2. Bio-Based Materials Used for the Stabilization of ACNs
2.1. Pectin
Pectin (PC), a polysaccharide polymer, is a viable solution for the stabilization of ACNs by way of the formation of non-covalent complexes
[47][41]. ACN metal chelate complexes solubilized in PC prevented the precipitation of ACNs in aqueous environments, making them a potential candidate for beverage-related applications
[48][42]. The most probable mechanisms responsible for ACN-PC binding appear to be ionic interactions between positively-charged ACN flavylium cations and free pectic carboxyl groups, and ACNs aromatic stacking on bound ACNs
[49][43]. The binding exhibits an increasing trend, as pH becomes more acidic, given that flavylium cation is more dominant and prevalent in the pH range of 1–3, with the converse increase in pH values causing a decrease in the concentration of flavylium cations
[50][44]. This trend can be seen in
Figure 2, where the effect of pH on the color stability of ACNs can be observed. PCs isolated from sugar beet, apple, and citrus improved the stability characteristics of ACN extracts from blackcurrant, and strawberry, making them suitable for food and beverage applications
[50][44].
Figure 2. A pictorial description of the pH effect on ACN color stability; changes in ACN color pigments due to the chemical alteration in nitrogenous compounds under various pH conditions.
Similarly, blueberry PC (chelator-soluble) enhanced the stability attributes of malvidin-3-glucoside (M3G), blueberry extract, and cyanidin-3-glucoside (C3G) during in vitro GI digestion simulation studies
[51][45]. ACNs with improved bioavailability influence the gut microbiota composition and increase the number of beneficial bacteria, lower inflammation, and alter the glycemic response, and other physiological responses
[52][46].
2.2. Proteins
The interaction of ACNs with protein can improve the overall stability of the ACN-protein complex, as well as its functional, and nutritional properties
[53][47]. ACNs and proteins combine owing to the inherent sensitivity of ACNs to alkaline oxidation, in turn yielding quinones
[54][48], which tend to form strong and fairly stable C-N or C-S covalent bonds by way of the nucleophilic addition of mercaptan and amino groups on the protein side chains
[55][49]. Given that the bioavailability of ACNs in the human body is markedly low
[9], combining ACNs with proteins can significantly improve the stability of these compounds, as well as enhance their absorption rates
[30].
ACNs from sour cherry (
Prunus cerasus L.) peel were encapsulated with whey protein isolates (WPI) and acacia gums, with an encapsulation efficiency of 70.30 ± 2.20%, and the in vitro digestibility analyses indicated that WPIs protected ACNs against gastric digestion, thereby facilitating their release in the small intestine
[56][50].
2.3. Lipids
Encapsulation of ACNs in emulsion droplets is a viable solution for enhancing their stability, and bioavailability. Studies have reported the encapsulation of ACNs in various types of emulsions, predominantly water-in-oil-in-water (W/O/W) emulsions
[57][51]. The W/O/W emulsions have different internal and external values of pH, which may slow down pH-induced color changes in the encapsulated ACNs
[58][52]. Phospholipids including lecithin isolated from the soybean, eggs, lecithin, and marine sources, as well as milk phospholipids, also form a major component of the liposome delivery systems
[59][53]. The presence of cholesterol along with phospholipids has also proven significant for the stabilization of ACNs
[60][54].
2.4. Biopolymer Complexes
Natural biopolymeric complexes, in particular, polysaccharides and protein matrices being utilized for the encapsulation of ACNs
[61][55]. The presence of electrostatic interaction-induced supramolecular structures within these macromolecules, their non-hazardous nature, and their generally recognized as safe (GRAS) status render them highly versatile vehicles for encapsulating and delivering ACNs in food-associated applications
[62][56]. Copigmentation intensifies and stabilizes the color of ACNs by protecting the flavylium cation of these pigments from nucleophilic attack
[63][57].
The biopolymers widely reported to be suitable for the copigmentation of ACNs include PC, dextran sulfate, guar gum, gum arabic, and whey proteins
[62][56]. For instance, in a study, gum arabic (0.05–5.0%) significantly improved the color stability of purple carrot ACNs when used for commercial beverage applications, with the highest stability levels occurring at intermediate gum arabic concentrations (1.5%)
[64][58].
3. ACN Delivery Systems
Different types of colloidal particles can be used as viable delivery systems for ACNs (
Figure 3 and
Figure 4), including, nanostructured lipid carriers (NLCs), solid-lipid nanoparticles (SLNs), liposomes, emulsions, and others
[65][59]. In encapsulation, highly sensitive constituents are enclosed within a wall material before delivering inside a system and could be formed by nanoencapsulation (<1 µm) or microencapsulation (1–1000 µm) depending upon the particle size
[66][60]. This significantly improves the stability, water solubility, and bioavailability of bioactive compounds
[67][61] and prevents the enzymatic and chemical degradation of ACNs
[68][62]. However, conventional microencapsulation has not proven effective in the context of stability studies for ACNs, primarily because of their bigger particle size and low encapsulation efficiency
[69][63].
Figure 3. Pictorial representation showing the interaction of polysaccharide-based wall material (pectin) with the ACNs color pigments as encapsulating agents to increase the stability of ACNs.
Figure 4. Formulation of different conventional and advanced delivery systems used for encapsulation of ACN pigments in different biopolymer complexes (carbohydrates, protein, lipids), and wall materials.
Different formulation and storage techniques related to encapsulation are summarized in
Table 1. Protein-based delivery systems are frequently used in microencapsulation owing to various favorable attributes, such as cost-effectiveness, porous structure, surface-active nature, and ability to self-assemble, bind water molecules, and form stable, biodegradable hydrogels
[70][64].
Table 1.
Methods for encapsulation of anthocyanin using different material and their outcomes.
Method |
Source |
Encapsulation Material |
Study Outcome |
Reference |
Spray drying |
Grape juice |
Maltodextrin, whey proteins, soybean |
120 days of ACNs stability at 35 °C using soybean with maltodextrin (10% degradation) |
[71][65] |
|
Pomegranate powder |
Gum arabic and modified starch |
Higher ACNs retention (60%) |
[72][66] |
|
Jussara |
Starch, inulin, and Maltodextrin |
ACNs color maintained together with light stability at 50 °C for 38 days |
[73][67] |
|
Barberry |
Gum arabica, Maltodextrin, and gelatin |
Microencapsulation efficiency of 92.8% was achieved during the study |
[74][68] |
|
Grape pomace |
Maltodextrins |
11 days’ half-life stability of encapsulated ACNs into apple pure matrix at 35 °C |
[75][69] |
|
Jussara |
Gum arabica, modified starch, whey proteins, or Soy protein isolate |
The use of the two polysaccharides with either one of the proteins resulted in high encapsulation efficiency and ACNs retention |
[76][70] |
|
Purple sweet potato |
MC grafted with cinnamic acid |
Maltodextrins with cinnamic acid improved ACNs stability over 30 days’ storage time in comparison to maltodextrin or free ACNs |
[77][71] |
|
Blueberry |
Whey proteins |
Encapsulated blueberry ACNs were more stable than blueberry extract |
[78][72] |
|
Purple maize |
Modified, normal, and waxy maize starches |
Acetylated starches had superior encapsulating power for ACNs |
[79][73] |
|
Black raspberry juice |
Maltodextrin, fenugreek gum, and microcrystalline cellulose |
Gum and cellulose increased the overall properties of the powders and ACN concentration |
[80][74] |
|
Roselle extract |
Maltodextrin, pectin, gelatin, Carboxy methyl cellulose, whey proteins, carrageenan, and Gum arabica |
Pectin showed better retention and release, throughout the storage period |
[81][75] |
|
Blueberry |
Maltodextrin and Gum arabica |
The use of an ultrasonic nozzle better protected the blueberry bioactive than the conventional nozzle and had similar results for AA and total ACN contents |
[82,[7683]][77] |
|
Jussara pulp |
Gelatin, Gum arabica, maltodextrin |
Optimization study pointed to 165 °C and 5% of carrier material as the best for ACNs retention. At 40 °C and 75% of relative humidity, ACNs half-life was 14 days when coated with GA |
[84][78] |
|
Saffron anthocyanin |
β-glucan and β-cyclodextrin |
Higher concentrations of ACNs were protected from adverse stomach conditions by encapsulation |
[85][79] |
|
Roselle extract |
β-cyclodextrin, soy protein isolate, gelatin |
Increased the thermal stability and encapsulation efficiency by 99% using the composite wall materials of purified roselle extract |
[86][80] |
Freeze-drying |
Raspberry |
Gum arabica and Soy proteins |
Increased retention of ACNs by 36% |
[30] |
|
Milk-Blackberry pulp |
Maltodextrin and modified starch |
High anthocyanin content and increased antioxidant capacity |
[87][81] |
|
Sour cherry |
Soy proteins and whey proteins |
SP showed higher encapsulation efficiency and higher anthocyanin content |
[88][82] |
|
Grape extract |
Acacia gum and whey proteins |
Improved encapsulation efficiency of ACN |
[89][83] |
|
Wine grape pomace |
Gum arabica and Maltodextrin |
Samples had 91% encapsulation efficiency |
[90][84] |
|
Saffron petal |
Cress seed gum, Gum arabica, Maltodextrin |
Cress seed gum had the same results for ACNs stability as other conventional wall materials however, lower color constancy |
[91][85] |
|
Cherry juice |
Maltodextrin and Gum arabica |
ACN retention was 90%, in comparison to liquid juice (11%) |
[92][86] |
Electrospraying |
Black carrot extract |
Gelatin and chitosan |
Faster release in the acetic acid medium with greater encapsulation efficiency |
[16] |
Ultrasonication |
Anthocyanin |
Lecithin |
Sustained release and high stability of anthocyanin |
[93][87] |
Electrospinning |
Sour cherry extract |
Lactalbumin and gelatin |
Increased bioaccessibility and stability of ACNs |
[94][88] |
Copigmentation |
Blueberry |
Chondroitin sulfate and kappa carrageenan |
Effective protection against degradation at low pH |
[95][89] |
|
Anthocyanin |
Guar gum |
High encapsulation efficiency and high kinetic stability |
[57][51] |
|
Blueberry and Elderberry |
Chondroitin and chitosan |
Improved chemical stability and stable color of anthocyanin |
[96][90] |
|
ACNs extract |
Gum arabica |
GA coating increased color stability and half-life of ACNs at high temperatures |
[97][91] |
|
Bilberry |
Dextran sulfate |
During storage in dark conditions at 4 °C, ACN content decreased by 12% as compared to extract (35%) |
[98][92] |
Supercritical CO2 |
Bilberry |
Soy lecithin |
Higher stability and encapsulation efficiency |
[99][93] |
Microwave |
Roselle and Red Cabbage |
Maltodextrin |
Encapsulated ACNs were able to improve margarine stability against phase separation and oxidation |
[100][94] |
Extrusion |
Haskap berries |
Calcium-alginate |
The increased residence time of microparticle gels in the stomach suggests a more controlled release of ACNs |
[101,102][95][96] |
|
Clitoria ternatea petal flower extract |
Alginate and calcium chloride |
Thermal stability with inhibition of carbohydrate and lipid digestion |
[26] |
Evaporation |
Cyanidin-3-glycoside extract |
N-trimethyl chitosan-coated liposomes |
Coating ACNs increase the antioxidant activity in rat’s cornea, with higher transepithelial transport |
[103][97] |
Gelation |
Jussara extract |
Alginate, chitosan, whey proteins, gelatin |
Alginate hydrogel beads and chitosan showed greater antioxidant capacity [higher protection] as compared to WP and gelatin |
[104][98] |
|
Black rice |
Maltodextrin, Gum arabica, whey proteins |
Whey protein isolates exhibited a greater release of anthocyanin in GIT with enhanced antioxidant activity |
[105][99] |
|
Mulberry |
Alginate and chitosan beads |
High ACN encapsulation efficiency |
[106][100] |
|
Purple rice bran |
Pectin, zein, and whey proteins |
Pectin-WP and Pectin-zein-WP capsules have the potential of slowly releasing delivery systems for ACNs |
[107][101] |
|
Blueberry |
Chitosan/cellulose Chitosan/sodium tripolyphosphate |
Cellulose nanocrystals had better ACN recovery and stability at pH 7 than sodium tripolyphosphate |
[108][102] |
Sol-gel technique |
Black carrot |
Silica (drug delivery system) |
Nanoparticles with ACNs were able to inhibit 87.9% of neuroblastoma cells |
[109][103] |
Coacervation |
Black rice |
Gelatin-acacia gum and chitosan- Carboxymethylcellulose |
Microcapsules can be applied for incorporating ACNs into nutraceuticals for controlled release |
[110][104] |
|
Blueberry |
Chitosan |
Chitosan was able to stabilize ACNs after simulated GI fluid assay and storage |
[111][105] |
|
Purple sweet potato |
Konjac glucomannan |
Extra chitosan oligosaccharides coat was needed to stabilize microspheres against stomach conditions and to release ACNs in the small intestine [in vitro] |
[112][106] |
Inclusion Complexes |
Bignay and duhat extract |
β-cyclodextrin |
Increased encapsulation efficiency and possess enzyme inhibitory properties |
[113][107] |
Yeast mediated encapsulation |
Chokeberry ACNs |
Saccharomyces cerevisiae [yeast] |
Yeast turned the ACNs efficiency around by 55% |
[114][108] |
These hydrogels, therefore, are not only suitable for encapsulating ACNs, ensuring stability, and controlled release, but also offer opportunities to be used for many food industry applications, such as thickeners (sauces, soups), texturizers (confections), and flavors (slow-release)
[115][109]. Nanoencapsulated complexes comprising CH hydrochloride, carboxymethyl CH, and β-Lg incorporated with ACN extracts also improved their bioavailability and stability
[116][110]. Another novel technique for encapsulation of ACN-rich extracts is by using niosomes, which have emerged as a suitable delivery system for ACNs, owing to the low toxicity and high biocompatibility attributes of these liposomal formulations
[117][111]. Molecular inclusion complexes are another approach to stabilize ACNs, whereby ACNs have been coupled with β-cyclodextrins (β-CDs), resulting in slower GI stabilization, as well as protecting the ACNs from polymerization and hydration reactions
[118][112]. This complexation with CDs increased thermal stability and reduced the degradation of ACNs, thereby protecting them in the difficult GI environment. Furthermore, the utilization of CDs as encapsulation materials for bioactive compounds may lead to enhanced solubility, greater permeability through intestinal membranes, as well as greater bioavailability of the encapsulated compounds
[119][113].
3.1. Nanoparticles
In a study
[120][114], ACN extracts encapsulated in β-Lg nanoparticles exhibited greater antioxidant activity and enhanced retention at various pH ranges: pH 6.8 (mouth), pH 6.9 (simulated gastric), and pH 2 (simulated intestinal), as compared to unencapsulated ACNs. Nanocarriers formulated using CH-PC complexes provided adequate protection against degradation by gastric juice and therefore, facilitated the release of ACNs in the small intestine
[121][115]. Nanocomposites containing amphiphilic peptide C6M1 significantly improved the stability of ACNs against increased pH, elevated temperatures, and metallic ions
[120][114].
Similarly, zein-ACN nanoparticles have been found to exhibit greater encapsulation efficiency and scavenging activity as compared to ACN extracts without nanocrystallization
[122][116]. SLNs made up of solid lipid shells [high melting lipid matrix], also have a better encapsulation efficiency, slow rate of degradation, superior stability, and low cost of production, as compared to nanoemulsions (NEs)
[123][117]. ACNs from red cabbage encapsulated in SLNs prepared by way of diluting the water-in-oil [W/O] microemulsions (MEs) containing ACNs in the aqueous phase exhibited greater stability under GI conditions, as compared to unencapsulated ACNs
[69][63]. However, encapsulating ACNs in SLNs might be challenging owing to their tendency to partition into the aqueous phase during preparation procedures
[124][118].
3.2. Liposomes
Liposomes have demonstrated the potential to protect and stabilize ACNs, ensuring their prolonged presence in the systemic circulation, and therefore, enhanced cellular uptake in the human body
[125][119]. Nanoliposomes, resulting from particle size reduction of conventional liposomes using ultrasound, membrane extrusion, or high-pressure homogenization, in particular, has emerged as an excellent delivery system for ACNs, owing to their amphipathic, non-immunogenic, and non-toxic characteristics, biodegradability, and biocompatibility
[69][63].
3.3. Emulsions
Multiple emulsions, or more colloquially, double emulsions, such as W/O/W emulsions, are garnering increasing interest in the context of encapsulation, enhanced retention, and improved protection of ACNs
[126][120]. Huang and Zhou
[127][121] encapsulated ACNs from black rice extract in a W/O/W emulsion, evaluated the changes in ACN concentration, and release attributes of the multiple emulsion by way of an in vitro simulated digestion study. The study reported a high microencapsulation efficiency of 99.45 ± 0.24%, and that the multiple emulsion-maintained encapsulations even after 2 h of exposure to gastric juice, thereby preventing the release of ACNs in the stomach environment
[127][121].
Likewise, owning a large droplet surface area, and a reduction in particle size have made NEs a proven solution for increasing the functionality of ACNs contained within
[128][122]. Furthermore, NEs provide greater stability against droplet aggregation and gravitational separation owing to their high surface-to-volume ratio, an attribute of critical significance from the perspective of shelf-life enhancement of various food and beverage industry products
[129][123].
3.4. Biopolymer Particles
Biopolymer particles comprise a dense framework, including supramolecular structures formed through electrostatic interactions, which can be used to encapsulate and deliver ACNs in the human body
[11]. The properties of the carrier type, or wall material, as well as the various interactions between the polymer system, and bioactive ensure that the release of the core components is initiated at a specific time and location within the human GI system
[45][124].
Among the carrier agents, the most commonly used biopolymers for ACNs encapsulation in recent years include maltodextrins (19.56%), gums (15.22%), milk proteins (13.04%), starches, and their derivatives (>9.78%)
[130][125]. Alginate, the polysaccharide isolated from various brown seaweeds, forms gels [through ionotropic gelation] in the presence of cations (Ca
2+, Ba
2+, Zn
2+) as crosslinkers, making these gels favorable options as delivery systems
[131][126]. Natural polymers, when compared to their synthetic analogs, are highly biocompatible and biodegradable, and can be used for the entrapment of both hydrophilic and hydrophobic drugs
[132][127]. A coacervated complex of CH and PC was used to encapsulate ACNs, with the subsequent in vitro analysis revealing that the bioaccessibility percentage of the coacervated ACNs formulation was markedly higher when compared to both the crude ACNs extract and the purified ACNs crystals
[133][128].
More recently, dietary fibers have gained prominence as biocompatible, biodegradable, relatively less toxic, and cost-effective colon-targeted delivery systems for various natural polyphenols with therapeutic potential
[134][129]. The various advantages imparted by these polymers include maintenance of the structural integrity of delivery systems, thereby shielding polyphenols from the harsh environmental conditions in the GI tract, facilitating the colonic microbe-mediated polyphenol biotransformation, strengthening dietary fiber-polyphenol interlinkages, improved colon-associated mucoadhesion, and resultant enhanced payload delivery, and synergistic prebiotic effects
[135,136][130][131] reported that hydrogels prepared using glucomannan and xanthan gums isolated from konjac [
Amorphophallus konjac] for encapsulation of ACNs offered superior protection against pH variations in the GI tract.
3.5. Copigmentation
Copigmentation stabilizes ACNs by way of the formation of a non-covalent molecular complex with a colorless organic or inorganic compound
[63][57]. The ‘sandwich’ complex that results, therefore, renders greater protection to the flavylium chromophore against the water molecule-induced nucleophilic substitution, thereby preventing the degradation of ACNs to colorless chalcone and hemiketal forms
[137][132]. PC-induced copigmentation of European bilberry (
Vaccinium myrtillus) ACN extracts extended stability to both ACNs and the resulting multiple emulsions
[62][56]. Furthermore, ACNs copigmented with PC or chondroitin sulfate (CS) (negative charge) combine with CH (positive charge), manifesting in the formation of polyelectrolyte complexes [PECs] that can be utilized as delivery systems and have been shown to enhance the biological function of ACNs
[138][133].