Wheat gluten, a bio-polymer to monitor carbon dioxide in food packaging: Electric and dielectric characterization

Abstract The use of wheat gluten as sensing material to detect carbon dioxide is a promising approach. The dielectric properties of wheat gluten are modified in contact with carbon dioxide gas at high relative humidity (90%) and at a temperature of 25 °C due to a structural change in the sensing material, where amino groups act as receptors to carbon dioxide molecules. In the present study, the effects of carbon dioxide on the electrical and dielectric properties of wheat gluten at 20% and 90% of relative humidity (usually found in food packaging) are determined and discussed. At 90% of relative humidity, a linear increase of the dielectric permittivity and dielectric loss was observed with a significant hysteresis which increased with the number of carbon dioxide treatment cycles. One of the significant results is the increase in the dielectric permittivity from 7.01 ± 0.07 to 12.02 ± 0.03 with a sensitivity of 31.38 ± 0.06 fF/%CO2 measured at 868 MHz. The developed sensor is sought to be integrated in the design of UHF-RFID (ultra-high frequency − radio frequency identification) systems working at 868 MHz.

Abstract: The use of wheat gluten as sensing material to detect carbon dioxide is a promising approach. The dielectric properties of wheat gluten are modified in contact with carbon dioxide gas at high relative humidity (90%) and at a temperature of 25°C due to a structural change in the sensing material, where amino groups act as receptors to carbon dioxide molecules. In the present study, the effects of carbon dioxide on the electrical and dielectric properties of wheat gluten at 20% and 90% of relative humidity (usually found in food packaging) are determined and discussed. At 90% of relative humidity, a linear increase of the dielectric permittivity and dielectric loss was observed with a significant hysteresis which increased with the number of carbon dioxide treatment cycles. One of the significant results is the increase in the dielectric permittivity from 7.01±0.07 to 12.02±0.03 with a sensitivity of 31.38±0.06 fF/%CO2 measured at 868 MHz. The developed sensor is sought to be integrated in the design of UHF-RFID (ultrahigh frequency -radio frequency identification) systems working at 868 MHz.

Abbreviations
Fabien Bibi received a Master Degree in Sensors and Associated Systems from the University of Montpellier (France) in 2012 and received his PhD at "l'Institut National de la Recherche Agronomique" (INRA) SupAgro in Montpellier, France in 2015. His PhD was entitled: Study of dielectric properties of vegetal proteins at high frequency for the development of green RFID bio-sensors to be interfaced with passive UHF RFID systems. His main research interests are in the development of capacitive and resistive sensors, to be coupled to RFID tags for intelligent packaging and food chain monitoring.

Introduction
The control of carbon dioxide concentration in food packages is important and necessary for the extension of food shelf-life. Production of carbon dioxide in food packages is not only due to the development of micro-organisms, but also to respiring products such as fruits and vegetables [1,2]. In the goal of maintaining high quality food products, the partial or complete elimination of carbon dioxide is essential in modified atmosphere packaging (MAP) [3]. Fresh products such as strawberries, cherries, broccoli and mushrooms benefit from a high carbon dioxide concentration level up to 20%, whereas other products for e.g. lettuce, tomatoes and pears suffer from carbon dioxide concentration of above 2% [4]. Carbon dioxide is not only an active gas in MAP, but is also known to be a marker of food degradation [5] and an indicator of microorganism growth, responsible for food spoilage [6]. Monitoring carbon dioxide in food packages would thus give a better control on the evolution of food metabolism and meet the consumers' demand for high quality food products. To reach this objective, a natural polymer has been selected according to its physico-chemical properties, electric and dielectric properties and its close mimicking behavior of food products: wheat gluten.
Wheat gluten has been broadly studied for its gas properties [2] and increasingly investigated for its unique mass transfer properties [7][8][9][10]. The high permeability of wheat gluten to carbon dioxide and (to a lower extent) oxygen resulted in a high permselectivity (ratio of carbon dioxide permeability to oxygen permeability) of the material [2], making them interesting for food packaging applications, such as fresh fruit and vegetable packages [10,4,11].

Version postprint
Comment citer ce document : Bibi, J. C. F., Guillaume, C., Gontard In addition to these properties, wheat gluten contains different types of bonds such as electrostatic interactions and hydrogen bonds [12][13][14][15] which interact with the matrix. These bonds give rise to strong dipole-dipole interactions, sharing of electron pairs between atoms (stable electronic configuration) and electrical interaction of low intensity between atoms and molecules [16][17][18][19]. Wheat gluten is thus considered as a polarizable material, having dielectric properties (permittivity and dielectric loss  [12,[20][21][22], but has never been studied as a potential film-based material for carbon dioxide monitoring, relying on its dielectric property variations at 868 MHz. The electric and dielectric properties of wheat gluten are also known to be sensitive to carbon dioxide [23] and could be used for monitoring packaging headspace in intelligent packaging systems.
Conforming to literature, the impact of carbon dioxide on polymers has been studied in several ways. FTIR spectroscopy has been performed on protein films exposed to high pressure carbon dioxide to evidence conformation changes in the films [24]. Under normal conditions, the permeability of carbon dioxide has been studied for modified atmosphere packaging applications [1,25] and for determining the permselectivity which is the ratio of carbon dioxide and oxygen permeability [1,2,26]. The study of the influence of relative humidity on the sorption and permeability of carbon dioxide gas in wheat gluten proteins [2,27] has also been performed.
Principally based on the development of sensors, Stegmeier et al. [28] studied the interaction of carbon dioxide and humidity with amino group systems for the development of carbon dioxide sensors working at room humidity. Endres et al. [29] studied and developed a synthetic polymer capacitive sensor system which suppresses the relative humidity effects. However, the 5 dielectric properties of natural polymers as a function of carbon dioxide concentration at high relative humidity in the objective to be used as sensors have not been studied.
In the present work, the impact of carbon dioxide on the electrical and dielectric properties of wheat gluten protein, used as sensor, is determined at low and high relative humidity value (20% and 90%), and at a constant temperature of 25°C. Interdigital capacitors (IDCs) were manufactured to have a high surface/thickness ratio and high exposure to the surrounding atmosphere. The IDC was placed in a climatic chamber where relative humidity, temperature, CO2 gas flow and pressure were controlled. It was essential to control and maintain constant relative humidity and temperature due to the sensitivity of wheat gluten to these parameters.
Electrical measurements performed with the IDC were used for identifying the dielectric permittivity and loss by retro-simulation of wheat gluten layer as a function of carbon dioxide concentration. The dielectric permittivity and loss obtained are further discussed in the objective of using wheat gluten for carbon dioxide monitoring coupled to UHF-RFID systems.
2 Materials and methods

Interdigital capacitor (IDC) sample
The design of the IDC system was performed using ANSOFT® software, which is a high performance electromagnetic simulation software. The IDC system design technique was already developed in a previous study [30]. In order to have an IDC system (figure 1 (a)) having   Wheat gluten coated IDC system was subjected to carbon dioxide treatment. The apparatus used is a climate control system provided by Hiden Isochema from England. The climate control system provides unique controlled climate, allowing command of atmospheric composition in terms of gases such as nitrogen, carbon dioxide and oxygen, controlling temperature, pressure, as well as relative humidity. The total control of the climate is possible due to sensors connected to the chamber: temperature sensor (±0.1°C), relative humidity sensor (±0.1%), gas flow rate sensor (±1%) and pressure sensor (±0.05%) were provided.
The apparatus is made up of two water baths, one equipped with a vapor generator, a chamber in which the atmosphere is controlled, an outer cylinder jacket, glove cuff, a heated power supply and a process control interface. The system is represented in figure 2. The carbon dioxide concentration was varied from 0% to 40%. Relative humidity was fixed in the chamber, first at 20% and then at 90%, and temperature was fixed at 25°C. The flow of gas (nitrogen and carbon dioxide) was fixed to 600 mL/min for 1 hour at each carbon dioxide concentration to fill rapidly the chamber, and then fixed to 200 mL/min for further processing of the experiment. To ensure complete equilibrium of wheat gluten coated IDC system to the surrounding atmosphere, the experiment was carried out on 3 days for each carbon dioxide concentration level. At the end of the allocated time, the automatic control of the system shifts to the next carbon dioxide concentration. Three increasing and decreasing carbon dioxide cycles were performed.
For electrical measurements, a coaxial cable, a Vector Network Analyzer (VNA), a hygrometer (Rotronic Hygromer) and a host computer were necessary. The calibration was performed over a frequency range of 30 MHz to 3000 MHz. It should be specified that the results presented in the scope of this work are at 868 MHz, being the working frequency of UHF passive RFID systems, with which wheat gluten is sought to be interfaced.
The coaxial cable was fixed to the extent possible to avoid stray capacitors due to cable movements after calibration, before connecting the IDC system. The latter was inserted into the climate control system where carbon dioxide gas concentration, relative humidity and temperature are controlled. A hygrometer was used to verify the relative humidity value. The temperature was fixed at 25°C during the whole experiment. In all cases, the IDC systems were positioned in the middle of the chamber to provide equal atmosphere interaction on the whole surface area of the sensor.
A host computer was connected to the VNA for data acquisition. The impedance real and imaginary components, as well as frequency were acquired at fixed time intervals (30 minutes for each measurement). These electrical properties were recorded (as triplicates) by a program developed on Labview software and data was saved in text files. The experimental set-up for electrical property measurements is given in figure 3 (a).
The IDC system is represented by the equivalent resistance capacitance (RC) circuit in Where R-P represents the reflection port, I the incident signal, R the reflected signal, p R & p C the resistance and the capacitance of the interdigital circuit, S R & S C the resistance and capacitance of the sensor,  the angular frequency and V the voltage applied to the circuit.
Where RT is Rp and RS in parallel, and where CT is CP and CS in parallel.

Identification of dielectric properties of materials by retro-simulation of IDC systems
The identification of the dielectric properties of wheat gluten was performed by simulation with ANSOFT® software using geometrical and substrate (FR4) parameters of the designed IDC system. The identification procedure was developed in a previous study [30] where the permittivity and dielectric loss were obtained by comparing the simulated impedance values to the measured values at the specified frequency. The only modification performed to the IDC system was the addition of the wheat gluten layer on top. This layer represented the dielectric whose permittivity and loss were varied and re-injected in the simulation procedure. By 3 Results and discussion 3.1 Effects of carbon dioxide on electrical properties of wheat gluten at low relative humidity value The impact of carbon dioxide was determined at low relative humidity (20%) on the wheat gluten network. The capacitance was calculated using the resistance capacitance model represented in figure 3 (b). As illustrated in figure 4, the capacitance value given by the VNA is stable for the increasing and decreasing carbon dioxide concentrations at 20% of relative humidity and at 25°C, and to a further extent, indicates that the dielectric properties (not determined in the present case) of wheat gluten are not influenced by carbon dioxide at low relative humidity. In its dry state, wheat gluten presents strong protein-protein interactions, instead of protein-water interactions, forming a dense network [35], probably due to high glutamine content (45%) linked together by hydrogen bonds (cross-linked glutamine). The 20% relative humidity results only in bound water content (3.6% dry basis) with the wheat gluten network. According to studies performed by Gontard et al. [4,36], at this water content, wheat gluten presents extremely low permeability to gases such as carbon dioxide and oxygen. The transport of carbon dioxide in the wheat gluten network is limited and the gas is consequently not able to bind with high energy sorption sites which are not accessible. Therefore, it can be assumed that wheat gluten does not show its most interesting dielectric properties at low relative humidity, having a gas barrier behavior.  [4,26,36], wheat gluten exhibits its most interesting properties at high relative humidity (90%). This aspect was also observed for dielectric properties of wheat gluten at 90% of relative humidity. Figure 5 illustrates the capacitance of the wheat gluten coated IDC system as a function of time from 0% to 40% of carbon dioxide at 90% of relative humidity and at 25°C. As it can be observed, the capacitance increases with carbon dioxide concentration, in a stepwise manner from 7.32±0.01 pF to 8.19±0.02 pF. The capacitance mimics perfectly the change in carbon dioxide concentration.
The standard deviation on the capacitance is low indicating that the measurements are repeatable. It was essential to keep relative humidity constant at 90%, as fluctuations in its value would obscure the effects of carbon dioxide on the electrical properties of wheat gluten. It is also noticeable on figure 5, that even after flushing with nitrogen gas at the end of the cycle, the process is not reversible in experimental conditions as the capacitance is higher compared to the beginning, indicating a non-reversible change in the structure of wheat gluten.  15 values reached at 0% of carbon dioxide at the end of the 3 rd cycle is 11.05±0.01, which is close to the permittivity value (11.97±0.13) at 40% of carbon dioxide. This drift indicates that carbon dioxide intake by wheat gluten at each cycle, associated with relative humidity causes further change in the protein layer. No gaps are detected at 40% of carbon dioxide for the 3 cycles, where the permittivity value reached remains around 11.97±0.13.
On an applicative point of view, the reversibility is not a crucial factor, to the extent that, with food fermentation, carbon dioxide content in packaging increases, followed by the disposal of the product. The sensor would therefore give information about the highest level of carbon dioxide reached. lateral groups or peptidic chain [27]. The formation of these bonds with the amide groups will modify the secondary structure of the protein [38].
As said previously, wheat gluten has a hydrophilic character and a high affinity to water molecules, at high relative humidity. It uptakes water considerably and the polymer matrix undergoes a significant swelling, which leads to a remarkable increase in the gas sorption values 17 together with their bonding with the polymeric chains, through hydrogen bindings, electrostatic bindings, covalent bindings and even hydrophobic bindings with chemical groups of aminoacid, will contribute in increasing the number of dipole-dipole interactions, as well as electrical conduction (for residual free charges) within the network.
High relative humidity is thus essential, to promote carbon dioxide solubility within the wheat gluten network, impacting the structure and consequently the dielectric permittivity and loss, both subjected to a linear increase. Figure 9: Fraction of dissolved carbon dioxide as a function of pH. Adapted from Chaix et al. and Hofland et al. [39,41] and Daniels et al. [40].
Comparing the physico-chemical properties of synthetic films such as polyimides, it was proved that their carbon dioxide permeability at the dry state (and at 25°C) had a higher value (2825 for wheat gluten [27] was noted, but not specified for polyimides. In agreement with permeability values, the same assumptions can be made. Polyimides are rather hydrophobic with molecular chain groups showing very low affinity to polar compounds. The solubility of water is lower than a few percents (mass basis).
Consequently, the polymer swelling is not significant and negligible in the presence of water, explaining the decrease in the permeability and sorption of carbon dioxide, as relative humidity increases. Table 1 summarizes different materials used as sensing element for the detection of carbon dioxide, as well as their detection principle. At a first glance, it can be observed that most of the materials found in literature, being polymers or metals, are based on a color change as detection principle. None of them are based on the dielectric phenomena that may eventually be altered in contact with the different forms of carbon dioxide. Several studies are focused on the potentiometric detection type and conductivity. They depend on the change in the concentration of ions, due to a change in pH [44], modifying the measured potential [44,45] or conductivity [46][47][48]. On the other hand, as specified by Neethirajan et al. [49], some potentiometric sensors display poor repeatability and reproducibility of potentiometric responses. Depending on the material used by the authors, some of them require extreme conditions for the correct operation of the sensor. For instance, 400°C is required as stated by Hong et al. [45]. This is not applicable in the agrifood sector where fresh produce are often stored at much lower temperatures. These sensors thus present some drawbacks. According to the studies, they are often bulky, have a complex detection system (Severinghaus concept) and need several elements for realizing a proper measurement. As such, the sensor developed by Varlan and Sansen [46], requires a reaction between carbon dioxide and bicarbonate solution, in a cavity covered with a gas permeable membrane. Admittedly, this system is very interesting as it allows performing carbon dioxide measurements, without having relative humidity interferences. In agrifood applications, this concept could be of main interest, as they target fresh produce containing high relative humidity within the packaging. This would thus give a measure of only carbon dioxide concentration. However, the system that needs to be set up in the packaging would alter its integrity due to potential cables needed for performing measurements. The use of a wireless detection system would be the ideal system to be established.

21
In the present study and for an application in real conditions, the selectivity should be taken with care, as the loss or gain in water and carbon dioxide production are two competing phenomena, rarely taking place simultaneously (relative humidity and temperature are imposed for storage in modified atmosphere packages) [10,11]. Consequently, the relative humidity in the packaging headspace remains constant while carbon dioxide concentration varies for one food product, and the sensitivity of the biosensor to the carbon dioxide gas should not be altered.
However, for other applications of carbon dioxide monitoring, where variations of relative humidity are noted, this aspect could be inadequate, for example in cases where water mass transfer occurs from the product to the atmosphere within the packaging [27]. As observed in table 1, the sensor's performance is highly dependent on factors like relative humidity, temperature or solvent concentration. Focusing on relative humidity, in cases where relative humidity is low, the sensitivity of the sensor is also low. These observations bring to the conclusion that relative humidity is essential for gas detection in particular cases.

Version postprint
Comment citer ce document : Bibi, J. C. F., Guillaume, C., Gontard  values. This indicates that the protein structure was modified. The irreversibility and hysteresis were even more pronounced with increasing cycles of carbon dioxide treatment. Water, so far, is an essential element for the sorption of carbon dioxide in the protein network as the dielectric constants were modified only at 90% of relative humidity. Further studies are still required for the evaluation of the full potential of using wheat gluten as carbon dioxide sensor.

Funding sources
Agence National de la Recherche (ANR) was the funding source.

Acknowledgement
Authors thank the "Agence Nationale de la Recherche" (a French funding agency) for funding this study within the framework of NextGenPack project. Special thanks are given to "l'Institut d'Electronique et des Systèmes" Montpellier for providing the necessary equipments for the smooth progress of the study.Carole Guillaume is an Associate Professor in Food Chemistry