Halochromic (pH-Responsive) Indicators Based on Natural Anthocyanins for Monitoring Fish Freshness/Spoilage

Abstract

Today, smart indicators especially based on pigments and natural biopolymers have developed significantly in laboratory and industrial scale. In addition to tracking the freshness and spoilage of the product, these intelligent systems inform the consumer about the quality of the packaged product without opening the package. On the other hand, they reduce food waste and minimize food poisoning. In this study, two halochromic smart indicators were constructed by encapsulating saffron petal and barberry anthocyanins in gelatin/chitin nanofiber films for indication of the freshness/spoilage of fish fillets. Insights into the molecular, structural, and optical properties of these indicators were obtained using X-ray diffraction, scanning electron microscopy, and infrared spectroscopy, and colorimetry analyses. The results showed that the indicators had smooth surfaces and that the pigments were evenly distributed throughout the biopolymer network. The barberry and saffron anthocyanin solutions underwent color changes from reddish to yellow for barberry and reddish to pink to violet to greenish and yellow for saffron anthocyanin after being exposed to different pH values (1–14). The change in appearance of halochromic indicators was quantified by measuring their color coordinates (L*, a*, b*). When applied for estimating fish spoilage, the color of the G/ChNF/BA and G/ChNF/SPA indicators turned from pink to yellow and from violet to green, respectively. After 3 days of storage, the pH and total volatile basic nitrogen of fish fillets reached 8.0 and 49.06 mg N/100 g, respectively. Therefore, a direct relationship between the increase in pH values, the increase in volatile nitrogen bases content, and the changes in the color of the smart indicator applied to monitor the fish was observed. The simulation tests showed that pH-responsive smart indicators can reveal visually fish fillets freshness in real time prior to the point of consumption.

1. Introduction

The main purpose of designing intelligent or smart indicators is to measure or indicate packed food products’ quality during the period of transport, storage, and sale [1,2]. Typically, they contain a sensing element that provides a measurable output when some aspect of the food quality changes, such as its freshness or safety. Halochromic indicators have drawn much attention for their extensive application in smart packaging systems because they can create a color response at a stable pH condition [3,4]. Changes in food quality are often accompanied by an alteration in pH, which can be detected by this kind of sensor [3,5]. An advantage of halochromic indicators is that they provide a visible indication of food freshness or safety that food manufacturers, distributors, or consumers can use to assess food quality [6,7,8]. Halochromic indicators suitable for food packaging and coating applications can be constructed from natural pigments, which are often more acceptable to consumers and cause less environmental pollution than synthetic dyes. Halochromic indicators have been constructed from pH-sensitive pigments extracted from various kinds of plants. For example, You et al. [9] prepared pH-intelligent Konjac glucomannan/carboxymethyl cellulose-based films by adding blackcurrant that have been used for measuring fish freshness. Gasti et al. [10] integrated pH-sensitive Phyllanthus reticulatus anthocyanin into a chitosan/methylcellulose matrix to produce smart films for monitoring the spoilage of fish fillets. Wu et al. [11] reported locust bean gum/polyvinyl alcohol-based colorimetric films produced via incorporation of betacyanins extract from the Celosia cristata L. flower, which showed good potential in measuring shrimp spoilage. The most important pH-sensitive pigments in these plants are anthocyanin-derived pigments, which are found in many kinds of fruits, vegetables, flowers, and other botanical sources [12]. Several kinds of anthocyanin-derived pigments became recently state of the art, as they are potentially more efficient intelligent indicators than anthocyanins, namely pyranoflavylium derivatives, flavylium dyes, and berry anthocyanin [13,14]. As well as their ability to change color with pH, many anthocyanins also exhibit good preservative properties, such as antioxidant or antimicrobial activity, which can prolong the storage time of packaged foods [15,16].

Previously, our group showed that halochromic films containing barberry and saffron anthocyanins could be used to indicate the spoilage of lamb during 3 days’ storage [15,16]. These films were constructed from biopolymer composites consisting of methylcellulose and chitosan nanofibers. In general, the functional performance of edible films and coatings can be modulated by using different kinds of biopolymers to construct them, such as chitosan [17], starch [18], methylcellulose [15], and gelatin [19]. In the current study, we focused on the utilization of films constructed from gelatin and chitin nanofibers. Gelatin (G) is a byproduct of the livestock industry that is produced through thermal and chemical hydrolysis of collagen [20]. It is a good candidate for creating edible coatings and packaging materials because it possesses good biocompatibility, biodegradability, and film-forming ability, and it is safe [19,21]. Even so, compared to synthetic polymers, gelatin films have several physicochemical limitations that currently restrict their commercial application, including weak mechanical performance, barrier performance, and processability. The introduction of chitin nanofibers (ChNFs) as a reinforcing nanofiller into the gelatin matrix can overcome some of these limitations [22]. Chitin is known as aminopolysaccharide polymer occurring in nature, and is derived from crustaceans, insects, and the cell walls of fungi. Typically chitin can be converted to chitosan through enzymatic or chemical deacetylation [23]. Chitin is obtained from byproduct of the food-processing industry such as shrimp and crab shells, that provides a large quantity of this biopolymer to be used in food and biomedical applications. One of its wide applications is the production of packaging films and coatings together with chitosan [24,25]. On the other hand, chitin and chitosan, in addition to their functional properties, improve the physical and mechanical properties of packaging films as nanofillers [26].

In this study, we used saffron petal anthocyanins (SPAs) and barberry anthocyanins (BAs) as pH-sensitive colorimetric indicators. These anthocyanins were encapsulated in composite biopolymer films constructed from gelatin and chitin nanofibers. After characterizing the molecular, structural, and optical/colorimetric properties of these indicators, we examined their ability for tracking the freshness of fish samples during 3 days’ storage at room temperature (RT). Our results provide valuable insights into the potential application of anthocyanin-loaded indicators as smart packaging materials.

2. Materials and Methods

2.1. Experimental Reagents

Barberry fruit was obtained from South Khorasan province (Iran), and saffron flowers were obtained from Khorasan-Razavi province (Iran). The gelatin (Type B, average molar mass = 80 kDa, bloom values = 200 g) and chitin nanofibers (purity > 99%, size~30–50 nm) were supplied from Sigma Aldrich (St. Louis, MI, USA) and Nano-Novin Polymer Company (Sari, Iran), respectively. Acetic acid, sodium hydroxide (NaOH), hydrochloric acid (HCl), and ethanol with high purity and glycerol were provided by Merck Co (Darmstadt, Germany). The fish samples were picked from an Azarmarjan aquatic market in Tabriz, Iran, and transported to the food microbiology laboratory in boxes with gel ice-packs.

2.2. Extraction of Anthocyanins

The barberry and saffron petal anthocyanins were prepared according to a method described in our previous research [15]. First, barberry fruits and saffron petals were dried in the shade using air flow and then ground and sieved. Then, 1 g of either crushed barberry fruit or saffron petals was added to 20 mL of 80:20 v/v distilled water/ethanol solution using magnetic stirring overnight at RT. After the filtration of final extracts, the water and ethanol were removed using a rotary evaporator at 37 °C to acquire concentrated extracts. The extracts were stored in a dark, airtight container at 4 °C prior to analysis and application.

2.3. Preparation of Halochromic Smart Indicators

Halochromic smart indicators were fabricated by casting method. First, 3.0 g of gelatin was dissolved into 100 mL water at 60 °C using mild stirring for 2 h. Simultaneously, 3.0 g of chitin nanofibers were dissolved in 100 mL of 1% acetic acid solution. Subsequently, the obtained chitin nanofibers solution, 1.8 g of glycerol, and 3% of anthocyanins were introduced into the gelatin solution through mild stirring for 2 h to completely mix. The final solution was cast onto a leveled dish (8 cm diameter) and dried for 2 days at RT. Finally, the indicators were kept at RT in the dark. G/ChNFs without anthocyanins were used as the control. The indicators containing barberry anthocyanin and saffron anthocyanin were named G/ChNFs/BAs and G/ChNFs/SPAs, respectively.

2.4. pH-Responsiveness of Anthocyanin Solutions and Halochromic Indicators

The color changes and pH-sensitivity of the anthocyanin solutions and halochromic smart indicators were investigated in buffer solutions (pH = 2–14) adjusted using HCl and NaOH. Digital photographs of the samples were recorded to provide insights into their pH-dependent color changes.

2.5. Color Coordinates of Halochromic Indicators

The color coordinates (L, a, b) of the halochromic smart indicators were measured using a Minolta Chroma Meter Model CR-400 (Minolta Co Ltd., Osaka, Japan). The Hunter color values, L* (lightness), a* (red/green), and b* (yellow/blue), of the indicators were determined using the instrument based on spectral reflectance measurements. The total color difference (ΔE*) was then computed using the following equation:

$$∆E=\sqrt{{(L_1^{\mathrm{*}}-L^{\mathrm{*}})}^2+{(a_1^{\mathrm{*}}-a^{\mathrm{*}})}^2+{(b_1^{\mathrm{*}}-b^{\mathrm{*}})}^2}$$

Here, $L_1^{*}$, $a_1^{*}$, and $b_1^{*}$ are the color indices of reference (white plate), and L*, a*, and b* are the color indices of each indicator in the film.

2.6. Characterization

2.6.1. Scanning Electron Microscopy (SEM)

Information about the surface morphology of the halochromic indicator films was obtained using (SEM) (Sigma VP, ZEISS, Jena, Germany) following gold coating at a working distance of ~5 mm.

2.6.2. Fourier-Transform Infrared Spectroscopy (FTIR)

Information about the molecular composition and interactions of the halochromic indicator films was determined by measuring their FT-IR spectra in a range from 4000 to 500 cm⁻¹ at 16 cm⁻¹ resolution using a Nexus-670 FT-IR spectrophotometer (Thermo-Nicolet, Madison, WA, USA).

2.6.3. X-ray Diffraction (XRD)

Information about the physical state of the halochromic indicator films was recorded by XRD analysis using an X’pert pro MRD diffractometer (PANalytical, Almelo, The Netherlands) within the 2θ angle range from 5° to 80° at RT.

2.7. Freshness Monitoring of Fish Fillet Samples

Halochromic smart indicators were used to estimate the freshness of fish filets. 25 g of fish filets were placed in a sterilized box. Then, halochromic smart indicators were attached to the inside of sterilized polyethylene terephthalate boxes that did not have direct contact with the trout fillets. The prepared samples were kept at 25 °C for three days. At 12 h intervals, the color change of the indicators was recorded by a digital camera. On each of the test days, pH changes and TVB-N values were also determined.

2.7.1. pH

The pH values of fish fillet sample were measured by a digital pH meter(Thermo Scientific, A211-pH meter, Tangerang, Indonesia) after homogenization of every 5 g of sample in 50 mL of distilled water.

2.7.2. Total Volatile Basic Nitrogen (TVB-N)

TVB-N levels were analyzed based on the study by Liu et al. (2020). Briefly, an analysis was performed using the Kjeldahl method with 5 g of homogenized fish sample added to 2 g of magnesium oxide powder in a balloon. TVB-N concentrations were measured in mg N/100 g of sample [27].

2.8. Statistical Analysis

Every experiment in this study was conducted 3 times (n = 3). The results and significant differences (defined as p < 0.05) were analyzed using an ANOVA model (using IBM SPSS Statistics 21, New York, NY, USA) and Duncan’s multiple range test, respectively.

3. Results and Discussion

3.1. Surface Morphology

The SEM micrographs of the G/ChNF film with and without anthocyanins are shown in Figure 1. The microscopy images of the pure G/ChNF films show that they had a fairly heterogeneous surface with some localized aggregates, which can be ascribed to the presence of chitin nanofibers that protruded out of the film matrix (Figure 1a). Interestingly, after both types of anthocyanin (BAs or SPAs) were incorporated into the G/ChNF films, the microscopy images indicated that their surfaces became smoother (Figure 1b,c). These results suggest that the barberry and saffron pigments had a good compatibility with the other components within the biopolymer-based indicators, i.e., the gelatin and chitin nanofibers. Even so, the surface morphology of the G/ChNF/SPAs smart indicator appeared to be somewhat smoother than that of the G/ChNF/BAs ones (Figure 1c). These changes in film morphology may be attributed to the plasticizing properties of the anthocyanins. A similar phenomenon in which the surface of the chitosan film became smooth and homogenous when mangosteen rind powder was added, was observed by Zhang, Liu [17]. According to earlier studies, anthocyanins have been reported to have a high compatibility with various types of biopolymers, including methylcellulose [15], and gelatin [19].

3.2. FT-IR Spectroscopy

Information about the molecular composition and interactions of the anthocyanin-loaded biopolymer films was assessed using FT-IR spectroscopy. This spectroscopy method is sensitive to the interactions of specific functional groups in materials. As shown in Figure 2, the IR spectra contained numerous absorption bands that could be related to specific functional groups associated with the film constituents. The major groups involved were hydroxyl groups (-OH) belonging to gelatin, chitin nanofibers, and anthocyanins, as well as amine groups (-NH₂) belonging to gelatin and chitin nanofibers. Interactions of these functional groups with other constituents in composite materials are known to alter their vibrations and, as a result, their spectral positions [17]. Overall, all the composite films exhibited fairly similar spectra with some minor variations depending on the specific constituents they contained. For the G/ChNF film, a characteristic peak was observed in the spectral pattern between 3500 cm⁻¹ and 3100 cm⁻¹ (~3252.56 cm⁻¹), which corresponds to O-H and N-H stretching vibrations of the amide A and amide B groups [28]. This peak is therefore sensitive to the interactions between chitin nanofibers and gelatin chains. Furthermore, the broad peak observed in this position may also reflect overlapping between N-H stretching peaks of both the amine and amide ΙΙ groups [29,30]. The position of these peaks shifted from ~3252.56 cm⁻¹ to 3272.33 cm⁻¹ and 3265.26 cm⁻¹ when barberry and saffron pigments were incorporated into the G/ChNF films, respectively, in accordance with their interactions among the functional groups of the film. The G/ChNF films with and without anthocyanins exhibited several characteristic absorption peaks, such as C-H stretching at ~2950 cm⁻¹–2800 cm⁻¹, C=O stretching at ~1626 cm⁻¹ (corresponding to the amide Ι group), and C-O stretching around ~1028 cm⁻¹ [15]. The peak observed around 1536.2 cm⁻¹ was attributed to the amide ΙΙ group, which arises due to N-H bending and C-N stretching [15]. Absorption peaks associated with carbonyl groups (1700 cm⁻¹–1600 cm⁻¹) are typically associated with the presence of acetic acid, which is used as a solvent to dissolve the chitin nanofibers. Additionally, the aromatic ring stretches observed in the halochromic smart indicators around 1500 cm⁻¹–1400 cm⁻¹ (~1444.5 cm⁻¹) confirmed the presence of anthocyanins. The above results are generally consistent with earlier research on similar materials used in the matrix of intelligent packaging films [15].

3.3. XRD Analysis

It is well known that the optical, mechanical, and barrier characteristics of packaging materials are influenced by the crystallinity of film matrices. For this reason, the effects of the barberry and saffron pigments on the crystallinity of the G/ChNF films were investigated using XRD analysis (Figure 3). The G/ChNF films exhibited two characteristic peaks around 2θ = ~8° and ~20°. The ChNF peaks have been observed as well as gelatin concentration peaks, which have been linked to gelatin α-helix and β-sheet [29,30]. The diffraction peak positions of the G/ChNF films were not significantly altered by the addition of the anthocyanins. This result suggests that the ordered regions in the G/ChNF films were not significantly changed by the addition of the barberry or saffron pigments. Based on this result, barberry or saffron pigments had no significant effect on the ordered regions in the G/ChNF films. Due to the homogeneous distribution of anthocyanins throughout the film matrix, neither the structure of the chitin nanofibers nor the structure of the gelatin molecules was significantly affected. The XRD patterns of methyl cellulose-chitosan nanofiber-saffron petal anthocyanin [15] and gelatin-κ-carrageenan-TiO₂ nanoparticles- saffron or red barberry [19] were also similarly changed.

3.4. pH-Sensitivity of the Anthocyanin Solutions and Halochromic Smart Indicators

Anthocyanin-loaded biopolymer films and solutions of anthocyanin were evaluated at different pH levels (Figure 4). Films containing BAs and SPAs were immersed in buffer solutions (pH = 2–14) prior to analysis in order to control their pH. According to Figure 4, the color of the anthocyanin-loaded G/ChNF films showed a similar dependence on pH to that of the anthocyanin solutions. pH was a factor in determining the film color based on the type of anthocyanin. In acidic conditions, saffron anthocyanin is reddish pink, while in alkaline conditions, it is greenish yellow. However, barberry anthocyanin is reddish/crimson in acidic conditions and yellow in alkaline conditions. Anthocyanin molecules change color due to pH-induced structural changes: flavylium cation (pH < 4); carbinol pseudo-base (pH 5–6); and chalcone (pH > 8) (Figure 5). Similar color changes in response to pH changes were also reported by many researchers when using different anthocyanins. For example, Bao et al. (2022) developed a colorimetric smart indicator based on blueberry anthocyanin [31]. The blueberry anthocyanin solution revealed color changes from pink at acidic conditions, to grey-pink and grey-blue in neutral conditions, and to grey-brown at alkaline conditions. In another study, Gasti et al. (2021) fabricated a colorimetric smart packaging film based on Phyllanthus reticulatus fruit anthocyanin [10]. The Phyllanthus reticulatus anthocyanin solution was red in acidic pHs and pink at pH 4–5. The anthocyanin color altered to grey in neutral pHs, and gradually converting to green in alkaline pHs.

The pH-sensitivity of the biopolymer films loaded with anthocyanin was demonstrated by these results. The color changes of smart indicators were similar to the color changes of anthocyanins solution, which proves the efficiency of anthocyanins as smart materials in development of intelligent packaging systems. Therefore, they may be useful to monitor the freshness of perishable foods when a pH change is associated with quality changes. Other researchers have reported that anthocyanins from other types of edible plants also exhibit halochromic behavior, including blueberry [31], black eggplant [32], purple sweet potato [33], and red cabbage [18].

3.5. Color Coordinates of Halochromic Smart Indicators

Evaluation of color coordinates is one of the most important parameters for smart packaging. Because they show the efficiency of anthocyanin used as a smart material for packaging systems. The color coordinates (L, a, b, and ΔE) of anthocyanin-loaded halochromic smart indicators are shown in Figure 6. Visually, the G/ChNF film had a slight yellowish appearance (b = +9.5). The films loaded with barberry and saffron anthocyanins exhibited the highest redness (a = +24.5) and blueness (b = −10.7) indices and appeared reddish and violet to the eye, respectively. Films became heavier after incorporating anthocyanins, which can be explained by absorption of more light and less scattering from their surfaces. The color changes can be attributed to the reddish, bluish, and yellowish colors of the BA and SPA solutions under acidic, neutral, and alkaline conditions, respectively. Other studies have reported similar color shifts for on locust bean gum/PVA composite film + cockscomb flower [11], carboxymethyl cellulose/poly-vinyl alcohol (PVA) + rose petal extract [34], and cassava starch + grape skins [35].

3.6. Fish Freshness/Spoilage Monitoring Using Halochromic Smart Indicators

There is often a change in the pBMH of perishable foods when they undergo spoilage. For instance, the pH of the muscular tissues in fresh meat and fish may change significantly as a result of biochemical reactions associated with protein and lipid degradation, as well as due to microbial growth. In general, TVB-N levels increase when fish spoils because volatile amines are formed. Hence, TVB-N levels are widely used to indicate fish spoilage. Additionally, fish pH values are affected by the production of amines during fish spoilage [36]. TVB-N and pH levels are shown in Figure 7A,B, respectively. TVB-N levels in the G/ChNF/SPA film increased over time from an initial level of 6.7 ± 0.1 mg N/100 g for fresh fish (Figure 7A). The level of TVB-N increased dramatically after 12 h from 18.16 ± 1.35 mg/100 g to 31.28 ± 2.03 mg/100 g at 24 h of storage. Finally, after 72 h, TVB-N levels reached 49.06 ± 0.95 mg/100 g. These changes were similar to those of the G/ChNF/BA film as shown in Figure 7. pH-responsive indicators change color due to nitrogenous gases released during storage, and the pH of the fish fillets increased from around 6.3 to 8.0 (Figure 7B). The pH values of >7 and TVB-N level of 25 mg N/100 g have been considered as a rejection limit for fish fillet samples. Within the first 24 h, the values of pH and TVB-N were higher than the standard limit. Ekrami et al. (2022) developed colorimetric indicators based on salep mucilage with saffron anthocyanin to monitor trout fish freshness, and the color change of indicators from yellow to brown [37]. Fathi et al. (2023) developed intelligent carboxymethyl chitosan films incorporated with saffron anthocyanin to monitor lamb meat freshness and observed the color change from pink to green [38]. Based on these results, both types of films loaded with anthocyanin might be suitable for monitoring fish spoilage. In this study, anthocyanin-loaded G/ChNF films attached above the headspace of a packaging container holding fish fillets were used to monitor the quality of the fish during storage at RT. Figure 8 shows the color changes of the films before and after storage. The G/ChNF/BA smart indicator changed from pink to yellow, whereas the G/ChNF/SPA smart indicator changed from violet to yellowish/greenish. Other kinds of biopolymer indicators containing anthocyanins have also been reported to be suitable for the real-time monitoring of the spoilage of fish and other seafood products [19,31,39]. Taken together, these results suggest that anthocyanins may be a good pH-sensitive indicator for application in food-packaging applications.

4. Conclusions

The design and introduction of smart packaging/indicators is one of the emerging technologies in the food packaging industry that has developed dramatically. these smart systems, in addition to being able to inform consumers and stakeholders about the conditions of packaged products and monitor and track the freshness of products in real-time, leading to reduce food waste. In this study, we have shown that halochromic indicators could be created by encapsulating natural anthocyanins from saffron petals or barberry anthocyanins in gelatin/chitin nanofiber films. Electron microscopy, X-ray diffraction, and infrared spectroscopy were used to examine the film morphology and molecular interactions. A notable result was the enhanced surface smoothness of the films induced by the anthocyanins, which plasticized the biopolymers within the matrix. As the pH increased from 1 to 14, the color of the films changed from reddish to pale peach to pink to yellow for the barberry anthocyanins and from reddish to pink to violet to green to yellow for the saffron petal anthocyanins. Following that, the anthocyanin-loaded films were successfully used to monitor fish spoilage during storage by using them as freshness indicators. Color changes in the smart indicators were attributed to changes in pH levels and to the nitrogen gasses released as muscle tissues degraded. After 3 days of storage, the pH and total volatile basic nitrogen of fish fillets reached 8.0 and 49.06 mg N/100 g, respectively. Therefore, these indicator/sensors may be useful for developing smart packaging systems for monitoring the freshness of foods. However, some limitations such as the low stability of anthocyanins in environmental conditions should be investigated in future studies to make the application of these smart materials practical on an industrial scale.