Application of Plant Extracts Rich in Anthocyanins in the Development of Intelligent Biodegradable Packaging: An Overview

Abstract

Consumers are increasingly opting for food with high quality, in addition to practicality, as there are changes in time, habits and preferences, demanding that the food and packaging industries adapt to a new lifestyle. Intelligent packaging provides consumers with real-time information about the quality and safety of packaged products. A critical analysis of the processes used to develop these packages was carried out. In this context, this review aims to analyze the concept of intelligent packaging, emphasizing the incorporation of extracts rich in anthocyanins, verifying its relationship with the development of new technologies and discussing current aspects of the scientific production process of the packaging. It was also highlighted that anthocyanin compounds are susceptible to pH variations. As an indicator of pH variation, a plant extract was necessary to incorporate into a solid matrix to immobilize the dye. The pH indicator film represents a simple and visual method to detect changes in food products. In this sense, technological processes and resources have been gaining prominence with the premise of offering quality, convenience and safety for consumers and companies.

1. Introduction

Due to changes in consumer demand for food quality and their concern about environmental changes, new food packaging approaches have been designed. Although traditional foods contribute to distribution logistics, maintenance and preservation of distribution, they are not enough to guarantee safety. Food safety is one of the major concerns on the food chain due to food spoilage, which can also result in high indices of food loss [1]. Thus, one of the main objectives of food engineering and technology is to provide products of better quality and better development for their useful life [2,3].

The dissemination of this idea was possible due to the discovery of the properties of some compounds with biological activity and their interaction with different polymeric materials. A new trend in food packaging is intelligent and biodegradable packaging, which can detect environmental changes inside the food packaging during storage. This new packaging can provide adequate information about the quality and safety of a product before its consumption and it can be easily degraded when discharged into the environment. This type of film uses response factors to monitor, track and provide feedback on important changes within or around the packaging system, providing information relevant to quality and safety [4].

Food packages classified as intelligent indicators include three main components: (a) it is capable of displaying qualitative information through rapid visual changes (e.g., a color change or an increase in color intensity); (b) it has sensors that provide quantitative results to users, usually by emitting physical or chemical signals; or/and (c) it has data carriers, such as barcodes and radio frequency identification (RFID) tags, which are intended for data storage and traceability [5,6]. Among the indicators incorporated into packaging, colorimetric indicators have stood out for their simplicity of the process and ease of communication with the consumer. Colorimetric indicators can be produced by combining natural colorants, such as anthocyanins, which are natural pigments that are non-toxic, soluble in water and sensitive to changes in pH. Anthocyanins are found abundantly in fruits and flowers and have a basic structure of three carbon rings with a central ring of flavonoid cations [7,8].

Several packages with pH indicators were developed using anthocyanins derived from various sources, such as red cabbage [9], purple sweet potato [10], blueberry [11], eggplant [12] and black carrot [13]. Regardless of the anthocyanin source used, the packages developed can provide immediate qualitative information through visual colorimetric changes caused by the structural alteration of the pigment and, consequently, indicate the freshness and the stage of deterioration of the food, presenting itself as a convenient, fast and nondestructive packaging [14].

Anthocyanins have an enormous potential to be applied as a pH indicator. To produce intelligent packaging, anthocyanin extract can be incorporated into a biodegradable polymer base to ensure consumer safety and environmental preservation. Unlike commercially used synthetic dyes, which are often harmful to human health and the environment, anthocyanins offer numerous advantages. Furthermore, the literature presents several papers supporting the importance of anthocyanins as an important compound, with various beneficial effects on human health [15]. Another important point is related to biodegradability and feasibility, since polymers derived from natural sources are currently considered potential substitutes for conventional polymers [16]. However, the properties of polymeric films prepared from natural sources must be improved, aiming to compete with petroleum-based films, especially regarding mechanical (Young’s modulus, elongation at break and tensile strength) and barrier (water vapor permeability) properties and those related to the affinity with water [17]. To overcome this limitation, biodegradable polymers can be blended to match characteristics and provide functionality for applications such as packaging. However, biodegradable polymer blends need careful post-consumer management and efficient design to allow biodegradation [18]. Starch [19], pectin [20], gelatin [21] and chitosan [22] are examples of biomaterials used to produce food packaging.

In this sense, this study aims to review the incorporation of extracts rich in anthocyanins to develop intelligent packaging. Several papers have reviewed the application of anthocyanins in intelligent packaging. Still, none of them made a complete review of anthocyanin’s compounds and their degradation, as well as the different sources and the use of new technologies for extraction. Also, different biodegradable materials are reviewed, discussing current aspects of the scientific systematization of the production process. To conclude, the article shows different applications and highlights results, which vary significantly when applied to different foods.

2. Anthocyanins

The term anthocyanin is of Greek origin, where “anthos” means flower and “kyanos” means dark blue. After chlorophyll, anthocyanins are the most important group of plant-derived pigments [23]. They are the largest group of water-soluble pigments in the plant kingdom and are found in more significant quantities in angiosperms [24]. More than 700 different anthocyanins can be found in nature, with the most common being pelargonidin, cyanidin, delphinidin, peonidin, petunidin and malvidin [25,26]. Despite being the largest group, the content of anthocyanins is significantly affected by the harvest season and the ripening stage of the plant [27]. For example, Lu et al. (2024) [26] indicated that anthocyanins are responsible for the red hue of leaves that can be seen in the autumn.

The functions of anthocyanins in plants are varied: they act as antioxidants, provide light protection, serve as a defense mechanism and play important biological functions. The vivid and intense colors they produce play an essential role in several reproductive mechanisms of plants, such as pollination and seed dispersal [28]. Narayan et al. (1999) [29] described anthocyanins as potent antioxidants compared to classical antioxidants, such as hydroxy anisole butylate, hydroxytoluene butylates and alpha-tocopherol (vitamin E). Additionally, anthocyanins are highly compatible with biological systems and are non-toxic, making them a promising alternative as a natural colorant in the food industry [30].

When added to foods as a coloring agent, anthocyanins help prevent auto-oxidation and lipid peroxidation in biological systems. The primary chemical structure of anthocyanins is characterized by a 2-phenylbenzopyrilium (flavylium cation) hydroxylated at the 3, 5 and 7 positions. The variations in anthocyanins arise from differences in the number and position of hydroxyl and methoxyl groups in the B-ring, as shown in Figure 1. Also, the wide range of anthocyanins described in the literature is related to the differences in the number and nature of the sugars attached, as well as their position of attachment. The number and nature of aliphatic or aromatic acids attached to the sugar residues also influence the characteristics of anthocyanin [31].

Anthocyanins are responsible for numerous shades of color in flowers, fruits and leaves [32,33]. The increase in hue results from a bathochromic change, characterized by the absorption band of light in the visible spectrum range changing from a shorter to a longer wavelength. This shift causes a change in color from orange/red to purple at acidic pH. The opposite shift is called a hypsochromic shift [34].

One example of this behavior is described by Tang et al. (2023) [35] when evaluating the anthocyanins of red cabbage. These authors describe that, when the pH value is below 3.0, the red cationic flavilium is the main form of anthocyanins. However, when the pH is raised to 6.0, the anthocyanins undergo structural changes to a neutral quinonoidal base, changing the color from red to purple. As the pH increases to 7.0, the color changes to blue, due to the presence of anionic and natural quinonoidal bases, changing to green when the pH goes beyond 7.0. Similarly, Zhang et al. (2023) [36] evaluated the anthocyanins in purple sweet potato and verified that under pH 1.0, these compounds were red, changing to red-pink until pH 6.0 and reddish-purple at pH 7.0. Between pH 9.0 and 11.0, the anthocyanins exhibited a blue-green color. This color evaluation can be seen in Figure 2.

2.1. Sources

Several plants have been proposed as sources of anthocyanin-based colorants. According to various studies performed over the years, there are at least 73 genera from 27 families that contain anthocyanins [38]. However, their utilization has been restricted by challenges, such as pigment stability, availability and economic factors [39].

Anthocyanins are responsible for the vibrant color of several plants, like red radish, red grape, red cabbage, purple potatoes, purple corn, black chokeberry, black carrot, blackcurrant, blackberry, elderberry, raspberry, strawberry and others [35,40]. These compounds, classified as secondary plant metabolites, are synthesized in the cytoplasm and stored in the cytosol. The most common types of anthocyanins are (a) cyanidin, which accounts for approximately 50% of anthocyanins and is responsible for magenta and crimson colors, (b) pelargonidin, which accounts for 12% of anthocyanins and is responsible for orange and salmon colors, (c) peonidin, which accounts for 12% of anthocyanins and is responsible for magenta color, (d) delphinidin, which accounts for 7% of anthocyanins and is responsible for the purple color and (e) petunidin and malvidin, which together account for 7% of anthocyanins and are responsible for the colors mauve and blue and purple, respectively [40,41].

It is widely known that there are several natural sources of anthocyanins available in nature. One plant that must be highlighted is the red cabbage (Brassica oleracea var. capitata rubra) where the number of anthocyanins is higher than other substances [42]. More than 30 anthocyanins can be found in red cabbage, with the major content being non-acylated or acylated forms derived from cyanidin-3-diglucoside-5-glucoside [43,44]. The extract obtained from red cabbage was already applied to film production and presented high sensitivity to mushroom freshness detection, having a stability of 50 days, when stored at 4 °C [45].

Another important plant, which is rich in anthocyanins and widely studied, is the grape (Vitis vinifera). According to Nogueira et al. [46] the main anthocyanin profile consists of derivatives of 3-glucoside and 3,5-diglucoside and trace amounts of pelargonidin. This author also applied the grape pomace extract (20 and 40%) to the production of arrowroot starch film and indicated that the final product has colors varying from pink for acidic pH, gray for neutral pH and green to yellow to basic pH, which is in accordance with the behavior described above.

Blueberry (Vaccinium spp.), according to Hu et al. (2024) [47] and Lu et al. (2024) [26], is considered the king of anthocyanins, due to the high content of these compounds, with major constituents comprising malvidins, petunidins, peonidins, delphinidins and cyanidins. Hu et al. (2024) [47] also indicated that the natural extract of this fruit has a high sensitivity to volatile nitrogenous compounds, making it an excellent indicator for intelligent packaging. The main highlight of this work was the evaluation of the stability of the produced film, which lasted 15 days under all the tested conditions.

Finally, another vegetable that is an excellent source of anthocyanins is eggplant (Solanum melongena). In this vegetable, the anthocyanins are concentrated in the skin, consisting of delphinidins, petunidins and malvidins [26]. Yong et al. (2019) [48] added eggplant extracts to a chitosan film and indicated that the addition improved the film thickness, light barrier and mechanical properties. The authors also indicated that, due to the high anthocyanin content, the films could be used to monitor milk degradation.

In this sense, we can state that anthocyanins, regardless of the source used, have proven to be an excellent natural dye, which can be used as a pH indicator in smart packaging. Furthermore, few studies have evaluated the stability of these compounds when applied in these packages, since in the studies evaluated, stability ranged from 15 to 50 days.

2.2. Anthocyanins Stability

According to Giusti and Wrolstad (2003) [39], the use of natural colorants in food systems is restricted due to their low stability in processing, formulation and storage conditions. In this context, Chen and Stephen Inbaraj (2019) [49] and Zhang and Jing (2020) [50] defined anthocyanin stability as the incapability of flavylium cations to change their structures into colorless forms of carbinol pseudobases and chalcones. The formation of these structures is the primary stage in the deterioration of anthocyanins.

Anthocyanins are highly unstable compounds and their stability can be influenced by several factors, like pH, temperature, light, the presence of oxygen, the presence of metal ions, enzymes and proteins. Also, they can be affected by ascorbic acid, sugar and sulfur dioxide [35,41,51]. But the anthocyanins’ stability can also be related to the structure of the molecule, as the stability increases with the number of methoxyls in the B ring and decreases as hydroxyls increase. Among the most common anthocyanins, the most stable is malvidin, followed by peonidin, petunidin, cyanidin and delphinidin. Changes in the structure, such as glycosylation and acylation of the sugars, also increase the stability; for example, the diglycosides are more stable than the monoglycosides [31].

According to Lima et al. (2011) [52], the structure of anthocyanins is another factor that influences their stability. The presence of sugars, acylated sugars, methoxyl and hydroxyl groups has a significant effect on these compounds. Anthocyanins with a greater number of hydroxyls are less stable than those with a greater number of methoxyls. Also, the degree of glycosylation is another structural characteristic that favors the stability of these molecules, with diglycosylated molecules being more stable than monoglycosylated molecules.

When considering external factors, the pH is one of the main ones that affects anthocyanin stability. At weakly acidic or neutral pH, these compounds are converted into colorless ionized quinoidal bases, which are less stable [35,53]. To explain the degradation of the anthocyanins, Chen et al. (2020) [54] proposed two possible routes: (a) the C-O bound present on the pyridine ring is opened, forming the intermediate chalcone-3-glycoside, which loses a sugar group forming a chalcone and then into a protocatechuic acid, or (b) the cyanidin-3-O-sophoroside (Cy-3-soph) suffers a successive loss of sugars, producing cyanogenic glycosides and these are then broken into photocatechuic acid. These changes in the structure result in different colors depending on the pH and is believed that these compounds are more stable in acidic environments [55].

Another factor that can influence the anthocyanin degradation is the temperature. These compounds are known to be highly stable in temperatures between 4 and 65 °C, as the color is accented at higher temperatures. However, when processed in temperatures higher than 70 °C, there is an acceleration in the degradation rate of the anthocyanins [56,57]. According to Zhang et al. (2022) [58] the temperature must be evaluated for anthocyanin stability, as during the heating, it is possible to visualize a logarithmic destruction of pigments. The thermal degradation mechanism involves the deglycosylation and reactions that open the ring to form chalcones [55]. The less stable chalcone structure of anthocyanins is formed at a temperature of 60 °C. These unstable chalcones eventually can be transformed into brown degradation products [58,59,60].

When evaluating the light influence, it is known that the exposure to light provides energy to the extract, passing it from a stationary or stable state to an excited or unstable state. When this happens, photochemical reactions arise that destroy the molecules. The oxygen turns this degradation even worse, Enaru et al. (2021) [40] indicated that oxygen can cause the degradation of anthocyanins through indirect oxidation, oxidizing constituents of the medium, or by the action of oxidizing enzymes, which can produce dark decomposition compounds or depigmented compounds. Chen et al. (2023 and 2018) [55,60] studied the photo-stability of red cabbage anthocyanin. They indicated that exposure to 72 h of simulated solar light has a significant negative effect, while samples maintained in the dark were more stable. One alternative to reduce the degradation of anthocyanins is the complexation of these compounds with another molecule. Liu et al. (2020) [61] indicated in their work that the use of proteins to enhance the stability of anthocyanins is a good method to enhance stability. On the other hand, the presence of sugar can either improve or degrade the anthocyanins. According to Akther et al. (2020) [62] and Slavu et al. (2020) [63], moderate sugar concentrations are effective in maintaining the anthocyanins, but increasing the sugar concentration may decrease stability and result in the formation of brown pigments.

Also, metal complexation is an alternative for stabilizing anthocyanins derived from fruits, but this phenomenon can result in diverse color variation, as metal copigmentation can alter the absorption spectrum [40,64]. Another alternative is copigmentation, which is the most promising technological process until now. Macromolecules, like gum arabic, xanthan gum, alginate, pectin, chitosan and modified starch, can serve as copigmentation agents to improve the stability of anthocyanins [60].

All these factors must be considered when developing intelligent packaging. According to Calva-Estrada et al. (2022) [65], light, heat, oxygen and high humidity in food packaging tend to reduce the stability of natural compounds and these factors can also cause conformational changes and affect film efficacy.

2.3. Anthocyanins Extraction

The extraction processes used in the industry involve methods such as maceration, solvent extraction, steam, cold pressing and compression [66,67]. However, the high cost of the processes and the environmental problems generated have made the development of emerging technologies attractive. Consequently, new extraction techniques have been developed to address these deficiencies, such as ultrasound-assisted extraction (UAE) and high-pressure extraction [66,68,69].

Due to the low thermal stability of anthocyanins, non-thermal extraction methods or those using low temperatures become interesting to avoid their degradation and bioavailability.

Table 1. Advantages and disadvantages of anthocyanins extraction methods.

Method	Advantage	Disadvantage	Reference
Solvent extraction	This is an extraction method of easy operation. Also, it is possible to use several solvents, such as deep eutectic solvents. Also, this extraction method has been related to good values of anthocyanin recovery.	The main disadvantage of this method is related to the high amount of waste generated. Also, it has a long time of reaction and low yield.	[70,71,72]
Ultrasound-assisted extraction	This method has as its main advantages faster mass transfer, the usage of reduced temperature, low solvent consumption, low installation cost and ease of operation.	The main disadvantage is the potential for inhomogeneous heating in the extraction process. Also, the intensity of the bubbles generated can affect the cells, enhancing the temperature and causing an enhancement of the temperature.	[73,74,75]
Microwave-assisted extraction	This method uses low amounts of solvent and sample, as well as short treatment times. Also, it has easy adaptation to industrial scale and low operating costs.	Some factors as the time, extraction power and effect of temperature on the sample must be carefully considered to avoid anthocyanin degradation due to excessive heat.	[76,77,78]
Pressurized liquid extraction	The use of high pressure allows working at high temperatures. These conditions increase the solubility of the anthocyanins.	The main disadvantages are related to the high cost of the equipment and the necessity of optimization of extraction parameters, such as temperature and pressure.	[79,80,81,82]
Supercritical fluid extraction	This method results in an extract of elevated quality, with high purity. Also, this method is considered more sustainable and environmentally friendly.	This method has a substantial initial investment. Also, it is a process that demands a high amount of energy due to supercritical conditions.	[83]
Enzyme-assisted extraction	The extraction is performed under relatively mild conditions. Also, this method has presented a higher yield and quality extract.	The major disadvantage is the long extraction time. Also, it is well known that the presence of enzymes can reduce the stability of anthocyanins, so stability evaluation must be performed.	[84,85,86]

shows the advantages and disadvantages of conventional and emerging extraction methods.

As can be seen above, each method has advantages and disadvantages that must be carefully evaluated according to the application of the extract, as well as the raw material selected. Also, it is necessary to evaluate the cost of implementation and processing and assess whether this is offset by the sales value of the product. More details of each method and some applications for anthocyanin extraction are described below.

2.3.1. Solvent Extraction (SE) and Deep Eutectic Solvent Extraction (DES)

The solvent extraction process is generally applied at high temperatures. However, it can also be performed at milder and room temperatures, having these characteristics as an advantage over anthocyanins. Despite this, the method is very time-consuming, lasting between three hours and three weeks. It is also unfeasible due to low yield, a large amount of plant material, high solvent consumption and environmental impact [70,71]. This technique uses solvents according to their polarity to extract the compounds of interest. In general, there is a saturation of the extraction solvent or a diffusion equilibrium between the solvent and the plant cell.

It is important to note that, even though presenting several disadvantages, this technique is one of the most used extraction methods due to its suitability for different scales, simplicity and low cost compared to other methods [67].

In general, extracting a solute from porous particles to a solvent during a diffusion process involves several steps, such as diffusion of the solvent into the porous solid, dissolution of the solute in the solvent, diffusion of dissolved solute to the surface of the particle and diffusion of dissolved solute from the particle surface to the solvent [87].

In addition to the extraction method, the choice of solvent is crucial for a better and faster extraction. With that in mind, green and efficient alternatives, such as using deep eutectic solvents (DESs) in extraction, have been emerging, showing favorable results compared to conventional solvents. A study by Bubalo et al. (2016) [88] demonstrated that the yields of phenolic compounds from the grape seed coat obtained with DES were higher than those obtained with water and conventional solvent using the maceration process.

A crescent number of studies have been performed using solvents for the extraction of anthocyanins. It is important to highlight three papers that elucidate the usage of deep eutectic solvents for anthocyanins extraction. Airouyuwa et al. (2024) [89] reviewed recent developments in the use of DES for sustainable green extraction and concluded that the use of DES is used as a more biocompatible and efficient alternative to conventional solvents. They indicated that the anthocyanins extracted using DES have clearly shown increased stability towards time-dependent degradation and are considered promising solvents for the extraction of anthocyanins. But it is important to highlight that, even being considered eco-friendly, ecotoxicity studies have shown that the eutectic mixtures are toxic to a certain extent for both aquatic and terrestrial so they cannot be labeled as “readily biodegradable”.

Also, Foroutani et al. (2024) [72] evaluated the stability, bioavailability and antioxidant properties of anthocyanins extracted by DES. These authors indicated that using these solvents improved the extraction efficiency of anthocyanins. They also indicated that these compounds presented higher stability, bioavailability and antioxidant activity. They also defined that among the solvents evaluated, the choline chloride-oxalic acid is the most efficient for anthocyanin extraction. The major problem indicated by these authors is the difficult separation of the target compounds from the solvent.

Kurek et al. (2024) [90] evaluated the impact of the extraction with deep eutectic solvent on anthocyanin degradation. They indicated that it is of critical importance to select the appropriate solvent and storage conditions to preserve the anthocyanin content and indicated that the combination of choline chloride and malic acid exhibited the highest browning index, while the combination of choline chloride and xylitol was the most effective solvent in preserving anthocyanins and minimizing browning.

So, based on these results, it is possible to conclude that the use of DES can be an efficient alternative for anthocyanin extraction, but the solvent selection must be carefully evaluated, as they can reduce the anthocyanin content or present separation problems.

2.3.2. Ultrasound-Assisted Extraction (UAE)

Ultrasound-assisted extraction (UAE) has been widely applied for the extraction of bioactive compounds from plant extracts. Many reported applications have shown that ultrasound-assisted extraction is a green and economically viable alternative to conventional food and natural product techniques. It offers several benefits, such as faster mass transfer, thus decreasing extraction time, reduced temperature, selective extraction, low solvent consumption, low installation cost and ease of operation [73,74].

However, one disadvantage is the potential for inhomogeneous heating in the extraction [91]. It is also important to highlight that the mechanism of cell disruption by ultrasonic waves is associated with the phenomenon of cavitation. This phenomenon results in the release of highly energetic shock waves, which cause the appearance of mechanical stresses, causing damage to the affected surface. The ultrasonic wave may cause an internal structural change in plant matrices due to the produced cavitation bubbles. This bubble breakage of cell walls releases anthocyanins into the solvent medium through diffusion and/or dissolution [92].

On the other hand, when the bubbles are smaller than the cells, they can generate disruptive shear stress without the need for cell movement. In this way, larger cells feel the turmoil of rupture more than smaller cells [93]. Much of the ultrasonic energy absorbed by the cell suspension is transformed into heat, so temperature control is necessary [75], otherwise, there may be a significant degradation of the thermolabile compounds.

The frequency of ultrasound in the extraction is effective because it breaks the micelle or matrices of the sample, facilitating the access of the solvent to the contained compounds. Furthermore, the power of ultrasound agitates the extraction solvent, thus increasing the contact between the solvent and the targeted compounds, significantly improving this extraction efficiency [69].

Heat and ultrasound-assisted extraction methods were applied to recover anthocyanins from Hibiscus sabdariffa. The extraction using ultrasound was approximately 2.5 times more efficient, allowing the recovery of higher values of anthocyanins when compared to the literature. These results can be used as a viable source of anthocyanins to produce bio-based colorants [94].

To establish environmentally friendly extraction methods for anthocyanins, DES was investigated as a green alternative to conventional solvents, along with high-efficiency ultrasound-assisted extraction. This approach utilizes DES as a green solvent and ultrasound as an alternative representing a good choice to design eco-friendly extraction methods for phenolic compounds from various sources [95].

To enhance extraction efficiency, combined approaches are being explored, such as unconventional technologies such as ultrasound and alternative solvents, aiming to obtain better extraction values and reduce environmental damage [96]. However, the generated extracts can still present problems of light stability, thermal stability, water solubility and bioavailability. Devi et al. (2024) [97] evaluated the ultrasound-assisted extraction of anthocyanin from black rice bran. Acidified ethanol and methanol were tested for extraction at different intervals of 10, 20, 30, 40, 50, 60, 70 and 80 min and the ultrasound power of 50–350 W. The study concluded that ultrasound-assisted extraction is an effective method for the extraction of anthocyanins and that prolonged extraction time and ultrasound power can lead to the decomposition of anthocyanins.

Albuquerque et al. (2024) [98] compared the heat-assisted and ultrasound-assisted extraction methods for the extraction of anthocyanins from Eugenia spp. peel and indicated that the usage of ultrasound resulted in higher extract yields. Although the higher yields, the extracts produced with the heat-assisted extraction were more concentrated in anthocyanins.

Using ultrasound extraction can be an alternative to enhance extraction yield, but it is important to perform more studies, as even when presenting a higher yield, it can present low selectivity for the desired compound. Additionally, more studies must be performed to evaluate the stability of these compounds and their ability to detect pH changes when applied in intelligent packaging.

2.3.3. Microwave-Assisted Extraction (MAE)

MAE is a promising emerging technique that has been used for the extraction of bioactive compounds, utilizing low amounts of solvent and sample, as well as short treatment times [76]. It aims to use heating by electromagnetic waves through an electric and magnetic field, with heating and cooling in short periods and under controlled conditions [99]. Other advantages of MAE are its easy adaptation to industrial scale and low operating costs [77].

The principle of this technique is based on the direct heating of molecules by ionic conduction and dipole rotation. The frequencies vary from 0.3 to 300 GHz, which correspond to wavelengths from 1 cm to 1 m [100]. Several factors can influence the quality of extraction by this method, such as time and extraction power, the effect of temperature on the sample, solvent composition, pre-leaching time, pH, particle size and sample moisture [78].

For successful MAE extraction, it is essential to choose a good solvent, which must have microwave absorption properties, such as permanent dipoles [101]. Samples and solvents with different polarities result in no heating of the sample and, consequently, the desired extraction is not obtained. Conversely, using a solvent with a very high microwave absorption capacity, we can heat the sample in seconds and thus degrade the compounds of interest. Therefore, several works study different solvents in different proportions to obtain the best performance [102].

For the extraction of anthocyanins in different raw materials, MAE has proven to be a viable alternative to conventional extractions. Elez Garofulić et al. (2013) [103] studied the isolation of anthocyanins and phenolic acids from marasca cherries (Prunus cerasus var. Marasca) and obtained greater efficiency in MAE compared to conventional extraction, with higher concentrations of anthocyanins and shorter extraction times in all solvents used. Furthermore, for extracting anthocyanins, the best results were at lower temperatures (60 °C) and shorter treatment times (6–9 min).

In another study, Gamage and Choo (2023) [104] evaluated the microwave extraction of black goji berries and compared it with hot water, ultrasound and pectinase-assisted extraction. The same authors indicated that pectinase-assisted has the best extraction yield, but all the techniques tested were equally effective in obtaining an extract with high anthocyanin content. These same authors evaluated hot water, ultrasound, microwave and pectinase-assisted extraction of anthocyanins from blue pea flowers and in these studies, microwave-assisted extraction was the best method to obtain an anthocyanin extract with a high extraction yield.

It is important to emphasize the different results when comparing extraction methods from different sources. A complete study must be performed to ensure that the compounds extracted will be able to show the pH differences in the intelligent packaging and if the compounds will be stable when used.

2.3.4. Pressurized Liquid Extraction (PLE)

Pressurized liquid extraction (PLE) is an environmentally friendly alternative to conventional extraction methods, such as Soxhlet solvent maceration. PLE can extract bioactive compounds from solid and semi-solid matrices, making it suitable for thermolabile compounds such as anthocyanins. A major advantage of PLE over low-pressure extraction methods is that high-pressure solvents remain liquid even when subjected to temperatures well above their boiling points, thus allowing work at high temperatures. These conditions increase the solubility of target compounds in the solvent and the kinetics of desorption of solid matrices [79,80,81].

The efficiency of PLE is influenced by diffusion and solubility. When the process is managed by diffusion, strong interactions between the matrix and analytes may hinder extraction due to long diffusion paths. The most important process parameters are solvent temperature and particle size. However, if the PLE is solubility controlled, the analyte-matrix interactions are quite weak and the extraction rate mainly depends on the compartmentalization of the analyte between the matrix and the extraction fluid. In both cases, the pressure is fundamental, as it facilitates the penetration of the solvent into the pores of the matrix and increases the yield [105,106,107].

Factors such as ethanol concentration, number of extraction cycles and temperature significantly impact the extraction process of anthocyanins present in jambolan. The increase in ethanol concentration (60–80%) increased the anthocyanin extraction due to the increase in medium polarity [108]. However, temperature control must be strict, as anthocyanins are thermolabile compounds, which can lead to their degradation. Also, there is a synergistic effect between solvent concentration and temperature, where higher temperatures lead to a reduction in the viscosity of the solvent, having a positive impact on the diffusion process and the solubilization of anthocyanins [109,110].

Bombana (2023) [111] evaluated maceration, ultrasound and pressurized liquid extraction for obtaining the anthocyanins present in the peel of guabiju (Myrcianthes pungens). In this study, the ultrasound-assisted extraction method obtained the highest amount of anthocyanins and the final by the PLE method was approximately 2.4 times more expensive than the other methods. It is important to note that, even having good results, PLE has several disadvantages, like its high cost. The PLE method presented lower extraction efficiency, yield and productivity, which could have been caused by the combination of the pressure and high temperature. Also, the color obtained in the PLE extract is different from the other extracts, that are next to red, which can influence the pH detection when applied in intelligent packaging.

2.3.5. Supercritical Fluid Extraction (SFE)

Supercritical fluid extraction (SFE) is a process that utilizes a fluid at pressures and temperatures beyond its critical point, modifying its capabilities as a solvent. Supercritical fluids have properties between liquids and gases; for example, the viscosity of the fluid is similar to a gas, but on the other hand, the density of the fluid is next to those found for liquids. Additionally, the diffusivity is intermediate between liquids and gases [112].

Most SFE applications developed nowadays seek to gain advantage of the mild critical temperature and pressure values of carbon dioxide (CO₂), which is a green solvent and is cheap and easily available. Also, it is a non-toxic, non-explosive and readily available solvent and does not lead to large chemical changes in biocompounds, preserving their biological properties. It is well known that the final product must be free of the extraction solvent, which is another advantage of CO₂, which is a gas at room temperature. This means that when the pressure of the system is relieved, the CO₂ evaporates, leaving the product solvent-free. Also, using the supercritical extraction with CO₂ can disrupt intracellular electrolyte balance by modifying cell membranes and removing essential cell constituents [112,113,114].

However, a primary disadvantage of using CO₂ as an extraction solvent is related to its very low polarity, which results in a very low extraction of high- or medium-polarity compounds [114]. To overcome this limitation, co-solvents like ethanol or water can increase the solvating power of the CO₂, allowing the extraction of polar compounds, like anthocyanins [112,113].

Numerous studies have reported that the usage of ethanol-water co-solvent enhanced the yield extraction of anthocyanins. This happens because the water of the co-solvent reacts with the CO₂, generating carbonic acid, which reduces the pH facilitating the penetration of the solvent and the removal of the anthocyanins from the vacuole and resulting in a highly stable extract [113].

Idham et al. (2022) [113] evaluated the extraction of anthocyanins from roselle calyces using supercritical carbon dioxide extraction and indicated that the co-solvent selection and the operation pressure were the most significant factors in the extraction. Also, when evaluating the stability of the extracts, the SFE is effective in protecting the anthocyanins and maintaining color stability.

Jiao and Pour (2018) [115] evaluated the extraction of anthocyanins from haskap berry pulp, which presented a high potential to be used as a green technology but relies on the use of a combination of water and supercritical CO₂. They also indicated that parameters such as pressure, temperature, time and co-solvent to biomass ratio have an influence on the extraction yield and must be evaluated to optimize the process.

Based on these findings, it is possible to conclude that, even with the use of a co-solvent, supercritical extraction can be a viable process to obtain extracts rich in anthocyanins. Also, some papers indicated that the use of this technique maintained color stability. But, even presenting all these advantages, it is necessary for further studies to evaluate the application of these extracts in intelligent packaging, ensuring the response of the compounds to the pH changes.

2.3.6. Enzyme-Assisted Extraction (EAE)

Enzyme-assisted extraction (EAE) has recently emerged as an innovative method for the extraction of anthocyanins from different plants. The EAE is performed under relatively mild conditions and, as its principal mechanism, the cell is ruptured through enzymatic hydrolysis, but it requires a long extraction time when performed at 40–50 °C [84,85].

The EAE process focuses on the breakdown of the cell wall and the release of the compounds from the interior of the cell. To make the cell rupture, enzymes, like cellulases, hemicellulases, xylanases, proteases, α–amylases, β–glucosidases and pectinases, can be used [86,116].

Domínguez-Rodríguez et al. (2021) [86] indicated that the extraction performed with enzymes has presented a higher yield and extract quality. Also, they indicated that the main advantages of this method are the reduced extraction time and lower solvent consumption. However, it is well known that better results for extraction depend on several factors, such as the type and concentration of the enzyme, the temperature and the pH [116].

Given these variables, it is important to evaluate the optimal conditions for each plant and enzyme studied, aiming to obtain a higher yield and quality of extract. Amulya and Islam [117] studied the enzyme-assisted extraction of anthocyanins from eggplant peel. The enzyme used in the process is cellulase and factors such as temperature, enzyme concentration and time were evaluated, showing that the process is an effective way to extract bioactives from eggplant peel.

Domínguez-Rodríguez et al. (2021) [86] also evaluated the enzyme-assisted extraction of non-extractable polyphenols from sweet cherry (Prunus avium L.) pomace. Three different enzymes were tested: Depol, Promod and Pectinase. The optimal extraction condition was obtained at a temperature of 70 °C and a pH of 10, but the reaction time varied from 40 min for Depol and Promod to 18.4 min for Pectinase. These optimal conditions allowed the extraction of higher content of proanthocyanidins.

González et al. (2022) [118] evaluated the comparison between ultrasound-assisted and enzyme-assisted extraction of anthocyanins from blackcurrant. Their results indicated that the composition of the extraction solvent has been the most influential variable. Also, no differences have been observed in anthocyanin yield with both methodologies.

Despite the promising results of EAE for extracting these compounds, further studies must be performed to evaluate the usage of these extracts in intelligent packages. It is well known that the presence of enzymes can reduce the stability of anthocyanins, so it is important to evaluate if the long storage of an extract containing enzymes and anthocyanins can result in reduced stability of the extract and if it is suitable to be used as a color change indicator in the packaging.

3. Intelligent Food Packaging

People’s lifestyles have become increasingly complex, leading consumers to eat more conveniently and quickly. Thus, for this reason, food industries have developed convenient packaging that meets this demand, without forgetting the basic function of food protection and the ease of communication between packaging and the final consumer [119].

Two new trends that stand out in packaging development: active and intelligent packaging. Active packaging has active agents (antioxidant compounds, oxygen and moisture absorbers, for example) that intentionally interact with the product, with the purpose of protecting, prolonging shelf life, preserving sensory properties, maintaining product quality and ensuring food safety [120].

Intelligent packaging, on the other hand, aims to facilitate the communication of the freshness state of the food to the consumer, that is, providing dynamic feedback on the real quality of the product. In this way, to inform the consumer about the current situation of the food, devices, such as indicators, sensors, or data carriers, are inserted or incorporated into the body of the package, so that they can interact with the internal and external components of the food and the environment in which it is located and provide, as a result, an immediate response (color change, for example) that correlates with the physical, chemical and biological properties of the food [121].

Several devices and equipment have been developed to inform the state of conservation of food, such as radiofrequency identity tags, time-temperature indicators, electrochemical sensors and electronic tongues and noses; however, these systems are complex and expensive [122]. Simpler devices can be used for this control, without any difficulty; one of them being the use of an indicator that shows the change in food pH [3,123]. Indicator packaging transmits to the consumer information related to the presence or absence of a substance, qualitatively or semi-quantitatively, through immediate visual changes. Indicator packages are divided into three categories—time-temperature indicators, freshness indicators and gas indicators—and usually appear in the form of an indicator card (label) and indicator film [124].

Freshness indicators are designed to monitor changes in characteristics related to the quality of the food and inform consumers [122]. The action principle of a freshness indicator is based on the detection of alterations in the concentration of substances inside the package. The appearance of these substances is related to microbial growth and colorimetric changes in this indicator allow the consumer to be informed of this behavior. Changes in the concentration of volatile nitrogen compounds, carbon dioxide, sulfide compounds and glucose can result in colorimetric changes in freshness indicators [5]. This measurement helps inform consumers about the quality of the food before consumption, making consumers aware of the safety of the food consumption [15].

For the development and implementation of indicator packaging in the food industry, this packaging must have a good colorimetric representation of changes inside the packaging, good sensitivity and speed of response to these changes. Colorants, such as chlorophenol red and bromophenol blue, have been used as sensors in commercial intelligent packaging due to their good responses to pH changes. However, these colorants are categorized as toxic and potentially harmful to human health, as they can migrate into the food during the monitoring time and result in toxicity and carcinogenicity problems [15,125,126].

In this sense, natural colorants can be an alternative to dyes, as they have properties such as biocompatibility, renewability, non-toxicity and ease of implementation and can be safely disposed of in natural environments [15,127,128].

Applications

There are about 600 million cases of gastrointestinal diseases due to the consumption of spoiled foods, with deaths from food poisoning reaching 420,000 per year [129]. In this sense, intelligent packaging has been developed for assessing the freshness of the products. Several studies that apply anthocyanin extracts from different plant origins in polymer matrices are being developed as potential smart devices [16,130,131]. According to studies, the range of concentrations of the anthocyanin extract varies from 0.1% to 66% (w/w) added on a polymer basis [27].

Another important variable is the polymer used, with polysaccharides being studied as a natural alternative to conventional synthetic plastics. Studies are performed using starch, gelatin, cellulose and algae [27,123,130,132,133,134,135,136,137].

The selection of the polymer is crucial for intelligent packaging, as color variation is dependent on these compounds. For example, for the detection in aqueous matrices, the substrate should not be soluble in water and for gas samples, must have the permeability of the analytes. In this sense, high porosity and large surface area can improve the sensitivity due to the high adsorption and diffusion of the compounds [138]. Neves et al. (2022) [27] reviewed several manuscripts and concluded that the pH change from acidic (2–3) to neutral or alkaline (7–11) is detected by the color change from red to blue when using starch films, red to green or pink to yellow when using films made of starch, polyvinyl alcohol (PVA) or carboxymethyl cellulose.

One application of this innovative packaging is for products that have their freshness considerably declined over time due to the incidence of food spoilage. This behavior is unable to be discerned with the naked eye by the consumers and some conventional packages make it challenging to identify changes in the food [139]. In the typical food degradation, the proteins are broken down into peptides and amino acids followed by their conversion into CO₂ and ammonia gases, resulting in a dramatic pH drop, which is detected by the colorimetric indicator and alerts consumers that food is unsafe for consumption [18,140,141].

Following these conditions, Wei et al. (2017) [142] produced films based on gellan gum incorporated with powdered purple sweet potato (Ipomoea batatas L.). In this case, colorimetric responses to pH changes were observed, and the packaging showed high antioxidant activity. In addition, the devices were efficient in the colorimetric transition due to pH changes caused by volatile basic compounds produced by Escherichia coli when this microorganism metabolized proteins in vitro.

Pourjavaher et al. (2017) [143] developed a film using cellulose nanofibers of bacterial origin incorporated with red cabbage extract (Brassica oleraceae L. var. Capitata f. rubra) to serve as an intelligent pH indicator device. In addition to determining the effect of incorporating the cabbage extract on thermal, mechanical, microscopic, structural and interaction properties with the polymeric matrix, the developed indicator was efficient in detecting pH variations in vitro, showing potential application in monitoring the food preservation conditions throughout its storage.

Ma et al. (2018) [16] developed films based on a blend composed of poly (vinyl alcohol) (PVOH) and chitosan nanoparticles incorporated with blackberry extract aiming to detect pH variations in solutions and foods. In addition to the films added with blackberry extract showing greater resistance compared to the control film, they were able to detect pH changes in fish samples during storage.

In general, berries, like blackberry and blueberry, are the most common source of anthocyanin used to produce intelligent packages [27,144,145]. This occurs because these fruits combine qualities, such as attractiveness and safety, with economic advantages, as they are easy to cultivate and have a global supply [27]. But still, there are some challenges in applying these compounds in intelligent packaging, which lies in ensuring the stability of the anthocyanins due to the temperature variation, presence of light and oxygen exposure [38].

Sani et al. (2021) [137] indicated that the increase in the activity of the microorganisms leads to the temperature of the packaging to increase. This warming, with O₂ present in the package, is the most important destructive factor of anthocyanins, hydrolyzing the glycosidic bond and resulting in pigment loss and the malfunction of the intelligent packaging sensor. Also, it is important to emphasize that it is known that natural colorants are slightly lower in sensitivity to pH changes, so it is necessary to perform several tests to ensure their functionality [126]. One potential solution to mitigate this issue is the combination of two anthocyanin indicators, which can help avoid false positives and negatives, thereby enhancing the accuracy of sensor results [146]. Other examples of innovative materials incorporated with anthocyanins to develop intelligent packaging can be seen in

Table 2. Application of intelligent packaging incorporated with anthocyanins in food freshness.

Source	Matrix	Food	Main Results	Reference
Grape peel	Rice starch and citrated starch	Shrimp	The porous starch matrix provided great mechanical resistance, higher than the commercial polystyrene foam available. The anthocyanins incorporated into the foam presented efficient colorimetric changes based on the pH (∆E approximately 40). Also, both films developed exhibited moderate antioxidant activity (between 2 and 16%).	[147]
Grape pomace	Arrowroot starch	Fish meat	The grape pomace extract has colors varying from purple and blue tones. When added to the arrowroot starch film, it was able to present a color change, from pink at acidic pH to green at basic pH. All firms presented color differences with values higher than 3, which is easily detected by the naked eye.	[46]
Blueberry	Wheat gluten proteins	Shrimp	The film developed presented improved water vapor barrier, mechanical and light blocking. Also, it was efficient to monitor shrimp degradation, changing color according to the pH. Also, this color variation is stable for 15 days.	[47]
Jambolan fruit extract	Laponite and montmorillonite nanoclay	Shrimp	Anthocyanins were stabilized into montmorillonite and showed antioxidant properties. They maintained a stable color for 60 days and allowed us to monitor the quality of fresh shrimp throughout their shelf life.	[148]
Roselle (Hibiscus sabdariffa)	Octenyl succinic anhydride starch and polyvinyl alcohol	Pork and Shrimp	The film produced presented good morphology, thermal and color stability, as well as pH sensitivity, changing from bright red to yellow with increasing pH from 2.0 to 12.0. The colorimetric changes can be visualized by the naked eye and represent the freshness loss.	[126]
Rose petal	Carrageenan hybridized with carbon dots	Pork and Shrimp	The film produced presented good thermal stability. The loss of freshness was successfully visualized with a color change from red to yellow, presenting a high potential for application in food packaging.	[149]
Aronia melanocarpa anthocyanins	Cassava starch and PVA films	Milk	Aronia melanocarpa anthocyanins improved the mechanical (from 10 to 25 MPa) and UV barrier properties of the starch/PVA films (between 60 and 80% at 800 nm). The colorimetric responses of the films to pH changes, combined with a rancid aroma from the milk, indicated the potential use of the produced material as intelligent packaging.	[150]
Blueberries extract	Chitosan films fortified by cellulose nanocrystal	Shrimp	Biodegradable packaging was prepared based on chitosan that was incorporated with blueberry extract and fortified with cellulose nanocrystals. Applying 9% cellulose nanocrystals improved the film’s mechanical properties. However, it showed a negative impact on its barrier properties. The developed films showed antioxidant and antimicrobial properties and colorimetric responses to pH variations, showing the potential to indicate shrimp freshness and delay its deterioration.	[151]
Sweet potato peel extract	Carrageenan films integrated with TiO2 carbon dots	Shrimp	Films manufactured with carrageenan and incorporating carbon dots doped with TiO2 and anthocyanins from sweet potato peel showed 100% UV protection, high antioxidant activity and antibacterial activity against strains of L. monocytogenes and E. coli. Due to their sensitivity to pH variations, the films produced showed the ability to monitor shrimp freshness in real time, serving as a good indicator and helping to provide safe food.	[152]
Grape skin	Soybean polysaccharide, glycerol and graphene oxide	Salmon	The film, added with anthocyanins, presented a reduction in the mechanical (from 27.2 to 2.7 MPa) and thermal properties and presented an increase in the MC (15.1 to 25.1%). The film exhibited a recognizable color alteration when the environment changed to alkaline, being a suitable option to indicate freshness changes.	[153]
Hyacinth bean (Lablab purpureus (L.)	Films based on guar gum (GG) and polyvinyl alcohol (PVA)	Shrimp and pork	GG/PVA-based films incorporated with hyacinth bean anthocyanins (HBAs) were developed to monitor the quality of meat products, such as shrimp and pork. HBA improved the uniformity and compactness of the films by forming hydrogen bonds with the film matrix. The application of 2% HBA enhanced the films’ mechanical, thermal, barrier properties and antioxidant activity. The developed films showed responses to pH variations during refrigerated storage of shrimp and pork, making them suitable for application in the intelligent packaging field.	[154]
Red grape	Cellulose/Salep-based intelligent aerogel	Beef	Aerogel based on cellulose/Salep incorporated with anthocyanins was developed to monitor the freshness of beef. The aerogel produced by freeze-drying showed high porosity (90.22–91.13%) and low density (13.55–16.08 mg/cm3), which influenced the sensitivity and stability of the color. During meat storage, the aerogels showed high sensitivity to pH variations due to the chemical breakdown of proteins and the release of volatile bases.	[155]
Black wolfberry, Roselle, Morning glory, purple potato, Rose, Carnation, Mulberry, Red cabbage and Grapes	Agar and polyvinyl alcohol	Salmon	Among all the anthocyanin sources, Roselle presented the highest absorption spectrum value. Also, when added to the hydrogel film, it was an effective freshness indicator, changing from red to green.	[156]
Coleus grass (Plectranthus scutellarioides) leaves	Chitosan and Fucoidan	Salmon	The addition of anthocyanins improved the antibacterial (from 10 to 25 mm) and antioxidant activity (from 20 to 80%) of the films. Also, the films produced are sensitive to ammonia gas produced during food degradation, even with a few anthocyanins.	[157]

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4. Conclusions and Future Scope

The development of intelligent packaging with colorimetric indicators or sensors depends on selecting raw material sources, formulation composition and manufacturing processes, requiring production and performance parameters standardization. Therefore, incorporating advanced processes and technological resources has stood out, addressing demands for quality, practicality and safety for both consumers and industries.

Among the most promising alternatives are intelligent packaging solutions containing anthocyanin extracts, which offer significant advantages over traditional packaging. These solutions enable efficient and practical monitoring of food conditions, facilitating the rapid detection of spoilage and the tracking of shelf life. In addition to providing economic benefits, these packages enhance food safety, reducing losses and waste.

Technological advancements are expected to continue improving the effectiveness and sustainability of these solutions. Moreover, raising awareness about the importance and potential of these technologies is crucial, particularly regarding public health protection and the promotion of safer and more sustainable food systems.