An Antioxidant and Intelligent pH-Sensitive Composite Film Based on Gelatin and Persian Gum Using Purple Carrot Extract

Arab, Mohammad Sadegh; Lashkari, Hannan; Niakousari, Mehrdad; Eskandari, Mohammad Hadi

doi:https://doi.org/10.1155/2023/2848780

Journal of Food Processing and Preservation

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Abstract Introduction Discussion Conclusion Data Availability Conflicts of Interest Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2023 | Article ID 2848780 | https://doi.org/10.1155/2023/2848780

An Antioxidant and Intelligent pH-Sensitive Composite Film Based on Gelatin and Persian Gum Using Purple Carrot Extract

Mohammad Sadegh Arab,¹Hannan Lashkari,²Mehrdad Niakousari,³and Mohammad Hadi Eskandari³

Academic Editor: Charalampos Proestos

Received30 Sept 2022

Revised16 Nov 2022

Accepted18 Nov 2022

Published31 Mar 2023

Abstract

The present research is aimed at developing novel intelligent pH-sensitive films based on Persian gum and gelatin using purple carrot extract. To this end, films were developed by mixing polymer solutions containing Persian gum (5%) and gelatin (5%), in equal proportions and partially replacing distilled water of solutions with purple carrot extract at 0, 15, 30, and 45%. Total anthocyanin content, antioxidant activity, color indices, and tensile strength of films were evaluated. Purple carrot extract incorporation in the film caused a slight reduction in the lightness and an increase in the anthocyanin content, DPPH inhibition, yellowness, redness, and tensile strength. The sample containing 45% extract (anthocyanin concentration was 2.39 mg/ml) was selected as the best pH-sensitive film based on mechanical and antioxidant properties and evaluated in terms of moisture content, moisture absorption, pH, heat seal ability, thickness, contact angle, FTIR, X-ray diffraction, and colorimetric tests. The sensitivity of the film to pH changes was assessed in the range of 2-12. The color of the film changed from pink to blue as a function of pH. The results proved that films based on Persian gum, gelatin, and purple carrot extract could be used as novel food grade biomaterials to monitor the freshness/spoilage of food.

1. Introduction

Nowadays, concerns about buying and consuming healthy and natural food have led to particular consumer attention in many areas of the food industry, including packaging. In the food packaging industry, due to the suitable mechanical and physical attributes of synthetic polymers, manufacturers have a great desire to produce and consume them [1]. However, there is an increasing tendency to utilize green and edible polymers to supply the packaging materials for the food industry. Green and biodegradable packaging are prepared from biopolymers including carbohydrates, proteins, and lipids, which results in increasing the stability of the food products. The advantages presented by these biodegradable materials are included water resistance, retaining aromatic compounds, reducing oxidation and discoloration, and antimicrobial activities [2].

Gelatin is an inexpensive biopolymer that presents an excellent performance and good properties as an edible film. However, gelatin has no adhesion ability and hence this property can be achieved with the help of other polymers (starch, Persian gum, CMC, and chitosan). The dispersion of gelatin can form a gel structure upon heating that is heat reversible. The melting point of the gelatin gel (less than 35°C) is lower than human body temperature, which makes it a widely used ingredient in food products to produce a unique taste and organoleptic properties [3].

On the other hand, Persian gum (PG), as an anionic hydrocolloid, is exudated from mountain almond trees [4, 5] which contains a certain amount of protein and natural water-soluble (~30%) and water-insoluble (~70%) fractions [6, 7]. Mahfoudhi, Chouaibi, Donsi, Ferrari, and Hamdi [8] reported that Arabinose, xylitol, galactose and uronic acid (46.8 : 10.9 : 35.5 : 6.0 mass ratio, respectively) with traces of rhamnose, mannose, and glucose were reported as the monosaccharides present in the polysaccharide chain and some minor protein content. High water holding capacity and emulsification properties, make it a proper choice in food formulations. It has been reported that the water-soluble fraction of PG is dissolved in water at low temperatures and capable to form a fragile film, while the insoluble fraction is unable to dissolve even in hot water and unable to form films due to precipitation during drying of film solution. Protein-based biodegradable films which are produced with gelatin are known as strong translucence films and suitable substitutes for synthetic films [9]. Although PG and gelatin are potential biopolymers to utilize in food packaging, the limited antioxidant activity of these films is a major factor in distinguishing them from active packaging materials. In this context, many attempts have been conducted to develop the functionalities in edible films based on the abovementioned biopolymers by the addition of components with antioxidant characteristics [10].

Anthocyanins are natural and edible pigments produced by plants and found in various parts of the plant such as stems, roots, leaves, flowers, and fruits. They are nontoxic and water-soluble compounds, responsible for the colors blue, purple, and red, and belong to the group of flavonoids [11]. Anthocyanins are glycosides or acyl-glycosides of anthocyanins that are located in cellular vacuoles. The most common anthocyanin types are the glycosides cyanidin, delphinidin, malvidin, polargonidin, peonidine, and petonidine. The application of this anthocyanin was increased due to their physiological, biological, and functional properties including antioxidants, anticancer, and anti-inflammatory activities, heart protection, a barrier against chemicals and antimutation [12]. These compounds are proper additives for the production of active and intelligent packaging and also can change their chemical structure to show different colors at different pHs; therefore, they can be exploited as a pH indicator to track food degradation. Various herbs such as sweet purple potatoes, red cabbage, black soybeans, blackberries, blueberries, and purple or black carrots are good sources of anthocyanins that are used for smart and active packaging [13].

Purple carrots (Daucus carota subsp. Sativus var. Atrorubens Alef) grow in the Middle East and Europe. The acylated anthocyanins in purple carrots are a natural pigment suitable for coloring nectars, beverages, jellies, and pastries. The predominant anthocyanins in purple carrots are cyanidin, which contains various nonacetylated sugars (two of which), or acetylated (three) with sinapic acid, ferulic acid, or coumaric acid [14].

There are a lot of research on the application of anthocyanins in the intelligent films. Wu [15] evaluated the active and intelligent gellan gum-based packaging films for controlling anthocyanins, Chen, Zhang, Bhandari, and Yang [16] studied the pH-sensitive films containing curcumin and anthocyanins, Vedove [17] evaluated smart packaging based on cassava starch and anthocyanin.

This study is aimed at preparing an intelligent and active pH-sensitive film with antioxidant activity based on the combination of Persian gum and gelatin using anthocyanin-loaded purple carrot extract (PCE). Initially, anthocyanins were extracted from the purple carrot. Then, the obtained PCE was incorporated into Persian gum and gelatin film-forming solution. The effects of PCE on the physicochemical and functional characteristics of composite Persian and gelatin film were investigated.

2. Method and Material

2.1. Materials

Bovine gelatin (bloom 200) was purchased from Gelatin Halal Co. (GHC, Tehran, Iran) and Persian gum was obtained from a local market in Shiraz (Fars Province, Iran). Glycerol as the plasticizer was prepared from Emboy Kimya (Turkey). Purple carrots were purchased from a local market in Bandar Abbas (Iran).

2.2. Extraction of PCE as a pH Indicator

The extraction method was carried out according to the method of Barnes, Nguyen, Shen, and Schug [18]. All purple carrot samples were kept in the freezer at -16°C for 24 h, then diced by knife. 10 g of purple carrot samples were added to the centrifugal tube containing 100 ml of a mixture of ethanol, distilled water, and HCl (70 : 30 : 1 v/v/v) and then stirred overnight. Finally, the samples were centrifuged and the supernatant was separated manually and filtered.

2.3. Film Preparation

To prepare film-forming solutions, gelatin (5%, w/v), Persian gum (5%, w/v), and glycerol (3%, w/v) were added to the distilled water. The solutions were mixed in equal proportions by a magnetic stirrer for 6 h. The films were prepared by pouring 20 ml of the mixture into the Petri dishes. In order to make a thin layer, the solutions were spread on the surface by a glass rod with 10 centimeter diameter and then put aside to dry at ambient temperature for 48 h and 24 h in the desiccator. To prepare the active films, 15, 30, and 45% of PCE were replaced with distilled water during the preparation of film-forming solution [19].

2.4. Anthocyanin Content

Total anthocyanins content of films specified by a pH differential method using two buffer systems: KCl buffer, pH 1.0 (0.025 M), and sodium acetate buffer, pH 4.5 (0.4 M). Briefly, 0.4 ml of film samples was blended with 3.6 ml of corresponding buffers and read against water as a blank at 510 nm (A₅₁₀) and 700 nm (A₇₀₀). Equation (4) was used to calculate absorbance (A): [20].

Total anthocyanin content (TAC) of samples (mg cyanidin-3-glucoside/100 ml of extract) was evaluated using the following equation:

where is absorbance, is the molecular weight of cyanidin-3- glucoside (449.2), is dilution factor (10), and is the molar absorptivity of cyanidin-3-glucoside (26,900).

2.5. Antioxidant Activity

Antioxidant activity of films was determined based on the radical scavenging assay [21]. For the concentration-dependent antioxidant assay, a preweighed amount of the prepared film samples was placed in 5 ml of 10 mM DPPH solution with methanol as solvent. After storing the sample solutions in darkness for 60 min, spectrophotometric methodology for reaction solution was evaluated at 517 nm. DPPH radical scavenging activity was calculated using

where is the absorbance of the blank cell and is the absorbance of the reaction solution.

2.6. Color Properties

Color variables of the films were determined using a Minolta colorimeter (CR-20, Konica Minolta, Inc., and Tokyo, Japan). The images were cut to and transformed into CIELAB color system, and their corresponding , , and parameters were analyzed using ImageJ 1.47n software (National Institute of Health, USA). The measurements were conducted in triplicates [22].

2.7. Mechanical Analysis

Film tape () was fixed between the grips of Instron universal instrument model no. 5543A (Instron Engineering Corp., Norwood, MA, USA). The tensile strength () was calculated by dividing the maximum peak force () by the film area () [23, 24].

where obtains by multiplying the film thickness (mm) by film width (mm).

2.8. Moisture Content

The moisture content of films was determined by following the procedure described by Tabatabaei [24] and Muñoz, Aguilera, Rodriguez-Turienzo, Cobos, and Diaz [25]. Films were weighed and dried in oven for 105°C to reach a constant weight. The moisture content of films was calculated using the following equation:

where is wet weight and is the dry weight of the film.

2.9. Moisture Absorption

A cut of films was placed in a desiccator at 0% relative humidity by using anhydrous calcium chloride for 6 days at room temperature, weighed, and placed in a vessel containing saturated K₂SO₄ with 97% relative humidity at room temperature, then weighed until the constant weight was gained. Moisture uptake was calculated from [22].

where is the initial weight and is the weight of the film after water absorption.

2.10. pH Test

For pH determination, the film was firstly mixed with 10 ml water and kept to absorb water. Then, the pH of the film solution was evaluated by a digital pH meter (model: Seven Compact S-220, USA) [26].

2.11. Film Thickness

Film thickness was measured by using high accuracy (0.001 mm) digital micrometer (iGaging, san Celemt, California) for at least ten randomized locations of films. This procedure was repeated three times for other independent films [22].

2.12. Heat Sealing Ability

Film samples were cut into a tape shape of . One tape was placed on the top of another and then heat-sealed using an automatic heat sealer (AH26 Mod: Go plus 8, IRAN). The width of the sealing area was equal to 1.5 mm. All sealed films were then conditioned at room temperature with for two days prior to analysis. The heat-sealing ability was measured using the peel and the lap shear tests according to Standard ASTM F-88 [27] with slight modifications, using a texture analyzer (Instron Engineering Corp., Norwood, MA, USA). Each tape of the sealed film was hitched to the clamp, in a manner that each end of the leg was kept perpendicularly to the force direction. The clamps were 50 mm apart and a statistical force of 100 N with a speed of 30 mm/min was applied. The ultimate force in which the tapes were detached was determined as sealing strength (Pa).

2.13. Contact Angle

When a droplet of liquid is placed onto a surface, the angle between the underside of the droplet and the adjacent curvature of the droplet is called the contact angle. The test was conducted using a camera to capture the picture of a placed droplet of liquids (~15 ml) onto the surface of films after 10 s. The contact angle was determined using ImageJ software (1.44p, National Institute of Health, USA). The replications for each liquid and sample were 9 times and 3 times, respectively [28].

2.14. Fourier-Transform Infrared (FTIR) Spectroscopy

FTIR spectra of films were collected in form of transmittance using a FTIR spectrometer (Thermo Nicolet Corp., Madison, WI, USA) at a step of 4 cm^-1 in a period of 400 to 4000 cm^-1 wavenumbers. The milled samples were blended with KBr (1 : 100, w/w) and formed into pellets before spectroscopy.

2.15. X-Ray Diffraction (XRD)

Crystalline characteristics of films were evaluated using Bruker AXS D8 Advance 191 X-ray diffractometer (Thermo Nicolet Corp., Madison, WI, USA). The operation was at 40 mA and 40 kV using Ni-filtered Cu Kα radiation. The diffraction pattern was obtained from at a scan speed of 0.1°/s.

2.16. SEM

The images of the samples were determined by a scanning electron microscope (TESCAN vega3, Czech Republic). SEM images were taken at a 20 kV accelerating voltage.

2.17. Determination of pH-Sensitivity of Film

In order to investigate color changes, the film was flooded with an aqueous media with different pHs from 2 to 12 for about 15 min. The color changes were recorded by Minolta colorimeter (CR-20, Konica Minolta, Inc., Tokyo, Japan). The color difference for the film at different pHs compared to a constant pH was determined through

2.18. Statistical Analysis

The design of the completely randomized analysis of variance (ANOVA) procedure was conducted using SPSS software (Version 16; SPSS Inc., USA). The comparison of the differences between mean values was also conducted using Duncan’s multiple range tests at .

3. Result and Discussion

3.1. Anthocyanin Content

Total anthocyanin content in film samples is shown in Figure 1. As expected, the film with 45% of PCE presented higher content in comparison to others.

3.2. Antioxidant Activity

Since free radicals are responsible for nutritional degradation, the antioxidant activity of composite films plays an important role in food packaging [29]. As depicted in Figure 2, the antioxidant activity of film samples was notably improved by increasing PCE (). This fact can be attributed to the increase in anthocyanin content in the films. A similar trend in free radical scavenging was reported by Yong, Wang, Zhang, Liu, Qin, and Liu [13] who fabricated chitosan film incorporated with anthocyanin-rich purple and black eggplant extracts.

3.3. Colorimetric

Visual and optical properties are important features of films that are used as packaging or as food coatings. The overall appearance of the food is directly related to the color of the packaging, and this can greatly affect the customer’s product friendliness [30]. The values of lightness (), green-red (), and blue-yellow () parameters of films are shown in Figure 3. The addition of PCE into the film matrix caused a remarkable () effect on this parameter. Control composite film was transparent with more greenness and blueness color, whereas with an increase in the content of anthocyanins, the significantly () decreased, while and significantly () increased. Therefore, PCE resulted in darker and more redness and yellowness composite films which were more intense at higher concentrations. A similar trend was also reported by Kurek [31] for chitosan film incorporated with anthocyanin-loaded blueberry extract. In general, more opacity has been reported with the incorporation of natural active ingredients such as pomegranate rind extract [32] and black eggplant extracts [13] into the film-forming solutions.

3.4. Mechanical Analysis

The mechanical strength and transportation capability play a key role in the selection of a polymer as a packaging material [33]. As shown in Figure 4, the tensile strength of films with PCE with 15, 30, and 45% concentration (67.8, 70.6, and 76.53 Pa, respectively) was higher than control film (65.2 Pa) (). The increase in tensile strength of samples incorporated with PCE might be due to H-bonds formed between OH and amine groups of gelatin and Persian gum and polyphenols in the extracts which interrelationship and interfacial interaction of polymer chains have a median effect on all mechanical factors [34]. A similar trend of mechanical strength was reported for agar-nanocellulose and zein-pomegranate peel extract films [35]. According to the results of physicochemical and textural properties, the film sample containing 45% extract was selected for more evaluation.

3.5. Moisture Content

As shown in Table 1, significant differences () were observed in the moisture content of control film and active film with 45% of PCE. Generally, the relatively low moisture contents in PCE films might be due to the interactions between amine and OH groups of Persian gum, gelatin, and anthocyanins in PCE, which might restrict the interactions of Persian gum and gelatin with water [10]. A similar result was also reported for chitosan film with eggplant extract represented [13], whereas Kurek [31] reported a slightly lower moisture content in blueberry and blackberry pomace extracts-loaded chitosan film. These contradictory results indicate the significant () effect of composition and the concentration of anthocyanins on their degree of bonding to biopolymer matrix and hence the moisture content of final films.

3.6. Moisture Absorption and pH

The water absorption isotherm provides useful information about the microstructure details of the films. The results of the Table 1 showed that PCE-loaded composite film presented more water abortion than control film. This fact might be attributed to higher accessible polar segments due to the presence of anthocyanins which resulted in a higher amount of absorbed water molecules [36]. A similar observation was also reported when grapefruit seed extract was added to chitosan film [37]. On the other hand, the results of pH measurements of film solutions showed a lower value after incorporating PCE which can be related to the presence of the functional acidic group of anthocyanin.

3.7. Heat-Sealing Ability

The sealing ability of films is important for food packaging to manufacture sachets and pouches [38]. As shown in Table 1, PCE-loaded film presented significantly () higher heat-sealing strength (83.33 Pa) than control film (81.7 Pa). Interaction of aldehydes, ketones, and anthocyanin with protein and hydroxyl group, additionally, hydrophobic groups of polyphenols are capable to associate with gelatin chains through hydrogen bonds and hydrophobic interaction. These macromolecules by creating cross-linkage and hydrogen bonds in proteins might result in a strong network in the gelatin matrix, resulting in the better heat-sealing ability of PCE-loaded composite film [39].

3.8. Film Thickness

Mechanical properties, heat-sealing ability, and water vapor/oxygen permeability are predominantly influenced by the film thickness. As illustrated in Table. 1, the thickness of PCE-loaded film and control film were 79.33 μm and 80.33 μm, respectively. Accordingly, no significant () difference was observed in thickness value after the incorporation of PCE. This observation suggested a good distribution of PCE in the film matrix. A similar observation was also reported for pectin/chitosan composite films incorporated with purple sweet potato, blueberry, and cranberry extracts [40]. Since numerous OH- groups are presented in anthocyanins might result in a firm interaction with gelatin and Persian gum chains in film and a compact network was formed.

3.9. Contact Angle

Contact angle describes the uniformity of surface, wettability, surface free energy, and also the placement of functional groups on the surface which are critical characteristics in packaging films [41]. As shown in Table 1, the contact angle of PCE-loaded film (76.64°) was significantly () more than the control film (66.2°). All hydrophilic samples have a contact angle of less than 90° and both samples had hydrophilic surfaces. However, less hydrophilicity of PCE-loaded film surface might be due to the dominant anthocyanin content, which led to forming polar sites to interact with Persian gum and gelatin chains, resulting in a lower exposure of biopolymers polar groups for the interaction with water molecules at the surface of films. This phenomenon was also reported with cassava starch-based film with anthocyanin [17].

3.10. FTIR Analysis

FTIR spectra and FTIR peak assignments are presented in Figure 5 and Table 2, respectively. The broad band at 3000-3500 cm^-1 was attributed to vibrational stretching of hydroxyl groups which reflects the water retention and hydrogen bonding in Persin gum and gelatin [7, 19]. Also, the peak in the range of 2800–3000 cm^-1 and a peak at 1411 cm^-1 were mainly ascribed to carbon-hydrogen and C=N bond stretching vibration, respectively [7, 42]. The absorption peak at 1655 cm^-1 was attributed to the double bond stretching vibrations between carbons (C=C) [24]. The peak around 1031 cm^-1 might be due to the interaction between hydroxyl groups of plasticizer and film polymers [43]. Moreover, it has been reported that the region in 1600–1700 cm^-1 is assigned to the amide I spectral region as well as accurate to the secondary structure of proteins which is predominantly a consequence of stretching bonds in C=O of peptide structures [19]. Other peaks at 2932, 1238, and 1020 cm^-1 might be attributed to carbon-hydrogen and carbon-oxygen single bonds [44]. The peak at 1031 cm^-1 was also ascribed to the stretching vibration bond of the C–O–C structure [4, 23].

3.11. X-Ray Diffraction (XRD)

Figure 6 represents the crystallography patterns obtained from the X-ray diffractometer. The presented broad peak of about 20° implied the amorphous structure of PCE-loaded and control film samples. Since no other significant peak was observed, it can be concluded that no expressive crystalline structure was detected. The peak diffraction of the composite film after incorporating PCE was lower than the control film. This finding was consistent with previous reports in which the diffraction patterns were decreased after the addition of anthocyanins-loaded extract to the film formulations [21, 45]. This fact can be attributed to the disruption of ordered network structures of the biopolymer matrix after incorporating extract [17, 21]. However, some reports showed higher crystallinity of anthocyanin-loaded films [13] which was related to the content and composition of anthocyanins added.

3.12. SEM

Figure 7 shows the surface images of composite films. The surface of the control composite film was relatively compact. Similarly, after the addition of PCE, a uniform/smooth surface without cracks, holes, and insoluble particles was observed, indicating the good compatibility and miscibility of ingredients. The cross-sectional images of two samples show smooth and continuous structure. Also, Gahruie [7] reported homogeneous microstructures of Persian gum/gelatin composite films at pH 4.18 and 5.6.

(a)

(b)

3.13. pH Sensitivity of Film

The pH response and of PCE-loaded film were shown in Figure 8 and Table 3, respectively. Film samples were shown pH-sensitivity and notable color changes as affected by different buffer solutions. By increasing pH values, the color of the film turned from pink to blue/purple. These color changes occur due to the structural changes of anthocyanin at different pHs. Anthocyanins exist basically in cationic form (red or pink flavylium cation) at pH 2–3. The purple color observed at high pH (pH 4–6) due to the generation of carbinol pseudobase and blue-colored observed at due to the formation of quinoidal bases [34]. Other research also showed pH sensitivity of biopolymer-based films incorporated with extracts of purple potato, red cabbage, blackberry pomace, and purple and black eggplant [13]. Changes in color of samples validated by measuring the of samples (Table 3). The difference () in the amount of indicates the color change of the film at different pH. The results proved that PCE-loaded film could act as a pH indicator.

4. Conclusion

Since the exploitation of green, nontoxic, and biodegradable packaging materials has been considered a global concern, the design of a proper packaging film to sustain the food product quality and protect it from environmental conditions and degradation is a promising field of study in the food industry. The aim of this study was to prolong the food products shelf-life by the potential of active Persian gum and gelatin composite film incorporated with PCE. The results showed that the antioxidant activity of film was successfully improved by incorporating a high concentration of extract. Moreover, incorporating PCE improved mechanical strength, heat-sealing ability, and moisture absorption. The result suggested that biodegradable films with PCE can be exploited as an assured food packing material with good radical scavenging ability. However, further research is needed to show validate the antioxidant activity and pH sensitivity of PCE-loaded films in real food packaging applications to monitor spoilage.

Data Availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

The financing of this case was provided by the student himself.

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Copyright © 2023 Mohammad Sadegh Arab et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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