**2. Materials and methods**

#### **2.1 Cross-linked film preparation**

Pectin (Pec) from criolla orange was dissolved in distilled water at ambient temperature under mechanical stirring at a concentration of 2 wt.% of solid with 1 vol.% of glycerin (GLY, Biopack Argentina). The pH of the pectin solution measured with a pH meter (Melter Toledo) was 3.2. After the pectin solution was homogeneous, it was spread on a leveled Petri dish and placed in an oven at 40°C for 24 h for slow solvent evaporation. Once a pectin film was formed, it was peeled off and submerged in a 0.1 wt.% CaCl2 (Merck) solution at 40°C without stirring (stagnant conditions). Diffusion of calcium ions occurred by a driving force of concentration gradient, allowing a moderate cross-linking effect in the polymer matrix. A contact time of 24 h was probed to obtain cross-linked pectin films. After cross-linking time, the pectin film was washed with a hydroalcoholic solution several times to remove excess calcium salt on the film surface. Cross-linked pectin with calcium was named Pec-Ca, and it resulted in a transparent and handling film used for mechanical and barrier characterizations. The same procedure and salt concentration were used in the case of FeCl3 salt (Merck), and the cross-linked film was called Pec-Fe. This last film was light brown and retained transparency and easy handling. Commercial pectin from citrus peel was supplied by Sigma Aldrich (galacturonic acid ≥74.0%, methoxy groups ≤6.7%), and it was used to prepare uncross-linked films. The same procedure as pectin from criolla orange was used to obtain the commercial pectin film, and it was called Com-Pec.

#### **2.2 Mechanical characterization**

CT3 Brookfield texture analyzer with a load cell of 50 kg and a resolution of 5 g was used to perform tensile strength assays at a speed of 5 mmmin<sup>1</sup> according to the ASTM D 882 requirements. For an experiment, samples were cut into rectangular pieces of 40 mm in length and 10 mm wide. Thickness was measured using a Köfer micrometer (precision 1 μm). To ensure complete relaxation of the polymeric structure once the films were peeled off, they were placed in a humidity chamber for 24 h at a relative humidity of 40% and room temperature (25°C) before they were measured. Then, the experimental procedure was carried out under the same humidity and temperature conditions. Typical curves of tension (*σ*) versus elongation (*ε*) were built for each sample, and they were used to determine the values of the mechanical parameters, such as young's modulus (E; MPa), tension at break (*σ*; MPa), elongation

at break (*ε*; %), and Tenacity (T; kJ�m�<sup>3</sup> ). Young's modulus was calculated from the slope of the σ–ε curves when a linear relationship between them was observed. σ and ε were calculated as the final points on the curves; this behavior indicated that rupture of the sample occurred, and tenacity was calculated as the area under the *σ*–*ε* curves before rupture [10]. The reported results are the average values from at least three film samples. The *σ*–*ε* relationship is given by the following equation:

$$
\sigma = E \times \mathfrak{s} \tag{1}
$$

where: *σ* ¼ *F=A* in MPa, *F* being the force in N and *A* the transversal area of the specimen in m<sup>2</sup> .

$$
\varepsilon = \frac{\Delta L}{L\_0} \times 100\tag{2}
$$

where: Δ*L* is the change in length and *L*<sup>0</sup> is the initial length of the specimen, which was 20 mm.

#### **2.3 Water uptake**

Polysaccharides in general and pectin are hydrophilic polymers able to absorb water from the environment to the detriment of the films' physical integrity. It is a matter of science to find ways to prevent water absorption for expanding the field of biopolymers application. Even reducing water uptake under acceptable values would represent a contribution to broadening polysaccharide film applications, for example, for food packaging. In this study, water uptake (WU) was determined gravimetrically. Weights of completely dried samples were measured directly. Film specimens were introduced into bottles containing 20 mL of distilled water and shaken at ambient temperature (25°C). At intervals of 24 h, films were removed from the medium, dried to remove excess water, and immediately weighed. The water uptake of the cross-linked films was calculated according to the following Equation [11]:

$$\text{WU} = \frac{W\_{24} - W\_0}{W\_0} \times 100\,\tag{3}$$

where: *WU* is the value of water uptake (%), *W*<sup>24</sup> is the weight of swollen film at a time "t = 24 h", *W*<sup>0</sup> is the weight of dried film at "t=0".

#### **2.4 Water vapor permeability**

Water vapor transmission rate (WVTR) was determined gravimetrically using a modified ASTM Method E 96–95. The film specimen was mounted on an acrylic permeation cell comprised of two chambers. The upper chamber was in contact with water vapor pressure, while the bottom chamber was filled with an adsorbent material. The film specimen was in between both chambers, acting as a barrier. Therefore, the driving force of the global process was the difference in water vapor pressure at both sides of the film specimen. Once the permeation cell was assembled, all systems were placed into a chamber with temperature and relative humidity control. The operational conditions are fixed at 37 � 2°C and 98% relative humidity (RH). Water vapor permeability (WVP) (ng�m�m�<sup>2</sup> �s �1 �Pa�<sup>1</sup> ) was calculated from [11]:

$$\text{WVP} = \frac{\text{WVTR} \times l}{\Delta P} \tag{4}$$

#### *Effect of Cross-Linking Agent on Mechanical and Permeation Properties of Criolla Orange… DOI: http://dx.doi.org/10.5772/intechopen.102976*

where: WVTR (ng�m�<sup>2</sup> �s �1 ) was measured through a film specimen; *l* (m) was mean film thickness, Δ*P* (Pa) was partial water vapor pressure difference across the two sides of the film specimen.

#### **2.5 Gas permeation**

Flexible packaging materials must fulfill some specific characteristics according to the food they will pack. Fruits and vegetables are a particular type of food because they continue breathing after harvesting. Fruits and vegetables need oxygen to breathe, converting carbohydrates into carbon dioxide and water vapor. Post-harvest respiration uses stored starch or sugar and will stop when these reserves are exhausted. Therefore, designing a film that can retard fruits and vegetable respiration by controlling oxygen permeability and nitrogen and carbon dioxide exchange is desired. This condition might modify the atmosphere around the fruits and vegetables, altering oxygen levels inside the packaging, retarding the production of ethylene, and, thus, limiting the physiological decay of the product [12, 13]. This modification also reduces ripening-induced quality degradation in texture or loss of bioactive compounds during storage.

On the other hand, a minimal amount of oxygen might let anaerobic fermentation process, leading to spoilage [14]. For that reason, studying gas permeation through pectin and cross-linked pectin films is necessary to define the applicability of these films to the packaging of fruits and vegetables. In this study, N2, O2, and CO2 permeability were measured at 30°C and 1 bar using a classical time lag apparatus. The effective membrane area was 11.34 cm<sup>2</sup> , and permeate constant volume was 35.37 cm3 . After the membrane degassing procedure, gas permeation measurements were carried out under high vacuum (*p* ≈ 10 torr) and 30°C for 10 h. The amount of gas transmitted at time "*t*" through the membrane was calculated from the permeate pressure (*p*2) readings in the low-pressure side of the permeation cell. Permeability coefficients (*P*) were obtained from the flow rate into the downstream volume upon reaching the steady-state as:

$$P = \frac{Bl}{T\_c p\_1} \frac{\text{dp}\_2}{\text{dt}} \tag{5}$$

where: the cell constant *B* = 11.53 (cm3 (STP) K)/(cm2 cmHg); high-pressure side *<sup>p</sup>*<sup>1</sup> (cmHg); membrane thickness *l*(cm), the slope of the *p*<sup>2</sup> versus *t* plot in steady-state d*p*2*=*d*t* (cmHg/s), the temperature of the permeation cell *Tc* (K). Permeability values were obtained in Barrer unit (*B*), i.e., 1 *<sup>B</sup>* = 10�<sup>10</sup> cm3 (STP) cm�cm�<sup>2</sup> �s �1 �cmHg�<sup>1</sup> and then converting Barrer to other units for comparison purposes.

Theoretical separation factors (*α*) were calculated from the relation between the permeation coefficients of pure *i*and*j* gases as:

$$a\_{i/j} = \frac{P\_i}{P\_j} \tag{6}$$

#### **3. Results and discussion**

#### **3.1 Mechanical properties**

Mechanical properties of pectin and cross-linked pectin films were evaluated through strength-strain curves of each sample. Besides, commercial pectin was also analyzed. **Table 1** shows values of young's modulus (*E*; MPa), tension at break


#### **Table 1.**

*Mechanical properties of films.*

(*σ*; MPa), elongation at break (*ε*; %), and tenacity (T; kJ�m�<sup>3</sup> ) of all samples. Results showed an increase in young's modulus and tension at the break with cross-linking, with the modulus being higher in the presence of calcium ions and retaining almost the same tension at break concerning Pec-Fe. An increase in elongation at break was observed in Pec-Fe, additionally with a higher energy absorption during the deformation process. Com-Pec resulted in a more deformable and resilient film reaching 30% of elongation with a tenacity one order of magnitude higher concerning the other samples. Mechanical results depicted a stiffness effect of cross-linking, more pronounced in Pec-Ca. This result might be because calcium ions better accommodate within pectin chains to form a stable egg box conformation regarding Fe ions. Although the ionic radius of calcium ions is higher than for iron ions, their divalent charge better interacts with two adjacent carboxylic acid units negatively charged within two entangled pectin chains. **Figure 1** shows the egg box model for Pec-Ca. Cybulska et al. [15] mentioned that the binding process in chain-to-chain pectin interactions with Ca2+ ions required a pronounced shift of one galacturonate chain concerning the other chain. Thus, the interaction of calcium ions with polygalacturonate chains may occur via oxygen atoms in the carboxylate group, in the ring, in the glycosidic bond, and the hydroxyl group of the next residue. Cross-linking formation at pH3.2 may be connected with the binding of calcium ions to pectin, hydrophobic interactions, and the formation of hydrogen bonds [16]. In this case, the interaction of Ca2+ with other oxygen atoms in galacturonic acid residues was suggested.

Furthermore, water molecules might also interact with ions, competing with carboxylic acid units to stabilize them. The calcium egg-box model is formed in a two-fold conformation of pectin chains. Remnant water molecules within the pectin matrix might provoke a polymorphic transition from two-fold to three-fold chain conformation, disrupting the egg-box configuration. This fact might explain results obtained by Pec-Fe, which showed lower young's modulus and higher elongations at break correlated with a more hydrated configuration. Regarding the structural possibilities of pectin-Fe, it could be like xanthan gum-Fe studied by Vazquez et al. [17].

**Figure 1.** *Calcium "egg-box" model for pectin, based on [15].*

*Effect of Cross-Linking Agent on Mechanical and Permeation Properties of Criolla Orange… DOI: http://dx.doi.org/10.5772/intechopen.102976*
