Valorización de subproductos y residuos de la industria del cangrejo rojo en base a su contenido proteico

Departamento de Ingeniería Química. Universidad de Sevilla VALORIZACIÓN DE SUBPRODUCTOS Y RESIDUOS DE LA INDUSTRIA DEL CANGREJO ROJO EN BASE A SU CONTENIDO PROTEICO “Valorisation of wastes and by-products from Red-Crayfish Industry based on their protein content” MANUEL FÉLIX ÁNGEL Sevilla, 30 de noviembre de 2015 3 Departamento de Ingeniería Química. Universidad de Sevilla TESIS DOCTORAL VALORIZACIÓN DE SUBPRODUCTOS Y RESIDUOS DE LA INDUSTRIA DEL CANGREJO ROJO EN BASE A SU CONTENIDO PROTEICO “Valorisation of wastes and by-products from Red-Crayfish Industry based on their protein content” Tesis Doctoral presentada por D. Manuel Félix Ángel. Dirigida por los Doctores: D. Antonio Francisco Guerrero Conejo y D. Alberto Romero García Los Directores El Doctorando Fdo. D. Antonio Guerrero Conejo Fdo. D. Alberto Romero García Fdo. D. Manuel Félix Ángel Sevilla, 30 de noviembre de 2015 5 D. ANTONIO FRANCISCO GUERRERO CONEJO Y D. ALBERTO ROMERO GARCÍA, PROFESORES DE LA UNIVERSIDAD DE SEVILLA INFORMAN: Que la presente Memoria titulada “VALORIZACIÓN DE SUBPRODUCTOS Y RESIDUOS DE LA INDUSTRIA DEL CANGREJO ROJO EN BASE A SU CONTENIDO PROTEICO” presentado por D. MANUEL FÉLIX ÁNGEL para optar al grado de Doctor con Mención Internacional, ha sido realizada en su mayoría en el Departamento de Ingeniería Química de esta Universidad bajo nuestra dirección, por lo que autorizamos su presentación. Y para que conste y en cumplimiento de la legislación vigente, firmamos el presente en Sevilla, a 30 de noviembre de 2015. D. Antonio Guerrero Conejo D. Alberto Romero García 7 Dr. D. Alfonso Mazuelos Rojas, Director del Departamento de Ingeniería Química de la Universidad de Sevilla: CERTIFICA: Que la Tesis Doctoral que presenta D. Manuel Félix Ángel ha sido realizada dentro de la línea “Reología aplicada y tecnología de fluidos complejos”. 9 INDEX 1. ACKNOWLEDGEMENTS. 15 2. SYNOPSIS. 21 3. BACKGROUND 31 3.1. Proteins 33 Amino acids 33 Peptides and proteins 35 Protein structure 35 Primary structure 36 Secondary structure 36 Tertiary structure 36 Quaternary structure 37 Protein separation and characterisation 38 Dialysis 38 Column chromatography 39 Ion-exchange chromatography 39 Size-exclusion chromatography 39 Affinity chromatography 40 High-performance liquid chromatography (HPLC) 40 Electrophoresis 40 Protein denaturation 42 Non-covalent forces in a protein 43 Mechanism of denaturation 46 Temperature-induced denaturation 46 Pressure-induced denaturation 48 Denaturation by small-molecular weight additives 49 Denaturation induced by pH 50 Techno-functional properties of food proteins 51 Protein solubility 52 Effect of pH 52 Salt concentration’s 52 Denaturation of protein 53 Gelation 53 Thermally irreversible (thermoset) gels 53 High Pressure Processing (HHP) in food processing 54 Water binding 55 Emulsification 57 Foaming 58 Acknowledgements 17 Como ya se dijo hace muchos años, no sólo de pan vive el hombre. Así, durante estos años en los que he realizado el trabajo de tesis doctoral en el Departamento de Ingeniería Química, no sólo he crecido profesionalmente, sino que además he estado rodeado de una familia que me ha acompañado en muchos momentos de mi vida, es por ello que ha llegado la hora de rendir tributo a todos ellos y agradecerles cada vez que han estado ahí para lo que les he necesitado. Por ello, quiero recrear el recorrido que podría haber hecho un día cualquiera a la entrada en el departamento. Tras abrir la puerta de entrada del departamento, y enfilarme en ese pasillo, que bien podría ser el de uno de esos hospitales que antaño llamaban de beneficencia, cabe recordar aquellas veces en las que tras entrar Pepe me ofrecía té verde o bien si estaba, como él dice, “condimentado”, un té moruno. Tras esta pequeña pausa en la que no sólo había degustado una bebida con fantásticas propiedades antioxidantes, sino que también discutimos sobre la burocracia de la Universidad, me adentro en el departamento donde paso por Secretaría donde se encuentra Montse y sus ayudas con todo los trámites administrativos (que no fueron pocos y van en aumento) que tenemos que realizar o a Manolo y todos sus paseos al Pabellón de Brasil, entregando todos los documentos para que estuvieran a tiempo. Finalmente, como no, ese rincón a la izquierda del departamento me recuerda a Felipe y sus mil quehaceres con las tareas de director de departamento y Antonio y su doble pantalla, que tanta utilidad tiene para trabajar juntos y evitar la tan conocida tortícolis y sus consiguientes masajes terapéuticos. Tras salir de ese rincón del Departamento con un caramelo que Montse me ha ofrecido, paso por la puerta del laboratorio 3 y no puedo dejar de recordar la salud de la maceta de Mari Carmen, que con sus mimos la hace estar resplandeciente, o la sonrisa de Jenny que se encuentra hablando con Luisma. Bueno como todo cambia, con Luis, que aunque parezca mentira, Luisma hace ya unos cuantos meses que se fue y con él el “Re-lio” del departamento. Acknowledgements 18 Como no recordar aquellas idas y venidas a ese laboratorio, donde tantas dudas se solucionaban, y donde otras tantas surgían como agua en proceso de licuefacción tras el terremoto de lo que somos, química. La misma que da forma a las endorfinas y oxitocinas, la misma que nos permite alcanzar la plenitud como ser humano, la misma que nos permite alcanzar aquello conocido como felicidad, la misma que ha permitido que ésta última madure en una gran amistad. Sin saberlo, comenzamos con 18 años una andadura juntos, el destino hizo que compartiéramos una parte de nuestras vidas, y será el tiempo el encargado de labrar cada uno de nuestros caminos. Entrando ya en el laboratorio 4 para imprimir unos artículos, saludo a Pablo, que me cuenta las nuevas travesuras de su peque, y a Carlos, sorprendiéndome al entrar en su despacho con una señal que indica, ¡Peligro Canguros! ¿Por qué si aquí no hay de esos? ¡Ahh!, es un recuerdo de cuando estuviste en Australia, me quedo mucho más tranquilo. Tras salir, Lucía me comenta una de esas aventuras deportivas que ha vivido el fin de semana, siempre me ha gustado el deporte, ciertamente éste no es más que un reflejo de esa vida que cuando somos pequeños nos espera fuera de las instalaciones deportivas, y como no, nos ayuda a madurar, a crecer. No puedo olvidarme que después de hacer deporte, hay que recuperar fuerzas y nada mejor que uno de esas cuñas, frescas por supuesto, que Aurora nos trae de Los Palacios. Antes de llegar al Laboratorio 6, me cruzo con Ana Martín que viene llegando escuchando música, y en la “L” me topo con Juan Carlos, con él comento la ruina hacia la que se dirige el Betis, recuerdo aquellos maratones de prácticas, dónde teníamos nuestro descanso para comernos nuestro abanico, ibérico por supuesto, y comentar el trascurso de esos pequeños momentos que construyen la vida. Pregunto por Paco, pero está en la Escuela, allí donde se pasa tantas y tantas horas, y donde también hemos compartido algunas horas en ese laboratorio de la edad moderna. Finalmente, Nieves me da con la mejor de las sonrisas el matraz de fondo redondo que le pedí el día anterior, pero mirando bien, hay alguien más, Nati, que además de adecentar todo aquello por donde pasa, tantas risas gasta en este lugar llamado “L”. Acknowledgements 19 Tras salir, me encuentro con José Antonio, que viene de su laboratorio 5 y me comenta sus últimos logros en el Battlefield, su inestabilidad en el pH del agua de la pecera o los últimos arañazos que le ha propiciado su gata. Nos dirigimos a visitar a Paco, a hacerle una de esas preguntas que no pueden ser otra cosa más que ocurrencia de José Antonio. Paco intenta comprenderla sin poner cara de “yo no entiendo nada”, tras lo que no puede hacer más que reir. Nuria nos escucha, y me comenta que este fin de semana va a pasarse por La Palma, MI PUEBLO!! ¡Cuidado! por allí hay gente peligrosa, le advierto. De camino al laboratorio 6, saludo a Mc. Alfaro y paso por el despacho de Cecilio, bueno despacho o confesionario, porque creo que lo que ese despacho no haya oído…, porque CEcilio siempre tiene 5 minutos para escuchar a cualquiera, para solucionar cualquier duda, para hacer aquello con lo que si todos hiciéramos el Mundo iría mejor: escuchar y ser amable, olvidando todos aquellos prejuicios, orgullos y recelos que nos prohíben ser felices. Tras todas las paradas en todos esos rincones que forma el departamento, se va haciendo tarde, así que ya tengo que entrar en el laboratorio 6, donde están Alberto y Víctor. Víctor está terminando uno de esos trabajos del Máster que tanta “emoción” genera, y Alberto preparándose una de esas clases que luego tanto éxito tiene entre sus alumnos, una de esas clases en las que alumnos suyos, que a la vez son conocidos míos, me paran y me comentan su magnífica escenificación. Ha llegado la hora de trabajar, con la ilusión de hacer las cosas bien, sin tener miedo al futuro, a sabiendas que tras la defensa del trabajo de tantos años se cerrará una etapa, pero con la ilusión que tras el cierre de una puerta se abre un ventanal. Durante estos años de tesis, también han formado parte de mí mis amigos y aquellas personas con las que he vivido, Dani, Mary, Javi, Moi, Juanjo, Jorge e Inma, así como con todos aquellos con los que he tomado una cerveza y he amenizado los fines de semana en la Palma, Ismael, Juandi, Juan, Jaime, fran, así como de otros tanto que han estado conmigo en las duras y en las maduras. Aquellas personas que por suerte o azar del destino he conocido en los otros lugares donde he llevado a cabo mi labor investigadora, a Heline y Raf en Bélgica, a Turid, Acknowledgements 20 Trude, Siri, Giancarlos en Noruega o a Adri, Espe, Isa, Ortega, Rocío o Inma en Huelva. Por último, no puedo olvidarme de mi familia, aquellos que han formado parte de mi vida desde que nací (puesto que soy el hermano pequeño), aquellos que me han visto alegre y enfadado, aquellos que me han guiado a lo largo de tantos años, aquellos que sé que nunca me abandonarán y siempre me querrán, aquellos a los que dedicaré unas líneas más adelante. No puedo olvidarme de aquellas personas que ya no forman parte de aquello que conocemos como familia, bien porque ya no están entre nosotros, como mis abuelos, bien porque actualmente no lo sean, pero lo serán. Aquellos en los que tendrá influencia este presente, que para entonces será pasado, aquellos en los que influiré e influirán en un futuro. A todos ustedes os tengo en mente en este preciso momento. 2. Synopsis. Synopsis 23 Every year food and agricultural industry produce a big amount of surpluses and wastes which are discarded or used as a low added value byproduct. A particularly relevant example of this fact is located in Andalusia (southern Spain) and it is associated to the red-swamp crayfish. This crustacean was introduced in the middle of the twentieth century and due to favourable weather conditions, abundant food and the lack of predators, it has undergone a fast widespread growth (Kirjavainen and Westman 1999). This rapid growth has contributed to the development of a strong local crayfish industry at the marshes of the Guadalquivir River (Geiger, Alcorlo et al. 2005). A currently attractive way to valorise these products is taking benefits from its relatively high protein content. Crustaceans constitute an excellent source of high-quality protein, rich not only in essential amino acids and lipids, including long-chain polyunsaturated fatty acids from ω-3, but also containing other components of functional value such as astaxanthin that possess a high antioxidant capacity, even higher than others important antioxidants such as β-carotene or vitamin E (Miki 1991). The quality of this protein concentrate will contribute to increase the functional properties in their products and derivatives. To obtain a useful crayfish protein concentrate for these three applications, a previous stage of characterization and optimization of different flours from crayfish pulp has been carried out. This stage was developed by PEVESA, who selected the temperature, pH, extraction procedure and proteindispersion fraction in order to obtain the phase with the highest content of soluble protein. This study is focused on different applications of crayfish protein systems derived from their functional properties, aiming at the achievement of high value-added crayfish-based products. The applications considered in this Synopsis 24 study will be: emulsification ability, gel formation and bioplastics processing, and. First of all, one application come from food industry, which is really interested in produce stable oil-in-water (o/w) emulsions containing a protein different to egg-yolk protein as the only emulsifier in order to produce food products such as mayonnaise and salad dressings. These alternative proteins would avoid the presence of cholesterol from yolk, the development of salmonella in yolk-containing food products or allergic reactions, which nowadays are more and more frequent. Other authors have used previously myofibrillar proteins such as actomyosin to produce emulsions and have demonstrated that actomyosin from hake had higher emulsifying activity and stability than the actomyosin from chicken and pork (Cofrades, Carballo et al. 1996). Protein from crayfish may constitute an excellent source of protein which may contribute to stabilize o/w emulsions. In addition, other functional property is the ability of proteins to denature form aggregates and adopt gel-structure after a controlled heating. This application contributes to design a wide variety of food systems with the desire rheological consistency, microstructure and texture. Crustacean meat contents mainly sarcoplasmatic and myofibrillar proteins. Sarcoplasmic fraction with a globular and relatively simple structure, show a weak gelation capacity and, therefore, a little contribution to the texture of processed foods. On the other hand, myofibrillar proteins, specially myosin and actomyosin, constitute multiples dominions that tend to form viscoelastic networks and gels with high consistency (Damodaran 1997). Finally, an interesting area of research is the antioxidant characterisation of these gels. Due to the presence of the above mentioned functional ingredients, these gels may constitute an excellent source of protein for the human nutrition. Synopsis 25 Finally, a currently attractive way to valorise these by-products is through the use of this protein concentrate as renewable resources in the manufacture of “green materials”, replacing hardly degradable plastic materials from oilbased synthetic polymers. Recently, some important applications for bioplastics are beginning to emerge in the areas of food-packaging, pharmaceutics, electronics, automotive industry and biomedicine. Thus, among other applications, bioplastics from proteins can be used in food packaging, fruit coating, encapsulation, textiles, absorbent materials or tissue engineering (Sharma and Luzinov 2012, Soroudi and Jakubowicz 2013). This wide variety of potential applications allows us to envisage an increasingly use of biobased-plastic materials in a near future. Thus, one of the main objectives of this study has been to evaluate the potentials of crayfish concentrate as emulsifier to obtain highly concentrated oil/water emulsions such as mayonnaise-like. First of all, it was necessary to optimize the processing and composition parameters that would lead to longterm stable emulsions. In this context, interfacial measurements were performed in order to facilitate prediction of emulsion stability. Relevant ultimate information on emulsion stability was obtained by characterizing the rheological properties and microstructure of crayfish-based emulsions. The rheological characterization has been focused on the linear viscoelastic properties of the emulsions, determined by means of small-amplitude oscillatory shear (SAOS). Microstructural parameters have been evaluated through droplet size distributions analysis. Microstructure has also been characterised by Confocal Laser Scanning Microscopy (CLSM). Another aditional objective has been to evaluate the gel ability and the bioactive potential of gels made from non-denatured crayfish protein concentrate at different pH values. To explore improvements in both, the gelation and bioactive ability ability, different hydrolysates were obtained Background. Proteins 33 3.1. Proteins Proteins are the intermediate in every process that takes place in a living being. Proteins also are the most abundant biological macromolecules, being considered even as biopolymers. All proteins are built from the same set of 20 amino acids which are covalently linked in a characteristic linear sequence. Each type of protein has a unique sequence of amino acids which provides each of them with distinctive chemical properties. This group of 20 precursor molecules may be considered as the alphabet used to write the protein structure and functionality. Proteins are found in a wide range of sizes, from relatively small peptides to huge biopolymer with molecular weight over the millions (Lehninger, Nelson et al. 2005). Amino acids All of the 20 amino acid are α-amino acids. Each of them have a carboxyl group and an amino group bonded to the same carbon atom (the α-carbon), and an “R” group. They differ from each other in their “R” groups, which vary in structure, size, and electric charge. For all the amino acids except for the glycine, the α-carbon is bonded to four different groups: a carboxyl group, an amino group, the “R” group, and a hydrogen atom. According to this structure, the α-carbon atom is a chiral centre. Thus, the four different groups can occupy two unique spatial arrangements due to the tetrahedral arrangement of the bonding orbitals around the α-carbon atom, such that each amino acid have two possible stereoisomer L and D. However, all the amino acids synthetized by living being are L-amino acid (Aluru 2005). Figure 3.1-1 shows the 20 amino acids classified by “R” group nature (non-polar, polar-uncharged, polar-positively charged, polar-negatively charged and aromatic). Background. Proteins 34 Amino acids in aqueous solution also can act as acids and bases. The amino and carboxyl groups of amino acids, along with the ionisable R groups of some amino acids, can work as weak acids and bases. When an amino acid loss an ionisable R group and is dissolved in water at neutral pH, it exists in solution as the dipolar ion, or zwitterion which can act as either an acid or a base. These kind of substances which have an amphoteric behaviour are named ampholytes (Lehninger, Nelson et al. 2005). The twenty amino acids are: Glycine, (Gly); Histidine, (His); Isoleucine, (Ile); Leucine, (Leu); Lysine, (Lys); Methionine, (Met); Phenylalanine, (Phe); Proline, (Pro); Serine, (Ser); Threonine, (Thr); Tryptophan, (Trp); Tyrosine, (Tyr); Valine, (Val). Figure 3.1-1: Chemical composition and structure of the twenty amino acids, classified by “R” group nature Background. Proteins 35 Peptides and proteins Peptides and proteins are the polymers of amino acids. Two amino acid molecules can be covalently bounded through an amide linkage, leading a dipeptide. Such a linkage is formed by removal of one hydroxyl-group from the carboxyl group of the starting amino acid and one hydrogen from the amino group of the followed amino acid. As a consequence, formation of a peptide bond is a condensation reaction where each peptide joining release a molecule of water. Figure 3.1-2 schematises this chemical reaction. Figure 3.1-2: Formation of peptide bond from two amino acids Three amino acids can be joined by two peptide bonds to form a tripeptide. In the same way, amino acids can be joined to form tetrapeptides, pentapeptides, etc. When a few amino acids are linked in this way, the structure is called an oligopeptide (from 6 to c.a. 20 amino acids). When many amino acids are joined, the product is called a polypeptide. Proteins have thousands of amino acid. In fact, polypeptides whose molecular weight is higher than 10.000 Da, are generally called proteins (Lehninger, Nelson et al. 2005). Protein structure For large macromolecules such as proteins, the task of describing and understanding structure is approached at several levels of complexity, arranged in a kind of conceptual hierarchy. For proteins, four levels of protein structure are commonly define (Aluru 2005). Background. Proteins 36 Primary structure The most important element of the primary structure is the sequence of amino acid residues. The differences in primary structure can be especially informative. Each protein has a distinctive number and sequence of amino acid residue. The function of a protein depends on its amino acid sequence. Secondary structure Secondary structure is referred to particularly stable arrangements of amino acid residues giving rise to recurring structural patterns. Thus, the term secondary structure refers to any chosen segment of a polypeptide chain, describing the local spatial arrangement of its main-chain atoms, without regard to its relationship to other segments. A regular secondary structure occurs when each dihedral angle (φ and ψ) remains the same. The most commonly structure are α-helix and βconformations. Where a regular pattern is not found, the secondary structure is sometimes referred to a random coil However, this conformation does no describe properly a structure. Tertiary structure Tertiary structure describes all aspects of the three-dimensional folding of a polypeptide. Thus, the overall three-dimensional arrangement of all atoms in a protein is referred to as the protein's tertiary structure. Amino acids that are far apart in the polypeptide sequence and are in different types of secondary structure may interact within the completely folded structure of a protein. The location of bends in the polypeptide chain and the direction and angle of these bends are determined by the number and the location of specific bend-producing conformation. Different Interacting segments of polypeptide chains are held in their characteristic tertiary location, such as Background. Proteins 37 several kinds of weak interactions or even by covalent bonds (e.g. disulphide bonds) between different protein segments. Quaternary structure When a protein has two or more polypeptide subunits, their arrangement in space is referred to as quaternary structure. Thus, the arrangement of proteins which contain two or more separate polypeptide chains, or subunits, in three-dimensional complexes constitutes the quaternary structure. Considering these higher levels of structure, it is very useful to classify proteins into two main groups: fibrous proteins (whose polypeptide chains are arranged in long strands or sheets) and globular proteins (whose polypeptide chains are folded into a spherical or globular shape) (Lehninger, Nelson et al. 2005). Figure 3.1-3 illustrate the four different conformations which are adopted by a protein: Figure 3.1-3: Conformations adopted by a protein. Background. Proteins 38 Protein separation and characterisation To study a protein in detail, the researcher must be able to separate it from other proteins in pure form and must have the techniques to determine its properties which allow to understand its behaviour and its relationship with other macro and micro molecular systems. Thus, in any protein purification, the first step is the proteins solubilisation into a solution, which is called a crude extract, is the separation. Several methods are available for separating one or more of the proteins from the crude extract. Usually, the extract is subjected to treatments that separate the proteins into different fractions based on a physical property such as size or charge. A separation based on protein solubility (which depends on pH, temperature and salt concentration, among other factors) is commonly used in an early fractionation step. For instance, the solubility of proteins is generally lowered at high salt concentrations. Thus, the addition of certain salts in the right amount can selectively precipitate some proteins, while others will remain in solution (Lehninger, Nelson et al. 2005). Dialysis A solution containing the protein of interest must usually be modified before the subsequent purification step. Dialysis is a procedure that separates proteins from small solutes because of the larger size of proteins. The partially purified extract is placed in a bag or tube made of a semipermeable membrane. When this is suspended in a much larger volume of buffered solution of appropriate ionic strength, the membrane allows the exchange of salt and buffer but not of proteins. Thus, dialysis retains large proteins within the membranes, allowing the concentration of other solutes in the protein preparation to change until they come into equilibrium with the solution outside the membrane. Background. Proteins 39 Column chromatography The most powerful methods for fractionating proteins make use of column chromatography technique, where a buffered solution (the mobile phase) percolates through it. The protein-containing solution, layered on the top of the column, percolates through the solid matrix. Individual proteins migrate faster or more slowly through the column depending on their physical properties. Ion-exchange chromatography This technique makes use of differences in the sign and magnitude of the net electric charge of proteins at a selected pH. The column matrix is a synthetic polymer containing bound charged groups. If those groups are bound with anionic groups from protein are, the column will be called cation exchangers column, and those with bound cationic groups are called anion exchangers. The affinity of each protein for the charged groups on the column is influenced by the pH (which determines the ionization state of the molecule) and the concentration of free salt ions in the surrounding solution. Separation can be optimized by gradually changing the pH and/or salt concentration of the mobile phase so as to create a pH or salt gradient. Size-exclusion chromatography This technique is also called gel filtration because it separate proteins according to size. In this method, large proteins emerge from the column sooner than small ones. The solid phase consists of cross-linked-polymer beads with engineered pores or cavities of a particular size. Large proteins cannot enter the cavities and take a short (and rapid) path through the column, around the beads. By the contrary, small proteins enter the cavities and are slowed by the longer path through the column. Background. Proteins 40 Affinity chromatography This technique is based on binding affinity. The beads in the column have a covalently attached chemical group called ligand (a group or molecule that binds to a macromolecule such as a protein). When a protein mixture is added to the column, any protein with affinity for this ligand binds to the beads, and its migration through the matrix is retarded. High-performance liquid chromatography (HPLC) HPLC makes use of high-pressure pumps that speed the movement of the protein molecules down the column, as well as higher-quality chromatographic materials that can withstand the crushing force of the pressurized flow. By reducing the transit time on the column, HPLC can limit diffusional spreading of protein bands and thus greatly improve resolution (Lehninger, Nelson et al. 2005). Electrophoresis Electrophoresis is one of the most commonly technique for the separation of proteins based on the migration of charged proteins in an electric field. This procedure is not generally used to purify proteins in large amounts, because usually are available simpler alternatives than electrophoretic methods, which often affect the structure and, as a consequence the function of proteins. Electrophoresis is however especially useful as an analytical method. Its advantage is that proteins can be visualized as well as separated, permitting a researcher to estimate quickly the number of different proteins in a mixture or the degree of purity of a particular protein preparation. Also, electrophoresis allows determination of crucial properties of a protein such as its isoelectric point and approximate molecular weight. Background. Proteins 41 Electrophoresis of proteins is generally carried out in gels made up of the cross-linked polymer polyacrylamide (PAGE). The polyacrylamide gel acts as a molecular sieve, slowing the migration of proteins approximately in proportion to their charge-to-mass ratio. Migration may also be affected by protein shape. In electrophoresis, the force moving the macromolecule is the electrical potential, E. The electrophoretic mobility, µ, of a molecule is the ratio of its velocity, V, to the electrical potential. Electrophoretic mobility is also equal to the net charge, Z, of the molecule divided by the frictional coefficient, f, which reflects in part the protein's shape. Thus: 𝜇=𝑉 𝐸=𝑍 𝑓 (3.1-1) Thus, as can be deduced from Equation 3.1-1 (3.1-1) the migration of a protein in a gel during electrophoresis may be regarded as a function of its size and shape. An electrophoretic method commonly employed for estimation of purity and molecular weight makes use of the surfactant sodium dodecyl sulphate (SDS), then the electrophoresis is called SDS-PAGE. SDS binds to most proteins in amounts roughly proportional to the molecular weight of the protein, about one molecule of SDS for every two amino acid residues. The bounded SDS contributes a large net negative charge, rendering the intrinsic charge of the protein insignificant and conferring on each protein a similar charge-to-mass ratio. In addition, SDS binding partially unfolds proteins, such that most SDSbounded proteins assume a similar shape. Therefore, electrophoresis in the presence of SDS separates proteins almost exclusively on the basis of mass (molecular weight), with smaller polypeptides migrating faster. After electrophoresis, the proteins are visualized by adding a dye, such as Coomassie blue, which binds to proteins but not to the gel itself. The molecular weight of each protein fractions is obtained by comparison with standard proteins whose molecular weight is known. If the protein has two or Background. Proteins 48 In addition, several proteins have been shown to undergo denaturation at cold temperatures. The cold temperature-induced denaturation of proteins is mainly due to a decrease in the stability of hydrophobic interactions at low temperatures. The fact that the hydrophobic and the conformational entropy are the two relevant forces, controlling thermodynamic stability of proteins tentatively suggests that the stability of proteins might be in some way dependent on the amino acid composition. Finally, factors such as water content of dry protein powders or the presence of small-molecular-weight substances affect thermal protein denaturation. Thus, as the water content is increased, the denaturation temperature of proteins decreases asymptotically towards a value that is similar to the denaturation temperature of the protein in a dilute solution. This is due to the plasticizing effect of water, which promotes segmental mobility in proteins. On the other hand, small-molecular-weight solutes, such as salts and sugars, generally leads to an increase in the denaturation temperature of proteins. Pressure-induced denaturation Proteins are inherently highly flexible. This high flexibility is the underlying reason for their marginal stability under physiological conditions. The flexibility of proteins arises because of unfilled spaces or cavities in the interior of the protein. These cavities are created by imperfect packing of the residues as the protein chain collapses on itself during folding, the partial specific volume (𝜈0), of a protein consists of EQ. (3.1-3): 𝜈0=𝑣𝑐+𝑣𝑐𝑎𝑣+∆𝑣𝑠𝑜𝑙 (3.1-3) where vc is the sum of constitutive specific volumes of atoms in the protein, Δvsol is the specific volume of cavities in the protein, and and Δvsol is the specific volume change because of the hydration process. The first term is Background. Proteins 49 constant for a given protein, while the last two terms are the main parameters affecting the specific volume of proteins. Under very high hydrostatic pressure, the collapse of the cavities formed as a result of imperfect packing of amino acid residues causes unfolding of the protein. In the unfolded state, elimination of the cavities decreases the volume, and hydration of the exposed hydrophobic residues also leads to a reduction in the volume of the solvent. Despite, pressure-induced gels are softer in texture than heat-induced gels, and feature the capability of retaining colour, flavour, vitamins and other nutrients that are destroyed to some extent in thermally processed foods and in heat-induced gels (Damodaran 1997). Denaturation by small-molecular weight additives Several small-molecular-weight solutes, such as urea, guanidine hydrochloride, surfactants, sugars, and neutral salts, affect protein stability in aqueous solutions. While urea, guanidine hydrochloride, and small-molecularweight surfactants destabilize the native conformation of proteins, sugars tend to stabilize the native structure. As for neutral salts, while certain salts termed as kosmotropes tend to stabilize protein structure (e.g. sulphate and fluoride salts of sodium), other salts, such as bromide, iodide, perchlorate, and thiocyanate, termed as kosmotropes, destabilize protein structure. The stabilizing or destabilizing effects of small molecular-weight additives on proteins is believed to follow a general mechanism. This is related to their preferential interaction with the aqueous phase and the protein surface. Additives that stabilize protein structure bind very weakly to the protein surface but enhance preferential hydration of the protein surface. Such additives are generally excluded from the region surrounding the protein and, Background. Proteins 50 as a result, their concentration near the protein is lower than in the bulk solution. This concentration gradient presumably creates an osmotic pressure gradient surrounding the protein molecule, sufficient enough to elevate the thermal denaturation temperature. In the case of additives that destabilize protein structure, the opposite seems to be true. That is, those additives that decrease the stability of proteins preferentially bind to the protein surface and cause dehydration of the protein. In such cases, water molecules are excluded from the region surrounding the protein and the concentration of the additive in this waterexcluded region is higher than in the bulk solvent. Favourable interactions of such additives with protein surfaces, particularly the non-polar surfaces, promote unfolding of the protein such that the buried nonpolar surfaces are further exposed for favourable interactions with chaotrope additives. Anionic surfactants such as sodium dodecyl sulphate (SDS), are potent denaturing agents with the skill of developing strong binding to hydrophobic groups in the crevices of protein molecules, which leads to destabilization and solubilisation of buried hydrophobic regions. Because of this high binding capacity, proteins in SDS solution become highly negatively charged. The resultant electrostatic repulsions between segments also play a role in protein unfolding as well as in preventing protein aggregation. Denaturation induced by pH With regards to pH-induced denaturation, proteins are either negatively or positively charged at neutral pH. Native proteins, at this physiological pH, present an equilibrium structure with a global minimum free energy that has already taken into account the pre-existing repulsive and attractive electrostatic interactions. However, at pH values far away from the neutral pH, changes in the state of ionization of various charged residues in proteins alter the electrostatic free energy, resulting in conformational changes. Most Background. Proteins 51 proteins are very stable at their isoelectric point (IEP), pH at which the net charge of the protein is zero and electrostatic repulsive interactions are at a minimum. However, proteins typically unfold at pH values far from the IEP, that is, below 4 and above 10. This unfolding is not simply because of changes in the ionization state of the charged residues on the surface of the protein, but is related to ionization of residues that are partially or fully buried in the protein (Hui 2006). Techno-functional properties of food proteins Functionality has been described as the set of non-nutritive roles that food constituents play in a food system. More formally, techno-functional properties are the physical and chemical properties that affect the behaviour of molecular constituents in food systems. Proteins in foods are multifunctional and may be the principal structural component in many food systems, including products from meat and poultry, eggs, dairy, cereals, and legumes. In fact, proteins contribute significantly to the sensory attributes and overall quality of food products. In this sense, protein functionality is considered critical for the improvement or existing food products or the development of new ones. An example is the use of less expensive protein sources as replacements in traditional food products. Use of less expensive proteins not only allows for cost reduction, but also can increase the utilization of food materials that previously might have been considered waste products. Techno-functional properties commonly associated with proteins include solubility, gelation, emulsification, foaming, and water-holding capacity (Damodaran 1997, Aluru 2005). Background. Proteins 52 Protein solubility The solubility of a protein, which is determined by its primary structure, is very often the key factor in delimiting its use in foods. If a protein has a polar surface due to the presence of polar amino acids, it will have good solubility in a polar solvent such as water. On the other hand, proteins with higher contents of hydrophobic amino acids, fewer charges on their surface, or those which contain many subunits tend to have limited water solubility (Damodaran, Parkin et al. 2007). Effect of pH Surfaces of proteins have net charges due to their amino acid content depending on the pH of their environment. A protein shows minimal solubility when it has a net charge of zero, that is, equal numbers of positive and negative charges on its surface. This is called the isoelectric point of the protein (IEP). There is minimal solubility because intermolecular repulsion is at a minimum and proteins will tend to aggregate. At pH values above the isoelectric point of a protein, it has a net negative charge. At pH values below the isoelectric point, it will have a net positive charge. In both cases the presence of pronounced surface charges will result in intermolecular repulsion and enhanced solubility. Salt concentration’s Solubility is also affected by the type and concentration of salts in a food system. As salts content increases proteins become more soluble, which is the so-called “salting-in” effect, attributed to the ability of salt ions to enhance the surface charges on proteins. In foods, sodium chloride is commonly used for this purpose. At high salt concentrations, usually above 1M, which is much higher than the concentration used in foodstuffs molar, protein solubility decreases. This effect is called “salting-out” and is thought to be due to salt competing with the proteins for available water for solvation. Background. Proteins 53 Denaturation of protein As described in section 3.1.5, when denaturation proceeds, protein molecules change in regard to surface charges, shape, size, and hydrophobicity. Most thoroughly denatured proteins are insoluble. In any case, denaturation always results in loss of solubility, and then it is undesirable in many food systems where solubility is important. However, denaturation may cause some desirable changes in some food systems. Therefore, the degree of protein denaturation required depends on the food application, but ensuring its exhaustive control over food processing is essential. Gelation A protein gel is a three-dimensional cross-linked network of protein molecules imbedded in an aqueous solvent. Most gels are very high in water content (up to 95-98%), and still have characteristics of solid or rigid food materials (Damodaran, Parkin et al. 2007). Gelation is based on the denaturation of proteins, followed by their intermolecular association to form matrices which trap water, fat, and other food ingredients. The formation of gels is influenced by heat, pH, pressure or shearing, and solvent conditions. Food gels are divided into two categories: thermally reversible and thermally irreversible gels (Damodaran 1997). Thermally irreversible (thermoset) gels Thermally irreversible or thermoset gels form chemical bonds that will not break during reheating of the gel and thus remain rigid if it is reheated. Most thermoset gels are the result of protein unfolding, followed by aggregation of the molecules into a cross-linked network. During this process, heated proteins partially unfold and form aggregates. As the “gel-point” temperature is reached, these aggregates unfold further and rapidly cross-link to form a gel. This network is generally formed via non-covalent bonds such Background. Proteins 54 as hydrophobic interactions and hydrogen bonds. Occasionally, disulphide bonds may be involved. High Pressure Processing (HHP) in food processing The effect of the high pressure on food preservation was study initially by Hopkins, Hite and Watson (1899). In 1899 some experiments were carried out in West University (Virginia), using high hydrostatic pressure to conserve juice, meat and fruit. These studies demonstrated that some microorganisms could be destroyed after 10 min at 658 MPa. At early 20th century, other research proved that egg white could be modified by high pressure. However, the research in this field did not undergo any substantial progress until significant improvements were achieved in the technology of hydraulic presses. This fact encouraged the renewed interest of researchers on HHP applications in the eighties. When a high pressure is apply to a food dipped in a liquid, the pressure placed on the sample is the same in all points. This is one of the advantages of this procedure, avoiding the differences found in the thermal treatment. In addition, compared to the thermal treatments, pressurisation and depressurisation cycles are faster, reducing processing time. As above mentioned, HHP can leads to the destruction of microorganisms without markedly altering the taste and flavour or the nutrient content of foods. In general, bacteria, which are in the logarithmic growth phase, are the most sensitive. Moderate pressures (300-600 MPa) involve the death of the vegetative cell. Usually a pressure of 400 MPa is apply for 5 min to reduce the population ten times (Hoover, Metrick et al. 1989). To destroy bacteria spores, a higher pressure is needed, however if the pressure is combined to a soft heating (60oC), spores are destroyed at about 400 MPa. Thus, a combination of high pressure and soft heating can suppose a synergic effect (Galazka and Ledward 1995). Background. Proteins 55 The enzyme activity strongly depends on pH, composition, temperature and pressure. Some enzymes can be disabled at 100 MPa, however other needs higher pressures (even 1,000 MPa) (Cano, Hernandez et al. 1997) In addition of microorganisms inactivation. HHP can also unfold proteins, solidify lipids, showing advantages in the preservation of sensory (colour, taste, flavour, texture, etc.) and nutritional properties (Tewari, Jayas et al. 1999). In addition, high pressure can induce conformational changes in proteins, which can involve important modifications of their techno-functional properties. As a consequence, proteins can undergo aggregation and gelation, depending on the pressure applied, as well as protein nature, composition, and environmental factors such as pH or ionic force. Because the aggregation and gelation are directly related to proteinprotein interactions, HHP processing will have a high influence on protein structure, since application of HHP may exerts a marked effect on noncovalent interactions (electrostatic, hydrophobic and hydrogen bonds). Thus, HHP treatment may lead to a breakdown of the tertiary and quaternary protein structure of globular proteins, but it has a very limited influence on secondary structure. In general, low pressures induce reversible changes such as proteincomplex dissociation, ligands, and conformational changes, whereas high pressures (> 500 MPa) generally involve irreversible protein modifications (Hereman, Van Camp et al. 1997). Water binding Water-binding capacity is the amount of water that is bound or retained by a protein under well-defined conditions. Thus, water binding is an important techno-functional property for several reasons: Background. Proteins 56 - Most foods contain high amounts of water and it is necessary to avoid chemical changes that might cause the formation of free water or drip loss. - Increasing the amount of water a product can hold effectively can increase the profitability of a given product - Both product yield and sensory quality are highly dependent on the proper moisture content of a finished food. Water is usually bounded to the surface of a protein by hydrogen bonding, which is sometimes called dipole bonding. Hydrogen bonding results from water’s interaction with the R group of amino acids which are dipoles. Water bound to the surface of proteins in this manner is called “monolayer” water and is very tightly associated with the protein. Other water associated with the protein or protein matrices can be trapped in capillary structures and pores. If water is not associated with the monolayer on the protein surface is called free water and moves unhindered throughout the food system. With regards to factors which Influence water binding, the most important are small polar molecules (e.g. sugars), temperature, salt content and pH. Small polar molecules and temperature will generally enhance water binding by proteins. In some cases temperature may induce formation of protein gels, which will enhance the binding of water by the system. Sodium chloride binds to charged groups on protein surfaces and weakens intermolecular bonds. This is a positive effect in systems which utilize muscle fibres as part of the structural elements of the food. Salt allows the muscle proteins to distance themselves from others within the muscle fibre and thus increase the number of sites for water to bind. The pH of a system markedly influences its ability to bind water. This is due to changes in the surface Background. Proteins 57 charges on a protein as the pH is altered. Water binding is the lowest at the isoelectric point (IEP) of a protein (Damodaran 1997). Emulsification An emulsion is a mixture of two immiscible liquids in which one is dispersed in the other in the form of droplets. The liquid in the droplets the dispersed is called internal, or discontinuous phase. In the same way, the surrounding phase is called the external or continuous phase. Emulsions in which the dispersed phase is a lipid are called “oil in water” emulsions (O/W). By the contrary, water in oil emulsions contain droplets of water dispersed in a lipid as continuous phase (W/O) (McClements 2004). When a liquid is exposed to other phase (e.g. air), the surface between them is in a state of tension. This state of tension is called interfacial tension (or surface tension, in the case of air) and it is a consequence of the attractive forces between molecules in the liquid that are enhanced by exposure to the other phase. The molecules “bunch” together to decrease their exposition to the air surface. The region of contact between two immiscible liquids is called the interface. The interfacial area plays an important role in emulsion formation. Thus, a considerable amount of mechanical energy is required in order to reduce droplet size and to increase interfacial area (Walstra 1993). However, as the interfacial area increases, the stability of the mixture decreases. Unfortunately, most emulsions are thermodynamically unstable and droplets tend to aggregate spontaneously in order to reduce the interfacial area. Therefore, emulsion stability, which is considered to be the primary requirement for the commercial application of emulsions, is in fact a kinetic concept such that an emulsion is considered stable when the number, size distribution, and arrangement of droplets do not undergo any discernible change over the storage time scale. Background. Proteins 64 Water Retention The ability of meat and meat products to retain moisture before, during, and after processing or cooking plays a crucial role in consumer acceptance of the product and is usually described in terms of water-holding capacity. Physico-chemically, the water in meat is present in either the bound or the free state. The bound water is tightly associated with proteins through charged groups and dipolar sites on the protein surface. Solubility Solubility of proteins is quite important for the manufacture of processed muscle foods. This is because most techno-functional properties of muscle proteins are related to protein solubility, and, in fact, some are achieved only when the proteins are in a highly soluble state. This is because most functional properties of muscle proteins are related to protein solubility, and, in fact, some are achieved only when the proteins are in a highly soluble state. Solubility of muscle proteins is a function of protein structures, the structure of myofibrils, pH, concentration (ionic strength) of salt added to meat, temperature, time of mixing meat with salt... Sarcoplasmic proteins are naturally soluble in muscle. However, solubilisation of myofibrillar proteins generally requires relatively high ionic strength (G > 0.4 M). Thus, protein solubility is highly dependent on the ionic strength of the extraction buffer. Extraction of myofibrillar proteins begin at an ionic strength close to 0.5 M, and it reaches a maximum at ionic strength 1.0 M. Thus, an increase in salt (NaCl) concentration to above 0.5 M (approximately 2% salt in meat), is widely used in processed meats (Xiong 2004). Viscosity of protein solutions Rheological properties, as related to flow and deformation, are important functional attributes of muscle proteins. The rheological behaviour of a Background. Proteins 65 protein suspension in muscle foods is often described in terms of viscosity. This is because the rheological properties of the aqueous protein phase can influence texture and stability. Proteins are charged polymers capable of binding water and causing fibre swelling by the uptake of water and loosening of the polypeptide matrix. As a consequence of swelling process, a protein increases its effective hydrodynamic volume, and, therefore, increases the resistance to shear. Finally, the myosin structure (the large length-to-diameter ratio of the rod portion), makes myosin highly viscous in salt solution. Because of its great viscosity and abundance in muscle, myosin is the major contributor to the rheological properties of the aqueous extract in salted meat (Xiong 2004). Gelation Gelation of proteins is a thermodynamic process that occurs widely in food processing using muscle proteins. A gel has been referred to as a continuous network of macroscopic dimensions immersed in a liquid medium and exhibiting non steady state flow. The importance of protein gelation to muscle foods has been demonstrated. Thus, myofibrillar proteins at the junction of meat particles were responsible for the meat binding and texture of cooked sausage products (Xiong 2004). Emulsification Emulsions from muscle proteins are stabilized through two mechanisms. The first mechanism is physical entrapment of fat globules within the protein matrix formed largely via protein-protein interactions. In the second mechanism, fat globules are stabilized by an interfacial protein film (membrane) that surrounds them. The interfacial film is interactive in the sense that it interacts with the viscoelastic continuous phase to further enhance the emulsion stability. Background. Proteins 66 In meat emulsions, salt-soluble proteins play the most critical role in forming interfacial films that encapsulate fat particles or oil droplets. The emulsifying capacity of different muscle proteins was found to follow the order of myosin > actomyosin > sarcoplasmic proteins > actin (Xiong 2004). Foaming Foaming of protein solutions is fairly common. Difficulties arise when the foam volume expands to the capacity of the container and proteins become denatured as a result of foam formation. The behaviour of proteins at the air/liquid interface is extremely important because the formation of a proteinbased flexible, cohesive film around air bubbles is essential for foaming capacity and foam stability. In fact, there is a relationship between the molecular flexibility of proteins, film properties, and foam stability. Flexible, disordered proteins are more surface-active than extensively cross-linked, stable, and compact globular proteins (Xiong 2004). Background. Interfacial Assessment 67 3.2. Rheology Definition The term "rheology" is originated from the Greek: "rheos" meaning "the river", "flowing", "streaming". Thus, rheology is literally "the science of flow". More specifically, rheology is the science of flow and deformation of matter and describes the interrelation between force, deformation and time. Therefore, it is a branch of physics since these variables come from the field of mechanics. However, rheological experiments do not merely show information about the flow behaviour of liquids, but also about the deformation behaviour of solids (Mezger 2014). Flow laws Isaac Newton was the first to express the basic law of viscosity describing the flow behaviour of an ideal liquid. Thus, Newton postulated that an ideal (Newtonian) fluid is a fluid in which the viscous stresses arising from its flow, at every point, are linearly proportional to the local strain rate. The constant of proportionality in Newton's Law is the viscosity of the fluid. EQ. (3.2-1) illustrates this behaviour: 𝜏=𝜂∙𝛾󰇗 (3.2-1) where τ represents the stress, η the viscosity and 𝛾󰇗 the strain rate Usually, a simple shear experiment, in which a fluid is confined between two plates (Figure 3.2-1) is used to define some fundamental rheological parameters (Two Plates Model). The upper plate with the (shear) area A is set in motion by the (shear) force F and the resulting velocity vmax is measured. The lower plate is stationary (v = 0). The gap size (y1 or y2) is the distance between the plates, and the liquid sample is sheared within this gap. It is assumed that the following shear conditions are met: Background. Interfacial Assessment 68 1) The sample adheres to both plates and does not slide or slip along them. 2) There are laminar flow conditions. Figure 3.2-1: Two-pate model representation Shear stress A force applied tangentially to an area being the interface between the upper plate and the liquid underneath, leads to a flow in the liquid layer. Shear stress (τ) precisely arises from the application of this tangential force (F) to the shear surface area (A), as the ratio between both variables: 𝜏=𝐹 𝐴 (3.2-2) Shear rate The shear stress (τ) causes the liquid to flow in a special pattern. A maximum flow speed is found at the upper boundary. The speed drops across the gap size (y) down to 0 (vmin = 0) at the lower boundary contacting the stationary plate. Laminar flow means that infinitesimally thin liquid layers slide on top of each other, similar to the cards in a deck of cards. Thus, one laminar layer is then displaced with respect to the adjacent ones by a fraction of the total displacement encountered in the liquid between both plates. The speed Background. Interfacial Assessment 69 drop across the gap size is named “shear rate” (γ ) and it may be mathematically defined by a differential function of the fluid velocity (v), as follows: 𝛾󰇗=𝑑𝑣 𝑑𝑦 (3.2-3) In addition, the shear rate may be defined as the time-derivative of the strain caused by the shear stress acting on the liquid layer: 𝛾󰇗=𝑑𝛾 𝑑𝑡 (3.2-4) Therefore, EQ. (3.2-3) and EQ. (3.2-5) may be combined to give a more general expression of Newton: 𝜏=𝜂∙𝑑𝑣 𝑑𝑦= 𝜂∙ 𝛾󰇗 (3.2-5) Dynamic viscosity From EQ. (3.2-1), the dynamic viscosity can be readily written as follows: 𝜂=𝜏𝛾󰇗 (3.2-6) For ideal-viscous fluids measured at a constant temperature, the value of the ratio of the shear stress (τ) to the corresponding shear rate 𝛾󰇗 is a material constant (η) (Newtonian fluids). Non-Newtonian liquids. Non-ideal liquids There are several kind of fluids which do not exhibit the ideal Newtonian behaviour where η is not constant. Various types of common flow behaviour can be observed in Figure 3.2-2. Background. Interfacial Assessment 70 Figure 3.2-2: Behaviour of different fluids: Newtonian (1), Pseudoplastic or Shearthinning (2), Dilatant or Shear thickening (3) and Pseudoplastic or Shear-thinning fluid with a yield point (4). Pseudoplastic fluids Many liquids show drastic viscosity decreases when the shear rate is increased from low to high levels. This means that for a given force more mass can be made to flow or the energy can be reduced to sustain a given flow rate (phenomena desirable in industrial processes). This behaviour is related to their internal structure. Many liquid products that seem homogeneous are in fact composed of several ingredients that may possess irregular shapes or may interact with each other. Other liquids may consist of polymer solutions with long entangled and looping molecular chains. At rest, all of these materials will maintain an irregular internal order and correspondingly they are characterized by a sizable internal resistance against flow (and as a consequence high viscosity). With increasing shear rates, matchstick-like particles suspended in the liquid will be turned lengthwise in the direction of the flow. Chain-type molecules in a melt or in a solution can disentangle, stretch and orient themselves parallel to the driving force. Particle or molecular alignments allow particles and molecules to slip past each other more easily. Spherical particles may be deformed to ovalshaped particles (smaller in diameter but longer). Moreover, there are several Background. Interfacial Assessment 71 other possible explanations for this behaviour, e. g. solvent layers may be stripped from dissolved molecules or from particles, which means, that the intermolecular interactions causing resistance to flow become reduced. Finally, for most liquid materials the shear-thinning effect is reversible (Mezger 2014) . Dilatant fluids Fluids are liquids which under certain conditions of stress or shear rate increase their viscosity whenever shear rate increases. Thus, the resistance to flow increase and may become so high that make impossible to pomp out the fluid. Dilatant flow behaviour is relatively common in highly concentrated suspensions. The particles are densely packed and the amount of liquid is just sufficient to fill the space between the particles. At rest or at low flowrate the liquid medium fully lubricates the particle surfaces and thus allows an easy positional change of particles when forces are applied (this suspension behaves as a liquid at low shear rates). At higher shear rates, particles will wedge others, causing general volume increases. Since the liquid is no longer sufficient to fill all voids and to keep the particle surfaces fully lubricated, the solution becomes more viscous. However, dilatant fluids are rare and this flow behaviour most likely complicates production conditions (Schramm 2000). Yield point A sample with a yield point begins to flow only if the external forces Fext acting on the material are larger than the internal structural forces Fint. Below the yield point the material shows elastic behaviour, exhibiting under load only a very small degree of deformation that does not remain after removing the load. Thus, When Fext < Fint the material is only deformed to a small degree. The sample does not begin to flow before Fext > Fint. Finally, the yield point is Background. Interfacial Assessment 72 also referred to as yield stress or yield value (Schramm 2000). There is a wide range of materials which appear to show this kind of behaviour, however Barnes (2000) stated that there is as much happening in terms of flow below as above the ‘yield stress’. Thus, Barnes showed a number of examples of such liquids, where the viscosity falls many orders of magnitude over a narrow range of shear stress, and indeed when approaching this critical stress region from regions of high stress it appears that the viscosity goes to infinity. However, careful and patient measurement below this stress shows that the viscosity is still finite, and eventually levels off to a constant, but very high value at low stresses. Fluids exhibiting such behaviour were named “very shear-thinning” or “yield stress” fluids and the critical stress was denoted as “apparent yield stress” (Barnes 2000). Thixotropic fluids Thixotropy is the change of viscosity with time of shearing, and is generally viewed as a troublesome property that one could well do without. In fact, thixotropy could be better seen as the result of a high degree of shear thinning, and comes about whenever a shear-induced change in microstructure takes time to occur. Microstructure is brought to a new equilibrium by competition between, on the one hand the processes of tearing apart by stress during shearing, and on the other hand build-up due to flow and Brownian motion induced collision, over a time that can be minutes. Then, when the flow ceases, the Brownian motion (the only force left) is able to slowly move the elements of the microstructure around to more favourable positions and thus rebuild the structure: this can take many hours to complete. The whole process is completely reversible. Background. Interfacial Assessment 73 Thixotropy is a function of time and shear rate, and therefore cannot be properly accounted for in experiments where both these variables are changed simultaneously. As a consequence, the best experiments to properly measure thixotropy are those where the sample to be tested is sheared at a given shear rate until equilibrium is obtained (Barnes 2000). Elastic Behaviour Nowadays instruments allow to characterize elastic behaviour in a range of very low deformations, and therefore without any destruction of the structure of materials to be tested. In order to define further rheological parameters, Two Plates Model will be used. This model is plotted in Figure 3.2-3, where shear strain is defined as follows: Figure 3.2-3: Shear deformation of a material using the Two Plates Model. Shear modulus When measuring an ideal-elastic solid at a constant temperature, the ratio of the shear stress and the corresponding deformation is constant if the measurement takes place within the reversible elastic deformation range F h s A ϕ 𝛾=𝑠ℎ (3.2-7) Background. Interfacial Assessment 80 Initially, there is an initial elastic response. Thereafter there is a so-called delayed elastic response where the deformation rate becomes slower and slower, ending up as a very slow but steady-state deformation at the longest times. This curve describes the creep response of most structured liquids and gels. Models for creep test In general, in a creep test, a simple elastic solid (a spring) shows an immediate response to give a constant deformation (strain). On the other hand, a simple Newtonian liquid (a dashpot) would show an ever-increasing strain, which displayed on a graph of strain against time would be a straight line starting at the origin, with the slope giving the shear rate 𝛾󰇗. Maxwell Model Thus, according to Maxwell model, the behaviour can be described by the EQ. (3.2-15). 𝛾=𝜏(1 𝐺+𝑡𝜂) (3.2-15) where, as defined previously, the strain is given by γ, the modulus by G, the time by t and the viscosity by η. Thus, at very short times, is characterised by an immediate elastic response, (γ = τ/G) and at very long times, when t >> η/G, by simple viscous behaviour, γ = σ t/η. Here η/G is called the relaxation time λ. Kelvin-Voigt model If a creep test is performed on a Kelvin-Voigt model, the strain gradually builds up to a constant value as described by EQ. (3.2-16), which is the solution of the model for the creep test. Background. Interfacial Assessment 81 𝛾=𝜏 𝐺[1−𝑒−𝑡 𝜆′ ⁄] (3.2-16) Here, λ’ is called the retardation time, since it characterises the retarded response of the model, and its value is again given by η/G. At very short times, the response is viscous, and 𝛾~𝑡∙ 𝜏/𝜂. Models for relaxation tests Relaxation tests consist on the application of a constant strain, and the monitoring of the consequent stress, which then decays away with time. Maxwell Model Thus, according to Maxwell model, the strain and the shear rate is as follows: 𝛾=𝛾𝑒𝑙+𝛾𝑣𝑖𝑠 (3.2-17) 𝛾󰇗=𝛾󰇗𝑒𝑙+𝛾󰇗𝑣𝑖𝑠 (3.2-18) After multiplying both equation sides by μ, we can obtain: 𝜇𝛾󰇗 =𝜇𝛾󰇗𝑒𝑙+𝜏 (3.2-19) Taking into account that 𝛾 󰇗𝑒𝑙=𝐺−1·𝑑𝜏 𝑑𝑡 ⁄, EQ. (3.2-19) can be expressed as follows: 𝜏=𝛾󰇗−𝜆𝑑𝜏 𝑑𝑡 (3.2-20) Kelvin-Voigt Model Thus, according to Kelvin-Voigt model, the stress applied is as follows: 𝜏=𝜏𝑒𝑙+𝜏𝑣𝑖𝑠 (3.2-21) Background. Interfacial Assessment 82 Introducing EQ. (3.2-11) and EQ. (3.2-12) in EQ (3.2-21), we can obtain the following expression: 𝜏=𝐺·𝛾+𝜇𝛾󰇗 (3.2-22) After dividing both equation sides by G, we can obtain: 𝜏 𝐺=𝛾+𝜆′𝑑𝛾 𝑑𝑡 (3.2-23) Oscillatory tests Oscillatory tests are used to examine all kinds of viscoelastic materials, from low-viscosity liquids to polymer solutions, and even rigid solids. This mode of testing is also referred to as "dynamic mechanical analysis" (DMA). To explain oscillatory tests, the Two Plates Model is used again. The bottom plate is stationary. When the wheel or disk is turning, the upper plate with the (shear) area is moved back and forth by the (shear) force. Figure 3.2-9 represents this movement: Figure 3.2-9: Oscillatory tests for a fluid between two parallel plates where F is the shear force, S the deflection path, ϕ the deflection angle and h the shear gap. Thus, instead of applying a constant stress leading to a steady-state flow, samples are subjected to oscillating stresses or oscillating strains. The stress may be applied as a sinusoidal time function (EQ. 3.2-24): s s h -F +F ᵩ ᵩ Background. Interfacial Assessment 83 𝜏=𝜏0∙sin (𝜔𝑡) (3.2-24) where τ is the stress applied, which is a sinusoidal function of the maximum stress applied (τ0), as well as, the frequency (ω) and time (t). Theoretical aspects of dynamic testing The Hookean Spring in oscillatory movement Figure 3.2-10 represents the movement of a wheel connected to the spring model. Figure 3.2-10: Spring subjected to an oscillatory movement The spring extends to a maximum strain (γ0) and contracts to its original length with a frequency equal to the angular velocity of the wheel (ω) and then the strain and the stress can be written as a function of time as follows: 𝛾=𝛾0∙sin (𝜔𝑡) (3.2-25) Thus, the stress function is defined by EQ. (3.2-26): 𝜏=𝐺𝛾0∙sin (𝜔𝑡) (3.2-26) For this case strain and stress are in-phase with each other. That is, upon deformation, the maximum stress and the maximum strain occur at the same instant in time. The newtonian dashpot model in oscillatory movement Figure 3.2-11 represents the movement of a wheel connected to the dashpot model: Background. Interfacial Assessment 84 Figure 3.2-11: Dashpot model in an oscillatory movement Consequently, if the spring is exchanged by a dashpot and the piston is subjected to a similar crankshaft action, the following equations apply: 𝛾󰇗=𝑑𝛾 𝑑𝑡=ω ∙ 𝛾󰇗∙ cos (𝜔𝑡) (3.2-27) Substituting this into the dashpot equation: 𝜏=𝜂∙𝑑𝛾 𝑑𝑡=𝜂∙𝜔∙𝛾0∙cos(𝜔𝑡) (3.2-28) For the dashpot the response of τ is 90° out of phase to the strain. This can also be expressed by defining a phase shift angle δ = 90° by which the assigned strain is trailing the measured stress. The above equation EQ. (3.2-28) can then be rewritten: 𝜏=𝜂∙𝜔∙𝛾0∙cos(𝜔𝑡)=𝜂∙𝜔∙𝛾0∙sin(𝜔𝑡+𝛿) (3.2-29) According to this equation, in-phase stress response to an applied strain is called “elastic”. A 90° out-of-phase stress response is called “viscous”. If a phase shift angle is within the limits of 0 < d < 90° is called “viscoelastic”. The Maxwell model in oscillatory movement Figure 3.2-12 represents the movement of a wheel connected to the Maxwell model: Background. Interfacial Assessment 85 Figure 3.2-12: Maxwell model in an oscillatory movement The stresses in each element are equal and the total strain is the sum of the strains in both the dashpot and the spring. The equation of state for the model is EQ. (3.2-30): 1 𝐺∙(𝑑𝜏 𝑑𝑡)+𝜏𝜂=𝑑𝛾 𝑑𝑡 (3.2-30) Introducing the sinusoidal function, we can obtain EQ. (3.2-31). 1 𝐺∙(𝑑𝜏 𝑑𝑡)+𝜏𝜂=𝜔∙𝛾0∙cos(𝜔𝑡) (3.2-31) The stress response to the sinusoidal strain consists of two parts which contribute the elastic sin-wave function with ϕ = 0° and the viscous cosinewave-function with ϕ = 90° The Kelvin-Voigt model in oscillatory movement Figure 3.2-13 represents the movement of a wheel connected to the Kelvin-Voigt model: Figure 3.2-13: Kelvin-Voigt model in an oscillatory movement Background. Interfacial Assessment 86 This model combines a dashpot and spring in parallel. The total stress is the sum of the stresses of both elements, while the strains are equal. The equation of state for the model is EQ. (3.2-32). 𝜏=𝐺∙𝛾+𝜂∙𝑑𝛾 𝑑𝑡 (3.2-32) Introducing the sinusoidal function, we can obtain EQ. (3.2-33). 𝜏=𝐺∙𝛾0∙sin(𝜔𝑡)+𝜂∙𝜔∙𝛾0∙cos(𝜔𝑡) (3.2-33) The stress response in this two-element-model is given by two elements being elastic when δ = 0, and being viscous when δ = 90°. Real viscoelastic samples Real viscoelastic samples are more complex than either the Kelvin-Voigt solid or the Maxwell liquid. Their phase shift angle is positioned between 0 < δ < 90°. G and δ are again frequency dependent: It is common to introduce the term Complex Modulus (G*) which is defined as EQ. (3.2-34) indicates: 𝐺∗=𝜏0 𝛾0 (3.2-34) G* represents the total resistance of a substance against the applied strain. It is quite important to note that for real viscoelastic materials both the complex modulus (G*) and the phase angle (γ) are frequency dependent. Complex numbers can be used to express the complex modulus in two other parameters. Thus, Complex modulus (G*) can be defined as EQ (3.2-35) shows: 𝐺∗=𝐺′+𝑖𝐺′′=𝜏0(𝑡) 𝛾𝐸 (𝑡) (3.2-35) Background. Interfacial Assessment 87 where the real part of the complex function is the elastic component, G’, and the imaginary part corresponds to the viscous component, G’’. As a consequence, the complex modulus G* (|G*|) results from the combination of both parts: |𝐺∗|=√𝐺′2+𝐺′′2 (3.2-36) Thus, G’ and G’’ may be expressed in terms of the measured quantities (τ0 and γ0) and the phase angle between them (δ): 𝐺′=𝐺∗∙cos𝛿=𝜏0 𝛾0∙ cos𝛿 (3.2-37) 𝐺′′=𝐺∗sin𝛿=𝜏0 𝛾0∙sin𝛿 (3.2-38) If a substance is purely viscous then the phase shift angle d is 90°: G’ = 0 and G’’ = G*. On the contrary, if the substance is purely elastic then the phase shift angle d is zero: G’ = G* and G’’ = 0 (Schramm 2000). It is also useful to define the tan δ EQ. (3.2-39). tan𝛿=𝐺′′ 𝐺′ (3.2-39) G' is known as the storage modulus. G’ is a measure of the deformation energy stored by the sample during the shear process. After the load is removed, this energy is completely available, now acting as the driving force for the reformation process which partially or completely compensates the previously obtained deformation of the structure. Materials which are storing to be whole deformation energy are showing completely reversible deformation behaviour since they are occurring finally with an unchanged shape after a load cycle. Thus, G' represents the elastic behaviour of a material. Background. Interfacial Assessment 88 On the other hand, G" is known as the loss modulus. G" is a measure of the stress energy used by the sample during the shear process and therefore, it is eventually lost by transformation into heat. Energy losing materials show irreversible deformation behaviour since they occur with a change of shape after a load cycle. Thus, G" represents the viscous behaviour of a material (Mezger 2006). Alternatively to the complex modulus G* one can define a complex viscosity, as EQ. (3.2-40) indicates: 𝜂∗=𝐺∗ 𝑖𝜔=𝜏0 𝛾0𝜔 (3.2-40) The complex viscosity (η^*) describes the total resistance to a dynamic shear. It can again be broken into the two components of the storage viscosity η’ (the elastic component) and the dynamic viscosity η’’ (the viscous component. The oscillatory response of real systems The most general response for G’ and G’’ of real samples (for structured systems) is shown in Figure 3.1-1. The exact values of the moduli and their position in the frequency domain will vary, however, this figure indicates the overall qualitative behaviour. A number of specific regions can often be differentiated, namely: a) The viscous or terminal region, where G’ predominates and viscous (flow) behaviour prevails. All materials have such a region, even solids (because they creep at long times), but the frequency where this is seen is often so low that most oscillatory instruments cannot detect this part of the curve. Background. Interfacial Assessment 89 b) The transition-to-flow region is so called because, when viewed from higher frequencies (where elastic behaviour dominates and G’ > G’’), the loss modulus G’’, describing viscous or flow behaviour, becomes significant. The point where the two moduli cross over is sometimes noted, and for a Maxwell model, this crossover frequency is given by the inverse of the relaxation time. c) The rubbery or plateau region is where elastic behaviour dominates. While in many cases we see what appears to be a flat plateau, there is always a slight increase of G’ with frequency, but it can be as small as a few percent increase in modulus per decade increase in frequency. The value of G’’ is of course always lower than that of G’, but sometimes it can be considerably lower. Figure 3.2-14: Regions in the viscoelastic spectrum of non-Newtonian liquids. d) A leathery or higher transition crossover region is also seen, where, due to high-frequency relaxation and dissipation mechanisms, the value of G’’ again rises, this time faster than G’. Once more at G’ = G’’, a crossover frequency can be defined, from which another characteristic time can be obtained. Background. Products from proteins 96 amount, the more space that the protein molecule has to spread out at the surface, and hence the greater the opportunity for unfolding to minimize the configurational free energy following adsorption. Early adsorbing proteins tend to exhibit a large loss of enzymatic activity, and are poorly exchangeable with the bulk phase after adsorption. Late adsorbing proteins tend to retain more enzymatic activity due to less unfolding and more participation in loosely held multilayers. The maximum adsorbed amount is determined by the rate of unfolding at the surface in relation to the rate of adsorption. Fast adsorption gives less time for protein molecules to spread out at the surface, and consequently the area occupied per molecule is lower and the adsorbed amount is higher. Adsorbed layers at fluid interfaces Almost everything described previously for protein adoption at solid surface is applicable when proteins are adsorbed at fluid-fluid interfaces. However, there are some differences. In particular, related to how protein molecules can penetrate further into the non-aqueous phase and can move more freely, on a liquid surface. Processes involving diffusion, reorientation and conformational reorganization will occur faster at air-water and oil-water interfaces than on solid substrates. In addition, another important difference is that in fluid-fluid interfaces is possible to perform additional experimental studies on the surface equation of state and on the surface rheological behaviour. Analytical surface equations of state developed for small-molecule adsorbed layers are unable to represent fully the properties of protein systems. However, some simple formula can capture most of the essential features of the behaviour. Nearly all protein adsorption studies are characterized by extremely non-ideal behaviour. The non-ideality arises from Background. Products from proteins 97 a combination of enthalpic and entropic contributions to the surface free energy as a result of complex intermolecular interactions and intramolecular rearrangements. Frumkin (1925) described a fairly simply equation equation that can account for both enthalpic and entropic terms: Π𝜔1 𝑅𝑇−ln(1−𝜃)−(1−𝑆−1)𝜃−𝐻 𝑅𝑇𝜃2 (3.3-3) where S is the ratio (ω1/ω2) of the solvent molar area (ω1) to the protein molar area (ω2), H is the enthalpy of mixing of a regular solution, R is the gas constant, T is the temperature and θ is the fractional surface coverage which may be expressed as 𝜃=𝜔2𝛤, in terms of the surface load (Γ). The first term is the surface pressure of an ideal surface mixture of equalsized adsorbed molecules. The second term (linear in θ) allows for the nonideal surface entropy of mixing of large and small adsorbed molecules (for S<<1). This term has the effect of greatly reducing the value of Π, especially at low values of θ. The third term (proportional to θ2) is related to intermolecular interactions in the adsorbed layer. For the normal case of net attractive protein-protein interactions (H>0), the combination of substantial enthalpy and entropy contributions reproduces the very strong deviation from ideality typically observed at low surface coverage. However, this equation breaks down at high surface coverage (θ→1) when θ is predicted to strongly diverge, whereas in practice Π reaches a saturation value corresponding to monolayer collapse and possible onset of multilayer formation. The theory breaks down because it is based on a twodimensional model of the adsorbed layer, whereas the real system is threedimensional. Regardless of the restriction of the two-dimensional model, other refinements can in principle be made to account for protein aggregation and Background. Products from proteins 98 reorientation in the adsorbed layer. In particular, a more realistic allowance for unfolding of protein molecules in the adsorbed layer can be made by replacing the single state of the adsorbed protein by a distribution of states with different values of the molar area ω2. However, such complex models have the disadvantage of increasing the number of adjustable parameters without providing a fully rigorous statistical mechanical description of the system under consideration (Dickinson and McClements 1995). Simulation model of a globular protein adsorbed layer A computer simulation model is available in which an adsorbed monolayer of globular protein molecules at a fluid–fluid interface is represented as a quasi-two-dimensional network of cross-linked rigid spherical particles (Wijmans and Dickinson 1998). The approach is motivated by the similarity in rheological properties between a globular protein adsorbed film and an extremely thin layer of bulk heat-set protein gel. The monolayer network structures produced by the model are qualitatively similar to those generated in two-dimensional simulations of aggregated particle gels formed from irreversibly bonding Brownian particles. However, the Brownian dynamics algorithm used to simulate the adsorbed layer properties is actually closer in detail to that used to simulate three-dimensional aggregated particle gel networks. The model neglects specific effects of changes in intramolecular interactions during or after adsorption, whilst accounting for associative intermolecular interactions between adsorbed particles through the formation of strong flexible bonds. Confinement of the particles to the interface is achieved with a steep potential well of finite width in the z-direction (perpendicular to the interface). Although particles are strongly adsorbed, they still have some freedom to distribute themselves around their equilibrium positions at Z=0. For this reason, the Background. Products from proteins 99 model of the layer is not completely two-dimensional (Wijmans and Dickinson 1998). Competitive adsorption Some of the complexity of protein absorption is partly due to the potential for competitive adsorption between proteins which are present. In mixtures involving globular proteins, the irreversibility of the adsorption events prevents equilibrium from ever being achieved. Although more easily denaturable (“soft”) proteins are expected to have greater surface affinity and exchangeability than more stable (“hard”) proteins, a useful rule of thumb is that the adsorbed layer will be dominated by the protein that presents itself first to the interface. In cases where both proteins are presented together in roughly equal amounts, the extent of dominance of individual components will depend on the residence times of individual protein molecules at the interface in relation to their rates of unfolding. Thus, the competition to occupy sites at the interface may prevent ‘early’ adsorbed species from ever achieving the ‘irreversible state’, in which case an initially adsorbing molecule may be displaced. The finiteness of the time required to achieve the irreversible state is a key point because it allows for multiple collisions of potentially competing molecules to take place before any chance of replacement becomes impossible. The protein interfacial composition at any given bulk protein composition ratio appears to be affected largely by the relative rates of arrival of the components at the interface and the molecular areas available to them at the time of arrival. In this sense the accumulation of protein molecules at the fluidfluid interface from the mixed solution is not a thermodynamically-controlled Background. Products from proteins 100 competitive adsorption process. This means that the composition of the interface cannot be predicted from any simple thermodynamic model When small-molecule surfactants are present in protein-containing systems, the adsorption of the protein is affected by the binding of surfactant to both the protein and the fluid interface In addition to the preferential binding of surfactant to the surface at high bulk concentrations, surfactant binding to hydrophobic sites on the protein may also reduce its surface affinity. Therefore the protein can be removed from the interface as a consequence of two distinct mechanisms: 1) The solubilisation mechanism: desorption of protein arises as a result of solubilisation into the aqueous phase in the form of a proteinsurfactant complex. 2) The replacement mechanism: displacement of protein arises because surfactant lowers the interfacial free energy more effectively than does protein (or protein-surfactant complex). Ionic surfactants usually bind strongly to proteins, such that competitive adsorption involving charged amphiphiles can be regarded as proceeding mainly by the solubilisation mechanism. With more weakly interacting nonionic surfactants, however, the replacement mechanism can be regarded as (Wijmans and Dickinson 1998). Interfacial Measurements Surface pressure-area The Langmuir adsorption model is the most common model used to quantify the amount of amphiphilic compounds adsorbed. This absorption is a function of partial pressure or concentration and temperature. For the sake of simplicity, this model considers the adsorption of an ideal gas onto an Background. Products from proteins 101 idealized surface. Thus, when a monolayer is fabricated at the gas-liquid or liquid-liquid interface, the film is named Langmuir film (Toth 2002). A useful method to characterise the Langmuir film is by obtaining the surface pressure/molecular area isotherms, measured by the Langmuir method. The measurement system consists of a plate fitted with two mobile barriers, and a device which is able to measure surface tension (e.g. Wilhelmy plate). Figure 3.3-2 illustrates the device measurement: Figure 3.3-2: Langmuir trough device Thus, the experiment consists of measuring the surface tension as a function of the mean molecular area perpendicular on the monolayer as is compressed by the two barriers. Figure 3.3-3 shows a typical pressure-molecular area curve for an amphiphilic monolayer adsorbed at the interface obtained when the barriers are moved towards each other. The different phases occurring over the adsorption isotherm are also represented in this figure. At the beginning, when the barriers are at their greatest distance from each other, the monolayer is in the gaseous phase where the hydrophobic groups are completely separated. Further compression (from right to left) forces the monolayer molecules into the liquid phase that causes a slight elevation of the surface pressure, starting with the so-called “lift-off” point. Further compression squeezes the amphiphilic molecules into a solid, which Background. Products from proteins 102 gives a steep rise in the surface tension. By further increasing the compression, the layer collapses into a many-layered structure (Toth 2002). Figure 3.3-3: Surface pressure Vs. Molecular area for an amphiphilic molecule Dilatational Droplet Tension When a drop of a fluid is in contact with other fluid, the drop exhibits a combination of effects coming from the surface tension (which tends to form a spherical drop) and the gravitational force (which tends to elongate the drop). In this way, the asymmetric drop can be expressed by using the Laplace equation: 1 𝑥𝑑(𝑥𝑠𝑒𝑛𝜃) 𝑑𝑥 =2 𝑏−𝑐𝑧 (3.3-4) Where x and z are the cartesian coordinates, b is the radius of curvature, Ѳ is the tangent angle and c is a constant related to the Capilar number, which is defined as : c=g·Δρ/γ, where g is the gravity acceleration, Δρ is the difference of density between both studied fluids and γ the surface tension. Mean molecule area [Å2] Surface pressure [mN/m] Gaseous Liquid Solid Collapse Background. Products from proteins 103 Interfacial Shear Rheology Two different kinds of interfacial rheological measurements can be distinguished by applying shear or dilatational deformations to a fluid-fluid interface. When the interface is sheared, both the area and the amount of surfactant in the interface remain constant and then it is possible to measure the required shear force applied to the plane of the interface. For most systems, shear rate thinning occurs and the observed viscosity is an apparent interfacial viscosity. For instance, values for globular proteins usually show a high experimental uncertainty, since the monolayer can yield or rupture and the measured “viscosity” will greatly depend on the rupture pattern. On the other hand, if the interfacial area is enlarged by dilatational deformation, leaving its shape unaltered, an increase in interfacial tension takes place, because the molar amount of adsorbed material per unit surface area (surface load, Γ) is decreased. This is usually expressed in the surface dilatational modulus, defined as follows: 𝐸𝑆𝐷=𝑑𝛾 𝑑ln𝐴 (3.3-5) where A is the surface area. For proteins ESD may be large and less dependent on time, because proteins adsorb more or less irreversibly. However, the concentration of protein at the interface has a large effect. In addition, changes in protein conformation upon adsorption and dilation can also affect the modulus. Surface rheological parameters of protein layers depend on pH, ionic strength, solvent quality, temperature. However, moduli and viscosities are usually at their maximum values near the isoelectric pH. In any case, the measurement of ESD is difficult and so is the interpretation of the results Background. Products from proteins 104 obtained from dilatational rheological measurements (Damodaran, Parkin et al. 2007). Background. Products from proteins 105 3.4. Products from protein Emulsions Emulsions are disperse systems consisting of two immiscible liquids. One of the phases (called the internal or disperse phase) is dispersed in the form of small droplets in a liquid medium (called the continuous phase). According to the hydrophobicity, some classes may be distinguished: oil-in-water (O/W), water-in-oil (W/O), and oil-in-oil (O/O). To disperse two immiscible liquids, it is needed a third component, namely, the emulsifier. The choice of the emulsifier is crucial in the formation of the emulsion and its long-term stability (Tadros 2013). Classification according to system structure: 1. O/W and W/O macroemulsions: Droplet size range of 0.1–5 μm with an average of 1–2 μm. They are kinetically stable. 2. Nanoemulsions: Droplet size range of 20–100 nm. Similar to macroemulsions, they are only kinetically stable. 3. Micellar emulsions or microemulsions: these usually have the size range of 5–50 nm. They are thermodynamically stable. 4. Double and multiple emulsions: these are emulsions-of-emulsions, W/O/W, and O/W/O systems. 5. Mixed emulsions: these are systems consisting of two different disperse droplets that do not mix in a continuous medium. Methods of Emulsification Several procedures may be applied for emulsion preparation, including: simple pipe flow devices (low agitation energy); static mixers and general Background. Products from proteins 112 1) Use of mixed surfactant films: In many cases using mixed surfactants can reduce coalescence 2) Formation of lamellar liquid crystalline phases at the O/W interface: As a result of multilayer structures, the potential drop is shifted to longer distances thus reducing the van der Waals attraction. Food emulsions Everything above described about emulsions is applicable to food emulsions, the only difference is that now the system is very complex, and proteins are usually used as emulsifiers instead of surfactants. Thus, at similar bulk concentrations (w/v), low molecular mass surfactants decrease the surface tension to a greater extent than the macromolecular surfactants. This difference is mainly related to differences in orientation and configuration of these surfactants at an interface. Although low molecular mass surfactants are more effective than proteins in reducing the interfacial tension, surfactant-based foams and emulsions are generally more unstable than those processed using proteins. This is because proteins, in addition to lowering interfacial tension, can form a continuous viscoelastic membrane-like film around oil droplets or air cells via non-covalent intermolecular interactions and via covalent disulphide cross-linking. Consequently, in foods, which contain both low molecular and macromolecular surfactants, the stability of colloidally dispersed phases is primarily dependent on protein films adsorbed at the interfaces. However, practical observations indicate that all proteins are not equally surface active, even though all are amphiphilic and a majority of them contain similar percentages of polar and nonpolar amino acid residues (Tadros 2013). Background. Products from proteins 113 The differences found in the surface activities of various proteins must be related to differences in their conformation and the susceptibility of those conformations to unfold at interfaces. Intuitively, the molecular factors that influence surface activity of proteins must be related to flexibility, conformational stability at interfaces, rapid adaptability of the conformation to changes in its environment, and to the distribution pattern of hydrophilic and hydrophobic residues in its primary structure, as well as on its folded surface. In addition, apart from the intrinsic molecular factors, the surface activity of a protein will be also dictated by several extrinsic factors such as pH, ionic strength, and temperature (Damodaran 1997). Emulsion characterisation Rheology of emulsions Emulsions generally show a rheological behaviour fitting into the category of complex fluids. However, they differ from other complex fluids in three main aspects: 1) The mobile liquid/liquid interface that contains surfactant or polymer layers. 2) The dispersed-phase viscosity relative to that of the medium has an effect on the rheology of the emulsion. 3) The deformable nature of the dispersed-phase droplets. Consequently, we can difference between interfacial and bulk rheology. Background. Products from proteins 114 Interfacial Rheology Mixed Surfactant Films According to the data available, there seems to be a relationship between the use of a mixture of surfactants and the enhancement of the stability of the emulsion. This could be due to the increase of interfacial dilatational elasticity ESD for the mixed film when compared to that one containing a single surfactant. However, other factors such as thinning of the film between emulsion droplets can also play a major role. Protein Films The viscoelastic properties of protein films at the O/W interface also correlates well with the stability of emulsion drops against coalescence. Some viscoelastic measurements can be carried out using creep-recovery tests. The stability of the emulsion was assessed by measuring the residence time t of several oil droplets at a planar O/W interface containing the adsorbed protein. Thus, Biswas and Haydon (1963) derived a relationship between coalescence time (τ) and interfacial parameters such as surface viscosity (ηs), instantaneous modulus (Go), and adsorbed film thickness (h) (EQ. 3.4-3). 𝜏=𝜂𝑠∙[3𝐶′ℎ2 𝐴−1 𝐺0−𝜙(𝑡)] (3.4-3) where 3C’ is a critical deformation factor, A is the Hamaker constant (Van der Waals body-body interaction) and φ(t) is the elastic deformation per unit stress. The equation analysis shows that viscoelasticity is necessary (eg. τ increases with increasing ηs) but not sufficient to ensure stability against coalescence. Film thickness seems to be the most important factor, such that to ensure stability of an emulsion h must be large enough. Background. Products from proteins 115 Bulk rheology Diluted emulsions For highly viscous oil droplets dispersed in low viscosity media such as water, dilute O/W emulsions (volume fraction φ ≤ 0.01) of non-interacting droplets behave as ‘‘hard spheres’’. In this case, the relative viscosity (ηr) is given by the Einstein equation (3.4-4): 𝜂𝑟=1+[η]ϕ (3.4-4) where [η] is the intrinsic viscosity (2.5 for hard spheres). For droplets with a viscosity comparable to that of the medium, the transmission of tangential stress across the O/W interface, from the continuous phase to the dispersed phase, causes liquid circulation inside the droplets. Energy dissipation is less than that for hard spheres and the relative viscosity is lower than that predicted by the Einstein equation. Thus, for an emulsion with viscosity ηi for the disperse phase and ηo for the continuous phase, the intrinsic viscosity can be defined as show EQ. (3.4-5). 𝜂=2.5 (𝜂𝑖+0.4𝜂0 𝜂𝑖+𝜂0) (3.4-5) When ηi >> ηo, the droplets behave as rigid spheres and η approaches the limit value of 2.5. In contrast if ηi << ηo (e.g. in foams), [η] = 1. If the volume fraction of droplets exceeds the Einstein limit, (φ >0.01), it must take into account the effect of Brownian motion and interparticle interactions. The smaller the emulsion droplets, the more important the contribution of Brownian motion and colloidal interactions. Brownian diffusion tends to randomize the position of colloidal particles, leading to the formation of Background. Products from proteins 116 temporary doublets, triplets, and so on. The hydrodynamic interactions are of longer range than the colloidal interactions, and they come into play at relatively low volume fractions (φ >0.01) resulting in ordering of the particles into layers and tending to destroy the temporary aggregates caused by the Brownian diffusion. This explains the shear thinning behaviour of emulsions at high shear rates. For the volume fraction range 0.01 < φ < 0.2, Bachelor (1977) derived the following expression for a dispersion of hydrodynamically interacting hard spheres: 𝜂𝑟=1+2.5𝜙+6.2𝜙2+𝜐𝜙3 (3.4-6) Where the first part corresponds to the Einstein limit (EQ. 3.4-4) while the third term accounts for hydrodynamic (two-body) interactions and the fourth term relates to multibody interactions. Concentrated emulsions Considering the rheology of concentrated emulsions, an expression for the fourth term in φ3 of EQ. (3.4-8) should be provided. Unfortunately, there is no theoretical rigorous treatment of this term and only semiempirical equations for intermediate volume fractions are available (Phan-Thien and Tanner 1999, Pal 2000). Two models were proposed by Pal (2001): 𝜂𝑟[2𝜂𝑟+5𝜆 2+5𝜆]1/2=𝑒[2.5𝜑 1−𝜑 𝜑∗] (3.4-7) 𝜂𝑟[2𝜂𝑟+5𝜆 2+5𝜆]1/2=[1−𝜑/𝜑∗]2.5𝜑∗ (3.4-8) Background. Products from proteins 117 where λ is the ratio of viscosities of disperse drops and continuous medium and φ* is the limit of closest packing of drops in free space. An increase in the concentration of drops in emulsions results not only in an increase in viscosity at low shear rates but also in the appearance of strong non-Newtonian effects leading to a shear rate dependence of the apparent viscosity. A remarkable transition from an almost Newtonian behaviour at low stresses to an anomalous flow with pronounced non-Newtonian effects may also takes place (Tadros 2013). Microscopy The human eye is able to resolve objects that are greater than 0.1 mm. Many of the structural components in food emulsions (such as droplets, surfactant micelles, fat crystals, gas bubbles or protein aggregates) are smaller than this limit and cannot be distinguished by humans. Several techniques are available to provide information about the structure, dimensions, and organization of the components within food emulsions, for example, optical microscopy, electron microscopy (SEM) or atomic force microscopy (AFM). These techniques have the ability to provide relevant information about complex systems in the form of “images” (McClements 2004). Conventional optical microscopy The optical microscope is one of the most valuable tools for observing the microstructure of emulsions. The optical microscope contains several lenses that direct light through the specimen and magnify the resulting image. The resolution is determined by the wavelength of light used and the mechanical design of the instrument, being the theoretical limit of resolution of an optical microscope about 0.2 μm. Nevertheless, it provides useful information about the size distribution of droplets in emulsions that contain larger droplets, and Background. Products from proteins 118 can often be used to distinguish between flocculation and coalescence, which is sometimes difficult using instrumental particle sizing techniques based on light scattering (McClements 2004) Laser Scanning Confocal Microscopy Laser Scanning Confocal Microscopy (LSCM) can provide extremely useful information about the microstructure of food emulsions. This technique has the advantage that provides higher clarity images than conventional optical microscopy, and often allows the generation of three-dimensional images of structures without the need to physically sectioning the specimen. The LSCM focuses an extremely narrow laser beam at a particular point in the specimen being analysed and a detector measures the intensity of the resulting fluorescence signal. 3D images can be obtained by focusing the laser beam at different vertical depths. The observation of the microstructure of multicomponent systems is often facilitated by using the natural fluorescence of certain components (such as proteins) or by using fluorescent dyes (McClements 2004). Scanning Electron Microscopy Scanning Electron Microscopy (SEM) is widely used to examine the microstructure of food emulsions, especially those that contain structural components that are smaller than the lower limit of resolution of optical microscopes. This technique can provide relevant information about the concentration, dimensions, and spatial distribution, whereas microstructure is not significantly altered by the sample Electron microscopes use electron beams, instead of light beams, to provide information about the structure of materials. These beams are directed through the microscope using a series of magnetic fields, instead of optical lenses, which used optical microscope. Electron beams have much Background. Products from proteins 119 smaller wavelengths than light and so they can be used to examine much smaller objects (about 0.2 nm) Static light scattering Droplet size analysis instruments that use static light scattering (also called laser diffraction) are based on the principle that a beam of light is directed through an emulsion, the laser is scattered by the droplets in a welldefined manner. A measurement of the extent of light scattering by an emulsion can be used to determine the droplet size distribution and concentration by using a mathematical model to relate the measured data to the particle characteristics. Food Gels Definition A food gel can be considered as a high moisture three-dimensional polymeric network that resists flow under pressure and is able to retain their distinct structural shape. Generally, a gel is a continuous network of interconnected particles or assorted macromolecules dispersed in a continuous liquid phase. The gelation is the phenomenon which involves the association or crosslinking of the polymer chains to form a three-dimensional network that immobilizes water within it. Food hydrocolloids are usually the most frequently used gelling agents in food products. A wide range of polysaccharides and proteins are nowadays available as food hydrocolloids derived from natural sources. Polysaccharides are incorporated because of their ability to control stability and texture of foods, as well as for their role in encapsulation and controlled release of active agents (flavours, functional ingredients, etc.) (Banerjee and Bhattacharya 2012). Background. Products from proteins 120 In food gels the liquid is invariably water and the molecular network consists of proteins or polysaccharides or a combination of both. The properties of the gel are the net results of the complex interactions between the water and the molecular network. The water, as a solvent, has influence in the nature and magnitude of the intermolecular forces that maintain the integrity of the polymer network. The polymer network holds the water, preventing it from flowing away. Unfortunately the complexity of these interactions, responsible for the useful functional properties of gels, makes it very difficult to predict quantitatively their physical properties, even for pure proteins or polysaccharides. In addition, food hydrocolloids are rarely pure and are often used with other ingredients that increase the degree of complexity of food systems. For this reason, their physical properties must generally be treated empirically (Damodaran 1997). Protein gelation Heat--induced gelation, pH-induced gelation or high pressure gelation are techniques widely used for the gelation of globular proteins and proceed through a series of transitions, such as denaturation (unfolding) of native proteins, aggregation of unfolded molecules, strand formation from aggregates, and association of strands into a network (Banerjee and Bhattacharya 2012). Heat-induced gelation Heat-induced gelation is probably the most important and common method to obtain gels. Gelation involves different steps from protein denaturation to formation of protein aggregates and association of aggregates to form a three-dimensional network: Background. Products from proteins 121 - The first step always consists of protein denaturation involving unfolding (at least partially) or dissociation of the molecules induced by thermal energy. As a consequence, some of the hydrophobic groups, which remained buried in the protein core under native configuration, become exposed to the aqueous phase above some temperature. - The second step takes place through hydrophobically driven proteinprotein interactions that lead to the association and aggregation of unfolded molecules to form complexes of higher molecular weight. In this stage, disulphide (-S-S-) bonds may also play an important role in combination with hydrophobic interactions. - Random association of aggregates to form a three-dimensional structure which extends to the whole system has been suggested as a third step. Meanwhile the continuous phase is entrapped within the network (Clark, Kavanagh et al. 2001). The reaction rate can be typically determined either by the unfolding or by the aggregation reaction, depending on the ratio of the reaction rates of the single steps (Banerjee and Bhattacharya 2012). Several variables, depending either on protein nature and composition or on environmental factors, such as pH or ionic strength may exert an important influence on protein-protein and protein-solvent interactions and as a result on these reaction rates, thus conditioning the type of gel network formed. Essentially, proteins can aggregate in two ways. One is by random aggregation which can lead to heterogeneous particulate network structures. The other is by linear aggregation that gives rise to fine stranded (string of beads) network structures. Many proteins (e.g. globular proteins) can form either type of gel network depending on the balance of forces. The Background. Products from proteins 128 In small-strain testing the strain must be low enough to avoid any unrecoverable structural change (e.g. SAOS tests). On the other hand, largestrain testing refers to deforming a sample above to the point of permanent structural change. This later group of tests often yields information that correlates with sensory evaluation (Schramm 2000). Small-strain testing. Oscillatory test Since gels exhibit viscoelastic behaviour, dynamic rheological tests to evaluate properties of gel systems are widely used for studying the characteristics of gels as well as gelation and melting. Figure 3.4-2 illustrates the change in modulus when the gel transforms its liquid-like structure to gellike structure. Figure 3.4-2: Viscoelastic response of a material If G’ is much greater than G’’, the material will behave more like a solid; that is, the deformations will be essentially elastic. However, if G’’ is much greater than G’, the energy used to deform the material is dissipated viscously and the material exhibits liquid-like behaviour. Three types of dynamic tests are usually used to obtain useful properties of gels, gelation, and melting: Background. Products from proteins 129 1) Frequency sweep studies in which G’ and G’’ are determined as a function of frequency, at fixed temperatures. 2) Temperature sweep tests in which G’ and G’’ are determined as a function of temperature at fixed frequency. 3) Time sweep in which G’ and G’’ are determined as a function of time at fixed frequency and temperature. Kinetics of Gelation Measurement of gelation kinetics requires a method for following the development of the gel network without significantly affecting the process by mechanical disturbance. The most widely used technique to study gelation kinetics, arisen after the development of controlled-stress rheometers, involves formation of the gel between the plates of an oscillatory rheometer (Hermansson 1986). With this type of instrument, provided that conditions are chosen appropriately, it is possible to monitor the evolution of the linear viscoelastic functions over the gelation process (Damodaran 1997). In fact, nowadays the most universaly accepted definition of the gel point comes from the determination of linear viscoelasticity properties associated to the development of a self-similar structure of the so-called critical gel (Winter 1987). Materials at the gel point exhibit a distinct rheological behaviour where the following power law equation holds for linear viscoelastic functions: 𝐺′(𝜔)=𝑘1·𝜔𝑛 (3.4-9) 𝐺′′(𝜔)=𝑘2·𝜔𝑛 (3.4-10) Background. Products from proteins 130 where n is the relaxation exponent and k1, k2 are constants related to this exponent and to the gel stiffness. As a consequence, the loss tangent is frequency independent at the critical gel. In practice, the gel point has been widely determined as the point at which tan(δ) is not a function of frequency. Gel Strength Many different instruments are available for measuring gel strength. Some simply results that provide empirical tests cannot be related to fundamental rheological quantities. However, there are other tests that can measure well-defined parameters such as shear modulus or rupture strength. A typical test measures the force applied for the gel versus displacement. In any case, as previously mentioned, modern rheometers are the most extended devices used to characterize gels. Moreover, among the different measurement techniques that rheometers can perform, SAOS measurements of viscoelastic properties using parallel plates or cone-plate geometries are preferred, since they allow monitoring the change in structure over gelation, which is based on the assumption that the material's behaviour is linear. Gel strength can then be related to linear viscoelastic functions (G', G'', tan δ, etc). Water holding capacity (WHC) Mainly, there are two causes which contribute to the water holding capacity of a material: the polarity (including surface charges) and the capillarity, which is the most important. For this functional property, myofibrillar proteins are the most important. Thus, water is able to be held mainly in the holes between actin and myosin filaments. Microscopy Microscopy techniques are widely used for the characterisation of the protein microstructure. Optical microscopy may be used, however LSCM and Background. Products from proteins 131 SEM, described above, are some of the most useful techniques for the visualization of gel microstructure. Nutritional properties Antioxidant properties of food gels Free radicals are generated through normal reactions within the body during respiration in aerobic organisms. The presence of these potentially toxic products can give rise to several diseases. Air pollutants and oxidants in tobacco can typically cause some harmful reactions in skin or can be absorbed to blood circulation, exerting some adverse effects. Additionally, UV radiation can be a producer of a variety of oxidants. The free radicals, which are physiologically produced, can provide a protection against infections. According to the free radical theory of ageing developed by Denham Harman, organisms age when free radicals accumulate in cells and cause harm over time. In this way, reactive species can cause damage in proteins, and mutations in DNA, oxidation of membrane phospholipids and modification in low density lipoproteins (LDL). Thus, antioxidant compounds can remove reactive species through enzymatic and non-enzymatic antioxidants. However, in certain circumstances the endogenous immune system fails to protect the body against reactive radicals on its own. This brings about the need for synthetic and natural antioxidants, which can prevent oxidative stress and its deleterious effects. Synthetic antioxidants are cost-effective and efficient but display some toxic and hazardous effects. In the areas of human nutrition, and biochemistry, natural antioxidants from food resources have been the focus of growing interest for their potential health benefits with little or no side effects (Sarmadi and Ismail 2010). Background. Products from proteins 132 Antioxidative peptides in food products Several peptides from protein ingredients have been found to possess antioxidant ability. Antioxidant peptides from foods contain 5-16 amino acid residues and are considered to be safe and healthy compounds with low molecular weight, low cost, high activity and easy absorption. They have some advantages in comparison to enzymatic antioxidants; that is, with simpler structure they have more stability in different situation and no hazardous immunoreaction. In addition, they present nutritional and functional properties beside their antioxidant activity. There are two different forms of antioxidant activity: either as hydrolysates of precursor proteins or as bioactive peptides. Hydrolysate is a mixture that is mainly composed of peptides and amino acids which are produced through protein hydrolysis by enzyme, acid or alkali treatment The exact mechanism underlying the antioxidant activity of peptides has not fully been understood, yet various studies have displayed that they are inhibitors of lipid peroxidation, scavengers of free radicals and chelators of transition metal ions. Antioxidative properties of the peptides are more related to their composition, structure, and hydrophobicity. Tyr, Trp, Met, Lys, Cys, and His are examples of amino acids that cause antioxidant activity. Amino acids with aromatic residues can donate protons to electron deficient radicals. This property improves the radical-scavenging properties of the amino acid residues. It is proposed that the antioxidative activity of Hiscontaining peptides is in relation with the hydrogen-donating, lipid peroxyl radical trapping and/or the metal ion-chelating ability of the imidazole group. In addition, SH group in cysteine has an independently crucial antioxidant activity due to its direct interaction with radicals. Nevertheless, protein linkage conformation and structural features of the peptides have been claimed to influence antioxidant ability (Sarmadi and Ismail 2010). Background. Products from proteins 133 Bioplastics The bioplastics industry is a strongly growing part of the plastics industry. With a global production capacity around 1.7 million tonnes per year in 2014, the volume in the market is still small compared to the overall plastics volume of 320 million tonnes/year. However, according to recent estimations, the share of bioplastics will increase up to 7.8 million tonnes of bioplastics (2-3 percent of plastics market) in 2019 (Plastics-Europe 2008). According to European-bioplastic Association, the term bioplastic encompasses a whole family of materials which have some differences with conventional plastics because they are bio-based, biodegradable, or both. Thus, two kinds of bioplastic materials can be distinguish according to this definition: - Bio-based materials, in which the material or product is derived from biomass, which can stem from either plant or animal sources. - Biodegradable polymeric materials, where the term biodegradable refers to a chemical process during which micro-organisms, which are available in the environment, convert materials into natural substances. Obviously, the process of biodegradation depends on the surrounding environmental conditions (moisture, temperature, light, O2), and on the material itself. Biodegradability is an inherent property of certain bioplastic materials that can benefit specific applications (Plastics-Europe 2008) Materials used in the bioplastic manufacturing Usually, a bioplastic material (of the biodegradable group) consists of a polymeric network formed by a biodegradable macromolecular substance (in this case is a protein) and a plasticiser. Background. Products from proteins 134 Natural raw-materials In recent years there has been a great interest to utilize renewable biomass in order to manufacture consumer goods which exhibit high-quality, cost-competitive and biodegradable, reducing the consumption and the dependence on petrochemical feedstock and diminishing environmental pollution (Rosentrater and Otieno 2006, Felix, Martin-Alfonso et al. 2014). Mainly, proteins and polysaccharides have been postulated as renewable biomass to manufacture biopolymers for many years (De Graaf 2000, Hernandez-Izquierdo and Krochta 2008). Polysaccharides are naturally extended, and are widely used for food industry. These compounds have been also used for bioplastics (e.g. starch and chitosan are good examples of polysaccharides used for this purpose). As regards proteins, they are a renewable, biodegradable resource with great potential to improve the quality and stability of a large range of food products by using a number of processing techniques (Romero, Cordobes et al. 2008, Jayasundera, Adhikari et al. 2009, Erni, Windhab et al. 2011). For a long time, proteins have been used to produce edible materials, but understanding of the precise physical and chemical mechanisms of protein interactions, they can be used to produce stable bioplastic materials (Hernandez-Izquierdo and Krochta 2008, Balaguer, Gomez-Estaca et al. 2011). Plasticisers Plasticizers are generally added to improve the processability of the protein network, as well as in order to modify the properties of the final structure, decreasing the glass transition and the brittleness. Usually, plasticizers consist of compounds which exhibit low-molecular weight, low volatility and that interact with the polymer chains producing swelling (Hernandez-Izquierdo and Krochta 2008). Background. Products from proteins 135 This type of compounds is widely used in polymer industries as additives. The primary role of such substances is to improve the flexibility and processability of polymers by lowering the second order transition temperature, the glass transition temperature (Tg). The council of the IUPAC (International Union of Pure and Applied Chemistry) defined a plasticizer as ‘‘a substance or material incorporated in a material (usually a plastic or elastomer) to increase its flexibility, workability, or distensibility’’. These substances reduce the tension of deformation, hardness, density, viscosity and electrostatic charge of a biopolymer, at the same time as increasing the polymer chain flexibility, resistance to fracture and dielectric constant. Other properties are also affected, such as degree of crystallinity, optical clarity, electric conductivity, fire behaviour and resistance to biological degradation (Vieira, da Silva et al. 2011). There are many plasticizer for protein-based bioplastics, such as: 1,4Butanediol, DATEMa, Dibutyl, Glycerol, Lactic acid, Octanoic, Palmitic acid, Sorbitol, Sucrose and Water (Hernandez-Izquierdo and Krochta 2008). For this study, Glycerol has been the chosen plasticizer. This is a widely used bioplastic, exhibiting hydrophilic properties with a low molecular weight, and high boiling point. Its high plasticizing effect has been attributed to the ease with which glycerol can insert and position itself within the 3-dimensional biopolymer network (di Gioia and Guilbert 1999). Methods for protein-based bioplastics manufacturing General approach Proteins offer a large range of possible physical and chemical interactions. This dual character is given because proteins can participate in non-covalent interactions such as ionic, hydrogen, and van der Waals bonding Background. Products from proteins 136 or in chemical reactions through covalent linkage (peptide and disulphide bonds). Usually, the formation of the protein network is divided in two main stages: Plasticization and protein interactions (Hernandez-Izquierdo and Krochta 2008). The plasticizing effect of small polar molecules has been described in terms of insertion and positioning within the 3-dimensional protein network. Mainly, there are four theories to explain this process: 1) The lubricity theory, where the plasticizer is acting as a lubricant to facilitate mobility of the chain molecules. 2) The gel theory, which considers the disruption of polymer-polymer interactions (weak physical interactions). 3) The free volume theory, which considers that the plasticizer increases the free volume and mobility of polymer chains (used to understand the effect of plasticizers in lowering the glass transition temperature). 4) The coiled spring theory, which explains plasticizing effects from the point of view of tangled-macromolecules. However, the various possible ways in which proteins may interact during thermoplastic processing are unclear. Thus, the reactivity of proteins depends on their physicochemical environment as well as on the thermomechanical treatment used (Hernandez-Izquierdo and Krochta 2008). Solvent casting For the formation of protein films or coatings, the protein has to be first dissolved in a proper solvent, which sometimes requires heating or pH Background. Products from proteins 137 adjustment, as well as the addition of some compounds which could improve film-forming or other properties Subsequently, the mixture is heated above the lipid melting point and then homogenized. Degassing is an important step to eliminate bubble formation in the final film or coating. Finally, the protein film or coating is formed by applying the prepared formulation to the desired casting or product surface and allowing the solvent to evaporate. Providing heated air at low humidity and high velocity increases drying rates (Krochta 2002). Thermo-mechanical processing Compression moulding Compression moulding technique use the combination of high temperatures, high pressures, short times, and low moisture contents in order to the transform the protein-plasticizer blends into viscoelastic melts. Then, the protein-based bioplastics are formed by cooling, increasing hydrogen, ionic, hydrophobic, and covalent interactions (Hernandez-Izquierdo and Krochta 2008). The use of higher compression moulding temperatures typically promotes a more extensive protein denaturation, and as a consequence higher cross-linking. Compression moulding can result in the formation of protein-based films or materials whose mechanical and barrier properties are dependent on the formulation and processing conditions used. This technology is suitable for investigating the thermoplastic properties of plasticized proteins as well as the properties of the resulting films and materials (Hernandez-Izquierdo and Krochta 2008). Background. Products from proteins 144 necking/yielding but either remains approximately constant or rises less steeply with increasing strain, depending on the extent of cold drawing. Cold drawing succeeds the yield point where material undergoes permanent deformation as a result of molecular slippage. Continuing extension of the narrow portion of the dumbbell specimen is achieved during drawing by causing the shoulders of the neck to travel along the specimen as it reduces from the initial cross-section to the drawn cross-section. At further elongations, the slope of the stress-strain curve increases again, due to “strain hardening”/“molecular orientation”, and finally material failures (Akay 2012). This type of curve is very useful since allows to classify the material. For instance, Figure 3.4-4 compares four different plastic materials: Figure 3.4-4: comparison between different curves stress-strain Thus we can say that (a) is a low ductility polymer, (b) is a ductile polymer, (c) is a ductile polymer capable of cold drawing, and (d) is a polymer with longrange elasticity (Akay 2012). Applications of protein bioplastics The main goal of biodegradable bioplastics is to achieve the replacement of existing synthetic, non-biodegradable products for these others at the Background. Products from proteins 145 lowest cost possible. Other goals may be also found for specific applications. For instance, edible films and coatings aim for improving food quality and shelf life by reducing the effect of moisture, oxygen, migration, etc., protecting food from microbes, maintaining food product integrity, and enhancing product appearance. However, this must be related to the cost of coating materials and to the cost of the coating process. Certain new edible film and coating materials made from proteins are targeted for replacing materials currently used in existing applications. However, compared to the large number of studies performed on film formation and properties, a relatively small number of application studies have been performed. For this reason, information available is generally lacking on approaches to coating foods, as well as the resulting effectiveness of edible films and coatings in food systems. This makes it very difficult for food processors to decide on the “value-added” merit of an edible film or coating relative to the additional cost involved (Krochta 2002). Composite materials A composite compound is a material made by combining two or more materials, and frequently they have very different properties. The two materials work together to give the composite unique properties. However, within the composite you can easily tell the different materials apart as they do not dissolve or blend into each other. Natural composites exist in both animals and plants from the early beginnings. Thus, wood is a composite which is made from long cellulose fibres (a polymer) held together by a much weaker substance called lignin. The two weak substances (lignin and cellulose) together form a much stronger structure. Background. Products from proteins 146 Nowadays, human have synthetized this kind of compounds. The first modern composite material was fibreglass. It is still widely used today for boat hulls, sports equipment, building panels and many car bodies. The matrix is a plastic and the reinforcement is glass that has been made into fine thread. On its own, the glass is very strong but brittle and it will break if bent sharply. The plastic matrix holds the glass fibres together and also protects them from damage by sharing out the forces acting on them. Some advanced composites are now made using carbon fibres instead of glass. These materials are lighter and stronger than fibreglass but more expensive to produce. They are used in aircraft structures and expensive sports equipment. Carbon nanotubes have also been used successfully to make new composites. 147 A Alberto y Antonio Por ayudarme a crecer junto a ellos, Por ser una pieza clave en mi formación como investigador, por todas esas horas de trabajo que hemos pasado juntos. 149 4. Materials and Methods Materials and Methods. Materials 151 4.1. Materials Crayfish powder The CF meat was separated from the shell by grinding and sieving and supplied as CF pulp by ALFOCAN (Isla Mayor, Sevilla, Spain). Figure 4.1-1 shows the process followed to obtain different protein fractions from crayfish (CF) meat. CF pulp was kept frozen until its use. After thawing at 4⁰C, CF pulp was homogenized and subjected to centrifugation at 15,000 x g for 15 minutes in a Centromix II-BL (Selecta, Spain), obtaining three different phases: a heavy phase, CF1P (c.a. 20 wt. %), an intermediate phase (CF2P) which is the aqueous phase (c.a. 70 wt. %) and a light phase, CF3P (c.a. 10 wt. %). The CF2P was the selected phase because it is the water soluble protein fraction and it represents the highest protein content. Finally, the intermediate phase (CF2) was freeze-dried in a BETA 1-8 LD Plus Series (CHRIST, Germany) to obtain a powder-fraction rich in proteins. This phase will be named CF2L. Figure 4.1-1 : Diagram of the procedure carried out in order to obtain the CF2L protein concentrate. Materials and Methods. Materials 152 The protein content of the CF2L was determined in quadruplicate as % N x 6.25 using a LECO CHNS-932 nitrogen micro analyser (Leco Corporation, St. Joseph, MI, USA) from Microanalysis service (CITIUS, University of Seville). In the same way, lipid, moisture and ash contents were determined according to A.O.A.C. (2000).Table 4.1-1 shows the elemental characterisation of CF2L system: Component wt. % Protein Moisture Lipid Ash 78.6 ± 0.5 6.8 ± 0.1 5.1 ± 0.3 9.5 ± 0.6 Table 4.1-1: Elemental characterisation of CF2L system First of all, it is remarkable the high protein percentage (ca. 80 wt. %). Hence, according to Pearson classification (1983), it must be considered as a protein concentrate. Furthermore, this system contents up to 5 wt. % of lipids despite centrifugation stage was carried out. That means that this stage is not able to a complete organic and aqueous phases separation, or in the manual procedure of separating both phases a certain amount of organic phase was adjoined together to aqueous phase. All systems studied have in common this protein concentrate, however for each product (emulsion, gel or bioplastic) some modifications have been considered: for emulsions a polysaccharide (a gum) have been used to provide stability. In addition, for gels three hydrolysed systems have been obtained at three different degree of hydrolysis and for bioplastics with a low-value crayfish flour have been evaluated in order to be cost-competitive with synthetic polymers. Materials and Methods. Materials 153 Materials for Emulsions The oil-in-water emulsions consist of a continuous phase of distillate water and dispersed phase of high oleic oil. To provide stability, apart from the initial crayfish protein concentrate (CF2L), a polysaccharide (xanthan gum) was used in order to improve the system stability. Gums have a high influence on the structural characteristics of food products, modifying the texture and the organoleptic properties, even being present at concentrations lower than 1 wt. %. For this reason, gums are nowadays widely used for fat replacement in many low-calorie products (Williams and Phillips 2003). Xanthan gum was discovered in the 1950s and nowadays have widely application in the food industry, where was introduced in the early 1970s. This gum is obtained from the genus Xanthomonas by aerobic fermentation Figure 4.1-2 shows the primary structure of the Xanthan gum (XG): Figure 4.1-2: Primary structure of xanthan gum . The xanthan gum is an anionic molecule which has a (1,4)-β-Dglucopyranose backbone and a trisaccharide side chain on every other glucose residue linked through the C3 position. The side chain consists of two mannopyranosyl residues linked on either side to a glucuropyranosyl uronic acid group. The inner mannose residue which is connected to the backbone may be acetylated while the terminal mannose residue may be pyruvated. The Results 256 confirm that DPPH measurements are not dependent on pH or on the hydrolysis degree. Finally, at pH 8.0 the lowest antioxidant activity was found for FC reagent. The degree of hydrolysis seems to favour FC activity in some extent at this pH, except for its highest value (i.e. CF2LH120) It is worth pointing out that the different behaviour of protein systems could be striking because it depends on the method used. In fact, many studies have been reported that the antioxidant activity depends on the nature of the reference compound (DDPH, ABTS, FC) (Wojdylo, Oszmianski et al. 2007, Wootton-Beard, Moran et al. 2011, Boulanouar, Abdelaziz et al. 2013). In any case, regardless of the pH studied and the different reference compounds used, there seems to be a general tendency of increasing antioxidant activity with the degree of hydrolysis, even though is not always significantly detected. An excessively high degree of hydrolysis seems to lose this ability. Figure 5.3-20 shows TCA-soluble peptides (free-amino acids and short peptides) before and after thermal processing. The proteolytic activity of enzymes, which are capable of breaking the chemical bonds of the muscle fibres during the cooking step, has been widely studied. This enzymatic process may be important since it can modify the characteristic texture of fresh fish or food products (Miller and Spinelli 1982, Greene and Babbitt 1990) The determination of TCA-soluble peptides has been carried out by means of measuring the absorbance of TCA-soluble peptide solution before and after gelation process at 280 nm. First of all, it can be observed significant differences between non-hydrolysate system (CF2L) and hydrolysates systems (CF2LH5, CF2LH25 and CF2LH120). These results could be expected since they can be a consequence of the hydrolysis carried out. This means that an Results 257 increase in pH values gives raise an increase in the number of amino acids and small peptides in the bulk solution. 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 AG 2.0 6.5 CF2L CF2LH5 CF2LH25 CF2LH120 CF2L CF2LH5 CF2LH25 CF2LH120 pH 8.0 BG Absorbance Figure 5.3-20: Proteolytic activity measured at 280 nm in all the systems studied (CF2L, CF2L5, CF2L25 and CF2L120), evaluated at three different pH values (2.0, 6.5 and 8.0), before and after gelation process. For CF2L system, a slight increase of TCA-soluble peptides at 20⁰C can be observed when an increase of pH takes place. Finally, after gelation process, TCA-soluble peptides do not increase in any case, for this reason we can discard proteolytic activity as a consequence of the thermal procedure followed. Regarding to hydrolysates systems, we could not found any pH dependence of TCA-soluble proteins on pH or hydrolysates systems, neither before gelation nor after gelation. Thus, proteolytic activity was not found in any system, and it is not pH-dependent. Finally, it is remarkable the decrease of TCA-soluble peptides found at pH 6.5 after gelation process for CF2L system. Hence, at this pH, the gel not only Results 258 exhibit better mechanical properties (higher gel strength and high WHC), but also the gel is able to hold in a greater extend free amino acids and short peptides in its structure. This can be related to the highest values of WHC found for CF2L system at pH 6.5. Results 259 5.4. Crayfish-based bioplastics An industrial low cost surplus obtained from crayfish (CF) (dried in a rotary furnace) was used in this section due to the restricted availability of CF2L protein concentrate, along with the high raw material required for protein-based bioplastics manufacturing. This way of proceeding allows us to study the optimisation of CF-based bioplastic. Eventually, at the end of this section, results were compared with CF2Lbioplastics processed by using the protocol obtained from CF-based bioplastics. The first stage for all protein-based bioplastics is to select the ration protein/plasticizer. This election is usually made based on the processability of the dough-like material obtained. Figure 5.4-1 shows both torque and temperature profiles as a function of mixing time for blends obtained at different CF/GL ratios, as well as their visual appearance after mixing. These results put forward the remarkable dependence of these parameters on the CF/GL ratio. Thus, a rapid increase in torque up to a maximum value takes place for the system having less amount of plasticizer, denoted as 80/20, which represents the CF/GL weight ratio. This evolution is readily followed by an asymptotic decrease towards a plateau value. In contrast, the system 70/30 shows a moderate growth in torque showing no maximum value but a slow tendency to the plateau value. The profile of the 60/40 system shows only a slightly initial increase in torque, being dominated by a constant torque value over mixing time. In general, the temperature evolution is very similar to the torque profile where an increase takes place excepting for the lowest CF/GL ratio. Both increases in torque and Results 260 temperature may be attributed to shear-induced exothermic reactions developed during the mixing process. 020 40 60 0 10 20 30 40 50 Torque (N·m) Time (min) 20 30 40 50 60 70 80 90 CF/GL Temp.(ºC) Torque (N m) SME(kJ kg-1) (60/40) 47901 (70/30) 33861 (80/20) 1331 Temperature (ºC) Figure 5.4-1: Images, SME and evolution with time of mixing torque and temperature for systems CF/GL (80/20, 70/30 and 60/40). As a consequence of the above-mentioned differences in torque profile, the specific mechanical energy (SME) employed for mixing is also quite different. This characteristic parameter of mixing may be defined as follows EQ. (5.4-1). (Redl, Morel et al. 1999): 𝑆𝑀𝐸=𝜔 𝑚∫ 𝑀(𝑡)𝑑𝑡 𝑡𝑚𝑖𝑥 0 (5.4-1) where ω (in rad/s) is the mixing speed, m (in g) the sample mass, M(t) (in Nm) the torque and tmix (in s) the mixing time. The values for the SME for these three systems are included in Figure 5.4-1. A remarkable increase in this parameter can be observed with the CF/GL ratio. This effect takes also place at the higher concentrations in spite of the fact that the plateau torque values are relatively close. Results 261 From the visual appearance observed in Figure 5.4-1, the 80/20 system does not seem to contain enough amount of plasticizer to obtain an easy-tohandle material, giving rise to a granulated powder instead of a homogeneous dough-like blend. This is evidenced by the fact that this is the system displaying the highest torque values, which correspond to the highest rheological consistency. On the other hand, the highest amount of glycerol in the 60/40 system provides a neat dough-like appearance to the blend. This is also consistent with the lowest torque values obtained (associated to a lower consistency) after the mixing process. The 70/30 blend shows intermediate appearance between both limits although it is closer to the most concentrated protein-based bled. In the present study, the system 80/20 has been discarded because the high energy required for mixing. Moreover, this system has proven to be hard to inject (results not shown). On the other hand, systems containing a low protein/glycerol ratio may be injected easily but the final bioplastic materials tend to exude the excess of glycerol. Exudation is a wellknown phenomenon described in the polymer/plasticiser literature and should be avoided in order to prevent contamination of the surrounding materials (e.g. in food packageing) (Rahman and Brazel 2004). This seems to be the case for the 60/40 system. As a result, the 70/30 system is selected as the most suitable to obtain a homogeneous and easily injectable CF/GL blend. In order to compare and evaluate the effect of chemical agents and synthetic polymer on crayfish-based bioplastic materials, a dual strategy was followed. On the one hand the presence of sodium sulphite (SS) or bisulphite (BS) as reducing agents, urea (U) as denaturing agent and L-cysteine (LC) as crosslinking agent were analysed as chemical modifiers. On the other hand, a polyester from fossil source, which has been widely used as the polymer matrix in the development of new materials, which is highly flexible, Results 262 biodegradable, biocompatible and easy to process, was used (polycaprolactone, PCL). Crayfish-bioplastic with chemical modifiers Blends characterisation Preparation of blends by thermoplastic mixing Figure 5.4-2 shows the evolution of torque as a function of time for different 70/30 CF/GL blends prepared using SS as reducing agent, U as denaturing agent, or LC as crosslinking promoter at different concentrations (3 and 30 mg/g protein for SS and U and 1 and 3 mg/g for LC). 0 5 10 15 20 25 0 5 10 15 20 25 30 AMOUNT OF ADDITIVES System 0 1g kg-1 3g kg-1 30g kg-1 CF/GL - - - CF/GL SS - - CF/GL U - - CF/GL LC - - Torque (N m) Time (min) Figure 5.4-2: Evolution with time of mixing torque for additive-containing system (SS, U and LC). Results 263 These results generally show that any of the additives used induces an anticipation of the torque profile, particularly at the highest additive concentrations. No differences in the torque profile are noticed at the two different concentrations of BS (data not shown). The highest amount of additive (corresponding to SS or U) also yields lower torque plateau values, which may be considered beneficial for the processability of blends, for example under injection moulding. Temperature-time profiles show the same type of evolution for all the additives and concentrations in spite of that an air stream was used as a cooling fluid. No differences are found in the final values for the highest SS or U content in this case. The only difference found between temperature and torque profiles is that the former undergo a further delay in time, which may be related to the air-cooling effect. It may be also noted that the system is not still properly mixed at times shorter than that one corresponding to the maximum torque. Therefore, as a general rule, the mixing time range for subsequent blend processing is selected once the plateau torque value is reached such that a good homogeneity degree is assured. Another interesting parameter to compare different mixing processes is the relative energy input (REI), which is defined as follows (5.4-2). 𝑅𝐸𝐼 (%)=𝑆𝑀𝐸𝑚𝑖𝑛(𝑎,𝑐) 𝑆𝑀𝐸𝑚𝑖𝑛(0)·100 (5.4-2) where 𝑆𝑀𝐸𝑚𝑖𝑛(0) and 𝑆𝑀𝐸𝑚𝑖𝑛(𝑎,𝑐) are the SME values at which torque reaches 95% of the maximum value for the profile of the additive free blend and additive at concentration c, respectively. Therefore, REI is an index of the reduction in energy input relative to that one required for mixing the additive free blend. Results 264 The values of SME20 and REI parameters are shown in Figure 3.1-1 for these additives. The SME values for BS are also included in this figure SME20 is the specific mechanical energy supplied, as defined in EQ. (5.4-2), after mixing for 20 min. As may be observed in this graph, an increase in additive concentration always leads to an apparent increase in SME20 parameter, as a consequence of the above-mentioned anticipation of the torque profile. If the same additive concentration is used, this increase is particularly remarkable for LC and, in any case, is only moderate when BS is used. 0 5000 10000 CF/GL 1g kg-1 3g kg-1 30g kg-1 (70/30) SME20 (kJ kg-1) Aditive SS BS ULC Figure 5.4-3: Specific mechanical energy (SME) employed for mixing at 20 minutes The values of REI parameters are shown in Figure 5.4-4 for these additives. Results 265 Aditive SS BS ULC 0 50 100 150 CF/GL 1g kg-1 3g kg-1 30g kg-1 (70/30) REI (%) Figure 5.4-4: Reduction energy index (REI) for each system. The results obtained with parameter REI confirm the relevant effect of the addition of LC at constant additive concentration. Thus, estimation tendencies from the results obtained indicate that the additive/protein ratio required to achieve a 50% REI would be 33 mg/g for U and 27 mg/g for SS, whereas a ratio as low as 4.4 mg/g would be required by using LC. Rheological characterisation of blends Figure 5.4-5 shows the dependence of linear viscoelastic functions on temperature for different blends containing 3 mg additive per g CF protein. The additive free (CF/GL) blend is also displayed in this figure. Results 272 Uniaxial tensile strength measurements Figure 5.4-9 displays the results of stress-strain curves obtained from tensile strength measurements for additive-free and additive-containing specimens at an additive/CF ratio. 0.00 0.01 0.02 0.0 0.2 0.4 0.6 0.8 System CF/GL CF/GL BS CF/GL SS CF/GL U CF/GL LC  (MPa) (mm/mm) Figure 5.4-9: Stress versus strain curves from tensile strength measurements for different CF/GL and additive-containing probes. All the curves exhibit a similar behaviour which consist of an initial linear elastic behaviour of high constant stress-strain slope yielding high values for the Young’s modulus (E), followed by a deformation stage with a continuous decrease in the stress-strain slope. A second constant slope is reached at the end of the plastic deformation stage. All the curves eventually reach a maximum value for the stress (σmax) and the elongation at break (εmax). Only Results 273 LC leads to an apparent enhancement of the tensile strength profile, also leading to a slightly shorter strain value. The values of stress-strain parameters (E, σmax and εmax) and their corresponding standard deviations are plotted in Figure 5.4-10 for the additive-free and additive-containing specimens at 3 mg/g additive/CF ratio. 0.5 1.0 1.5 2.0 2.5 max max E max (%) max (MPa) , 0 20 40 60 80 100 LC 130ºC LC 600s LC U SS BS E (MPa) CF/GL Figure 5.4-10: Parameters from tensile strength measurements: Maximum stress (σmax), elongation at break (εmax) and Young’s’ modulus (E) for different CF/GL and additive-containing probes. This figure puts forward once again that additive LC is the only one that improves parameters E and σmax over the additive-free system, under the same processing conditions. On the other hand LC-added specimens exhibit lower values for εmax. The rest of additives lead to similar or even lower values of the three parameters. Figure 5.4-10 also shows the values for LC-added specimens moulded at longer time or higher temperature. As may be Results 274 observed, an increase in the packing time from 200 s up to 600 s does not yield any noticeable change in tensile parameters. This result indicates that after 200 s packing time, the probe is already closed by the solidified blend. On the other hand, an increase in the mould temperature leads to remarkable changes in tensile parameters. Thus, the maximum stress and above all the elongation at break undergo an apparent increase in value (ca. 12 and 70%, respectively), whereas the Young’s modulus clearly decreases (ca. 30%) by increasing the mould temperature from 100 to 130 0C. Interestingly, it is the elongation at break the property that undergoes the most remarkable enhancement by favouring heat-induced crosslinking. In this way, the material exhibits higher toughness, in spite of being less strong. In fact, as stated by Lagrain et al. (2010), increasing the elongation at break of glassy, amorphous polymers typically goes at the expense of the elastic modulus. This compensation may also affect to the bending elastic properties of the bioplastic, thus explaining the small dependence of DMA profiles on moulding temperature. Moreover, this behaviour is similar to that one found for other elastomeric materials such as rubber-based blends (Zarate-Ramirez, Martinez et al. 2011) and is consistent with the results from DMA measurements that show an extension of the rubbery plateau obtained at high temperature. Results 275 Crayfish-bioplastic with a synthetic polymer. Composite materials A synthetic biodegradable polymer (PCL) were used to improve mechanical properties of CF-based bioplastic. This study can be considered as an alternative to the use of additives (previous section). Blends characterisation Preparation of blends by thermoplastic mixing Figure 5.4-11 exhibits torque and temperature profiles as a function of mixing time for different CF/GL/PCL blends maintaining the same CF/GL ratio at ca. 2.3, using different PCL content, where the blend without PCL is used as the reference system. 020 40 60 0 5 10 15 20 25 30 35 40 45 50 CF/GL/PCL Torque(Nm) Temp. (ºC) (70/30/0) (63/27/10) (49/21/30) Time (min.) Torque (Nm) 20 30 40 50 60 70 80 90 100 110 Temperature (ºC) Figure 5.4-11: Evolution of torque and temperature over the mixing process for crayfish flour/glycerol/polycaprolactone (CF/GL/PCL) systems: at constant CF/GL ratio: (70/30/0), (63/27/10) and (49/21/30) Results 276 These results put forward the relevant dependence of torque and temperature on the CF/GL/PCL ratio. Thus, a rapid increase in torque up to a maximum value takes place, followed by an asymptotic decrease towards a plateau value. Temperature profiles generally follow an increase towards a plateau value. The time required to reach the plateau values for both variables (torque and temperature), which is roughly the same, is clearly dependent on the CF/GL/PCL ratio. This coincidence may be seen as a consequence of the development of exothermic crosslinking reactions during the mixing processes that involve both an increase in temperature and consistency (reflected in torque). Both profiles show also an initial induction period, being more evident for those blends displaying the slowest evolution. It is worth mentioning that a torque peak appears when unmelted PCL is present in the blend. Thus, the PCL melt point, which is reached at about 55 0C, is coincident with the maximum torque value. In other words, the torque does not start to decrease until the PCL melting point is exceeded. As may be also observed in this figure, an increase in PCL content leads to a faster torque and temperature kinetics and, as a consequence, to an anticipation of both profiles. The effect also gives rise to a general increase in torque and temperature values at any time. Figure 5.4-12 exhibits torque and temperature profiles as a function of mixing time for different CF/GL/PCL blends. This figure shows both profiles for blends containing 10% PCL at different CF/GL ratios. As may be observe in this figure, the behaviour is quite similar to the previously found for systems at constant CF/GL ratio. Again, the temperature reached is above the PCL melting point, which ensures a homogenous mixing. However, the behaviour found for the system (60/30/10), seems to have an excess of glycerol, which lead to delay the plateau value. Results 277 020 40 60 0 5 10 15 20 25 30 35 40 45 50 CF/GL/PCL Torque(Nm) Temp. (ºC) (65/25/10) (63/27/10) (60/30/10) Time (min) Torque (Nm) 20 30 40 50 60 70 80 90 100 110 Temperature (ºC) Figure 5.4-12: Evolution of torque and temperature over the mixing process for crayfish flour/glycerol/polycaprolactone (CF/GL/PCL) systems: at constant PCL concentration: (63/27/10), (60/30/10) and (65/25/10). Thermal characterization of blends Heat flow patterns obtained from Differential Scanning Calorimetry (DSC) measurements are shown in Figure 5.4-13 and Figure 5.4-14. Figure 5.4-13 shows the thermogram for CF flour and for the reference system (CF/GL/PCL, 70/30/0), as well as the profiles corresponding to CF/GL/PCL blends at constant CF/GL ratio, as a function of PCL content. On the other and, Figure 5.4-14 displays the DSC results for CF/GL/PCL blends containing 10% PCL as a function of the CF/GL ratio. Results 278 25 50 75 100 125 System Symbol CF Flour CF/GL (70/30) CF/GL/PCL (63/27/10) CF/GL/PCL (49/21/30) Exo Up Heat Flow (W/g) Temperature (ºC) Figure 5.4-13: DSC profiles for crayfish flour and systems at constant CF/GL ratio: (70/30/0), (63/27/10) and (49/21/30) (A) 25 50 75 100 125 150 System Symbol CF/GL/PCL (65/25/10) CF/GL/PCL (63/27/10) CF/GL/PCL (60/30/10) Heat Flow (W/g) Temperature (ºC) Exo Up Figure 5.4-14: DSC profiles for systems at constant PCL concentration: (63/27/10), (60/30/10) and (65/25/10). Results 279 CF flour displays a typical endotherm of a fairly denatured protein system. This profile exhibits an endothermic first peak at 68 0C, a glass transition (Tg) at ca. 92 0C, as well as a broad endothermic event between the Tg and 130 0C. The first thermal event can be attributed to the physical ageing effect, which was previously reported for this CF flour (Farahnaky, Guerrero et al. 2008). Physical ageing is a general phenomenon that occurs over time in glassy or partial glassy polymers below their Tg and is a manifestation of the nonequilibrium nature of the glassy state (Strink 1978, Anon 1997). The glass transition at around 90 0C is consistent with previous results reported by Farahnaky et al. (2008) for crayfish flour and Hashimoto et al. (2004) for fish muscle proteins. On the other hand, the broad endothermic event may be related to the huge variety of protein fractions of different molecular weight that constitute the CF flour a reported in a previous paper (Romero, Cordobes et al. 2011). With regard to the endotherms obtained for the reference system the two endothermic events vanish. The total disappearance of the first endothermic peak, most probably as a consequence of mixing, confirms its physical ageing-driven nature. However, the glass transition remains roughly at the same temperature for all the blends studied. In addition, it is worth mentioning that all the systems containing PCL display an apparent endothermic peak at ca. 55 0C, which is attributed to the PCL melting point. This peak becomes more pronounced with increasing PCL content. The results obtained from DSC measurements also confirm the suitability of the temperature selected for the cylinder and mould, since the former (60 0C) is higher than the endothermic peak corresponding to the PCL melting whereas the later temperature (100 0C) is higher than the glass transition in Results 280 order to favour mobility and temperature-induced protein crosslinking (in combination with pressure). Injection moulding process Table 5.4-2 shows the conditions selected for the injection moulding process for each of the blends studied. T (0C) Pressure (MPa) Time (s) Pre-injection cylinder Injection Packing stage 60 60 100 100 0.1 0.1-50 50 20 100 <1 20 200 Table 5.4-2: Injection moulding parameters. The processing parameters (temperature, pressure and time) values for the injection moulding process used in this study, which are similar to those used in the previous section. The values for the processing parameters at the pre-injection cylinder are selected to ensure a blend viscosity low enough to facilitate its injection into the mould. The residence time selected for the packing stage (right after injection) has been 220 s since no further enhancement has been noticed by increasing this period. In addition, exposition to high temperatures for a long time typically leads to protein degradation (i.e. via Maillard-type reactions) (Fayle, Gerrard et al. 2002). Biocomposite characterisation Dynamic Mechanical Temperature Analysis Figure 3.1-1 shows the values of the elastic modulus, E’ from DMA temperature ramp test measurements. The reference system is compared Results 281 with those specimens with the same protein/plasticiser ratio (CF/GL/PCL: 63/27/10 and 49/21/30). As may be observed, all the specimens studied show a similar profile for E’ at low and medium temperature. In this way, an increase in temperature leads to a decrease in elastic modulus (E’) that tends to reach a plateau value. However, it is remarkable that PCL containing samples exhibit higher elastic modulus provided that temperature remains below the PCL melting point. At the melting point the three samples show rather coincident values that undergo a decrease with increasing temperature. In fact, there is a region where the PCL-free specimen shows slightly higher E’ values. Eventually a temperature is reached above which the decrease in E’ found for the reference specimen becomes faster whereas the PCL-containing composites tend to reach a plateau region. -20 0 20 40 60 80 100 120 140 104 105 106 107 108 109 1010 System E' (Pa) CF/GL/PCL (70/30/0) CF/GL/PCL (63/27/10) CF/GL/PCL (49/21/30) E' / (Pa) Temperature (ºC) Figure 5.4-15: Storage modulus from DMA temperature ramp measurements performed at constant frequency (6.28 rad/s) and heating rate (3 0C/min) for CF/GL/PCL probes at constant CF/GL ratio: 70/30/0, 63/27/10, 49/21/30. Results 288 0 5 10 200 400 600 800 (49/21/30) (63/27/10) (55/15/30) (65/25/10) (60/30/10) (40/30/30) 2.6 2.3 2 0% PCL 10% PCL 30% PCL Toughness (kJ/m3) 1.3 Ratio CF/GL (70/30/0) Figure 5.4-22: Toughness from stress-strain curve for all CF/GL/PCL systems: (70/30/0), (65/25/10), (63/27/10), (60/30/10), (55/15/30), (49/21/30) and (40/30/30). As may be observed, higher values in E’ and especially in εmax and toughness are obtained by increasing PCL from 0 to 30%. Moreover, an increase in the CF/GL ratio yields a different behaviour depending on PCL content. Those specimens containing 10 wt. % PCL display a maximum value in E as well as a minimum value in εmax. As a result, a moderate increase in toughness takes place. It should be also taken into account that an increase in the CF/GL ratio also involves a decrease in the PCL/CF ratio. These results suggest that both ratios exert opposite effects, where the dominant effect seems to be the former at low CF content and the latter at high CF content. This balance can explain the occurrence of the maximum in E and minimum in εmax. On the other hand, an increase in PCL content up to 30 wt. % may lead to high values in E that exhibit a continuous increase with CF/GL ratio, but lead to particularly high values in εmax that become less important for the highest Results 289 CF/GL ratio studied. The latter effect is so important in this case that toughness follows the same evolution. The final decrease in εmax observed in Figure 5.4-21 and Figure 5.4-22 is probably because the specimen becomes brittle as a consequence of the high amount of polymer (CF+PCL). It is also worth mentioning that a comparison of the elastic bending modulus (E’) and the Young’s modulus (E) in Figure 5.4-17 and Figure 5.4-20, respectively, put forward the similar behaviour of both parameters. In fact, both parameters reflect the contribution of CF protein and PCL to the elastic response at small strain under bending or uniaxial tension deformations. All these results reveal that both polymers play a significant role on the elastic properties. In order to assess the contribution of each polymer (CF and PCL) a comparison between two systems containing the same polymer/GL ratio (40/30/30 and 60/30/10) may be carried out. It is apparent that the PCL yields higher contribution to the elastic response, particularly under uniaxial tensile tests. This dominant contribution of PCL can be extrapolated to the plastic deformation region since the 40/30/30 specimen shows much higher value in εmax. The toughness also put forward this effect. X-Ray Diffraction (XRD) Figure 5.4-23 shows the X-ray diffraction spectra of the reference system and systems containing 10 wt. % PCL. This figure reveals the characteristic pattern of PCL in its crystalline structure with the well-developed peaks at 2θ = 21.5 ⁰ and 23.7 ⁰ in accordance with Vertuccio et al. (2009) that also reported a shoulder peak at about 22.0 ⁰. A further peak may be also observed at 29.7 ⁰, showing much lower intensity. Results 290 10 15 20 25 30 0 50 100 150 200 250 2(Degree) Intensity (Counts) Symbol System CF/GL/PCL (70/30/0) CF/GL/PCL (65/25/10) CF/GL/PCL (63/27/10) CF/GL/PCL (60/30/10) Figure 5.4-23: XRD for the reference system (70/30/0) and for CF/GL/PCL probes at constant PCL concentration: 10 wt. % of PCL: (65/25/10), (63/27/10) and (60/30/10) As for the reference PCL-free specimen, the diffraction spectrum displays a broad peak that unfortunately is located at the same position of the two main peaks of PCL. This figure also shows how the incorporation of PCL up to 10 wt. % does not lead to any modification in the location (2ϴ value) of the PCL peaks, but a progressive broadening effect takes place for both peaks as the protein content increases. According to Ungar (2004), X-ray diffraction peaks broaden when the crystal lattice becomes imperfect. Peaks broaden either when crystallites become smaller than about a micrometre or if lattice defects are present in large enough amount and there are two important causes: size and strain broadening. Therefore, in accordance with this statement, the results obtained for composites containing 10 wt. % PCL suggest that CF protein either inhibits the development of the PCL crystalline phase or favours the presence of lattice defects. Results 291 Figure 5.4-24 shows the X-ray diffraction spectra for systems containing 30 wt. % PCL: (55/15/30), (49/21/30) and (40/30/30). 15 20 25 30 35 0 200 400 600 800 1000 1200 1400 Symbol Systen PCL CF/GL/PCL (55/15/30) CF/GL/PCL (49/21/30) CF/GL/PCL (40/30/30) Intensity (Counts) 2(Degree) Figure 5.4-24: XRD for probes at constant PCL concentration: 30 wt. % (55/15/30), (49/21/30) and (40/30/30) This figure shows how the diffraction spectra of the composites containing 30 wt. % PCL neither yield any particular modification in the location (2ϴ value) of the PCL peaks. This fact means that the interplanar distance (dhkl) also remains unaltered, and as a consequence, the lattice structure does not suffer any change, regardless of the protein content of the composite specimen. These results suggest that amorphous sections are responsible for the protein-polymer system compatibility. Conversely, it can be observed that an increase in the total amount of synthetic polymer leads to remain unaltered crystalline sections. It is also worth pointing out that the crystalline phase of the PCL is well developed for composites containing 30 wt. % PCL, which suggests that the selected processing conditions do not affect to Results 292 the PCL crystalline morphology. In addition, these layers do not exhibit any broadening effect (neither size nor strain), thus indicating that the development of PCL crystalline phase is preserved after processing, which may well be related to the improvement of mechanical properties of these composites containing high percentage of PCL. CF and CF2L protein-based bioplastic comparison In order to compare both protein systems (CF and CF2L) the ratio of protein-content to plasticiser was kept constant. Then, if the protein content of the CF concentrate is 64.2 ± 0.9 wt. %, the total amount of proteins in the bioplastic probes is about 45 wt. %. Thus, if the protein content of the CF2L system is 78.6 ± 0.9 wt. %, the ratio protein/plasticiser should be 60/40. For this reason, the systems compared were: CF/GL (70/30) and CF2L/GL (60/40), being all injection conditions for the new system that one selected from the previous study for CF protein concentrate. Dynamic mechanical analysis and tensile test were performed to characterise the mechanical response of the new bioplastic. Dynamic Mechanical analysis Figure 5.4-25 shows a comparison between systems containing different crayfish powder (CF and CF2L) at the same protein/plasticiser ratio: Results 293 -40 -20 0 20 40 60 80 100 120 140 104 105 106 107 108 109 CF2L/GL (60/40) CF/GL (70/30) E' / (Pa) Temperature (ºC) Figure 5.4-25: DMTA for the reference system CF/GL (70/30) and the system with CF2L protein concentrate CF2L/GL (60/40). As may be observed, CF-based system has higher elastic modulus in the overall temperature interval studied. However, both bio-based plastic systems (CF and CF2L), exhibit similar behaviour. The increase of temperature leads to decrease the elastic modulus, until c.a. 100 oC where certain thermosetting potential seems to appear. The dramatic decrease of elastic modulus observed for CF/GL (70/30) probes was not found for CF2L/GL (60/40) probes, however elastic modulus for CF/GL (70/30) is never below CF2L/GL (60/40) modulus. Uniaxial tensile strength Figure 5.4-26 shows the tensile strength measurements for systems CF and CF2L at the same protein/plasticiser ratio (70/30 and 60/40, respectively): Results 294 0.00 0.01 0.02 0.03 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Stress (MPa) Strain (mm/mm) CF2L/GL 60/40 CF/GL 70/30 Figure 5.4-26: Tensile test for systems CF/GL and CF2L/GL at the same protein/plasticiser ratio (70/30 and 60/40, respectively) Parameters obtained from these measurements are shown in Table 5.4-3: System Elongation at break (%) Max. stress (MPa) E (MPa) CF/GL (70/30) CF2L/GL (60/40) 1.9 ± 0.2 2.9 ± 0.3 0.60 ± 0.12 0.62 ± 0.15 62.2 ± 4.5 38.4 ± 5.1 Table 5.4-3: Parameters from tensile strength measurements for CF/GL (70/30) and CF2L/GL (60/40) systems. Results from tensile tests show that both systems have comparable maximum stress value, however CF probes are more rigid, and as consequence these probes exhibit lower elongation at break and higher Young’s modulus. Both probes have comparable parameters. Results 295 A la vida Por todos esos momentos que me has dado, Por este ciclo que no estaba programado, pero que elegí sin saber a dónde llegaría ¿Qué hubiera sido de mí sin tus enseñanzas? 297 6. Conclusions Conclusions 304 9. The highest antioxidant activity was obtained against ABTS and the lowest when FC was used, since this reagent is specific for phenol compounds. No particular influence of pH was found for DPPH or FC activity. However, pH exerts a dramatic increase on the activity against ABTS (around one decade). In general hydrolysates systems exhibit a higher antioxidant activity except against DPW,. This behaviour is remarkable for ABTS at pH 8.0 where antioxidant activity reaches twice the value for CF2L. Once again, a high degree of hydrolysis is not desirable, because antioxidant activity decreases. Conclusions 305 6.4. Conclusions from Crayfish bioplastic 10. From the experimental results, it may be concluded that monitoring the torque over mixing of protein-based flour, additives and plasticizer it is useful to select the more suitable conditions (e.g. mixing time and formulation) in terms of energy efficiency. The addition of reducing agent (BS or SS), denaturing agent (U) or crosslinking promoter (LC), always yields an increase in energy efficiency at the mixing stage, leading to a remarkable reduction in the linear viscoelastic properties of blends, which is also important to select suitable operation conditions for injection moulding processing. 11. As for bioplastics, the additive developing a greater effect on mechanical properties is LC, which provides specimens showing a higher value for the Young’s modulus, as well as a remnant thermosetting potential for further processing at high temperature. However, it is the maximum elongation the property that is remarkably enhanced by increasing the thermosetting temperature, which takes place at the expense of the Young’s modulus. The maximum stress increase only when the temperature of the mould increase. 12. The combination of techniques such as mixing rheology of CF/GL/PCL, controlling torque and temperature profiles, and DSC is very useful for selecting suitable injection moulding parameters for processing blends. 13. CF/GL/PCL biocomposites show a remarkable enhancement in mechanical properties as compared to CF/GL bioplastics, even when crystalline structure remains unaltered. Furthermore, the protein/plasticiser ratio also plays a relevant role. Thus, both polymers play a significant role on the elastic properties. However, the PCL yields a dominant contribution to the Conclusions 306 elastic response, particularly under uniaxial tensile tests, and confer a higher ability to absorb energy before rupture. 14. 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Appendix T¼Z ε max 0 sðεÞ$dε(1) Regarding the tensile properties obtained from tensile tests applied to CF/GL, and CF/GL/PCL biocomposites, Fig. 6BeD show the values of the Young's Modulus, strain at break and toughness, respectively. As may be observed, higher values in E0and especially in ε max and toughness are obtained by increasing PCL from 0 to 30%. Moreover, an increase in the CF/GL ratio yields a different behaviour depending on PCL content. Those specimens containing 10 wt.% PCL display a maximum value in E as well as a minimum value in ε max . As a result, a moderate increase in toughness takes place. It should be also taken into account that an increase in the CF/ GL ratio also involves a decrease in the PCL/CF ratio. These results suggest that both ratios exert opposite effects, where the dominant effect seems to be the former at low CF content and the latter at high CF content. This balance can explain the occurrence of the maximum in E and minimum in ε max . On the other hand, an increase in PCL content up to 30 wt.% may lead to high values in E that exhibit a continuous increase with CF/ GL ratio, but lead to particularly high values in ε max that become less important for the highest CF/GL ratio studied. The latter effect is so important in this case that toughness follows the same evolution. The final decrease in ε max observed in Fig. 6C and D is probably because the specimen becomes brittle as a consequence of the high amount of polymer (CF þPCL). It is also worth mentioning that a comparison of the elastic bending modulus (E0) and the Young's modulus (E) in Figs. 4A and 6B, respectively, put forward the similar behaviour of both parameters. In fact, both parameters reflect the contribution of CF protein and PCL to the elastic response at small strain under bending or uniaxial tension deformations. All these results reveal that both polymers play a significant role on the elastic properties. In order to assess the contribution of each polymer (CF and PCL) a comparison between two systems containing the same polymer/GL ratio (40/30/30 and 60/30/10) may be carried out. It is apparent that the PCL yields higher contribution to the elastic response, particularly under uniaxial tensile tests. This dominant contribution of PCL can be extrapolated to the plastic deformation region since the 40/30/30 specimen shows much higher value in ε max . The toughness also put forward this effect. 4. Concluding remarks From the experimental results, it may be concluded that mixing process of protein-based flour, plasticiser and synthetic polymer can be controlled by monitoring the torque and temperature profiles in order to select the most suitable mixing time. The combination of techniques such as mixing rheology and DSC are very useful for selecting suitable injection moulding parameters for processing blends. In this way, it could be claimed that a bioplastic exhibiting desirable properties can be made by means of injection moulding, using a suitable formulation (crayfish flour, glycerol and PCL) and selecting proper thermomechanical processing conditions (injection pressure, temperature and residence time in the preinjection chamber and temperature in the mould). From the mechanical characterisation of the CF/GL/PCL biocomposites, it can be pointed out the remarkable enhancement in mechanical properties obtained for PCL containing systems even when crystalline structure remains unaltered. Furthermore, as regards the biocomposite formulation, not only the contribution of PCL is important but also the protein/plasticiser ratio plays a role. Thus, both polymers play a significant role on the elastic properties, however, the PCL yields a dominant contribution to the elastic response, particularly under uniaxial tensile tests and confer a higher ability to absorb energy before rupture. Fig. 6. Results from uniaxial Tensile Strength measurements for the specimens studied 70/30/0, 65/25/10, 63/27/10, 60/30/10, 55/15/30, 49/21/30 and 40/30/30: (A) Tensile stressstrain curves (only for specimens at constant CF/GL ratio: 70/30/0, 63/27/10 and 49/21/30); (B) Young's modulus (E); (C) strain at break (Ɛ Max ); (D) Toughness (T). M. F elix et al. / Composites Part B 78 (2015) 291e297296 The present work demonstrates the feasibility of designing renewable and biodegradable composites, which may be regarded as an alternative to conventional plastic materials, containing an important amount of CF, thereby finding value-added applications of these by-products of the crayfish industry. Acknowledgements This work is part of a research project sponsored by Andalousian Government, (Spain) (project TEP-6134) and by “Ministerio de Economía y Competitividad”from Spanish Government (Ref. MAT2011-29275-C02-02/01). The authors gratefully acknowledge their financial support. The authors also acknowledge to the Microanalysis Service, X-Ray and Functional Characterisation Service (CITIUS-Universidad de Sevilla) for providing full access and assistance to the LECOeCHNSe932, D8 Discover (Bruker) equipment and DSC Q20 Calorimetry (TA instruments), respectively. References [1] Plastics_Europe. The Compelling facts about plastics. An analysis of plastics production, demand and Recovery for 2006 in Europe. EUPC (the European Plastics Converters), EPRO (European Association of Plastics Recycling and Recovery Organisations), &EuPr (the European Plastics Recyclres); 2008. [2] Ray SS, Bousmina M. Biodegradable polymers and their layered silicate nano composites: In greening the 21st century materials world. Prog Mater Sci 2005;50(8):962e1079. [3] Rosentrater KA, Otieno AW. Considerations for manufacturing bio-based plastic products. J Polym Environ 2006;14(4):335e46. [4] Tummala P, Liu WJ, Drzal LT, Mohanty AK, Misra M. Influence of plasticizers on thermal and mechanical properties and morphology of soy-based bioplastics. Industrial Eng Chem Res 2006;45(22):7491e6. [5] Averous L. Biodegradable multiphase systems based on plasticized starch: a review. J Macromol Science Polymer Rev 2004;C44(3):231e74. [6] De Graaf LA. Denaturation of proteins from a non-food perspective. J Biotechnol 2000;79(3):299e306. [7] Irissin-Mangata J, Bauduin G, Boutevin B, Gontard N. New plasticizers for wheat gluten films. Eur Polym J 2001;37(8):1533e41. [8] Koch K, Gillgren T, Stading M, Andersson R. Mechanical and structural properties of solution-cast high-amylose maize starch films. Int J Biol Macromol 2010;46(1):13e9. [9] Cuq B, Gontard N, Guilbert S. Proteins as agricultural polymers for packaging production. Cereal Chem 1998;75(1):1e9. [10] Jerez A, Partal P, Martinez I, Gallegos C, Guerrero A. Rheology and processing of gluten based bioplastics. Biochem Eng J 2005;26(2e3):131e8. [11] Zheng H, Tan ZA, Zhan YR, Huang J. Morphology and properties of soy protein plastics modified with chitin. J Appl Polym Sci 2003;90(13):3676e82. [12] Fernandez-Espada L, Bengoechea C, Cordobes F, Guerrero A. Linear viscoelasticity characterization of egg albumen/glycerol blends with applications in material moulding processes. Food Bioprod Process 2013;91(C4):319e26. [13] Arshad MU, Jamil N, Naheed N, Hasnain S. Analysis of bacterial strains from contaminated and non-contaminated sites for the production of biopolymers. Afr J Biotechnol 2007;6(9):1115e21. [14] Maiborodin IV, Beregovoi EA, Shevela AI, Kuznetsova IV, Barannik MI, Manaev AA, et al. Morphological tissue changes after the implantation of a biodegradable material on collagen basis. Morfol St Petersbg Russ 2013;144(6):63e8. [15] G omez-Martínez D, Partal P, Martínez I, Gallegos C. Gluten-based bioplastics with modified controlled-release and hydrophilic properties. Industrial Crops Prod 2013;43(0):704e10. [16] Geiger W, Alcorlo P, Baltanas A, Montes C. Impact of an introduced Crustacean on the trophic webs of Mediterranean wetlands. Biol Invasions 2005;7(1): 49e73. [17] Romero A, Cordobes F, Puppo MC, Guerreroa A, Bengoechea C. Rheology and droplet size distribution of emulsions stabilized by crayfish flour. Food Hydrocoll 2008;22(6):1033e43. [18] Romero A, Cordobes F, Guerrero A, Puppo MC. Crayfish protein isolated gels. A study of pH influence. Food Hydrocoll 2011;25(6):1490e8. [19] Sharma S, Luzinov I. Water aided fabrication of whey and albumin plastics. J Polym Environ 2012;20(3):681e9. [20] Genadios A. Proteins based films and coting. New York: CRC Press; 2002. [21] Jerez A, Partal P, Martinez I, Gallegos C, Guerrero A. Egg white-based bioplastics developed by thermomechanical processing. J Food Eng 2007;82(4): 608e17. [22] Gonzalez-Gutierrez J, Partal P, Garcia-Morales M, Gallegos C. Effect of processing on the viscoelastic, tensile and optical properties of albumen/starchbased bioplastics. Carbohydr Polym 2011;84(1):308e15. [23] Filipczak K, Wozniak M, Ulanski P, Olah L, Przybytniak G, Olkowski RM, et al. Poly (epsilon-caprolactone) biomaterial sterilized by E-beam irradiation. Macromol Biosci 2006;6(4):261e73. [24] Wu CS. Physical properties and biodegradability of maleatedpolycaprolactone/starch composite. Polym Degrad Stab 2003;80(1):127e34. [25] Iannace S, Deluca N, Nicolais L, Carfagna C, Huang SJ. Physical characterization of incompatible blends of polymethylmethacrylate and Polycaprolactone. J Appl Polym Sci 1990;41(11e12):2691e704. [26] Corradini E, Mattoso LHC, Guedes CGF, Rosa DS. Mechanical, thermal and morphological properties of poly(epsilon-caprolactone)/zein blends. Polym Adv Technol 2004;15(6):340e5. [27] Etheridge RD, Pesti GM, Foster EH. A comparison of nitrogen values obtained utilizing the Kjeldahl nitrogen and Dumas combustion methodologies (Leco CNS 2000) on samples typical of an animal nutrition analytical laboratory. Animal Feed Sci Technol 1998;73(1e2):21e8. [28] Felix M, Martin-Alfonso JE, Romero A, Guerrero A. Development of albumen/ soy biobased plastic materials processed by injection molding. J Food Eng 2014;125:7e16. [29] Martin-Alfonso JE, Felix M, Romero A, Guerrero A. Development of new albumen based biocomposites formulations by injection moulding using chitosan as physicochemical modifier additive. Compos Part B-Engineering 2014;61:275e81. [30] Fayle SE, Gerrard JA. Chemistry RSo. The maillard reaction. Royal Society of Chemistry; 2002. [31] ISO U-E. Plastics - determination of tensile properties - part 2: test conditions for moulding and extrusion plastics Switzerland. International Organization for Standardization; 2012. [32] Farahnaky A, Guerrero A, Hill SE, Mitchell JR. Physical ageing of crayfish flour at low moisture contents. J Therm Analysis Calorim 2008;93(2):595e8. [33] Strink CE. Physical aging in amorphous polymers and other materials. Elsevier; 1978. [34] Anon. Thermal characterization of polymeric materials edited by Edith a. Turi Polym Test 1997;16(5):523. [35] Hashimoto T, Suzuki T, Hagiwara T, Takai R. Study on the glass transition for several processed fish muscles and its protein fractions using differential scanning calorimetry. Fish Sci 2004;70(6):1144e52. [36] Zarate-Ramirez LS, Martinez I, Romero A, Partal P, Guerrero A. Wheat glutenbased materials plasticised with glycerol and water by thermoplastic mixing and thermomoulding. J Sci Food Agric 2011;91(4):625e33. [37] Jerez A, Partal P, Martinez I, Gallegos C, Guerrero A. Protein-based bioplastics: effect of thermo-mechanical processing. Rheol Acta 2007;46(5):711e20. [38] Romero A, Beaumal V, David-Briand E, Cordobes F, Anton M, Guerrero A. Interfacial and emulsifying behaviour of crayfish protein isolate. Lwt-Food Sci Technol 2011;44(7):1603e10. [39] Aithani D, Mohanty AK. Value-added new materials from byproduct of corn based ethanol industries: blends of plasticized corn gluten meal and poly(- epsilon-caprolactone). Industrial Eng Chem Res 2006;45(18):6147e52. [40] Vertuccio L, Gorrasi G, Sorrentino A, Vittoria V. Nano clay reinforced PCL/ starch blends obtained by high energy ball milling. Carbohydr Polym 2009;75(1):172e9. [41] Ungar T. Micro structural parameters from X-ray diffraction peak broadening. Scr Mater 2004;51(8):777e81. M. F elix et al. / Composites Part B 78 (2015) 291e297 297 679 Research Article Received: 3 February 2014 Revised: 21 April 2014 Accepted article published: 6 June 2014 Published online in Wiley Online Library: 25 June 2014 (wileyonlinelibrary.com) DOI 10.1002/jsfa.6747 Development of crayfish bio-based plastic materials processed by small-scale injection moulding Manuel Felix,*Alberto Romero, Felipe Cordobes and Antonio Guerrero Abstract BACKGROUND: Protein has been investigated as a source for biodegradable polymeric materials. This work evaluates the development of plastic materials based on crayfish and glycerol blends, processed by injection moulding, as a fully biodegradable alternative to conventional polymer-based plastics. The effect of different additives, namely sodium sulfite or bisulfite as reducing agents, urea as denaturing agent and L-cysteine as cross-linking agent, is also analysed. RESULTS: The incorporation of any additive always yields an increase in energy efficiency at the mixing stage, but its effect on the mechanical properties of the bioplastics is not so clear, and even dampened. The additive developing a greater effect is L-cysteine, showing higher Young’s modulus values and exhibiting a remnant thermosetting potential. Thus, processing at higher temperature yields a remarkable increase in extensibility. CONCLUSION: This work illustrates the feasibility of crayfish-based green biodegradable plastics, thereby contributing to the search for potential value-added applications for this by-product. © 2014 Society of Chemical Industry Keywords: bioplastic; crayfish protein; mixing blends; rheology; tensile strength test INTRODUCTION The freshwater red-swamp crayfish (Procambarus clarkii)was introduced into Europe in the early 1960s. Since then, this species has undergone a fast widespread growth due to its resistance to fungal disease as well as to favourable weather conditions, abundant food and a lack of predators.1This fact has driven the development of a strong local crayfish industry at the marshes of the Guadalquivir River in Spain. This development has also led to the generation of a large amount of crayfish surpluses, as is frequently the case in the fish and shellfish industry.2According to recent estimations by the Food and Agriculture Organization the total world output of fish represented 156 million tonnes in 2011. Unfortunately, a high proportion of this amount is not eventually used for human food consumption. Thus, according to the USDA, up to 45% of fish and shellfish that enters the USA retail food market is not eaten and, as a consequence, ends as wastes. Therefore, a search for any value-added application to these surpluses is becoming a real priority. With regard to crayfish surpluses, some applications based on the functional properties of crayfish protein fraction have previously been assessed. Thus, interfacial properties of crayfish protein isolate (CFPI) have been studied3–5in order to address the good performance of crayfish protein in emulsion stabilisation.6,7 Thermal properties of crayfish flour8and CFPI in aqueous solution have also been studied in order to assess thermally induced enhancement of emulsion stability9as well as the potential of crayfish in the manufacture of surimi-like products based on its ability to form a gel.10,11 A currently attractive way to valorise these by-products is through their use as renewable resources in the manufacture of ‘green materials’, replacing difficult-to-degrade plastic materials made from oil-based synthetic polymers. Nowadays, some important applications for bioplastics are beginning to emerge in the areas of packaging, food production, pharmaceutics, electronics, automotive industry and biomedicine. Thus, among other applications bioplastics can be used in food packaging, fruit coating, encapsulation, textiles, absorbent materials or tissue engineering.12–14 This wide variety of potential applications allows us to envisage an increasing use of biobased-plastic materials. Thus, according to European Bioplastics, the production capacity for bioplastics is predicted to increase from approximately 700 000 tons in 2010 to 1.7 Mtons by 2015.12 However, like other bio-based innovations, bioplastics have struggled to achieve market share such that, at present, bioplastics constitute less than 0.5% of world plastics consumption.15 Therefore, it is still necessary to intensify the efforts in research and development, and in innovation in this field. Some biopolymers can directly replace synthetically derived materials in traditional applications or they simply possess unique ∗Correspondence to: Manuel Felix, Departamento de Ingeniería Química, Universidad de Sevilla, Facultad de Química, 41012 Seville, Spain. E-mail: [email protected] Departamento de Ingeniería Química, Universidad de Sevilla, Facultad de Química, 41012 Seville, Spain J Sci Food Agric 2015; 95: 679–687 www.soci.org © 2014 Society of Chemical Industry 680 www.soci.org M Felix et al. properties that could open up a range of new commercial opportunities. In any case, it is essential that bio-based materials exhibit suitable physico-chemical properties. In this sense, cellulose, starch, polysaccharides and protein have become increasingly competitive in recent years as substitutes for petrochemicals, in view of the increase in oil production costs. Both environmental and economic factors are expected to entail the development of new plastic materials such as those using by-products with high protein content.16 In this respect, proteins are exceptionally versatile materials, both in the sources from which they can be obtained and in the wide variety of possible modifications, which can be helpful in tailoring their properties to the particular requirements of a specific application. Proteins present significant advantages in that they are derived from a sustainable resource and can be processed in much the same way as conventional synthetic polymers.13 However, proteins are generally mixed with a plasticiser in order to reduce intermolecular forces among polymer chains, increasing mobility and reducing the glass transition.17 Traditionally, protein films18–20 are processed by a casting method; however, classical polymer processing techniques (compression moulding or extrusion) are being increasingly used in this field.21–24 Among them, injection moulding is a fairly attractive operation that has not received due attention yet. Thus, studies on protein-based biodegradable polymeric materials processed by injection moulding are scarce.25,26 Typically in this process, polymeric materials are subjected to suitable thermal conditions, being injected at high pressure into the mould cavity. Optimisation of processing conditions is essential to achieve the desire properties of the final product. This is particularly relevant in protein-based materials that require thermoplastic mixing with a proper plasticiser but benefit from a predominant thermoset character upon injection moulding.26 Besides, additives such as reducing agents may be helpful in order to reduce the average molecular weight of protein aggregates, thus facilitating both mixing and moulding processes. Thus, some authors studied the influence of reducing agents on the properties of thermo-moulded wheat gluten bioplastics,27 soy protein isolate,28–30 starch31 or flax fibres.32 The effect of adding a denaturing agent, such as urea has also been analysed on bioplastics processed by extrusion33 or compression moulding.34–36 As for the use of L-cysteine as cross-linking agent, Sun et al.37 have recently evaluated its performance on thermo-moulded gluten-based plastics. However, no information about crayfish-based bioplastics with or without reducing, denaturing or cross-linker agents has been found. The overall objective is to evaluate the potential development of plasticised crayfish bio-based plastic materials by means of a conventional and highly versatile polymer processing technique, such as injection moulding, as an alternative to moulded materials based on polymers derived from fossil fuels. A further objective is to analyse the effect of using different additives on the properties of crayfish-based products, using glycerol as the plasticiser. The additives assessed in this study were sodium sulfite or bisulfite as reducing agents, urea as denaturing agent and L-cysteine as cross-linking agent. A small-scale plunger-type injection moulding machine was used in this study to obtain crayfish-based specimens from crayfish/glycerol/additive blends, previously mixed by means of a mixing rheometer that allows the torque and temperature to be recorded during mixing. Rheological and differential scanning calorimetry measurements of these blends were also carried out in order to obtain information that may be used in the selection of suitable processing parameters for injection moulding operations (e.g. temperature and residence time in the pre-injection cylinder as well as the temperature of the mould). EXPERIMENTAL Materials Crayfish flour was obtained from ALFOCAN S.A. (Isla Mayor, Seville, Spain). The protein content of the crayfish flour, determined in quadruplicate as % N ×6.25 using a LECO CHNS-932 nitrogen micro analyser (Leco Corporation, St Joseph, MI, USA), was 642 ±9gkg −1. Glycerol (used as plasticiser) and additives (sodium sulfite, sodium bisulfite, urea and L-cysteine) were purchased from Panreac Química, S.A. (Barcelona, Spain). Free sulfhydryl groups Free sulfhydryl groups of protein samples were determined using the method developed by Beveridge et al.38 Samples were suspended (10 g L−1) in buffer containing 0.086 mol L−1 Tris–HCl, 0.09 mol L−1glycine, 4 mmol L−1EDTA and 8 mol L−1 urea, at pH 8. Dispersions were stirred at 25 ∘C during 10 min at 500 rpm in a thermomixer and then centrifuged at 15 000 ×g (10 min, 10 ∘C). Supernatant was incubated with Ellman’s reagent [5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB); 4 g DTNB L−1 methanol]. Absorbance at 412 nm was measured in a Genesis-20 spectrophotometer (Thermo Scientific, Waltham, MA, USA). The molar extinction coefficient of 3-thio-6-nitrobenzoate (TNB; 13 600 M−1cm−1) was used. Protein concentration of extracts was determined by the Bradford method. Sample preparation Blends with different crayfish flour/glycerol (CF/GL) ratios were manufactured by a thermomechanical procedure which consisted of two stages. Firstly, selected blends containing 700 g kg−1crayfish flour and 300 g kg−1glycerol (denoted 70/30) were mixed in a two-blade counter-rotating batch mixer Haake Polylab QC (ThermoHaake, Karlsruhe, Germany) at 25 ∘Cand50rpmfor60 or 20 min, monitoring the torque and temperature during mixing. Secondly, the dough-like materials obtained after mixing were subsequently processed by injection moulding using a MiniJet Piston Injection Molding System II (ThermoHaake) to obtain bioplastic specimens. Two moulds were used to prepare two type of specimens: (1) a 60 ×10 ×1 mm rectangular-shaped specimen, to be used for both dynamic mechanical temperature analysis (DMTA) experiments and transparency measurements, and (2) a dumb-bell-type specimen defined by ISO 527–2:1993 for determining the tensile properties of plastics. Characterisation of blends The most suitable processing variables such as temperatures in the pre-injection cylinder or in the mould were selected after performing temperature ramp tests and differential scanning calorimetry measurements (results not shown). Rheological measurements Dough-like materials were characterised by small amplitude oscillatory shear measurements, using a controlled-strain rheometer, in order to select the optimum conditions for injection moulding: (Mars II from Haake, Karlsruhe, Germany). The geometry used has been a plate and plate geometry (diameter, 25 mm) with a rough surface and a gap between plates of 1 mm. Low viscosity wileyonlinelibrary.com/jsfa © 2014 Society of Chemical Industry J Sci Food Agric 2015; 95: 679–687 681 Crayfish bio-based plastic materials processed by injection moulding www.soci.org Dow Corning 200 fluid was used as sealant to avoid sample drying. Strain sweep small amplitude oscillatory shear tests were also performed in order to establish the linear visco-elasticity range. Temperature ramp tests were carried out at 5 ∘Cmin −1from 20 to 100 ∘C and time sweep tests were performed for 1800 s at a selected constant temperature. Linear visco-elastic properties (G′, G′′) were monitored at a constant frequency of 6.28 rad s−1.Allthe systems studied had the same thermo-rheological history before performing any rheological test. Characterisation of bioplastics Dynamic mechanical temperature analysis DMTA tests were carried out with a RSA3 (TA Instruments, New Castle, DE, USA), on rectangular probes using dual cantilever bending. All the experiments were carried out at constant frequency (6.28 rad s−1) and strain (between 0.01 and 0.3%, within the linear visco-elastic region). The selected heating rate was 3 ∘Cmin −1.All the samples were coated with Dow Corning high vacuum grease to avoid water loss. Tensile strength measurements Tensile tests were performed by using an Insight 10 kN Electromechanical Testing System (MTS, Eden Prairie, MN, USA), according to ISO 527–2:1993 for tensile properties of plastics. Tensile stress and elongation at break were evaluated from at least three duplicates for each product using type IV probes and an extensional rate of 100 mm min−1at room temperature. Statistical analysis At least three replicates of each measurement were carried out. Statistical analyses were performed using a t-test and one-way analysis of variance (ANOVA, P<0.05) by using the statistical package SPSS 18 (SPSS, Chicago, IL, USA). Standard deviations from some selected parameters were calculated. RESULTS AND DISCUSSION Thermoplastic mixing of blends Figure 1 shows both torque and temperature profiles as a function of mixing time for blends obtained at different CF/GL ratios, as well as their visual appearance after mixing. These results show the remarkable dependence of these parameters on the CF/GL ratio. Thus, a rapid increase in torque up to a maximum value takes place for the system having less amount of plasticiser, denoted as 80/20, which represents the CF/GL weight ratio. This evolution is readily followed by an asymptotic decrease towards a plateau value. In contrast, the system 70/30 shows a moderate growth in torque showing no maximum value but a slow tendency to a plateau value. The profile of the system 60/40 only shows an initial increase in torque, although very slight, being dominated by a constant torque value over mixing time. In general, the evolution of temperature over time is very similar to the torque profile where an increase takes place excepting for the lowest CF/GL ratio. Both increases in torque and temperature may be attributed to shear-induced exothermic reactions, by S—S bonds formation, developed during the mixing process. As a consequence of the above-mentioned differences in torque profile, the specific mechanical energy (SME) input for mixing is also quite different. This characteristic parameter of mixing may be defined as follows:39 SME =𝜔 m∫tmix 0 M(t)dt(1) where 𝜔(in rad s−1) is the mixing speed, m(in g) is the sample mass, M(t)(inNm)isthetorqueandtmix (in s) is the mixing time. The values of the SME parameter for these three systems are included in Fig. 1. A remarkable increase in this parameter can be observed with the CF/GL ratio. This effect takes also place at the higher concentrations in spite of the fact that the plateau torque values are relatively close to each other. From the visual appearance observed in Fig. 1, system 80/20 does not seem to contain enough amount of plasticiser to obtain an easy-to-handle material, giving rise to a granulated powder instead of a homogeneous dough-like blend. In fact, this is the system displaying the highest torque values, which correspond to the highest rheological consistency. On the other hand, the highest amount of glycerol in the 60/40 system provides a neat dough-like appearance to the blend. This is also consistent with the lowest torque values obtained (associated with a lower consistency) after the mixing process. The 70/30 system shows intermediate behaviour between both limits, although it is closer to the most concentrated protein-based blend. In the present study, the 80/20 system has been discarded because of the high energy Figure 1. Mixing torque and temperature profiles and images for crayfish flour/glycerol (CF/GL) systems (80/20, 70/30 and 60/40). J Sci Food Agric 2015; 95: 679–687 © 2014 Society of Chemical Industry wileyonlinelibrary.com/jsfa 682 www.soci.org M Felix et al. AB Figure 2. Evolution of mixing torque (A) and temperature (B) as a function of mixing time or additive-containing system (sodium sulfite, urea and L-cysteine). SS, sodium sulfite; U, urea; LC, L-cysteine. input required for mixing. Moreover, this system has proven to be difficult to inject (results not shown). On the other hand, systems containing a low protein/glycerol ratio may be injected easily but the final bioplastic materials tend to exude the excess glycerol. Exudation is a well-known phenomenon described in the polymer–plasticiser literature and should be avoided in order to prevent contamination of the surrounding materials (e.g. in food packaging).40 This seems to be the case for the 60/40 system. As a result, the 70/30 system was selected as the most suitable to obtain a homogeneous and easily injectable CF/GL blend. Figure 2 shows the evolution of torque (Fig. 2A) and temperature (Fig. 2B) as a function of time for different 70/30 CF/GL blends prepared using sodium sulfite as reducing agent, urea as denaturing agent, or L-cysteine as cross-linking promoter at different concentrations (3 and 30 g kg−1protein for sodium sulfite and urea or 1 and 3 g kg−1for L-cysteine). These results generally show that any of the additives used induces an anticipation of the torque profile, particularly at the highest additive concentrations. No differences in the torque profile are noticed at the two different concentrations of sodium bisulfite (data not shown). The highest amount of additive (corresponding to sodium sulfite or urea) also yields lower torque plateau values, which may be considered beneficial for the processability of blends, for example under injection moulding. Temperature–time profiles show the same type of evolution for all the additives and concentrations in spite of the fact that an air stream was used as a cooling fluid. No differences are found in the final values for the highest sodium sulfite or urea content in this case. The only difference found between temperature and torque profiles is that the former undergo a further delay in time, which may be related to the air-cooling effect. It is interesting to notice that although all the additives in Fig. 2 led to an apparent anticipation in torque and temperature profiles, different mechanisms are involved depending on the additive used. Urea is a widely used denaturing agent that induces disruption of physical interactions among protein molecules. On the other hand sodium sulfite, acting as a reducing agent, induces disruption of S—S bonds. As a consequence, protein–plasticiser mixing is favoured in both cases resulting in a faster kinetics over the additive-free system. L-Cysteine is an amino acid that provides a free sulfhydryl group/molecule and therefore facilitates formation of S—S bonds with protein segments to form cystine groups.41 In this way, L-cysteine initiates the formation of exothermic S—S bonds, progressively increasing the temperature, which leads to anticipation of both torque and temperature profiles. In fact, this additive gives rise to the fastest anticipation for the same additive content. In addition, the highest temperatures reached by the L-cysteine-containing blend suggest the occurrence of a higher cross-linking degree as compared to the other systems. It may be also noted that the system is not still properly mixed at times shorter than that that corresponding to the maximum torque. Therefore, as a general rule, the range of mixing times for subsequent blend processing is selected once the plateau torque value is reached such that a good degree of homogeneity is assured. Another interesting parameter to compare different mixing processes is the relative energy input (REI), which is defined as follows: REI (%)=SMEmin (a,c) SMEmin (0)×100 (2) where SMEmin(0) and SMEmin(a,c) are the SME values at which the torque reaches 95% of the maximum value for the profile of the additive free blend and additive aat concentration c, respectively. Therefore, REI is an index of the reduction in energy input relative to that that required for mixing the additive free blend. The values of SME20 and REI parameters for all these additives are shown in Fig. 3A and B, respectively. The SME values for sodium bisulfite are also included in this figure. SME20 is the specific mechanical energy supplied, as defined in Eqn (1), after mixing for 20 min. As may be observed in Fig. 3, an increase in additive concentration always leads to an apparent increase in the parameter SME20, as a consequence of the above-mentioned anticipation of the torque profile. If the same additive concentration is used, this increase is particularly remarkable for L-cysteine and, in any case, is only moderate when sodium bisulfite is used. The results obtained with parameter REI confirm the relevant effect of the addition of L-cysteine at constant additive concentration. Thus, estimation tendencies from the results obtained indicate that the additive/protein ratio required to achieve a 50% REI would be 33 g kg−1for urea and 27 g kg−1for sodium sulfite, whereas a ratio as low as 4.4 g kg−1wouldberequiredbyusing L-cysteine. wileyonlinelibrary.com/jsfa © 2014 Society of Chemical Industry J Sci Food Agric 2015; 95: 679–687 683 Crayfish bio-based plastic materials processed by injection moulding www.soci.org A B Figure 3. (A) Specific mechanical energy (SME) after mixing for 20 min and (B) reduction in energy input (REI) for different crayfish flour/glycerol (CF/GL) blends (additive-free, sodium bisulfite, sodium sulfite, urea or L-cysteine). BS, sodium bisulfite; SS, sodium sulfite; U, urea; LC, L-cysteine. Temperature ramps Figure 4 shows the dependence of linear visco-elastic functions on temperature for different blends containing 3 g additive kg−1 crayfish protein. The additive free blend is also displayed in this figure. All the additives studied induce a reduction in G′and G′′ in the experimental temperature range. As for tan 𝛿profiles, all of them show a similar elastic-dominant behaviour characterised by the presence of a broad distribution showing a maximum value. This maximum has been also found at 70 ∘C by differential scanning calorimetry measurements for the CF/GL system (results not shown), being associated to a glass-like transition of the protein fraction. Most of additive-containing blends show this smooth thermal induced glass transition in the same temperature range than the additive free system (70–83 ∘C).However,ashift in this event towards higher values (80–87 ∘C) takes place when L-cysteine is used as additive. It should be mentioned that the cooling–setting stage after mixing leads to noticeable differences between the visco-elastic properties of additive-free and additive-containing CF/GL blends. Once again, the additive showing the poorest effect is sodium bisulfite. However, the cooling–setting stage does not induce any apparent difference in G′or G′′ among the other three additives (Fig. 4A), in spite of the fact that the expected mechanism would be different. Thus, sodium sulfite is expected to reduce the visco-elastic properties of CF/GL blends by breaking some disulfide bonds, although the effect does not seem take place at a high extent. A similar mechanism should explain the behaviour found for sodium bisulfite-containing blends, which becomes more important above 60 ∘C. In contrast, urea may show a double effect. Firstly, as reported by Verbeek and Van den Berg,42 urea may play the role of a plasticiser at a concentration as low as that used in this study, not showing any cross-linking effect. Secondly, urea, as a denaturing agent, tends to inhibit the recovery of physical interactions over the cooling–setting stage. In fact, the latter effect seems to be more relevant than the former, as no differences in the tan 𝛿peak can be detected in Fig. 4B. According to this figure, the L-cysteine-containing blend is the only one showing the tan 𝛿peak at higher temperature than the additive-free system. This effect may be related to a higher cross-linking extent, as previously mentioned (Fig. 2B). However, this higher degree of cross-linking does not involve any increase in G′and G′′.On the contrary, the results are similar to those shown by ureaand sodium sulfite-containing blends. An explanation for this effect should be related to the inhibition of physical interactions development over the cooling–setting stage. In any case, the addition of any of the additives used leads to a potential enhancement of the moulding processability of CF/GL blends, where rheological properties are regarded as the key AB Figure 4. (A) Storage modulus (G′) and loss modulus (G′′), and (B) loss tangent (tan 𝛿), at 1 Hz and 5 ∘Cmin −1, as a function of temperature for different crayfish flour/glycerol (CF/GL) blends (additive-free or 3 g kg−1sodium bisulfite, sodium sulfite, urea or L-cysteine). BS, sodium bisulfite; SS, sodium sulfite; U, urea; LC, L-cysteine. J Sci Food Agric 2015; 95: 679–687 © 2014 Society of Chemical Industry wileyonlinelibrary.com/jsfa 684 www.soci.org M Felix et al. Table 1. Standard values of injection moulding parameters for the pre-injection cylinder, injection and packing stage Parameter T (∘C) Pressure (MPa) Time (s) Pre-injection cylinder 60 0.1 100a Injection 60–100 0.1–50 <1 Packing stage 100 or 130 50 20 100 or 130 20 200a aFor a system containing L-cysteine, 130 ∘C and 600 s were also used. factor. In other words, additives contribute to facilitate injection of CF-based blends. Similar results were found by Pallos et al.43 for thermoformed wheat gluten modified by reducing agents and additives. Injection moulding process Table 1 shows the conditions selected for the injection moulding process for each of the blends studied. Processing parameters for the pre-injection cylinder are selected taking into account that the elastic properties of CF/GL blends should be reduced to some extent (typically G′should be in the order of 1 ×106Pa or below). On the other hand, the temperature should not be increased excessively in order to prevent thermally induced protein cross-linking effects before the injection stage. For the same reason, the residence time in the cylinder should not be long. Under such premises the parameters selected for the first stage have been 60 ∘C and 100 s. As for the mould processing conditions, the temperature has to be high enough to ensure the normal development of thermal and pressure-induced protein cross-linking reactions. On the other hand, exposure to high temperatures for a long time typically leads to protein degradation (i.e. via Maillard-type reactions). Therefore, 100 ∘C and 20 s have been selected as the moulding temperature and time, respectively. The injection pressure selected in this case has been 50 MPa. Once the blend has been injected into the mould, a further stage is performed at the same temperature for a residence time long enough (10 s) to allow for the development of protein cross-linking to achieve the final network structure. The residence time selected has been 200 s since no further enhancement has been noted by increasing this period. Mechanical characterisation of bioplastics Dynamic mechanical temperature analysis Figure 5 shows the values of the elastic modulus, E′, as a function of temperature (from −30 ∘C to 140∘) obtained from dynamic mechanical analysis (DMA) measurements for additive-free CF/GL specimens and CF/GL probes containing 3 g kg−1additive. Figure 5A compares the influence of different additives (sodium bisulfite, sodium sulfite, urea and L-cysteine) for specimens moulded at 100 ∘C for 200 s, whereas Fig. 5B shows the effect of different moulding conditions for L-cysteine-containing specimens. As may be observed in Fig. 5A, all the specimens show similar profiles for E′andalsoforE′′ (data not shown), undergoing a remarkable decrease with increasing temperature. The decrease tends to reach a plateau value at high temperature. In some cases the E′profile eventually evolves to an increase in value, indicating a certain thermosetting potential. DMA profiles for the additive-free, urea and sodium bisulfite specimens do not display any significant difference, in spite of the effect observed on the visco-elastic properties of their corresponding blends (Fig. 4A). Sodium sulfite-containing specimens show lower E′values at low temperature but higher values in the high temperature region as compared to the reference (additive-free) system. This system also evolves in a more gradual way than the other bioplastic specimens, particularly at high temperature, showing no thermosetting potential region. This behaviour may be associated with the ability of sodium sulfite to impair disulfide bridges. Thus, the results obtained after application of the procedure devised by Beveridge et al.38 indicate that the concentration of free sulfhydryl groups in crayfish flour is increased from 19 ±3 mmol kg−1to 300 ±17 mmol kg−1after addition of 3 g sodium sulfite kg−1 protein. As for L-cysteine specimens, they show a rather similar DMA profile at low and medium temperature but there is also an apparent increase in E′at high temperature, which reveals that this system still exhibits a marked remnant thermosetting potential for further processing. AB Figure 5. Storage modulus (E′) values from dynamic mechanical analysis (DMA) temperature ramp measurements carried out at 1 Hz and 3 ∘Cmin −1 for different crayfish flour/glycerol (CF/GL) systems: (A) additive-containing (sodium bisulfite, sodium sulfite, urea or L-cysteine) specimens; and (B) L-cysteine-containing specimens processed at different moulding temperature (100 or 130 ∘C) and packing time (200 or 600 s). The additive-free CF/GL is included as a reference. BS, sodium bisulfite; SS, sodium sulfite; U, urea; LC, L-cysteine. wileyonlinelibrary.com/jsfa © 2014 Society of Chemical Industry J Sci Food Agric 2015; 95: 679–687 685 Crayfish bio-based plastic materials processed by injection moulding www.soci.org This increase may be a consequence of different cross-linking reactions involving S—S bond formation, SH–SS interchange44 and also non-disulfide bonds. Thus, Rombouts et al.45 reported a heat-induced reduction in 𝜖-amino groups, being most likely the result of isopeptide bond formation in combination with Maillard and/or other heat-induced reactions. These authors also found that lysineand glutamine-containing peptides from high molecular weight glutenin sub-units also induced the formation of isopeptide bonds, thereby demonstrating that cross-linking did not solely depend on the availability of cysteine or cystine residues. In this sense, it is interesting to note that crayfish protein is rich in the amino acids glutamine and lysine.46 In order to explore this potential L-cysteine-containing specimens were processed using different moulding conditions over the packing stage, increasing either the packing time in the mould at 100 ∘C and 20 MPa (up to 600 s) or the moulding temperature at 20 MPa over 200 s (up to 130 ∘C). DMA results for these new L-cysteine-containing specimens are shown in Fig. 5B. As may be observed, the increase in moulding time does not lead to any noticeable change in the DMA profile, which suggests that the time selected for the former specimens is long enough to complete the cross-linking stage. On the other hand, an increase in moulding temperature leads to a plateau value for E′in the high-temperature region. However, this change in moulding conditions does not drive any particular enhancement in the visco-elastic bending properties below 60 ∘C. This behaviour is somehow unexpected since the occurrence of the above-mentioned thermosetting potential typically involves an enhancement of mechanical properties.21,47,48 A possible explanation for this lack of enhancement may be found in the fact that some degradation of specimens has been observed at 130 ∘Cand above. Probably, this degradation is related to other components (e.g. lipids content) rather than protein but it may produce some alterations of mechanical properties. Thus, using a defatted crayfish protein concentrate instead of crayfish flour would be a better choice in order to process at higher temperatures. Measurements of the uniaxial tensile strength Figure 6A displays the results of stress–strain curves obtained from tensile strength measurements for additive-free and additive-containing specimens at an additive/CF ratio. All the curves exhibit a similar behaviour which consist of an initial linear elastic behaviour of high constant stress–strain slope yielding high values for the Young’s modulus (E), followed by a deformation stage with a continuous decrease in the stress–strain slope. A second constant slope is reached at the end of the plastic deformation stage. All the curves eventually reach a maximum value for the stress (𝜎max) and the strain at break (𝜖max). Only L-cysteine leads to an apparent enhancement of the tensile strength profile, also leading to a slightly shorter strain value. The values of these parameters (E,𝜎max and 𝜖max) and their corresponding standard deviations are plotted in Fig. 6B for the additive-free and additive-containing specimens at 3 g kg−1additive/CF ratio. This figure indicates once again that additive L-cysteine is the only one that improves parameters Eand 𝜎max over the additive-free system, under the same processing conditions. On the other hand L-cysteine-added specimens exhibit lower values for 𝜖max.The remainder of the additives lead to similar or even lower values of the three parameters. Figure 6B also shows the values for L-cysteine-added specimens moulded at longer time or higher temperature. As may be observed, an increase in the packing time from 200 s up to 600 s does not yield any noticeable change in tensile parameters. This result indicates that after 200 s packing time the sprue is already closed by the solidified blend. On the other hand, an increase in the mould temperature leads to remarkable changes in tensile parameters. Thus, the maximum stress and, above all, the strain at break undergo an apparent increase in value (approx. 12% and 70%, respectively), whereas the Young’s modulus clearly decreases (approx. 30%) by increasing the mould temperature from 100 to 130 ∘C. Interestingly, the maximum elongation is the property that undergoes the most remarkable enhancement by favouring heat-induced cross-linking. In this way, the material exhibits greater toughness, in spite of being less strong. In fact, as stated by Lagrain et al.,49 increasing the elongation at break of glassy, amorphous polymers typically goes at the expense of the elastic modulus. This compensation may also affect the bending elastic properties of the bioplastic, thus explaining the small dependence of DMA profiles on moulding temperature. Moreover, this behaviour is similar to that found for other elastomeric materials such as rubber-based blends50 and is consistent with the results from DMA measurements that show an extension of the rubbery plateau obtained at high temperature. AB Figure 6. Results from tensile strength measurements for different crayfish flour/glycerol (CF/GL) systems: additive-free or 3 g kg−1additive-containing specimens (sodium bisulfite, sodium sulfite, urea or L-cysteine). (A) Stress--strain curves, and (B) parameters from tensile strength measurements: maximum stress (𝜎max), strain at break (𝜖max) and Young’s modulus (E). BS, sodium bisulfite; SS, sodium sulfite; U, urea; LC, L-cysteine. J Sci Food Agric 2015; 95: 679–687 © 2014 Society of Chemical Industry wileyonlinelibrary.com/jsfa 686 www.soci.org M Felix et al. CONCLUSIONS From the experimental results, it may be concluded that monitoring the torque over mixing of protein-based flour, additives and plasticiser it is useful to select the more suitable conditions (e.g. mixing time and formulation) in terms of energy efficiency. Characterisation of the rheological properties of blends (particularly their dependence on temperature) is also important to select suitable operation conditions for injection moulding processing. The addition of reducing agent (sodium bisulfite or sodium sulfite), denaturing agent (urea) or cross-linking promoter (L-cysteine), always yields an increase in energy efficiency (i.e. a decrease in REI value) at the mixing stage, leading to a remarkable reduction in the linear visco-elastic properties of blends. In contrast, the effect of the additives on the mechanical properties of the final bioplastic material is not so clear, particularly for the results from DMA measurements. The additive developing a greater effect is L-cysteine, which provides specimens showing a higher value for the Young’s modulus, as well as a remnant thermosetting potential for further processing at high temperature. This potential typically involves an enhancement of mechanical properties.21,47,48,51 However, in this study the maximum elongation is the property that is remarkably enhanced by increasing the thermosetting temperature. This moulding temperature-driven enhancement is clearly at the expense of the Young’s modulus, such that the effect on the bending elastic modulus is dampened. The present work puts forward the feasibility of developing crayfish-based green biodegradable plastics, thereby finding potential value-added applications for this protein concentrate by-product. However, much research is still needed in order to further explore the potential of crayfish in this field. Thus, from the results obtained, it seems to be an advisable strategy to investigate the possible synergetic effects resulting from the combination of the different additives studied. This combination could be studied by incorporating each additive at the mixing stage, according to the following sequence: (1) urea, to facilitate disruption of physical interactions; (2) sodium sulfite, to induce breakage of disulfide bonds; and (3) L-cysteine to anticipate and improve mixing efficiency and to promote subsequent cross-linking at the moulding stage. ACKNOWLEDGEMENTS This work is part of a research project sponsored by Andalusian Government (Spain) (project TEP-6134) and by the ‘Ministerio de Economía y Competitividad’ from Spanish Government (Ref. MAT2011-29275-C02-02). The authors gratefully acknowledge their financial support. The authors also acknowledge the Microanalysis Service (CITIUS-Universidad de Sevilla) for providing full access and assistance with the LECO-CHNS-932 equipment. 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