Chapter 6 Indentation Technique: Overview and Applications in Food Science Israel Arzate-Vazquez, Jorge Chanona-Pérez, Germin A. Rodriguez-Castro, Ariel Fuerte-Hernindez, Juan V, Méndez-Méndez and Gustavo F, Gutiérrez-Lipez 6.1 Indentation Technique 6.1.1 Definition Indentation technique or instrumented indentation testing (IIT) involves the appli cation of force with a known geometry indenter (tip) on the surface of a material; during the test the force and the depth of the tip into the material surface are con- tantly monitored (Fischer-Cripps, 2006b). Currently, indentation instruments can control the maximal load applied and the depth of the indenter on the surface. The indentation technique is mainly used for evaluation of mechanical properties such as hardness and elastic modulus, but also can include studies of the fracture resis tance of brittle materials, mechanical mapping, dislocation behaviors, yield strength and residual stresses, oblaining information about plastic and elastie deformation, viscoelastic behavior, or performance of time-dependent materials (e.g, polymers and biological materials: Fischer-Cripps 2006b: Lueca etal. 2010). This technique is very versatile and has some advantages which are mentioned below: |. Arzate-Vizquez (5) J. V. Méndez Méndez Centro de Nanociencias y Micro y Nanotecnologias, Instituto Poligenico Nacional, Lie Enrique Emo s/n, Unidad Profesional Adolfo Lipez Mateos, Cal. Zacateneo CP.OT738 México, DF, Mexico e-mail iarzate@ipn my, alext=26@yahoo.com.mx 41. Chanona-Pétee» GF, Gutéree-Lopee Departamento de Ingeniria Biogumica, Escucla Nacional de Ciencias Bioliicas, Insttto Politéenico Nacional, Carpio y Plan de Ayala sn, Col. Santo Tams, CP. 11340 México, DF, Mexico G.A. Rodriguez-Casto “A, Fuert-Hernindez Inattato Politéenico Nacional, SEPI-ESIME, Unidad Profesional Adolf Liner Mateos, Col. Zacatenco, C.P, 07738 México, DF, Mexico ‘© Springer Sciences Business Media New York 2015 81 H, Hemandez-Sinchez, GF. Gubirez-Loper (eds), Food Nanoscience and Nanotechnology, Food Engincering Series, DOI 101007/978-3-319-13596-0.62 I. Arzate-Véequez et al ‘+ Iisa quick and nondestructive test. + The sample size is small ‘+ Itis possible to evaluate mechanical properties of coatings and thin films of mi cro- and nanometric thickness. ‘+ Measurement is local, allowing the characterization of phases present in the ma- terial ‘+ Mechanical mappings can be performed along the samples. + Sample preparation is minimal and in the ease of metallic materials i is required that the samples are polished just asin the case of bone samples. Due to the importance and increasing use of the indentation technique in materi- als science it was necessary to develop international standards; an example is the international standard ISO 14577 “Metallic materials-instrumented indentation test for hardness and materials parameters,” which was published in ISO 2002. The standard consist of three parts, where the part | refers tothe test method, part 2 to the verification procedures and calibration of testing machines, and part 3 is the calibration of reference blocks. ISO 14577-1 establishes ranges of indentation technique based on the maximum load applied and the maximum depth of pen- etiation (Table 61). Subsequently, the Part 4 of the standard was developed that is aimed specifically atthe indentation of coatings and thin films, this was called ISO 14577-4 “Test method for metallic and nonmetalic coatings ISO 2007.” The development of ISO 14577-4 is currently of great importance because it provides details to consider when applying the indentation technique to characterize coatings and thin films, which are manufactured using nanotechnology tools (e.g., chemical ‘vapor deposition CVD). "As mentioned above, there are great advances inthe application ofthe technique in metallic materials, however recently in biological materials the use of technique is ineeasing and therefore it is necessary to establish protocols and international standards to consider specifi issues in these samples that nre mare complex dc to its muliphasie composition and heterogeneous microstructure. ‘There area wide varity of indentation instruments with different features inthe ‘market, typically the monitoring of displacement of indenter is done by capacitance or inductance method, while the generation force is done by electrostatic force, magnetic coils, or by piezoelectric element (Ebenstein and Pruitt 2006). ndentation instruments ae capable of applying load in the order of N to wN which include the three ranges indentation defined by ISO 14377, additionally the instruments count ‘with optical microscopes or heads atomic foree microscopy through which the r- ‘Table 6.1 Ranges of indentation technique as a function of maximum load applied and maximum depth of penetration (based in ISO 14577 Range imam oad (F ‘Maximum depth (max) Macro 2N1030KN Micro Less than 2 {Greater than 200 nm Nano 7 ‘Les than 200 am6 Indentation Technique: Overview and Aplications in Food Science 83 — Fig. 6.1 Nanoindentation tester (TTX-NHT, CSM Instruments, Switzerland) located inthe Centro de Nanociencis y Micro y Nentocnologins sidual footprint after indentation test can be captured. Figure 6.1 shows a nanoin- ddententation tester with optical microscope coupled toa digital camera (NNH-TTX, (CSM Instruments, Switzerland), 6.1.2 Measurement Method Instrumented indentation systems allow the application of a specified force or dis- placement history, in such a way that force and displacement, P and h, respec~ tively, are controlled and measured simultaneously over a complete loading cycle ‘The process of IIT involves an indenter tip, normal to the sample surface, which is driven into the sample by applying an increasing load up toa specific value. The in- enter is withdrawn gradually and the load decreases until full or partial relaxation ‘occurs in the material, Figure 6.2 illustrates the indentation process steps. ‘The main purpose of the instrumented indentation technique is to calculate the contact area (4) of the residual footprint as a function of the load applied by the indenter. In order to estimate the size of the contact area (4) itis necessary to know the geometry of the indenter used, because it has to calculate the contact depth (J) which is related to the contact area. Therefore, the indenters or tips play an impor- tant role in the indentation technique.4 I. Arzate-Viaque7 et 1 . C Vi | _vt | ws | wt rp | Fig. 62 Indentation process steps: approaching the indenter (a), contact point between the indenter and surface (b), application of the load (),inreasing load up to the maximum load (Pq) (2) ad unload the indenter (@) The basic geometries ofthe indenters include pyramidal and spherical. Berkov- ich and Vickers are the most pyramidal indenters commonly used. The Berkovich indenter has a pyramidal geomemry with triangular base and an angle of 03.03" be~ tween the axis of the pyramid and the three faces; on the other hand the Vickers indenter has a pyramidal geometry with square base with an angle of 136° between ‘opposite faces of the pyramid (Fischer-Cripps 2006b, 201 ¢; Lueca et al, 2010), ‘The pyramidal indenters are mainly used for hard materials (metals and ceramics), but also can be used for biological materials (e.g. bones and teeth) (Ebenstein and Pruitt, 2006; Lucea et al. 2010). The spherical and conical-spherical indenters are recommended for soft materials (c.g, polymers, elastomers, biological materials, and foods) because they minimize plastic deformation and stress concentration and avoid damaging the sample (Ebenstein and Pruitt, 2006). This kind of indenters cean be designed in different sizes of 200 jum to 1 pm of diameter. Figure 6.3 shows three-dimensional (3D) representations of the pyramidal (Berkovieh and Vickers), spherical, and conical indenters. Another type of indenters that is rarely used is the cylindrical flat punch. Diamond is the material most used for the manufacture of indenters, however they can also be made of tungsten carbide and sapphire (Lucea, etal, 2010). b | © a Fig. 63 3D representations of the indenters most commonly used in the indentation technique: Berkovic (a), Viekers (b, spherical (e) and conical (@)6 Indentation Technique: Overview and Aplications in Food Science 85 6.1.3 Analysis of Data (Hardness and Elastic Modulus) ‘When instrumented indentation test is used to estimate the elastic modulus and hhardness of material, itis necessary to obtain load versus depth displacement mea ‘surements. Depth measurement and knowledge of the shape of indenter are used to calculate the contact areas, instead of measurement ofthe size of impressions. There are different methods to analyze the load-displacement data that are used to com- pute hardness and elastic modulus of materials, Oliver and Pharr (1992) proposed a method of analysis for the load-displacement curves, known as compliance. Fig~ lure 6.4 is a schematic illustration of load-depth indentation measurements, where the deformation during loading is assumed to be both elastic and plastic (Hardness printing form) and the elastic displacement is recovered during unloading. The pa- rameter P is the load and h the displacement relative to the initial surface. From the load-penetration curve, P,.. is the maximum load, f,. is the maximum displace~ ‘ment, $s the contact stiffness determined as the slope of the upper portion of the unloading curve during the inital stages of unloading (P/dii), and h, is the perma- nent depth of penetration after the indenter is fully withdrawn. Doemer and Nix (1986) determined the hardness (77) and elastie modulus (E) considering that the contact area remains constant as the indenter is withdrawn, Fig. 64 A schematic illusta- tion of load-penetration Pause measurements foranindenta- \ tion est Pay Indentation load (P) he Ban Displacement (h)86 I. Arzate-Viaque7 et However, Oliver and Pharr (1992) showed that unloading curves are distinctly curved and usually well fitted by the power law relation: (hh (6.1) P ‘where a and m are power law fitting constants. From these data, the initial unload= ing slope (contact stiffness, 8) is estimated by analytically differentiating Eq. (6.1) and evaluating the result at the maximum indentation depths dp s-(# = Bh gue — hd"! (62) (%),., Binh gun =) 62) From Eq, (6.3) is calculated the contact depth i, (see Fig. 6.5) under the maximum indentation force: 63) where « is a constant which depends on the indenter geometry, 0.72 for conical indenters (Hay and Pharr 2000) ‘The projected contact area, 4, under the maximum indentation force considering fa sharp conical indenter is obtained by the indenter tip included angle, 0, and the estimated contact depth, that is A=[d)amnoF. 4) According to assumption that the compliances of the specimen and the indenter tip ccan be combined as springs in series, the effect of a nonrigid indenter on the load~ Pp Indenter ‘Surface under Fig. 65 Cross setion ofan indentation6 Indentation Technique: Overview and Aplications in Food Science 87 cisplacement curve can be accounted for by detining a reduced modulus calculated by $88 " 2p ah) TA. (60) and. where v and vj, E and £, are Poisson's ratios and elastic modulus of the material evaluated and the indenter material, expectively. Hardness is defined as the ratio of indentation load and projected contact area, as following: Pv 6) H A ‘This definition of hardness is different fom that used in an imaging indentation test, In the later case, the area isthe residual area measured after the indenter is e- ‘moved, while inthe instrumented indentation test the area isthe contact area under ‘maximum load. A conventional hardness test with zero residual area would give infinite hardness, while a nanoindentation test would give a finite hardness. 6.1.4 Parameters and Viscoelastic Behavior Some materials exhibit a time-dependent mechanical behavior or viscaclastic (© g lass, metal or ceramics at high temperatures, polymers, plastics, composites: Che- na etal. 2013; Lisnyaka etal. 2008); however, most biological materials exhibit diferent degices of viscoelasticity (8 tissues, organs of plants and animals, foc ‘materials; Isaksson et al. 2010). Viscoelasicity is a time-dependent mechanical property of the materials with sensitivity to load rate and deformation applied. coelastic behavior can be quantified in a material by the phenomena occurring in this when loads and deformations are applied, such as modulus of elasticity which is dependent upon the rate of loading, ereep, relaxation, and hysteresis. By indentation technique is possible to determine the viscoelastic properties such a ereep and relaxation in different kinds of biological materials (Wu et al. 2011). Inorder to represent the ereep behavior, the sample is subjected to a loading rate until reaching a maximum value, in which the load is kept constant during a certain period of time, and finally applying an unloading rate until this reaches zero, Indenter depth within the period where the load is kept constant represents the creep (Fischer-Cripps 2004), and is given by the following equation88 I. Arzate-Véequez et al Fig. 6.6 Typical curve of indentation of contcal bone (a) and graph of load applied (segmented line) and depth indentation (continuous line) in funtion of ime (b) showing of creep indentation Cr= bet x 100, (68) ‘where fi, isthe indentation depth at time f, when the force is kept constant, and /, is the indentation depth at the time r, of holding the constant test force, as shown, in Fig. 6.6b. Figure 6.6a represents the typical curve during a nanoindentation test of cortical ‘bone in osteons lumbar vertebrae pig, showing the relationship between the applied force and the indentation depth. While in Fig. 6b, the segmented line plot repre sents the application of force and the continuous line plot corresponding indentation depth (creep) both in function of time. The parameters used were: maximum load of 10 mN, loading rate of 120 mN/min, unloading rate of 120 mN/min, and 10 s pause at maximum load. Otherwise, in order to represent the relaxation behavior, an indentation depth ratio is applied in the sample, to reach a maximum value which is kept constant dur- ing a certain period of time and finally applying an unloading rate of the indentation reaching a value of zero. Indenter force is included in. the period where indentation depth is kept, represents the relaxation of stresses in the material, and is given by the equation: 69) where F, is the force at time f, when the indentation depth is kept constant, and F, is the force at time ¢, in the moment the indentation depth maintained was removed, as shown in Fig. 6.76. Figure 6.7a represents the curve during a nanoindentation test in an annulus porcine lumbar intervertebral disc, showing the relationship between indentation depth and force. Whereas in Fig. 6.7, segmented line plot represents the inden- tation depth applied and continuous line plot represents the corresponding force6 Indentation Technique: Overview and Aplications in Food Science 89 Fig 6.7 Indentation curve controlling of depth in annulus an! stop of load applied (continu fine) and Upside time (b) showing relaxation indentation in (serene ie) i Fs (relaxation) both in function of time. The parameters used were: maximum depth of 1000 nm, loading rate of 2500 nm/min, unloading rate of 2500 nmv/min, and 30s pause at the maximum indentation depth. ‘Therefore, from the above it can be concluded that from indentation curves con- {rolling the applied load and the indentation depth the creep and relaxation indenta- tion can be calculated which are parameters that are associated with viscoelastic bbchavior. The following briefly describes the term viscoelasticity to have a greater understanding of this behavior present in biological materials, ‘The two basic components of the viscoelastic behavior are viscosity and elastic- ity, When sudden load is applied to a viscoelastic material, it exhibits an elastic ‘behavior presenting a deformation, which is represented by a spring in a mathemati- cal model. The elastic behavior is described by Hooke’s Law, where the stress is directly proportional to the strain but independent of the strain rate: o=Ee, (6.10) where @ isthe stress, E the elastie modulus, and & the strain. Immediately after this, the material behaves as a viscous liquid, which is rep- resented by a dashpot using a mathematical model. This behavior is described by [Newton's law, in which the stress is directly proportional to the strain rate but inde pendent ofthe same strain: de ae 6.10 " (Fi ) : Cc Where o isthe stress, 1 is the coefficient of viscosity ofthe fluid, and 4 isthe strain rae Ifa viscoelastic material is subjected to a higher load rate, the slope of the stress— strain curve willbe steeper and therefore, the elastic modulus willbe higher, this characteristic is typical of viscoelastic materials, Ina viscoelastic material, the ereop ‘occurs when a sudden load is applied and this is Kept constant fora certain time90 I. Arzate-Viaque7 et ab ak 3 al el 3 Fig. 68 Diagram of different models representing the viscoelastic behavior based in eeep and relaxation: Maxwell model a), Kelvin-Voigt model (b), and Three-Element Model (€) ‘The typical curve shows a steady state asymptotically (Fig. 6.8, C2). Relaxation ‘occurs when in a previously deformed material, the stresses to which its subjected decrease asymptotically through @ certain time, whereas the deformation is kept constant (Fig. 6.8, C3). ‘A viscoelastic material also exhibits a phenomenon called hysteresis when sub- jected to cyclic loading and unloading. During this process, the material presents a loss of energy, which is represented by the arca between the loading and unloading curve, Viscoelastic materials can be represented by constructing mathematical mod- els that include arrangements between springs and dashpots. The springs are used to represent the behavior elastic solid, whereas the dashpots represent the behavior ofa viscous fluid. From serial and parallel arrangement between these elements, it is possible to construct empirical models that represent more accurate biological ‘materials such as bone, cartilage, tendons, and ligaments ‘One ofthe basic mathematical models is the Maxwell model, which is construet- ced by a series arrangement of a spring and a dashpot (Fig. 6.8, Al), the governing equation for the model is shown in Fig, 6.8, A4 from which is possible to determine the viscoelastic behavior of the material. In a creep tes, this madel adequately ex- plains the instantaneous deformation, which is represented by the spring, this being true for biological materials; however, the creep is linear and does not conform to the reality for this type of material (Fig. 6.8, A2), whilst relaxation is exponential, adjusting to the real behavior of biological materials (Fig. 6.8, A3). ‘Another basie model is the Kelvin. Voigt model which is constituted by a parallel arrangement of a spring and a dashpot (Fig. 6.8, BI), the governing equation for the6 Indentation Technique: Overview and Aplications in Food Science o ‘model 1s shown in Fig. 6.8, 84, trom which it possible to determine the viscoelastic ‘behavior ofthe material Ina creep test in this model, the element represented by the spring does not present an instantaneous deformation, wich isnot true tor biolog cal materials; however, the model explains adequately ereep behavior within the viscous element (Fig. 6.8, B2), whilst not having a relaxation phenomenon, which is not true for biological materials (Fig. 6.8, B3). Although these models are useful for understanding, the viscoelastic behavior does not represent any real material known. However, through a combination of these, it is possible to construct more complex models able to represent more ef- ficiently the behavior of different types of biological materials (Yuya et al. 2010; Lukes et al. 2008). One such model is the Three-Element model, which consists of ‘spring and a Kelvin—Voigt model in series (Fig. 6.8, C1), the governing equation of this model is shown in Tig. 6.8, C4, from which it is possible to determine the viscoelastic behavior ofthe material. This model is one ofthe most used to deseribe the viscoelastic behavior of biological materials, as described more realistically (Fig. 6.8, C2 and C3). 6.2 Mechanical Characterization of Biological Materials by Indentation During the last decades thore has been inereasing use of indentation technique of bi- ological materials, This fact is due to the advantages this technique has, which have ‘been mentioned above. In the literature, there are several works in which indenta- tion technique has been employed for evaluating mechanical propenies of biologi- cal materials to micro- and nanolevels. Table 6.2 has listed some of the published ‘works using indentation technique in biological materials, in the table the aim of the research, the mechanical parameters assessed and type of indenter used can be seen. As scen in Table 6.2, the Young's modulus or elastic modulus is one of the ‘more evaluated parameters due to which the biological materials present elastic or viscoelastic behavior. Additionally, it ean be seen that both pyramidal and spherical indenters are used forthe examination of samples In general, biological tissues that have been characterized by tis technique can be grouped into soft tissues (e2., cartilage, arteries, polymers, hydrogels) and min- cxalized tissues (€., bones, tecth, bioceramics). Some types of samples that have been characterized include bones (Albert etal. 2013), teoth (Fung-Ang etal. 2009), canilage (Franke et al. 2011), turtle carapace (Achrai and Wagner 2013), black coral skeleton (Juarez de la Kosa et al. 2012) and wings dragontlies (Lon etal. 2007) The use of indentation technique allows to elucidate and establish correlations of| ccomposition-function type, which makes ita powerful and useful tol for the analy- sis of biological samples. In the literature, critical review articles are found about the nanoindentation in biologic materials. Ebenstein and Prt (2006) reviewed the most important aspects of the technique and cited some examples published. They note that the nanoinden-2 I. Arzate-Viaque7 et Table 6.2 Studies of mechameal chaaetenzation o bologial mates by indentation echnuque Biological | Objective of work | Mechanical param- | Indenter type | References materi ers evaluated Aiular por | Use of dynamic | Storage E'and Betkovieh | O. Franke ranoindentation at loss modulus tal, 2011 diferent frequency | (1.9-7.5 MPa) ranges Poly(dimethyl | Investigate the use| Young's modulus | Spherical | Kohn and siloxane) | ofsurfacunis 10 | (0.71-8.34 MPa) hensicin, (PDMS), | eliminate adhesion 2013, poly(ethylene | between the tp and )(PEG) | the sample Black coral | Evaluate the ‘Young’smodu- | Spheroconical | Juirez de a stotoon | morhanical popere |e (8 58 667 Roce otal ties in diferent zones | GPa) hardness 2012 ofthe sample (13295-157,59 MPa) “Tune carapace | Correlate the ‘Young's modulus (02 |Beskovich | Achrai and microscale arehi-- (0 18.2.GPa) hardness Wagner, tecture with (0.007-0.67 GPa) 2013 the mechanical properties tation technique has emerged as a powerful tool for the evaluation of mechanical properties at the nano- and microscale in tissues and other biomaterials. Besides, they discuss the operating principle of the technique and mention that most poly- ‘mers and biomaterials exhibit viscoelastic or time-dependent behavior. A highlight that they deepen isthe hydration factor in hiological samples, hecause the evaluated properties depend on the state of hydration of the sample, so itis very important to consider the evaluation of these materials. In another work, Lucca et al. (2010) presented a review of the measurement ‘method, the instruments used, and the procedures for analysis of results. Also, a sec tion of work allocated to the utility of the indentation in biological materials. They ‘mention that in general the biological materials show small values of the elastic ‘modulus, exhibited viscoelastic behavior, and that the mechanical properties de pend of their hydration status. As well they mention some studies that have been conducted in various biological materials ‘Moreover, using the indentation technique it is possible to evaluate the fracture toughness in biomaterials and hard tissue such as bones. Kruzic etal. (2009) studied four indentation techniques to evaluate the fracture toughness in cortical bone: the Vickers indentation fracture (VIF) test, the cube comer indentation fracture (CCIF) test, the Vickers crack opening displacement (VOCD) test, and the interface in- entation fracture (IIF) test. Among the conclusions of the work indicate that it ‘ust take into account the spatial resolution when considering biomaterials and hard biological tissues. Finally, mention that organic component of the hard tissue ‘may influence the evaluation of material properties.6 Indentation Technique: Overview and Aplications in Food Science 93 In short, the importance of knowing the articles published and advances in the characterization by indentation technique in biological material, are useful to estab- lish basic considerations that should be taken info account when examining food materials which are likely to have similar characteristics to some biological materi- als mentioned above, 6.3 Examples of the Use of Indentat in Food Science n Technique ‘There are few reports in the literature regarding the use ofthe indentation technique to evaluate mechanical properties In foods. Below are discussed some oF the Works that have been published, 6.3.1 Micromechanical Properties of Hen's Eggshell Severa et al. (2010) describes the applicability of the indentation method for de- termining micromechanical properties of hen’s eggshell, In methodology, they se lected eggshell fragments extracted from different locations and were subsequently ‘embedded in resins, Resin tablets were polished in order to obtain flat surfaces with low roughness values. Roughness has a considerable effect on the measurements of the mechanical properties of the samples evaluated. The elastic modulus was de- fined as the main parameter for evaluation of the samples. The test variables were: ‘maximum load of 12 mN, loading and unloading rate of 72 mN/min, and pause of 20s. A Berkovich diamond tip was used in all the experiments. Figure 6.9a shows 4 typical curve of indentation (force vs. penetration depth) on eggshell. In each sample, the experiment was performed in a matrix of 9 <24=216 indentations in the material cross section as shown in Fig, 6.96. restate oe) Fig. 69.» Typical curve of ientation (ld vs, pentane) b Optical age of eggbell ros section shows a matrix of indents (9 24216) (Severa etal. 2010)oy I. Arzate-Viaque7 et ‘From the results, igh vanations in the values of Young’s modulus at dividual points were found, This fact is attributed to the chemical composition (concen- tration and crystal orientation of CaCO,). The Young's modulus values were not significantly different and ranged from 47.4 to 53 GPa, They conclude that the indentation technique proved to be appropriate, easy to use, and a powerful tool for local evaluation of mechanical properties of eggshell 6.3.2 Fracture Behavior in Eggshell Currently our research group is conducting a study of facture toughness in eggshell ‘with the purpose of establishing the fracture behavior using a Berkovich indenter. Eggs from a national trademark were randomly selected for the experiment. The ceggs were then washed with distilled water to remove dirt. Subsequently, equato- rial sections of eggshell were obtained and embedded in epoxy resin. Once the resin was hardened after 24h, the tablets were polished with sandpaper of different aan sizes and alumina to achieve a miror finish, Figure 6.10 shows a diagram of gashell preparation to evaluate the fracture by indentation texhnigu. The sutface roughness of the samples was evaluated after polishing with an AFM (diMultimode V, Veeco, USA) and Ra values (arithmetic average roughness) of 6.54:+0.45 nm \were observed, A nanoindentation tester (TTX-NHT, CSM Instruments, Switzer land) with a Berkovich diamond indenter was used. Preliminary results showed that high values of maximum loads generated frac- tures in the cross section ofthe egashell. Figure 6.11 shows an optical image of a ma === | Fig. 6.10 Diagram of eggshell preparation for evaluation by indentation technique6 Indentation Technique: Overview and Aplications in Food Science 95 Fig. 6.1 Optical image of residual footprint of indenta- corey coy tion on eggshell showing wo types of facture: preexisting cracks and deflection crack residual footprint indentation showing two types of fracture patterns: preexisting cracks and crack deflection. Itis necessary to continue fo conduct more experiments to elucidate properly the fracture pattern and to calculate the fracture toughness by established models in the literature for biomaterials. Another result observed from preliminary results is the presence of the indentation size effect (ISE) which establishes the values of hard- ness and Young's modulus are based on the maximum load, 6.3.3, Biopolymers and Edible Films Biopolymer membranes and edible films have been characterized mechanically by indentation technique. Edible films are films made of biopolymers (polysaccha- rides, proteins, lipids) that act as gas exchange membranes and confer protection to the foods in order to extend their shelf life (Lin and Zhao, 2007) Moreover, due to the growing pollution problem the need to design biodegradable packaging has arisen which incorporates biomolecules (polysaccharides) in their composition sig nificantly reducing the time of biodegradation, Based on the studies published in the literature, this type of membranes are the most evaluated through indentation technique and Table 6.3 shows some of them. Lower values of elastic modulus are present in these materials seen in Table 6.3 Additionally, the values of the mechanical properties are strongly influenced by the ‘membrane composition and moisture content, The kind of tip that was used in all the work was pyramidal, although, as mentioned above, for this type of materials spherical indenters are recommended, ‘The advantages of this technique for the characterization of films are obtaining reliable values of hardness and elastic modulus even when the films have small thicknesses and it can carry out localized measurements. When performing the characterization of these materials, the state of hydration should be considered be-