained with the method of the invention show a swelling capacity, expressed as a percentage water absorption property, comprised in the range from 70 to 150% w/w. Also advantageously, said film of the invention has a contact angle, measured by OCA 15plus (Dataphysics) and a 2pl water drop, higher than 60°, preferably from 65° to 85°, more preferably it is 73°.

In this way, the film of the invention ensures that it can be used in environments with presence of water or that are humid, as the contact angle and the reduced swelling demonstrate the low hydrophilic nature thereof.

Said bio-nanocomposite material of the invention on the one hand ensures the listed important properties, on the other hand, it was synthesised by means of a specific process that is simple, fast and without the use of toxic solvents and difficult and drastic reaction conditions, which require a large amount of energy.

In a further aspect thereof, the present invention concerns the synthesis of a bio- nanocomposite material comprising the steps of: a) providing larvae and/or pupae of Black Soldier Fly (BSF) Hermetia illucens, fed with the organic fraction of urban solid waste (OFMSW); b) extracting proteins from larvae and/or pupae, thus obtaining at least one protein extract; c) mixing said at least one protein extract of step b) with at least an adduct of an allotrope of sp² carbon with at least a pyrrole derivative, so as to obtain a uniform mixture; d) adding to the mixture of step c) at least an aliphatic hydrocarbon compound selected from: i) C2-C21 containing at least an oxygenated functional group selected from the group consisting of hydroxyl, epoxide, ether, acid, ester, carbonate; ii) polyethylene glycol (PEG) with a molecular weight in the range from 0.2 to 35 kDa; and iii) polytetrahydrofuran (poly-THF) with a molecular weight in the range from 0.25 to 2.9 kDa, thus obtaining a bio-nanocomposite material; wherein in phase b) the protein extract is a mixture of muscle proteins with a molecular weight higher than 5 kDa, obtained from the organic material produced by grinding the larvae and/or pupae of the Black Soldier Fly insect (BSF) Hermetia illucens dried and frozen, degreasing in soxhlet with organic solvents, filtrating of the resolubilized degreased fraction at a pH in the range from 9 to 11 and precipitating the protein content at a pH in the range from 3 to 5.

Preferably, the pH of step b) is pH = 11 for the filtration at basic pH, and pH = 4 for the precipitation at acidic pH.

Preferably, the protein extract of step b) is a mixture of muscle proteins with a molecular weight higher than 10 kDa, more preferably with a molecular weight higher than 20 kDa.

Said protein extract of step b) was extracted after degreasing with an aliphatic hydrocarbon having a boiling point of less than 100 °C, preferably less than 70 °C, more preferably less than 50 °C, even more preferably being petroleum ether.

The protein extract from step b) was characterised by means of a BCA assay, and an amount of protein within the extract in the range from 70 to 90% by weight, preferably 80% was estimated.

The protein extract was titrated by means of TNBSA assay, and an amount of primary amine groups comprised in the range from 0.4 to 0.6 mmol per gram of extract, preferably equal to 0.5 mmol per gram of extract was estimated.

The protein extract was also analysed by SDS-PAGE, revealing a heterogeneous population of molecular weights comprised in the range from 10 kDa to 250 kDa, with the presence of a band at higher intensity and thus preferred in the range from 70 to 80 kDa, preferably at 75 kDa.

By means of nano LC-MS, it was observed that the protein extract of step b), obtained as described above, comprises mainly muscle-type proteins, specifically belonging to the myosin, actin and tropomyosin family. In particular, the proteins identified, following a comparison on the “Insecta” database, are the following: myosin heavy chain (muscle), Actin-87, L-lactate dehydrogenase, Tropomyosin-2, Tropomyosin-1 , Tropomyosin Lep, Muscle-specific protein 20.

Preferably, in step c) the mixture is in water.

Optionally and preferably, after step b) and before step c), there is a step b’) of denaturation of the protein extract of step b) by reduction of the sulfur bridges -S-S- with methods known in the sector at a pH in the range from 2 to 5, and a subsequent cross-linking of said protein extract at a pH in the range from 8 to 12.

The known methods in the sector mentioned above comprise, for example, the use of hydrides, of mercaptans or mechanical methods such as for example ultrasonication.

Preferably, a compound (C1-C5) comprising at least one -SH group and optionally at least one -OH group is used according to the present invention; more preferably, said compound (C1-C5) comprising at least one -SH group is selected from the group consisting of 2-mercaptoethanol, 1 -propanethiol, 1 ,4-dithiothreitol, 1 -hexanthiol, even more preferably being 2-mercaptoethanol.

Preferably prior to step d), there is a step c') of sonication of the at least one protein extract with the at least one adduct of an allotrope of sp² carbon with at least one pyrrole derivative for a time in the range from 5 to 20 minutes, preferably from 8 to 15 minutes, more preferably of 10 minutes. This step allows the control of the dimensions of the at least one adduct of an allotrope of sp² carbon, breaking the macroscopic aggregates and ensuring the homogeneity of the mixing with the at least one protein extract.

This method, further to allowing the synthesis of a bio-nanocomposite material, brings about the intrinsic advantage of enabling the reduction, the recycling and the reuse of organic waste (OFMSW), which was previously only treated by composting and anaerobic digestion.

The known composting systems normally require precise and sometimes costly and complicated pH, moisture and temperature conditions, controlled passages of air injection and subsequent control of the nitrogen/carbon balance, and specific treatment plants with portions for controlling the toxin and odour gas emissions.

Even in the case of the anaerobic digestion, specific pH, temperature, waste quality and mixing system are required to allow the bacteria to work efficiently. For a high- performance and low-cost waste digestion, doing a selective sorting of OFMSW is necessary.

Thus, the method of the present invention not only allows to easily obtain a bio- nanocomposite material, but also the efficient re-use and efficient valorisation of the OFMSW, through step a) in which said OFMSW is used to feed the BSF larvae.

In a further aspect thereof, the present invention concerns a method for obtaining a bio-nanocomposite film, comprising the steps of: e) providing the bio-nanocomposite material of the invention; f) pouring the bio-nanocomposite material from step e) into a mould; g) drying the bio-nanocomposite material of step f) in the mould at room temperature.

Preferably, in step g) the drying takes place for a time from 24 to 96 hours, preferably from 48 to 80 hours, more preferably it is of 72 hours.

Still preferably in step f) the mould used is flexible to facilitate the removal of the film of the invention after drying in step g).

More preferably, the mould is made of silicone, to avoid adhesion of the dry film, and further facilitate the removal of the film.

The invention will be further described in the following experimental part, which is intended for illustrative and non-limiting purpose, which gives examples of preparation of the bio-nanocomposite material of the invention, and its mechanical resistance tests.

EXPERIMENTAL PART

Materials and Methods

The protein extracts came from the insect Black Soldier Fly (Hermetia lllucens) reared on Organic Fraction of Municipal Solid Waste (OFMSW) and were produced by the University of Insubria. The protein extracts came from both the larvae and from the pupae of said insect. The BCA reagents (A and B) came from ThermoFisher Scientific. The Laemmli buffer (2x) came from BioRad. The conductive carbon black was from Imerys. Unless otherwise stated, the remaining reagents came from Sigma-Aldrich.

Example 1 - Characterisation of protein extracts

1.1 BCA assay

The BCA test was used to estimate the amount of proteins in the extracts. The ThermoFisher Scientific's BCA protocol was followed (PierceTM BCA protein assay kit, catalogue numbers 23225 and 23227).

Suspensions of 1 mg/mL were prepared by dissolving the extract in dbkO adjusted with NaOH up to pH = 9-10. The extracts were further solubilised by protein denaturation by heating the suspensions up to 80 °C for 20 minutes.

100 pl of extract solution were added to 2 mL of working reagent (50 parts of reagent A, 1 part of reagent B) in a 2 mL test tube. dH2O was used as a blank solution (i.e. , 100 pL of dF were added to 2 mL of working reagent in a 2 mL test tube). The tube was stirred gently to homogenise the solution and the whole was incubated at 37 °C for 30 minutes. The absorbance values were read on a SmartSpec Plus spectrophotometer (BioRad) at 562 nm. The analyses were performed in triplicate for each replicate tested.

The protein extract of step b) was characterised by means of a BCA assay, and a protein content within the extract of 80% was estimated.

1.2 NH2 titration assay

The amount of -NH2 functional groups was estimated using the TNBSA test. Glycine solutions at known concentrations (from 200 to 0.75 pg/mL) were used to generate a standard calibration curve. The larvae extracts were solubilised (160 pg/mL) in 0.1 M NaHCOs buffer (pH = 8.5) by heating them for 20 minutes at 80 °C. 2-fold and 4- fold dilutions were also prepared to ensure that the results fell within the linear zone of the standard curve. 75-pL of solubilised extract were mixed with 75-pL of 0.01 % TNBSA in a transparent 96 multi-well plate. After 2 hours of incubation at 37 °C, 90 R.H., the plates were placed on an automatic absorbance reader (TECAN). The absorbance data were taken at 348 nm. Only the data with absorbance values fallen in the linearity region were considered as valuable. The analyses were carried out in triplicate for each replicate tested. The amount of -NH2 groups was reported as moles/100 g of extract.

The protein extract was titrated by means of TNBSA assay, and an amount of primary amine groups equal to 0.5 mmol per gram of extract was estimated.

1.3 SDS-PAGE

SDS-PAGE was used to separate the proteins from the extracts based on their molecular weight (MW).

Suspensions of 5 mg/mL were prepared by dissolving the extract in dH2O adjusted with NaOH up to pH = 9-10. The extracts were further solubilised by protein denaturation by heating the suspensions through a 20-minute ramp rate up to 80 °C.

A 10 pl volume of each sample (corresponding to approx. 50 pg of proteins) was mixed with 10 pl of Laemmli buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue and 0.125 M Tris-HCI, pH approx. 6.8). The electrophoresis plate on SDS-polyacrylamide gel (PAGE) was composed by a stacking gel (125 mM Tris-HCI, pH 6.8, 0.1 % SDS) with a large-pore polyacrylamide (4%) fused onto the resolving gel (12% acrylamide gradient in 375 mM Tris-HCI, pH 8.8, 0.1 % SDS buffer). The cathode and anode compartments were filled with Trisglycine buffer, pH 8.3, containing 0.1 % of SDS. The electrophoresis was at 100 V until the dye front reached the bottom of the gel. Staining and decolourisation were performed with Colloidal Coomassie Blue and 7% acetic acid in water, respectively. The SDS-PAGE gels were scanned with a Versa-Doc imaging system (Bio-Rad). The densitograms were obtained by processing the images with the Imaged, NIH software.

The protein extract was also analysed by SDS-PAGE, revealing a heterogeneous population of molecular weights comprised in the range from 10 kDa to 250 kDa, with the presence of a band at higher intensity and thus preferred at about 75 kDa, as shown in Figure 1 .

1.4 Nano LC-MS

The various sampling lanes of the SDS-PAGE gels were cut into 18 pieces along the migration path, based on the positions of the peaks of the corresponding densitograms (the images were processed and analysed using the Imaged, NIH software after acquisition). The proteins were reduced by 10 mM dithiothreitol (DTT) and alkylated by 55 mM of iodoacetamide. The gel pieces were reduced in acetonitrile and dried under vacuum; the proteins were digested overnight with bovine trypsin. The tryptic mixtures were acidified with formic acid up to a final concentration of 10%. 5 pl of tryptic digest for each band were injected into a capillary chromatographic system (EasyLC, Proxeon Biosystems, Odense, Denmark). Peptide separations occurred on a homemade 10-cm capillary column of fused silica sprayed in reverse phase RP (10 cm x 75 pm inner diameter), packed with 3 pm ReproSil 100C18 (Dr Maisch GmbH, Germany). A gradient of eluents A (H2O with 2% (v/v) ACN, 0.1 % (v/v) formic acid) and B (ACN with 2% (v/v) H2O with 0.1 % (v/v) formic acid) was used to achieve separation, from: 8% B (at 0 min, 0.2 pL/min of flow rate) to 50% B (at 80 min, 0.2 pL/min of flow rate). The LC system was connected to an LTQ-Orbitrap mass spectrometer (ThermoScientific, Bremen, Germany) equipped with a nano-electrospray ion source (Proxeon Biosystems). The full-scan mass spectra were acquired in the LTQ Orbitrap mass spectrometer with the resolution set to 60,000. For accurate mass measurements, the lock-mass option was used. The acquisition mass range for each sample was from m/z 350 to 1500 Da and the analysis was carried out in triplicate. The four most intense double- and triple-charge ions were automatically selected and fragmented in the ion trap. The target ions already selected for MS/MS were dynamically excluded for 60 s.

By means of nano LC-MS, it was observed that the protein extract comprises predominantly muscle-type proteins, specifically belonging to the myosin, actin and tropomyosin family. In particular, the proteins identified, following a comparison on the “Insecta” database, are the following:

Myosin heavy chain (muscle), Actin-87, L-lactate dehydrogenase, Tropomyosin-2, Tropomyosin-1 , Tropomyosin Lep, Muscle-specific protein 20.

Example 2 - Preparation of bio-nanocomposite films from BSF proteins

2.1 Synthesis of 2-(2,5-dimethyl-1 H-pyrrol-1-yl)-propane-1,3-diol (serinol pyrrole, SP)

6.46 g (0.071 mol) of powdered serinol (PM = 91 .11 g/mol) were poured into a 100 ml round-bottomed flask fitted with a magnetic stirrer and heated to 155 °C for 15 minutes under stirring, then 8.10 g (0.071 mol) of 2,5-hexanedione (PM = 114.14 g/mol) was added drop by drop to the liquid serinol. The obtained solution was stirred for 2 hours in the same flask fitted with a reflux condenser to prevent the evaporation of the sample. The condenser was then removed and the solution heated for 30 minutes. After this time elapsed, the reaction was interrupted simply by cooling to room temperature (R.T., 20 ± 2 °C). The pure product 2-(2,5-dimethyl- 1 H-pyrrol-1 -yl)-propane-1 ,3-diol (SP) was obtained as a dark amber viscous liquid (11.64 g, 97% yield).

2.2 Functionalisation of the conductive carbon black (CBC) with serinol pyrrole to form a CBC/SP adduct

2.00 g of conductive carbon black (CBC) were poured into a 100 ml round-bottomed flask; then, 50 ml of acetone were added. Subsequently, the suspension was sonicated through a bath sonicator apparatus (Ultrasonic Cleaner 2200 S3, Sonica) for 10 minutes. 0.47 g of SP were subsequently weighed in a glass vial, solubilised in 8 mL of acetone and poured into the CBC/acetone suspension. The mixture was finally sonicated through a bath sonicator apparatus for a further 10 minutes. Subsequently, the acetone was removed with a rotavapor machine (Rotavapor Rl I, BLICHI Switzerland) at 40 °C and 300 mbar. The dried sample was transferred into a 250 ml two-neck round-bottomed flask. The flask was fitted with a magnetic stirrer and placed in an oil bath at 180 °C under stirring (300 rpm). In addition, an air flow is introduced from the secondary neck of the flask while an air condenser with sintered septum is added to the main neck. The system is allowed to stir for 2 hours. The reaction is then carried out pure, without solvents or catalysts. A Soxhlet extraction is then performed on the obtained powder to remove the unreacted PyC fraction (T = 90 °C, t = 16 h).

2.3 Production of the base film: BSF protein + Glycerol

The BSF protein extracts were weighed and dispersed at a concentration of 50 mg/mL in dF . The volume of the suspensions was set for each test at 10 ml using a 10 ml beaker. The extracts were solubilised by adding 500 pL of 1 M NaOH, bringing the pH from 7 to 9-10. The suspensions were subsequently heated through a 20-minute ramp-rate heating from R.T. to 80 °C, with the temperature constantly monitored. After this time elapsed, the suspension was brought to its initial volume (10 mL) by adding fresh dH2O. Glycerol (50% by weight of the extract) was then added and mixed by stirring for 10 minutes at 400 rpm. The suspension was finally poured into 25x25 mm or 25x50 mm silicone moulds for film pouring. The pouring volume was calculated so as to provide the same height reached by the suspension in each mould used (2 ml for 25x25 mm moulds and 4 ml for 25x50 mm moulds).

The films were obtained by peeling them off the moulds after 72 hours of drying at room temperature (RT, 25 °C).

2.4 TGA/DTA analysis of the base film: BSF protein + Glycerol

The mass loss and the heat flow of the protein films were measured on an SDT Q600 V20.9 Build 20 TGA/DTA instrument. In short, 10 mg of each powder or film sample was collected and carefully transferred into glass vials. The samples were heated from 30 °C to 900 °C at a heating speed of 10 °C/min using both nitrogen (N2) and air as the atmosphere. An alumina plate was used as reference material for the DTA tests.

Figure 2 shows the results of the thermal characterisation by TGA/DTA on the protein film which show its stability up to a temperature of 150 °C. If this temperature is exceeded, degradation phenomena compromise its integrity. In particular, it can be noted an initial weight loss equal to 10-15% of the weight of the film which can be associated with dehydration. Subsequently, a complete dehydration and the degradation of the glycerol reduce the mass of the film to 60% with respect to the total starting weight. This value is in accordance with the amount of glycerol added during the preparation of the film equal to 50 php (equivalent to 33% on the total). Above 250 °C, there is a gradual degradation of the oxygenated groups of the protein matrix, until reaching an exothermic peak associated with the combustion of the polypeptide chains in the presence of oxygen at a temperature of 550 °C.

2.5 ATR - FTIR analysis of the base film: BSF protein + Glycerol

The samples (i.e. both extracts and protein films) were studied using a Nicolet iS5 FT-IR Spectrophotometer imaging system (Thermofisher) equipped with Veemax II ATR with variable angle (KBr crystal) in a nitrogen (N2) atmosphere. The IR absorption spectrum was recorded in the ATR image mode in the 600 cm’¹ and 4000 cm’¹ regions based on 128 scans and a 4 cm’¹ resolution. Background subtraction was performed before each measurement.

From the infrared spectroscopy on the protein film shown in Figure 3, the incorporation of the plasticiser, in this case glycerol, into the protein matrix of the extract can be observed. In particular, the presence of the signal associated with hydrogen bridges in the 3500-3000 cm’¹ region and the two characteristic peaks of the polyamides in the region between 1700 and 1500 cm’¹ can be noted. No signals associable with a covalent bond between the plasticisers and the protein matrix were observed, indicating that the interaction between the two components is supra- molecular. In particular, the polar groups characteristic of glycerol and of the protein matrix seem to interact via hydrogen bridges.

2.6 Production of the bio-nanocomposite material: BSF protein + Glycerol + CBC/SP

250 mg of BSF protein extracts were weighed into a 10 ml beaker. Subsequently, different amounts (5, 10, 15, 20, 22.5, 25, 35.5 and 50 mg) of CBC/SP adduct were added to give 2, 4, 6, 8, 9, 10, 15, 20 parts per hundred protein (php) of bio- nanocomposites, respectively. 5 ml were added and the extracts were subsequently solubilised by adding 300 pl of 1 M NaOH under magnetic stirring (400 rpm), bringing the pH from 7 to 9-10. The entire mixture was sonicated for 10 minutes at room temperature (approx. 25 °C) to ensure homogeneity and the breakage of the macroscopic aggregates. The obtained suspensions were subsequently heated with a 20-minute heating ramp from room temperature (approx. 25 °C) to 80°C, by constantly mixing and monitoring the temperature. After this time elapsed, the suspension was brought to its initial volume (5 mL) by adding dH2O. Glycerol was then added (50 php) and mixed under stirring for 10 minutes at 400 rpm. The suspension was finally poured into a 25x50 mm silicone mould for film pouring. The pouring volume was calculated to provide the same height reached by the suspension for each mould used (5 ml for 40x40 mm moulds).

The films were obtained by peeling them off the moulds after 48 hours of drying at room temperature (around 25 °C) and one of them is shown in Figure 4.

Example 3 - Characterisation of the film of bio-nanocomposite material of the invention

3.1 Electrical conductivity measurements of the film of bio-nanocomposite material of the invention

The electrical conductivity of the films (o) was assessed by measuring the surface electrical resistance (Rsh) of a defined film region and their thickness (t). The direct measurement of the resistance of the sheet was standardised using the device shown in Figure 5. The measurements of the surface resistance were performed on a TackLife digital multimeter (DM02A) by positioning the two probes in dedicated suitable stations on the device in Figure 6.

The measurements were carried out in 3 different positions for each film (left, centre, right). The average triplicate value was calculated and considered as the average surface resistance of the coating.

The mass resistance of a resistor of length L, width W and thickness t is given by the following equation: where p is the mass resistivity. Considering the geometry imposed by the device depicted in Figure 2, the length (L) and the width (W) of the delimited portion of the coating, representing the resistance of the system, are equal to 1 cm. Therefore, the L/W ratio is dimensionless. Considering L/W = 1 , the previous equation becomes: p L p

R = - ‘ — = - [Ohm] t W t ^{L J}

The units of this equation are always Ohm, however R = p/t represents the resistance between two opposite sides of a square of conductive film with an area of 1 cm². To distinguish between mass resistance and surface resistance, the p/t ratio is given by the units of ohm/n and is called surface resistance, Rsh. Thus, the resistance of the sheet can be defined as:

Rsh

The surface resistance is also known as surface resistivity. Consequently, the surface resistivity was calculated using the following equation.

The electrical conductivity was obtained as the inverse of the surface resistivity.

The thickness of the films was measured on a thickness gauge (TG3388, EptaTECH) as the average of multiple measurements carried out on different regions of the films.

The graph in Figure 7 shows the improvement in electrical conductivity as a function of the concentration of CBC/SP adduct added in Example 2.4, i.e. , 5, 10, 15, 20, 22.5, 25, 35.5 and 50 mg of CBC/SP adduct to give 2, 4, 6, 8, 9, 10, 15, 20 parts per hundred proteins (php) of bio-nanocomposites, respectively.

An increase of an order of magnitude is clearly visible around 10 php. From now on, we will refer to this point as the percolation threshold, i.e. the minimum threshold below which there is no electrical response from the film of the bio-nanocomposite material.

In order to be able to verify the influence of the presence, within the film of bio- nanocomposite material, of the carbon adduct, i.e. , the conductive carbon black CBC with respect to the carbon adduct functionalised with serinol pyrrole (CBC/SP), two films of bio-nanocomposite material containing BSF + 15 php of CBC/SP and BSF + 15 php CBC were prepared.

Figure 8A highlights an increase, up to reaching a doubling, in the electrical conductivity of the film of bio-nanocomposite material containing BSF + 15 php CBC/SP, i.e., the one in which the conductive carbon black is functionalised with serinol pyrrole, as per Example 2.2, with respect to the one containing BSF + 15 php unfunctionalised CBC.

This behaviour can be attributed to an increased compatibility of the functionalised CBC/SP filler with the BSF protein matrix. CBC itself tends to aggregate when it is not uniform and well dispersed. Consequently, when not functionalised, CBC does not provide a continuous conductive network in the presence of a protein matrix, as is the case instead with the CBC/SP adduct, i.e. , CBC functionalised with SP.

Figure 8B highlights local defects in a film of bio-nanocomposite material with CBC not functionalised with SP. The defects are absent in the film of bio-nanocomposite material in which the SP-functionalised CBC adduct, CBC/SP, was used.

Consequently, the functionalisation of the carbon black CBC with serinol pyrrole improves its dispersion capacity and thus uniforms the CBC within the BSF protein matrix. This ensures a uniformity of the resulting electric field, thereby increasing up to doubling the electrical conductivity and thus the performance of the single layer of bio-nanocomposite material.

3.2 Bending tests, compression and tensile conductivity tests of the conductive layer of the film of bio-nanocomposite material of the invention

The bend tests were performed in a static configuration. The bio-nanocomposite films obtained in Example 2, having CBC/SP concentrations of 9, 10 and 15 php, were perforated in rectangular pieces of standardised dimensions, i.e. 40 x 7 mm (Figure 9A).

The multimeter clamps were connected to the end points of the films of bio- nanocomposite material. The prepared films of bio-nanocomposite material were left hanging, subjected to gravity, on metal cylinders (Figure 9B) with different radii of curvature, respectively 10 mm, 8 mm, 7 mm, 5.5 mm, 4 mm, 3 mm from right to left in Figure 9B, while still connected to the clamps of the multimeter. The phenomenon that occurs is shown in Figure 10.

The electrical resistance was measured using a TackLife digital multimeter (DM02A) shown in Figure 11 A-D.

The mechanisms involved in the compression and tensile tests of the conductive layer of the film of bio-nanocomposite material are shown in Figure 12A-B.

Figure 12A depicts the set-up of the compressive resistance test, where the conductive layer of bio-nanocomposite material is facing and in contact with the metal cylinder. Due to the effect of force of gravity, said film of bio-nanocomposite material is therefore subjected to a compressive force from the edges towards the centre of the film, indicated as F_c in Figure 12A.

Figure 12B depicts the set-up of the tensile test, where the conductive layer of bio- nanocomposite material is facing outwards and not in contact with the metal cylinder. Due to the effect of the force of gravity, said film of bio-nanocomposite material is therefore subjected to a tensile force from the inside towards the edges of the film, indicated as Ft in Figure 12B.

The individual samples of film of bio-nanocomposite material subjected to the two compression and tensile tests by means of the metal cylinders of different radii were subsequently tested to verify the electrical conductivity before and after the mechanical stress.

The results of the graphs of Figure 13A-C show the variation of the electrical resistivity as a function of the radius of curvature both in compression and in tension. Figure 13A, i.e. the result of the resistivity measurements of a film of bio- nanocomposite material, obtained according to Example 2 and having a concentration of the CBC/SP adduct of 9 php (just below the percolation threshold), before and after compression and tension, shows two curves with symmetrical trend.

In this region, the equilibrium between the BSF protein matrix and the CBC/SP adduct is highly sensitive to the variations in structure, therefore, the bending can change the uniformity of the layer and thus the uniformity of the electric field and thus provide a more or less conductive layer when subjected to compression or tensile modes, respectively.

The bio-nanocomposite films with a CBC/SP concentration of 9 php were therefore not extremely stable under mechanical stresses.

However, the possibility of varying the conductivity in a predictive and reproducible manner is useful in the fields of flexible electronics, where it is important that the structural variations lead to an electrical variation that provides specific output signals.

Increasing the concentration of CBC/SP adduct in the tested bio-nanocomposite films, i.e. passing to 10 php and 15 php, the results of the electrical properties of the films before and after the mechanical stress disclosed in Figure 13B and 13C an almost constant trend in the conductivity of the films, thus demonstrating their stability under both compression and tension.

The results obtained are reported in Table 1 below.

Table 1

Table 1 shows the normalised resistivity of the bio-nanocomposite films with the three different CBC/SP adduct concentrations as a function of the radius of curvature in both compression and tension.

Table 1 does not report the mechanical test values of the sample of film of bio- nanocomposite material with a concentration of 15 php of CBC not functionalised with serinol pyrrole SP, as the film was found to be so little elastic that it did not survive either the compression or tensile tests. That is, the weight of the probes of the multimeter led to the breakage of the sample. This did not occur for any of the films with increasing concentration from 9 to 15 php of CBC/SP adduct.

Also in this case, like above for the electrical conductivity tests, this behaviour can be attributed to an increased compatibility of the functionalised CBC/SP filler with the BSF protein matrix. The absence of a coupling agent (like serinol pyrrole) leads to a worse interaction between the CBC and the polypeptide chains of the BSF proteins, thus compromising the mechanical properties of the final film.

Consequently, the functionalisation of the carbon black CBC with serinol pyrrole not only improves its conductivity, but, by improving the uniformity of the final film, significantly improves its mechanical properties, to the point of providing an elastic and resistant film. 3.3 Swelling tests (Sw %) of the film of bio-nanocomposite material of the invention

The swelling behaviour of the films was tested in aqueous solutions at room temperature (RT, 20 ± 2 °C). Dehydrated films (80 °C, 24 h) were immersed in a BAC/dH2O solution (100 pg/mL) to prevent the growth of microbes, algae or fungi after several days of immersion.

The preliminary tests were performed by immersing the films in both pure dhkO and BAC/dH2O solution, without finding any statistical differences in the swelling profiles during the first week. Then, it was confirmed that BAC should not influence the swelling behaviour of the tested films of bio-nanocomposite material.

The films were removed from the solution and gently blotted with blotting paper before weighing them. The weights were taken at 5 and 30 minutes, then after 1 , 2, 4, 6 hours and finally every 24 hours for several days to create reliable swelling profiles. The swelling parameter was calculated as follows: 100

A graph highlighting the differences in the water absorption profiles of 4 films of bio- nanocomposite material is presented in Figure 14. In particular, films of bio- nanocomposite material were tested with a CBC/SP adduct concentration of 2, 8, 10, 20 php. The results clearly indicate a decrease in water absorption as the adduct content increases. This result suggests a decrease in the structural mesh size of the protein as a matrix with a uniformly dispersed CBC/SP adduct, due to the presence of a higher amount of adduct.

3.4 Contact angle

The contact angle (C.A.) measurements were performed on an OCA 15plus contact angle system (Dataphysics). 2-pL drops were dispensed through a 500pL Hamilton syringe onto the target surface at a speed of 1 -pL/sec. For C.A. measurements, the baseline was set as the linear segment connecting the two end points of the fall. The analyses were carried out in triplicate for each side of the film tested.

3 films were tested, specifically having CBC/SP adduct concentrations of 2 php, 10 php and 20 php. Figures 15A-C show an increase in the contact angle, which is indicative of hydrophobicity of the surface of the film of bio-nanocomposite material where the drop is deposited, as the concentration of the CBC/SP adduct increases, thus demonstrating a marked water resistance of the film of bio-nanocomposite material of the invention.

Claims

1 . A bio-nanocomposite material comprising:

- at least a protein extract;

- at least an aliphatic hydrocarbon compound selected from: i) C2-C21 containing at least an oxygenated functional group selected from the group consisting of hydroxyl, epoxide, ether, acid, ester, carbonate; ii) polyethylene glycol (PEG) with a molecular weight in the range from 0.2 to 35 kDa; and iii) polytetrahydrofuran with a molecular weight in the range from 0.25 to 2.9 kDa; and

- at least an adduct of an allotrope of sp² carbon with at least a pyrrole derivative of Formula (i) wherein

- R1-R4 are independently selected from the group consisting of hydrogen, C1-C3 alkyl, linear or branched C2-C10 alkenyl or alkynyl, aryl, linear or branched C1-C10 alkyl-aryl, linear or branched C2-C10 alken-aryl, linear or branched C2-C10 alkyn-aryl and heteroaryl;

- W is selected from the group consisting of hydrogen, linear or branched C1- C10 alkyl, linear or branched C2-C10 alkenyl or alkynyl, aryl, linear or branched C1-C10 alkyl-aryl, linear or branched C2-C10 alken-aryl, linear or branched C2-C10 alkyn-aryl, heteroaryl, carboxyl and a group of Formula wherein

- M, J and Q are independently selected from the group consisting of hydrogen, hydroxyl, amine, linear or branched C1-C10 alkyl, linear or branched C2-C10 alkenyl or alkynyl and a group selected from: wherein:

- R5-R19 are independently selected from the group consisting of hydrogen, linear or branched C1-C10 alkyl, linear or branched C2-C10 alkenyl or alkynyl, aryl, linear or branched C1-C10 alkyl-aryl, linear or branched C2-C10 alken- aryl, linear or branched C2-C10 alkyn-aryl, heteroaryl or carboxyl;

- R14-R16 are selected from -OCH3 and -OCH2-CH3;

- Ris is selected from -CH2-SH and -CH2-CH2-S-CH3;

- T is oxygen or sulfur, with the proviso that when T is sulfur m' is an integer from 1 to 4 and when T is oxygen m' is an integer from 1 to 2;

- I’, p’, q’, r’ are independently integer from 0 to 12; and

R20 is selected from the group consisting of hydrogen, alkyl, aryl, benzyl, amine, alkyl-amine, aryl-amine, benzyl-amine and amino-aryl; and

R21-R25 are independently selected from the group consisting of hydrogen, linear or branched C1-C10 alkyl, linear or branched C2-C10 alkenyl or alkynyl and 1 -(4-aminocyclohexyl) methylene, wherein said at least a protein extract is extracted from the Black Soldier Fly (BSF) Hermetia illucens, and wherein said allotrope of sp² carbon has a surface area higher than 60 m²/g, measured by BET in accordance with the standard ASTM D3037.

2. The bio-nanocomposite material according to claim 1 , wherein the at least one allotrope of sp² carbon is selected from the group consisting of carbon black, fullerene, single-walled or multi-walled carbon nanotubes, graphene and graphite with a number of stacked graphene layers up to 1000, preferably it is carbon black.

3. The bio-nanocomposite material according to any one of claims 1 -2, wherein said at least a pyrrole derivative is selected from the group consisting of: 2- (2,5-dimethyl-1 H-pyrrol-1 -yl)ethan-1 -ol (ethanolpyrrole, EP), 2-(2,5-dimethyl- 1 H-pyrrol-1 -yl)-propan-1 ,3-diol (serinol pyrrole), 3-(2,5-dimethyl-1 H-pyrrol-1 - yl)propan-1 ,2-diol (iso-serinol pyrrole), 2,5-dimethyl-1 -(3- (triethoxysi ly l)propy l)-1 H-pyrrole, a-hydro-co-hydroxypoly(oxy-1 ,4-butanedyl) or (poly(1 ,4-butanediol)), O,O'-Bis(2-aminopropyl) polypropylene glycol-b- polyethylene glycol-b-polypropylene glycol) or (O-(2-aminopropyl)-O'-(2- methoxyethyl)polypropylene glycol, a-hydro-co-hydroxypoly (oxy-1 ,4- butanedyl) or (poly(1 ,4-butanediol)), preferably it is 2-(2,5-dimethyl-1 H-pyrrol- 1 -yl)-propan-1 ,3-diol (serinol pyrrole). The bio-nanocomposite material according to any one of claims 1 -3, wherein said at least an adduct of an sp² carbon allotrope with at least a pyrrole derivative is in an amount higher than 10 php, parts by weight of adduct per 100 parts by weight of protein, preferably it is in an amount in the range from 10 to 30 php, parts by weight of adduct per 100 parts by weight of protein, more preferably it is 15 php parts by weight of adduct per 100 parts by weight of protein. The bio-nanocomposite material according to any one of claims 1 -4, wherein said at least an aliphatic hydrocarbon compound is i) a C2-C21 aliphatic hydrocarbon compound containing at least an oxygenated functional group selected from the group consisting of hydroxyl, epoxide, ether, acid, ester, carbonate, preferably containing at least two oxygenated functional groups. The bio-nanocomposite material according to claim 5, wherein said at least an aliphatic hydrocarbon compound C2-C21 contains at least a hydroxyl oxygenated functional group, preferably said at least an aliphatic hydrocarbon compound C2-C21 containing at least one oxygenated functional group is selected from the group consisting of: glycerol, isopropanol, cardanol, 2- amino-1 ,3-propanediol (serinol), isoserinol, serinol pyrrole, epoxidized castor oil, epoxidized soybean oil, polyethylene glycol, poly (tetrahydrofuran), aldaric acid, glucaric acid, triglycerides where the fatty acids are preferably lauric, myristic, palmitic, oleic, linoleic, linolenic, stearic, adipic acid ester, sebacic acid ester, phthalic acid ester, ethylene carbonate, propylene carbonate, glycerol carbonate, and their mixtures. A film obtained from the bio-nanocomposite material according to any one of claims 1 -6. The film according to claim 7, having a conductivity comprised from 1x1 O’² to 5 Q’¹*m’¹, preferably from 0.8 to 3 Q’¹*m’¹. The film according to claim 7 or 8, having an electrical conductivity retention capacity, when subjected to mechanical stress, comprised in the range from 90 to 100%, preferably from 98 to 100%, more preferably is 100%. A method of synthesis of a bio-nanocomposite material comprising the steps of: a) providing larvae and pupae of Black Soldier Fly (BSF) Hermetia illucens, fed with the organic fraction of urban solid waste (OFMSW); b) extracting proteins from larvae and pupae, thus obtaining at least a protein extract; e) mixing said at least a protein extract of step b) with at least an adduct of an allotrope of sp² carbon with at least a pyrrole derivative, thus obtaining a uniform mixture; c) adding to the mixture of step c) at least an aliphatic hydrocarbon compound selected from: i) C2-C21 containing at least an oxygenated functional group selected from the group consisting of hydroxyl, epoxide, ether, acid, ester, carbonate; ii) polyethylene glycol (PEG) with a molecular weight in the range from 0.2 to 35 kDa; and iii) polytetrahydrofuran (poly-THF) with a molecular weight in the range from 0.25 to 2.9 kDa, thus obtaining a bio-nanocomposite material; wherein in phase b) the protein extract is a mixture of muscle proteins with a molecular weight higher than 5 kDa, obtained from the organic material produced by grinding the larvae and/or pupae of the Black Soldier Fly insect (BSF) Hermetia illucens dried and frozen, degreasing in soxhlet with organic solvents, filtering of the resolubilized degreased fraction at a pH in the range from 9 to 11 and precipitating the protein content at a pH in the range from 3 to 5.