Smart edible films based on mucilage of lallemantia iberica seed incorporated with curcumin for freshness monitoring

Abstract The objective of the present work was first to develop a smart packaging system based on Lallemantia iberica seed gum (LISG)/curcumin and, subsequently, investigate its physicochemical characteristics and biological activity. Finally, the response of LISG/curcumin films against pH change and the spoilage of shrimp were tested. The barrier properties and mechanical performance of the films improved as the curcumin concentration increased. FT‐IR analysis revealed the formation of physical interaction between LISG and curcumin. LISG/curcumin films showed a continuous and steady release of curcumin. The incorporation of curcumin into LISG matrix imparts antioxidant and antibacterial/mold activity to the films. A strong positive correlation was observed between total volatile base nitrogen (TVBN) content of shrimp and a* (redness) during storage time (Pearson correlation = 0.975). Eventually, LISG/curcumin film could be a promising smart packaging system capable of detecting food spoilage.

acid (Fathi et al., 2018). Sadeghi-Varkani et al. (2018b) optimized the formulation of biodegradable films based on LISG. The optimal formulation of LISG-based film was found to be the biopolymer concentration of 1.2% and glycerol concentration of 35%. In this condition, the fabricated films had highest barrier and mechanical performance. In another study, Sadeghi-Varkani et al. (2018a) developed a nanocomposite film based on LISG matrix incorporated by TiO 2 nanoparticles. The authors observed that LISG nanocomposite had excellent physico-mechanical characteristics and thus could be used in packaging applications to extend the shelf life of foods.
In the present work, for the first time, a smart packaging system formulated by LISG/curcumin was developed and its physicochemical and biological properties were investigated. Finally, the capability of the developed packaging system to monitor the freshness of shrimp was tested.

| Materials
Lallemantia iberica seeds were purchased from a local market in Sari, Iran. The extraction of mucilage from the seeds was carried out as described by Fathi et al. (2018). All the chemicals were obtained from Sigma-Aldrich. Microbial cultures were supplied from Himedia.

| Smart film preparation
A dispersion containing 1% (w/v) Lallemantia iberica seed gum (LISG) in deionized water was prepared on a magnetic stirrer at ambient temperature and stored for 12 hr at refrigerator to be hydrated com- pletely. An ethanolic solution of curcumin (0.2%-0.6% w/v) was also prepared, followed by the addition of tween 80 (150 mg ml -1 ), as emulsifier under stirring. Afterward, the curcumin dispersion was added dropwise to the LISG dispersion with rate of 1 ml min -1 . Then, in order to evaporate ethanol from the dispersion, rotary evaporation (at 40ºC under vacuum condition) was carried out. Finally, the resulting dispersion was casted on teflonated Petri dishes (15 cm diameters), dried in an air dryer at 25°C for about 16 hr, peeled and stored in desiccator for further study.

| Morphological properties
Field emission scanning electron microscopy (FESEM) was employed to elucidate the morphology of LISG film with and without curcumin. First, the film samples were coated with gold and then their morphological properties were investigated using FESEM (Tescan, JMIRA3 LM). The instrument was operated at an accelerating voltage of 4 kV.

Films thickness
The films' thickness was measured by a hand-held digital micrometer. Five film positions were selected to determine the thickness of the film. The mechanical and barrier properties of the films were computed using the average thickness.
Water solubility (WS), moisture content (MC), and water vapor permeability (WVP) WS of the films was determined as reported by Gontard et al. (1994) with slight modifications. Briefly, the pieces of the film (2.0 × 2.0 cm 2 ) were weighted, and dissolved in 50 ml of distilled water at 25ºC for 24 hr using an incubator equipped with a heating unit. Finally, the remaining pieces were filtered through Whatman nº 1 filter paper and dried at 105°C until reaching a constant weight (W1). WS was determined using the following equation: in which, W 0 indicates the initial dry weight.
In order to measure MC of the pieces of the film, first they were conditioned, and then their weight loss after heating at 105°C until reaching constant weight was recorded. MC value was calculated as follows (Zolfi et al., 2014): Water vapor permeability of the films were determined as follows: The films were sealed over beakers containing silica gel (0% RH). Initially, the beakers were weighed every 9 hr and then weighted every 24 hr for 4 days. Following formula was used to calculate WVP of the films: here, Δm/Δt is weight gain per unit of time (g s -1 ), X shows the films' thickness (mm), and A is exposed the area of the films (m 2 ) (ASTM Standard 1989).

| Mechanical properties
Mechanical performance of the developed films was evaluated using M350-10CT Machine (Testometric Co.) as described by ASTM standard assay of D882-91 (ASTM, 1991). The initial grip separation and crosshead speed were set at 50 mm and 50 mm min -1 , respectively. All the tests were carried out in five times. Mechanical parameters including tensile strength (TS) and (1) (2) MC = Water weight loss Moist film weight × 100 (3) WVP = Δm AΔt X Δp elongation at break (EB) were determined from the stress-strain curve.

| FT-IR analysis
The FT-IR analysis was performed to investigate the chemical structures of LISG, curcumin, and LISG/curcumin films. 5 mg samples were grounded and pressed into a pellet with potassium bromide powder. The scanning was conducted in the wavenumber range of 550-4000 cm -1 .

| Release rate
The release rate of curcumin from LISG/curcumin film was determined as described by Kang et al. (2018). The films were cut with dye cutting press (2 × 2 cm) and then immersed in ethanol solution (5 ml).
2.5 ml of the solution was taken at various time intervals (0.5, 1, 3, 6, 9, 12, and 15 hr) and the absorbance was read at 428 nm using a UVvis spectrophotometer (Agilent Cary 100 Series). A standard curve was plotted, and the curcumin concentration in ethanolic solution was calculated according to the standard curve.
Different models, including zero-order diffusion, Higuchi's diffusion, and Korsmeyer's Peppas models, were employed to describe the release profile of curcumin.

Antimicrobial and antimold activity
The antibacterial and antimold activities of the films were tested using zone inhibition method. The test was conducted against Escherichia coli O157:H7EDL 933, Bacillus cereus PTCC 1247, Bacillus subtilis PTCC 1023 (ATCC 6633), and Penicillium expansum. For this purpose, the film pieces (10 × 10 mm) were placed on solid nutrient agar inoculated by the tested bacterium, followed by incubating at 37°C for 2 days. The diameter of inhibition zone of the disk was recorded as the antibacterial potential of the samples.

DPPH radical scavenging activity
DPPH activity of the samples was tested according to the earlier described assay (Lai et al., 2010) with slight modifications. The films were immersed for 5 days, and then 2 ml of the resulting solution mixed with 2 ml of methanolic solution of DPPH (150 μM). The mixture was shacked vigorously, followed by keeping in the dark for 30 min at 37ºC. Finally, the absorbance was recorded at 517 nm using a UV-vis spectrophotometer (Agilent Cary 100 Series). The scavenging effect was calculated as follows:

Films response to pH changes
The color change of the smart films in facing the media with different pHs (3, 7 and 11) was recorded using a digital camera (Kodak M853).

| Statistics
SSPS software (Version 16) was used to analyze the experimental data. In order to compare the mean values, Duncan test was used.

| Film microstructure
The physical and mechanical properties of films are mainly dependent on interfacial adhesion between filler phase and polymer matrix as well as the microstructure of films (Fabra et al., 2011). In the present study, the microstructure of the neat films and those incorporated by different concentrations of curcumin were evaluated by FESEM analysis (Figure 1). It can be observed that in the neat film, there are some cracks and cavities that can increase the gas permeability of the films. However, in the films incorporated by curcumin especially those containing 0.6% curcumin, the entire surface of the films covered by micrometric curcumin crystals which can act as an obstacle for the diffusion of gasses. Moreover, addition of 0.6% curcumin resulted in a denser structure of LISG film. Thus, it is expected that LISG/curcumin (0.6%) has the lowest water vapor permeability and most mechanical performance which will be evaluated in the following sections. This observation is due to good compatibility between curcumin and LISG and filling of interspace of the films by curcumin microcrystals.

| Physical properties
The moisture content of the films is a crucial factor that affects the stability of the films and their brittleness. Furthermore, low content of moisture leads to the protection of films against physical and microbial spoilage (Thakur et al., 2016). The moisture content of LISG-based film as a function of curcumin concentration is given in Table 1. When the curcumin concentration increased from 0% to 0.6%, the MC decreased significantly. This observation is related to the hydrophobic nature of curcumin (Rostami & Esfahani, 2019). This trend is in agreement those reported by Rostami and Esfahani (2019) who can effectively protect foods against physical and microbial spoilage. On the other hand, high level of WS can be considered as a desirable property, as it imparts a high degree of biodegradability (Stuchell & Krochta, 1994). As presented in Table 1, with increasing curcumin concentration from 0% to 0.6%, WS of the films decreased significantly which has been attributed to the hydrophobic nature of curcumin (Rostami & Esfahani, 2019). Pereira and Andrade (2017) indicated that as the curcumin concentration increased, the WS of chitosan-based films decreased. Likewise, Wu et al. (2018) indicated that the WS of films based on gelatin reduced significantly as the curcumin concentration increased.
Water vapor permeability is one of the main property of the packaging system because it changes the quality and shelf life of foods (Abdollahi et al., 2013). The WVP of packaging materials should be as low as possible in order to preserve the quality of foods over storage period (Zhou et al., 2009). The effect of curcumin incorporation on the water barrier of LISG films is given in

| Mechanical characteristics
The mechanical performance of the developed films was evaluated by two mechanical parameters including tensile strength (TS) and elongation at break (EB). The results are presented in Table 2. With increasing curcumin concentration from 0% to 0.2%, no significant change was observed in TS and EB, but beyond this point, TS increased significantly. The mechanical performance of films is mainly dependent on the microstructure of films and intermolecular force (Shahbazi, 2017). Therefore, the observed effect may be attributed to the strong interaction between the curcumin and polymer structure by H-bond formation, which could enhance the mechanical performance of the film (Bonilla et al., 2014;Liu et al., 2018). Interestingly, the TS of the developed films was near to LDPE as a synthetic film.
On the other hand, as the curcumin concentration decreased, EB showed a decreasing trend. This effect is due to the smaller molecular length of curcumin than LISG, which reduces the deformation of the developed films . The results are in agreement with those reported in previous studies Rostami & Esfahani, 2019). The results of mechanical analysis demonstrated that the mechanical characteristics of LISG film improved significantly with incorporation of curcumin (p ˂ .05), which indirectly exhibited good compatibility between curcumin and LISG matrix.

| FT-IR analysis
The FT-IR spectroscopy is commonly used as a useful tool to examine the impact of the additive incorporation on the chemical structure of polymer-based films by observing the alterations happened in the absorption frequency of the functional groups (Najafi et al., 2020). The FT-IR spectra of curcumin, neat LISG film, and LISG/curcumin film are presented in Figure 2. In LISG spectrum, the peak located in the wavenumber range of 3,000 to 3,500 cm -1 is assigned to OH groups and the main one around 2,900 cm -1 is associated with the C-H stretching vibrations of alkane groups in LISG chain (Sinha et al., 2010). The signal at 1,057 cm -1 is related to O-acetyl groups (Dokht et al., 2018;Percival, 1962). The wavenumber around 1,430 cm -1 is attributed to the symmetrical stretching of carboxylate groups in the uronic acid structure (Vinod et al., 2008). The FT-IR spectrum of curcumin was also recorded (Figure 2). The peak at 3,300 cm -1 is due to the presence of phenolic hydroxyl groups.
The diagnostic peak at 1,600 cm -1 is assigned to the symmetric aromatic ring stretching vibrations (Parveen et al., 2019). In conclusion, it can be seen that there was no considerable change in the FT-IR spectrum of LISG after curcumin incorporation, suggesting the formation of physical interaction between LISG and curcumin.

| Release rate of curcumin
The release profile of curcumin in ethanol from LISG/curcumin film is presented in Figure 3. When the contact time with ethanol increased from 0 to 6 hr, the curcumin released rapidly which may be due to the release of curcumin present on the film's surface. The release rate reached equilibrium after 9 hr immersion. LISG/curcumin film can be used as a promising packaging system because the curcumin released at a low rate that leads to restricting the bacterial growth for a prolonged time and preventing oxidation of food products over storage period.
The values of coefficient of determination (R 2 ) and root mean square error (RMSE) for the models used to describe the release behavior of curcumin from LISG/curcumin films are given in Table 3. It can be observed that Peppas model was the best model to fit control release data. It has been reported that n parameter in Korsmeyer's Peppas equation is measure of the transport mechanism of bioactive compounds, where n ˂ 0.43 shows Fickian diffusion, 0.44 ˂ n ˂ 0.85 demonstrates Anomalous transport and n ˃ 0.85 reveals Case-II transport (Siepmann, & Peppas, 2012). As presented in Table 3, the n value is <0.43. Therefore, the release of curcumin from LISG/curcumin film in ethanol obeyed Fickian diffusion.  Thanzami et al., 2015). The antioxidant activity of LISG/curcumin film was significantly more than neat film (p ˂ .05). The antioxidant activity of curcumin has been related to the existence of the ortho-methoxy groups of curcumin. Overall, the developed film can be introduced as a good candidate for packaging of the foods sensitive to oxidation.

| Film response to pH changes
pH is commonly employed to recognize bacterial growth. To date, several smart packaging systems detecting pH have been fabricated (Kiryukhin et al., 2018;Nopwinyuwong et al., 2010;Pacquit et al., 2007;Rukchon et al., 2014;Salinas et al., 2012). The sensitivity of curcumin to pH change and the presence of carbon dioxide and ammonia have been documented in previous studies (Wannawisan et al., 2018). The response of LISG/curcumin films in contact with acid, neutral, and alkali liquids is depicted in Figure 4a. It can be observed that the developed films had the ability to sense pH changes.  darker. An increase in pH of medium from 3 to 7 was accompanied by a slight increase in a* value, but with further increase of pH, the value of a* showed a significant increase, indicating the intensification of the red color. These results are in agreement with those observed in previous studies (Musso et al., 2017;Pereira & Andrade, 2017). Overall, LISG/curcumin films can be employed to measure the pH increase produced by the basic spoilages.

| Shrimp spoilage trial
Microorganisms are the main reason for the spoilage of animal-based protein foods. Total volatile base nitrogen (TVBN) is commonly used as an indicator for evaluating the seafood spoilage. The production of TVBN reduces the pH of the medium. As mentioned above, LISG/curcumin film had the ability to sense pH changes, and thus the developed film was used as an indicator for monitoring the freshness of shrimp ( Figure 5). The value of a* (redness) as a function of storage time is presented in Figure 6. It can be observed that a* value of LISG/curcumin film increased from 5.7 to 9.9 when the storage time increased to

CO N FLI C T S O F I NTE R E S T
We declare that there are no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data available on request due to privacy/ethical restrictions.