Impact of MG²⁺ and Tara Gum Concentrations on Flow and Textural Properties of WPI Solutions and Cold-Set Gels

Abstract

Cold-set gels of whey protein isolate (WPI) and of WPI plus tara gum (TG) were produced through the addition of magnesium chloride to heat-denatured (HD) WPI and WPI+TG solutions. The flow behaviour of the WPI, TG, and WPI+TG samples was analyzed: WPI solutions exhibit Newtonian behavior while the behavior of the TG and WPI+TG solutions can be described through the Cross and Carreau models. The mechanical characterization of the cold-set gels was done through puncture tests, and the Young's modulus for each cold-set gel was obtained. Statistical analysis of the mechanical data was made according to the Marquadt-Levenberg algorithm.

Keywords:

• Cold gelation
• Viscosity
• Texture
• Whey protein isolate
• Tara gum
• Magnesium

INTRODUCTION

Whey proteins are well known for their functional and nutritional properties, which lead to their incorporation in many food products.[1] One of the most important features of whey proteins is their gelling capability, and for this reason many studies have been concerned with the gelation process. Food protein gels are usually formed during heating, and consequently this process is known as heat-set gelation.[2–4] More recently, an alternative process called cold-set gelation, according to which the gelation occurs at ambient temperature, has been studied.[5–11] The cold gelation of whey proteins has two major steps. First, a native solution of proteins is heated above 65°C for protein denaturation and aggregation. During heating, the processing conditions, like heating temperature and time, the environmental conditions, like pH, mineral content, and ionic strength, and, finally, the protein concentration must be carefully manipulated in order to ensure that the electrostatic repulsion is high enough to prevent the formation of a gel. In a second step, the gelation of the denatured solution is induced, at ambient temperature, usually by the addition of a salt whose cations screen the protein charges and lead to the decrease of the electrostatic repulsion between the molecules. In the case of divalent cations, salt addition may also improve the formation of salt bridges between negatively charged protein groups which means that much less salt concentrations is needed to obtain a gel. The salt used in the present study was magnesium chloride, since Mg²⁺ is an alternative to the most common used cations, like sodium,[5,11–13] calcium,[5,6,8,11,12,14] or iron.[15] Besides, magnesium is a very important element for human health, since it is essential for nerves, muscles, and most other body cells to function. Magnesium is also a vital catalyst in enzyme activity, especially the activity of those enzymes involved in energy production; it also assists in calcium and potassium uptake.[16]

In recent years, considerable interest has also been devoted to the study of protein-polysaccharide mixtures, particularly in order to increase their applicability in the food industry.[17] Proteins and polysaccharides are the most important gelling and thickening agents; therefore, the interactions between these two kinds of biopolymers are of great importance in determining the processes of structure formation and the mechanical properties of new products. In this paper, besides the study of cold-set gels of WPI, mixed gels of WPI and tara gum were also analysed. Tara gum is a galactomannan that is obtained from the seeds of the tara shrub, Caesalpinia spinosa, which is a native of the northern regions of Africa and South America. It is described as having a backbone of (1–4)-linked β-D-mannopyranosyl units one third of which possess a single unit side chain of (1–6)-linked α-D-galactopyranose.[18] Applications for TG are ice cream, cheese, bakery products, desserts, sauces and meat products.[19]

The behavior of simple whey protein or mixed whey protein plus polysaccharide systems has been essentially studied in solution and during gel formation using rheological small deformation techniques.[12] However, an important view of the food structure is related to the study of the breaking properties in large deformation, according to which the textural attributes are decisive for quality and consumer acceptance of the product. The objective of the present work was to analyse the impact of WPI and TG concentrations on the flow behaviour of WPI and WPI+TG native solutions and heat-denatured (HD) dispersions, as well as on the mechanical response of the cold-set gels obtained through the addition of Mg²⁺ to HD-WPI (or HD-WPI+TG) dispersions. The flow behavior of the WPI and WPI+TG solutions in the native form and after denaturation was analyzed in light of the Cross and Carreau models.[20,21] The large deformation behavior of simple gels of WPI and mixed gels of WPI+TG was assessed trough puncture tests. Values of fracturability, resistance to puncture, rupture strain, rigidity, and Young's modulus were obtained for each gel. A statistical analysis of the data was done in order to assess the influence of protein, tara gum, and magnesium concentrations on the gel properties.

MATERIALS AND METHODS

Materials

Whey protein isolate powder (LACPRODAN DI-9224, Lot no, 50M160218) was kindly supplied by Arla Foods Ingredients (Videbaek, Denmark). The protein content, determined by the Kjedahl method, was 93.5 wt%. According to the suppliers, the WPI total solids content was 94% (6% moisture), the ash content was 4.5% and the cation content was: sodium, 0.5%; phosphorus, 0.2%; chloride, 0.1%; potassium, 1.2% and calcium, 0.1% (all quantities are expressed in mass percentage). The tara gum was kindly supplied by Carob S.A (Spain), with the commercial reference no. 1760. The magnesium chloride, in the form of a hexahydrate salt (MgCl₂.6H₂O), the sodium azide (NaN₃) and the potassium hydroxide (KOH) were obtained from Riedel-de-Haën (Sigma-Aldrich Laborchemikalien, Seelze, Germany), with weight purity greater than 99, 99, and 85%, respectively.

Methods

As it was stated above, this work intends to analyse the impact of WPI and TG concentrations on the viscoelastic behaviour of WPI and WPI + TG native solutions and heat-denatured (HD) dispersions, as well as on the mechanical response of the cold–set gels obtained through the addition of Mg²⁺ to HD-WPI (or HD-WPI+TG) dispersions. Preliminary studies allowed us to define the upper and lower limits of the WPI, Mg²⁺ and TG concentrations that would produce cold-set gels after heat denaturation of WPI or WPI + TG solutions at 75°C for 20 min. Based on those studies an experimental design was implemented.

Experimental Design and Statistical Analysis

The Box-Behnken experimental design[22] was employed to investigate the interactive effect of WPI (6.0, 7.5, and 9.0 wt%), Mg²⁺ (25, 50, and 75 mM), and TG (0.20, 0.35, and 0.50 wt%). The experimental design consisted of 15 experimental points (with three independent variables and three levels, including three replicates at the central point) performed in random order (Table 1). The measured responses were fracturability, rupture strain, rigidity and Young's modulus.

Table 1 Box-Behnken experimental design matrix

	WPI concentration		TG concentration		Mg^2+ concentration
Runs	Coded X1	Uncoded WPI (wt%)	Coded X2	Uncoded TG (wt%)	Coded X3	Uncoded Mg^2+ (mM)
1	−1	6.0	−1	0.20	0	50
2	−1	6.0	0	0.35	−1	25
3	−1	6.0	0	0.35	1	75
4	−1	6.0	1	0.50	0	50
5	0	7.5	−1	0.20	−1	25
6	0	7.5	−1	0.20	1	75
7	0	7.5	0	0.35	0	50
8	0	7.5	0	0.35	0	50
9	0	7.5	0	0.35	0	50
10	0	7.5	1	0.50	−1	25
11	0	7.5	1	0.50	1	75
12	1	9.0	−1	0.20	0	50
13	1	9.0	0	0.35	−1	25
14	1	9.0	0	0.35	1	75
15	1	9.0	1	0.50	0	50

The results obtained from Box-Behnken experimental design were analysed by multiple regressions, using the least squares regression methodology, to fit the following second-order equations to all dependent variables:

(1)

where, Y is the measured response variable; b ₀, b_i , and b_ij are constant regression coefficients; and X_i and X_j are the independent variables, namely, WPI concentration, TG concentration and Mg²⁺ concentration. This methodology is an empirical modeling technique devoted to the evaluation of the relationship of a set of controlled experimental factors and observed results. It requires a prior knowledge of the process to achieve statistical model. A detailed account of this technique has been outlined.[22–26] All calculations were done using SPSS 14.0 software (SPSS Inc., Chicago, IL, USA). This software uses the Marquardt-Levenberg algorithm to find the parameters that give the ‘best fit.’

Preparation of WPI, TG, and WPI+TG Solutions

WPI solutions (6.2, 7.7, and 9.2 wt%, in protein) were prepared in beakers by weighing the appropriate amount of powder and adding distilled water, without reaching the final solutions weights. After 3 h of gentle mixing, the pH was adjusted to 7.0 with KOH 1M and/or HCl 1M. Two drops of sodium azide solution (2.0 wt%) were added in order to prevent bacterial growth. Finally, distilled water was added until the final weights were reached and the systems were kept under mixing for one more hour.

The tara gum stock solution was prepared as follows: the required amount of the powdered gum was gradually added to the appropriate amount of distilled water in order to obtain a solution with 1 wt% in TG. After 1 hour of vigorous stirring at room temperature, the solution was heated at 90°C for 30 min in a water bath, under continuous stirring. The non-dissolved material was removed by centrifugation at 30000 g for 1 h, at 25°C, and the concentration of the solution was determined from its dry matter content. TG solutions with 0.21, 0.36, 0.51, and 0.67 wt% concentrations were prepared from this stock solution by appropriate dilution with distilled water. To prepare the solutions of WPI+TG, the WPI powder was dispersed in the appropriated amount of tara gum stock solution. Then, distilled water was added, without reaching the final solutions weights, and from this point onward, the procedure was the same as that described above for WPI solutions.

Heat Denaturation and Cold-set Gel

The native solutions were denatured by heating at 75°C for 20 min, in a water bath. These conditions are known to provide a partial unfold and aggregation of the protein molecules without gelation.[10] After heating, the dispersions were cooled in an ice bath in order to stop the denaturation process immediately. The aggregates formed during this stage remained soluble and stable dispersions were obtained which were stored at room temperature overnight. Then, 39.0 g of each dispersion were put inside beakers and gelation was induced through the addition of 1.00 g of magnesium chloride solution with the appropriate salt concentration. The gels were placed in a chamber of controlled temperature (25°C) during 48 h before being subjected to the texture tests.

Viscosity of the Solutions

The rheological measurements were performed at 25°C using a controlled stress rheometer AR-G2 (TA Instruments) fitted with a cone-and-plate geometry (4° cone angle, 60 mm diameter, 107 μm gap). The flow curves of the WPI, TG and WPI+TG native solutions and heat-denatured dispersions were determined by subjecting each sample to consecutive 60 seconds steps of constant shear stress. This step duration ensured the record of steady state shear rates and the corresponding viscosities (shear stress over shear rate) allowed us to build the steady state flow curves. All experiments were performed over a shear rate window that ranged from the lowest operational limit of the rheometer to a value that ensures the ‘destruction’ of the sample. In all experiments, the samples were covered with a thin layer of paraffin oil to prevent evaporation.

Penetration Tests on the Cold-set Gels

The measurements were performed with a TA–XT2 Texture Analyzer (Stable Micro Systems, Godalming, Surrey, UK), at room temperature. The plunger was cylindrical with a 10 mm diameter, and operated with a speed of 0.1 mm s⁻¹. Tests were made directly in the beakers where the gels were penetrated down to 80% of the initial gel height. This deformation value was obtained through preliminary tests in order to ensure the rupture of the gel. To avoid friction, the plunger was lubricated with paraffin oil before each test. Data of force, distance and time were recorded, at a sampling rate of 50 pps (points per second). Taking into account that the texture tests have a great variability, eight replicates of each gel were tested being the results reported as the average. The following parameters were determined: fracturability [N], which is defined as the force at the first peak in the force vs. distance curve; rigidity [N m⁻¹], which is the ratio between force and distance, at the fracturability point; puncture resistance [N], which is the force at the second peak in the force vs. distance curve; and rupture strain, which is the strain at the rupture point. If there is no second peak, the puncture resistance is equal to fracturability.[27]

The measured force, F, and the depth of penetration, d, were manipulated in order to obtain the apparent Young's modulus, E, of each cold-set gel. The measured force, F, and the force due to buoyancy, F_b , were used to calculate the force corrected for buoyancy, F_cb .[28] The E value is given through the slope of the initial linear increase of F_cb plotted as a function of the strain (δ = d / L, where L is the gel height). The low penetration velocity of the probe (0.1 mm/s) and the high sampling rate (50 points per second) of the reported experiments, allowed us to record “quasi-static” forces resulting from the very slow deformation of the gels, thus ensuring that the “time” factor during the experiment was not a problem, as it is stated by Oakenfull and Tanner.[29]

RESULTS AND DISCUSSION

Flow Behaviour of the Solutions

Flow curves were performed at 25°C in order to investigate the shear, WPI and TG concentration effects on the apparent viscosity, η_ap. The WPI native solutions and heat-denatured dispersions exhibited a clear Newtonian behaviour, in the shear rate window analysed. The experimental values are reported in Table 2, and, as it would be expected, the viscosity increased with the WPI concentration, since a raise in the WPI concentration means a higher number of macromolecules in solution, and, consequently, higher viscosity values. The heat-denatured dispersions presented always higher viscosity values than the correspondent native solutions, meaning that the unfolding of the protein molecules was effectively induced during the heating process.

Table 2 Values of apparent viscosity for native (η_ap ^ND) and heat-denatured (η_ap ^HD) WPI solutions, at 25°C

CWPI (wt%)	ηap ^ND (Pa.s)	ηap ^HD (Pa.s)
6.0	0.0020 ± 0.0001	0.0029 ± 0.0002
7.5	0.0021 ± 0.0001	0.0049 ± 0.0003
9.0	0.0032 ± 0.0002	0.0068 ± 0.0003

The flow curves corresponding to the TG solutions manifested typical rheological characteristics of a thickened fluid: a zero shear viscosity plateau at low shear rates and a shear-thinning regime at higher shear rates (Fig. 1). Nonlinear models namely, the Cross and Carreau models,[20 –21] were used to describe the apparent viscosity. These models describe the dependence of the apparent viscosity on the shear rate, , according to the Eqs. (2) and (3), respectively:

(2)

(3)

where, η₀ is the zero-shear rate viscosity, η_∞ is the infinite-shear rate viscosity, τ and λ are time constants; and m and k are dimensionless constants. Since the high shear rate Newtonian viscosity was never approached, the above equations were simplified by assuming , i.e., by taking the infinite shear viscosity equal to zero. The adjustable parameters obtained for both models are reported in Table 3. From the results, it can be seen that both models represent, with good accuracy, the experimental data. However, the Cross model provided a slightly better fit with an average relative deviation (ARD) lower than 3% while the Carreau model typically showed higher ARD values. As expected, the values of η₀ increased with increasing concentration. Besides, shear thinning appears at lower shear rates when concentration increases; this means that the rate of formation of new entanglements diminishes as concentration increases which is in accordance with the increase of the time constants with concentration in TG. For the range of TG concentrations used in this work, it was possible to obtain a viscosity master curve, after performing a concentration dependent shift using the 0.20% TG solution as the reference (Fig. 2). Both vertical and horizontal shifts were necessary to obtain the superposition of the different curves. This procedure showed a dependence on TG concentration, as can be observed in Fig. 3: the value of the horizontal shift, α, increased with TG concentration, which is consistent with the increasing degree of entanglement of the systems.

Table 3 Cross (τ, m) and Carreau (λ, k) adjustable parameters for steady simple shearing, obtained for TG solutions, at 25°C

CTG (wt%)	η0 (Pa.s)	τ (s)	m (−)	ARD* (%)	η0 (Pa.s)	λ (s)	k (−)	ARD* (%)
0.20	0.042	0.03	0.53	1.4	0.039	0.22	0.17	4.8
0.35	0.252	0.05	0.59	1.8	0.236	0.30	0.19	4.8
0.50	0.978	0.08	0.62	2.9	0.922	0.36	0.22	3.8
*Average relative deviation,

Figure 1 Flow curves for tara gum native (ND) solutions (open symbols) and heat denatured (full symbols) dispersions, for different concentration values, at 25°C. Symbols: (□) 0.20 wt%, (△) 0.35 wt%, (○) 0.50 wt%, and (⋄) 0.65 wt%. Full line represents predictions of the Cross model and dotted lines those of the Carreau model.

Figure 2 Shear rate/concentration superposition for tara gum, native (ND) solutions (open symbols) and heat denatured (full) dispersions, at 25°C. Master curve at the reference concentration of 0.20 wt% TG. Symbols: (□) 0.20 wt%, (△) 0.35 wt%, (○) 0.50 wt% and (⋄) 0.65 wt%.

Figure 3 Horizontal (open symbols) and vertical (full symbols) shift factors as a function of the tara gum concentration, used to obtain the flow master curves of Fig. 2.

The analysis of the flow curves correspondent to the WPI+TG solutions, with a WPI concentration of 7.5 wt% and a TG concentration that varied from 0.2 to 0.5 wt%, shows that the Newtonian behaviour exhibited by the WPI solutions was irremediably changed by the introduction of tara gum. The behaviour of the mixtures was conditioned by the presence of the galactomannan, as it can be seen in Fig. 1. Taking this into account, the flow curves of the WPI (native or heat denatured) + TG mixtures were correlated in light of the Cross and Carreau models, like was done before for TG solutions. The obtained parameters are reported in Table 4.

Table 4 Cross (τ, m) and Carreau (λ, k) adjustable parameters for steady simple shearing, of the native and heat-denatured mixed solutions of WPI+TG, at 25°C

CWPI (wt%)	CTG (wt%)	η0 (Pa.s)	τ (s)	m (−)	ARD (%)	η0 (Pa.s)	τ (s)	m (−)	ARD (%)
Native solutions
7.5	0.20	0.066	0.54	0.39	9.2	0.074	0.45	0.69	2.2
7.5	0.35	0.466	0.39	0.52	6.7	0.495	0.27	1.22	3.9
7.5	0.50	2.126	0.16	0.81	8.6	2.159	0.15	1.68	3.8
Heat-denatured solutions (75°C/20 min)
7.5	0.20	0.074	1.82	0.21	2.6	0.076	0.34	0.84	2.2
7.5	0.35	0.511	0.39	0.50	7.4	0.517	0.24	1.23	4.8
7.5	0.50	2.458	0.14	0.86	8.8	2.505	0.14	1.69	3.9
*Average relative deviation, .

Adopting the same procedure used for TG solutions, it was also possible to obtain a master flow curve for the mixtures WPI+TG, taking as reference the flow curve correspondent to 7.5 wt% WPI + 0.20 wt% TG. The vertical and horizontal shifts for the native and the correspondent heat-denatured mixtures of WPI+TG are the same, what is a consequence of the zero-shear viscosities and the time constants being analogous. These results are plotted in Figs. 2 and 3.

Textural Characteristics of the Cold-set Gels

The puncture tests made on the cold-set gels allowed us to determine several mechanical properties for each of them. The values of fracturability, puncture resistance, rupture strain, rigidity and Young's modulus are reported in Table 5. According to the statistical procedure described in a previous section, the second-order polynomial model regression coefficients (Eq. 1) were determined for each response analysis, taking as independent variables the WPI, TG and Mg²⁺ concentrations. The results obtained are reported in Table 6a. In all cases, the total regression is significant (p ≤ 0.05).

Table 5 Mechanical properties of the cold-set WPI and WPI+TG gels

CWPI (wt%)	CTG (wt%)	CMg ^2+ (mM)	Fractura-bility (N)	Puncture resistance (N)	Rupture strain (−)	Rigidity (N m^−1)	Young's modulus (kPa)
6.0	0.20	50	0.08 ± 0.01	0.11 ± 0.02	45 ± 3	7 ± 0.5	1.84 ± 0.21
	0.35	25	0.11 ± 0.01	0.10 ± 0.01	41 ± 3	7 ± 0.4	0.71 ± 0.08
		75	0.16 ± 0.03	0.24 ± 0.02	61 ± 4	14 ± 1.2	3.09 ± 0.28
	0.50	50	0.28 ± 0.03	0.40 ± 0.03	57 ± 5	17 ± 1.5	6.86 ± 0.35
7.5	0.20	25	0.17 ± 0.02	0.24 ± 0.02	71 ± 7	11 ± 1	3.03 ± 1.29
		75	0.31 ± 0.03	0.36 ± 0.03	65 ± 5	39 ± 3	4.44 ± 1.31
	0.35	50	0.47 ± 0.04	0.64 ± 0.05	35 ± 4	60 ± 5	17.52 ± 1.51
	0.50	25	0.57 ± 0.03	0.83 ± 0.05	78 ± 8	30 ± 2	11.28 ± 1.42
		75	1.06 ± 0.06	1.31 ± 0.08	69 ± 6	97 ± 6	16.51 ± 1.48
9.0	0.20	50	0.61 ± 0.04	0.74 ± 0.05	45 ± 4	53 ± 3.4	13.07 ± 0.37
	0.35	25	0.78 ± 0.04	1.12 ± 0.06	39 ± 4	86 ± 6.8	19.60 ± 0.49
		75	1.41 ± 0.09	1.86 ± 0.09	41 ± 5	134 ± 9.3	21.40 ± 1.52
	0.50	50	2.06 ± 0.12	2.69 ± 0.12	57 ± 5	130 ± 8.2	48.58 ± 2.64

Table 6a Regression coefficients of fracturability, puncture resistance, rupture strain, rigidity and Young's modulus

	Fracturability (N)	Puncture resistance (N)	Rupture strain (−)	Rigidity (N m^−1)	Young's modulus (kPa)
b 0	7.25	8.96	45.5	111	26.8
b 1 (CWPI)	−1.58	−2.03	35.0	−35.7	−12.3
b 2 (CTG)	−12.4	−15.05	−603	−174	−180
b 3 (CMg ^2+)	−2.40 × 10^−2	−2.20 × 10^−2	−1.42	−1.63	1.20
b 11 (CWPI×CWPI)	8.30 × 10^−2	0.110	−2.06	1.72	0.545
b 22 (CTG×CTG)	4.44	4.52	917	−539	−51.5
b 33 (CMg ^2+×CMg ^2+)	−6.80 × 10^−5	−8.53 × 10^−5	2.40 × 10^−2	−6.00 × 10^−3	−1.20 × 10^−2
b 12 (CWPI×CTG)	1.39	1.84	1.74 × 10^−6	74.4	33.9
B 13 (CWPI×CMg ^2+)	4.00 × 10^−3	4.00 × 10^−3	−0.121	0.273	−4.00 × 10^−3
b 23 (CTG×CMg ^2+)	2.30 × 10^−2	2.40 × 10^−2	−0.202	2.60	0.255

The analysis of variance reported in Table 6b, shows that the linear WPI and TG concentrations are significant terms (p ≤ 0.05) for all the mechanical parameters determined. The linear Mg²⁺ concentration is significant only for the fracturability, puncture resistance and rigidity. The cross-products C_WPI×C_TG, C_WPI×C_Mg2+ and C_TG×C_Mg2+ are significant terms for all response analysis with four exceptions: C_WPI×C_TG and C_TG×C_Mg2+ are not significant for the rupture strain, C_TG×C_Mg2+ is not significant for puncture resistance and C_WPI×C_Mg2+ is not significant for Young's modulus.

Table 6b analyses of variance (p value) of fracturability, puncture resistance, rupture strain, rigidity and Young's modulus

	Fracturability (N)	Puncture resistance (N)	Rupture strain (−)	Rigidity (N m^−1)	Young's modulus (kPa)
CWPI	<0.01	<0.01	<0.01	<0.01	<0.01
CTG	<0.01	<0.01	0.02	<0.01	<0.01
CMg^2+	<0.01	0.01	0.06	<0.01	0.10
CWPI ×CTG	<0.01	<0.01	0.87	0.01	0.01
CWPI × CMg^2+	0.01	0.03	0.01	0.02	0.53
CTG × CMg^2+	0.03	0.07	0.27	0.03	0.04

The rupture strain seems to present a critical value at the middle point, which means that increases with WPI concentration until 7.5 wt% and then starts to decrease; with Mg²⁺ and TG concentrations, the rupture strain firstly decreases till 50 mM and 0.35 wt%, respectively, and then reverts this tendency. The Young's modulus increases with the Mg²⁺concentration till 50 mM, and then starts to decrease. These results show that cold-set gels with a large spectrum of mechanical properties have been produced by manipulating their composition.

CONCLUSIONS

The cold gelation of whey protein isolate and whey protein isolate plus tara gum solutions is an interesting alternative to the traditional heat gelation for application in the food industry. The obtained gels have a large spectrum of textural properties, depending on the protein, salt or TG concentrations. These findings are of great practical importance, since the utilization of these gels in very different products can be envisaged. The viscoelastic behaviour of the WPI solutions is Newtonian, while for the TG solutions and for the WPI +TG native solutions (or HD dispersions) a typical rheological behaviour of a thickened fluid was observed, that could be described through the Cross and Carreau models. The viscosity of heat-denatured dispersions is higher than that of the correspondent native solutions, which ensures that protein unfolding was induced during the heating process. According to the statistical analysis of the results, it was verified that the linear WPI and TG concentrations are significant terms (p ≤ 0.05) for all the mechanical parameters determined, but the linear Mg²⁺ concentration is significant only for the fracturability, puncture resistance and rigidity.