Time-temperature indicator (TTI) films are among the most promising intelligent packaging systems for the food industry. TTI films can safely indicate for the manufacturer or the final consumer, in real-time, the conditions of food processing, transportation, storage and deterioration. Nevertheless, it has the advantage of being of low cost and visually indicate the food conditions. The main idea behind TTIs is that many food products deteriorate due to changes in the temperature, which cause chemical reactions and microbial growth that can be detected by a TTI film. Several types of TTI, such as colorimetric \cite{Pacquit2007,Wu2013,Pereira_2015,Yoshida_2014,Zhang2014}, radiofrequency \cite{Length2010}, photochromic \cite{Kreyenschmidt2010}, bacterial growth kinetic sensor \cite{Zhang2013}, intelligent inks \cite{Mills2005}, oxygen indicators \cite{Vu2013,Eaton2002}, as well as nanotechnology sensor systems have been developed and successfully tested both in academy and in the industry. Among these systems, colorimetric TTI systems which can provide a response via a colour change according to pH changes of the product, providing information about the conservation and the actual quality of a food in a visual and intuitive way, have grown in importance and diversity, both in industry and academy, mainly due their low cost, simplicity and reliability. Besides their simplicity, the common feature of such systems is that they are based on biodegradable polymeric films and pH indicator dyes, thus not requiring expensive analytical instruments. Furthermore, there is a great interest of the food industry, retailers and consumers for environment friendly systems that can safely be applied as an accurate intelligent food-package.
As colorimetric TTIs based on carbohydrate polymers as a supporting matrix with indicator dyes have been developed \cite{Zhang2014,Nopwinyuwong2010,Pereira_2015,Golasz_2013,Yoshida_2014,Kuswandi_2012} and well-stablished in the literature, this section will report the techniques of preparation and characterization of such systems as well as the perspectives of future development and real applications.
Colourimetric TTIs are based on chemical, physical and enzymatic processes\cite{Arias_Mendez_2014} and have wide application in smart packaging, with applications to chilled or frozen foods. In smart packaging, this kind of sensors change their colour due to a change in a critical state, for example, the growth of micro-organisms fires changes in pH of the food, which in turn induces a colour change in a indicator dye, allowing the packaging to provide visual information about the product.
These films are basically prepared by the casting technique, which has low cost and high efficency for film preparation. Briefly, composite hydrocolloid films are prepared by suspending an amount of biopolymer in distilled or deionized water. The suspension is poured in acrylic or glass plates and dried until constant weight in order to obtain the films. Among the most diverse biopolymers that have been applied to smart and active packaging, biopolymers such as starches, gums, pectin, gelatin and chitosan are the polymers that have greater stability, due to their ability to form networks structured and thermally stable copolymers. Moreover, these polymers can form stable hydrocolloid suspensions and the casting of hydrocolloid suspension films has been successfully applied in the development of smart films prepared with polyaniline\cite{GARCIA_2004}, Cassava Starch/Glycerol\cite{Golasz_2013}\cite{Kuswandi_2012}, Chitosan\cite{Yoshida_2014} ,Chitosan/PVA\cite{Silva_Pereira_2015}\cite{Pereira_2015} and Chitosan/Starch films\cite{V_sconez_2009}. Hence, casting is a versatile, unexpensive, rapid and simple technique of film peparation that is well established in the literature. Although other techniques such as nancomposites\cite{Qureshi2012}, Layer-by-Layer\cite{Brasil_2012} and polymeric matrix \cite{Marek_2013} are also used to develop smart polymer films, the simplicity of casting technique, which does not requires complicated laboratorial instruments makes the casting technique the choice for the development of new and improved smart polymer films. Another advantage of the casting technique is that it allows the easy incorporation of micromolecules into the polymeric film by justing adding molecule such as natural pigments that act as sensors, into the film-forming solution.
There are several pigments, both natural\cite{Chigurupati_2002}\cite{Zhang_2014}\cite{Silva_Pereira_2015}\cite{Pereira_2015} and artificial\cite{Kim_2012}\cite{Salinas_2012}, that are used for the purpose of sensing dyes and there are many research focusing the application of such pigments in alternative thin films sensors\cite{Veiga_Santos_2010}\cite{Shahid_2013}\cite{Silva_Pereira_2015}\cite{Pereira_2015}. These pigments are usually solubilised in water, ethanol or a mixture of both and then added to the film-forming solution in an amount variating from 1 to 2-25%, forming very sensitive final films.
Although biopolymers have the advantage of being non-toxic and biodegradable, which is interesting for the development of environment-friendly packaging, they have the disadvantage of having high water vapour permeability and low mechanical/thermal resistance, which surely limits the use of biopolymers in a wide range of applications, including smart food packaging. In order to overcome these limitations, biopolymers are often combined as blends or copolymers with better features when compared to pure biopolymers. Thus, in order to evaluate the potential use of bipolymers and copolymers blends in food packaging applications, they should be characterized both physically and chemically. Among the most important features that are expected from a good packaging polymer, we can cite the spectroscopical, optical, thermal and mechanical features. A good biopolymer for packaging applications should have chemical and physical features that are comparable with those of commercially available packaging polymers, including its thermal stability, water vapour permeability and mechanical resistance.
As the physical and chemical properties of a polymeric film depends on its chemical structure, the first step is the spectrocopical charcterization of the films. The chemical structure of the polymeric films are often characterized by means of UV-Visible, Fast Fourier Transform Infrared and Raman spectroscopy. These techniques provide a picture of the functional groups present in the chemical structure and their interaction, such as the presence of hydrogen bonds and groups crosslink, since crosslink and hydrogen bonds can affect some blend properties, as for example mechanical resistance.
Optical, Atomic Force Microcoscopy (AFM), Scanning Electronic Microscopy (SEM) and X-Ray Driffracity (XRD) are techniques widely applied in polymeric film morphological characterization, which is important to find defects in the microstructure of polymeric matrix that can affect the film properties. Microscopy analisys can show the presence of granules in the film surface, the homogenity and smoothness of the film surface, as well as the presence of porous and cracks. Hence, microscopy is a useful tool to study the structural integrity of the polymeric matrix.
Differential Scanning calorimetry (DSC) and Thermogravimetric Analysis (TG) are thermoanalytical techniques that provide a clear picture on the thermal stability and the interaction of polymer blend components, since the interaction between the components of the blend causes a change in the melting point of each component.
The interaction of the polymeric matrix with water and humidity can be measured by the water Vapour Permeability (WVP), that is a measure of how fast the polymeric matrix absorbs humidty and Swelling and deswelling studies, which indicate the behaviour of the polymeric matrix when interaction with pure water. Thst is, how fast the polymeric matrix absorbs water. Both WVP and Swelling index are often gravimetrically determined. For WVP, the most used standard is the ASTM E-96-97. Briefly, the samples are maintained in a environment with controlled humidity and temperature and during assay the samples are weighted in a time interval, until the weight become constant and the changes in weight are plotted as a function of time. The final WVP is calculated according to Equation \ref{eqn:wvp}:
\[WVP = \frac{w}{\theta}\cdot\frac{24 \cdot t}{A \cdot \Delta p} \label{eqn:wvp}\]
where w is the weight gain; \(\theta\) is the time during which w occurred (hr); t is the sample thickness (mm); A is the test area (m\(^2\)); and \(\Delta\)p is the vapour pressure difference (kPa).
For Swelling Index (SI) assay, the samples are cut into slices and then kept in a desiccator with silica-gel until constant weight. After this procedure the samples are weighed and then subjected to immersion in beakers containing distilled water for different time intervals at room temperature. At each time interval, samples are removed, dried and weighed. The Swelling Index (SI%) is calculated according to Equation \ref{eqn:swell}:
\[SI\% = \frac{Final Weight - Initial Weight}{Final Weight} \cdot 100 \label{eqn:swell}\]
Good mechanical properties is expected for application of thin polymeric films in food packaging. Tensile strength, elongation at break and Young’s modulus are measured to determine the mechanical properties of these films. It is expected that a good biopolymer film for food packaging applications has mechanical properties comparable to those of commercially available polymers. These mechanical properties are measured using the method described in ASTM D1708-10, which is suitable for determining the mechanical properties of plastics or the traction of films with thicknesses ranging from 0.0025 mm to 2.5 mm, which is sufficient for the application in food packaging.
A colourimetric sensing film should have its colour parameter evaluated. The colour parameter indicatas how the film colour changes and if these changes can be detected with naked eyes. Thus, this is an imporant parameter to be evaluated. Briefly, thee colour parameters of the film is determined with a UV-Vis colour measurement spectrophotometer. Tests are performed in triplicate and the total colour values of L* (lightness), a* (red - green) and b* (yellow - blue) are registered. The colour differences are obtained according to Equation \ref{eqn:colour}:
\[\Delta{E} = ((\Delta{L}*)^2 + (\Delta{a}*)^2 + (\Delta{b}*)^2)^\frac{1}{2} \label{eqn:colour}\]
Where \(\Delta{L}* = L* - L_0*\), \(\Delta{a}* = a* - a_0*\) and \(\Delta{b}* = b* - b_0*\). \(L_0*\), \(a_0*\) and \(b_0*\) are the initial colour values of sensing films.
Smart biopolymer sensors has been sucessfully applied in food packaging applications. Wu et al. has developed a TTI based on urease that is able to indicate temperature changes from 5 to 30 \(^{\circ}\)C through colour changes. \cite{Wu_2013}. The literature also reports applications of colourimetric TTI to indicate meat and fish quality\cite{Golasz_2013, Shukla_2015, Zhang_2014, Silva_Pereira_2015}. Lee et al. \cite{Lee_2005} describe an oxygen indicator which is activated by UV colorimetrically using TiO2 particles by reducing photosynthesis, triethanolamine methylene blue in encapsulated in polymer. Freshness indicators, another application example, is successfully operating knowledge of the quality indicators of volatile compounds produced by microorganisms such as carbon dioxide\cite{Nopwinyuwong_2010}, volatile nitrogen bases\cite{PACQUIT_2007}, biogenic amines and toxins.