https://orcid.org/0000-0002-3379-0278
Abstract
Chrysin is a hydrophobic flavonoid with multiple health benefits. The various applications of chrysin are challenged by its poor solubility, instability and loss of bioactivity. Casein–chrysin complex and casein–polysaccharide–chrysin complexes have developed to overcome these limitations. Very high encapsulation efficiency of 98.23 ± 0.22% was achieved with casein–inulin–chrysin complex. The chrysin was able to form a stable casein–polysaccharide–chrysin complex suspension with a hydrodynamic diameter of 382.3 nm, zeta potential value of −12.3 mV and a Polydispersity Index (PDI) of 27.7. The antioxidant activity of chrysin increased about threefold after encapsulation. The release of chrysin from its encapsulated complexes to different buffers in the pH range of 3 to 10 was studied at 1:10 ratio. At the end of 48 h, only 6%–8% of chrysin was released in the pH range 3–4, 33%–58% at pH 5–9 and 62% at pH 10. The chrysin encapsulated in casein–inulin–chrysin complex was able to overcome the rapid release of chrysin from the casein–chrysin complex. The results indicate the successful development of a stable encapsulated chrysin complex which can overcome the various limitations of chrysin in its potential applications.
Introduction
Chrysin (5,7-dihydroxy-2-phenyl-4H-chromen-4-one and 5,7-dihydroxyflavone) belongs to the flavone class of polyphenolic compounds. Propolis, honey and Passiflora species are some of the major sources of chrysin (Stompor-gorący et al., 2021). Chrysin exhibits many biological and pharmacological activities including antioxidant, anti-inflammatory, anticancer, hepatoprotective, neuroprotective, antiallergic and antiviral activities; but its applications in the food industry are limited by its low solubility and the loss of biological activities over time due to instability (Ferrado et al., 2020). Encapsulation is a process of enveloping one sensitive core substance within another substance, which has been suggested to improve the bioavailability and stability of chrysin during storage and processing. The following attempts have been made so far by researchers to encapsulate chrysin in various wall materials: β-cyclodextrin (Chakraborty et al., 2010), poly lactic-co-glycolic acid-poly ethylene glycol (PLGA-PEG) (Mohammadinejad et al., 2015), nanolipid carriers (Sabzichi et al., 2017), PLGA-PEG nanoparticles (Firouzi-Amandi et al., 2018), cationic and amphiphilic polymers (Davaran et al., 2018), Bovine Serum Album (BSA) (Nosrati et al., 2018; Ferrado et al., 2020), soya and egg phospholipids (Kim et al., 2019), polycaprolactones (Ghamkhari et al., 2019), chitosan (Siddhardha et al., 2020) and nanoemulsions with edible oil (Ting et al., 2021). Most of these aim at drug-delivery and therapeutic uses. Reports on encapsulation of chrysin with biocompatible wall materials for potential food-based applications are scarce.
Food-based applications of chrysin can benefit from encapsulation with wall materials such as casein, which is an important and safe coating material with high consumer acceptance (Tang, 2021). Casein, a major milk protein, is Generally Recognised As Safe (GRAS) and is used in numerous food applications (Głąb & Boratyński, 2017). The present work has considered the use of casein and various food-grade polysaccharides for the encapsulation of chrysin, because encapsulation of hydrophobic bioactives using complexes of casein and polysaccharides/gums/pectins/polymers has been said to increase the encapsulation efficiency (EE) and bioactive's stability (Abd El-Salam & El-Shibiny, 2020). The present work attempts encapsulation of chrysin to form stable casein–chrysin and casein–polysaccharide–chrysin complexes, without harming its antioxidant activity and achieving slower chrysin release from the encapsulated complex.
Materials and methods
Sodium caseinate was obtained from SRL Chemicals, India. Tri-potassium citrate monohydrate, starch, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium phosphate monobasic, sodium phosphate dibasic, sodium bicarbonate and sucrose were obtained from Merck, India. Potassium phosphate dibasic anhydrous, carboxymethyl cellulose sodium salt (medium viscosity), calcium chloride dihydrate, sodium carbonate and inulin were obtained from Loba Chemie, India. Pectin, chitosan and xanthan gum were obtained from HiMedia, India. Chrysin was obtained from TCI, India. Ethanol, laboratory-grade was obtained from Changshu Hongheng Fine Chemicals. Gum arabic was obtained from Sigma, India. Deionised water was used for all experiments unless otherwise specified.
Encapsulation of chrysin to form casein–chrysin and casein–polysaccharide–chrysin complexes
Stock solutions of sodium caseinate, tri-potassium citrate, dipotassium hydrogen phosphate, calcium chloride and different polysaccharides were prepared by dissolving them in water and chrysin in absolute ethanol. Casein–chrysin complex was prepared by the reassembly method as described by Ghatak & Iyyaswami (2019). 0.01 mg mL−1 of chrysin was added drop-wise into 0.5% (w/v) sodium caseinate, 1 M tri-potassium citrate, 0.5 M dipotassium hydrogen phosphate and 0.1 M calcium chloride. pH was adjusted to 6.8 using 1 N HCl/NaOH and stirred for 1 h at room temperature to attain equilibrium and centrifuged at 8385 g for 20 min. The unencapsulated chrysin in the supernatant was analysed by measuring the absorbance at 316 nm in a UV spectrophotometer (Eppendorf BioSpectrometer kinetic, Hamburg, Germany), and the EE was calculated using eqn 1:
To investigate the impact of different polysaccharides, namely, Carboxymethyl Cellulose (CMC), chitosan, gum arabic, inulin, pectin, sucrose, starch and xanthan gum on the formation of casein–chrysin–polysaccharide complex and EE, individual polysaccharides (at 0.1% w/v concentration) were added to the casein and salt mixture. The effects of different parameters like sodium caseinate concentration (in the range 0.25%–1.25% w/v), polysaccharide concentration (0.1%–1% w/v), pH (4–8) and chrysin concentration (0.005–0.05 mg mL−1) on the EE were studied.
Statistical analysis
The experimental data were statistically analysed for their significance using One-way Analysis of Variance (Anova) method by utilising the OriginPro 2023b software. Statistical significance was determined by considering a P-value of less than 0.05. All the experiments were conducted in triplicates, and the data obtained were presented as the mean ± standard deviation. Tukey analysis was used for mean comparison.
Characterisation of the encapsulated chrysin
HPLC analysis
HPLC analysis was done to confirm the intactness of chrysin after encapsulation. The encapsulated chrysin was subjected to chromatographic analysis using HPLC (Shimadzu LC-20 AD; C-18 column (4.6 mm ID * 250 mm)) (Shiseido Co., Ltd, Japan CAPCELL PAK C18 MGII S5) after reverse extraction of chrysin from the encapsulated pellets with 1:20 water and stirring for 4 h at room temperature. A binary isocratic elution method was followed with mobile phase containing 85% acetonitrile and 15% water; flow rate was 0.6 mL min−1 with 10 min run time, and column temperature was 30 °C. The analyte was measured at 268 nm using a PDA detector (SPD-M20A).
Particle characterisation and antioxidant activity
Dynamic light scattering of the various samples (Table 1) was measured using (Anton Paar 3 Litesizer™, Graz, Austria) 500 particle size analyser. The antioxidant activities of pure and encapsulated chrysin were evaluated by DPPH assay based on Ghatak & Iyyaswami (2019). A mixture of methanol and the DPPH solution in equal volumes was used as a control. The antioxidant activity (AOA) of the samples was measured using eqn 2.
Particle size distribution, zeta potential, Polydispersity Index (PDI) and antioxidant activity of encapsulated chrysin
| Sample | Particle size (nm) | Zeta potential (mV) | Polydispersity Index (%) | Antioxidant activity (%) |
|---|---|---|---|---|
| Pure chrysin† | 7.50 ± 0.63e | |||
| Casein + Chrysin† | 230.9 | −14.3 | 27.6 | 14.36 ± 0.19d |
| Casein–chitosan complex with chrysin† | 256.1 | −11.1 | 24.0 | 17.54 ± 1.09c |
| Casein–gum arabic complex with chrysin† | 237.4 | −11.1 | 28.8 | 25.16 ± 0.10a |
| Casein–inulin complex with chrysin† | 225.9 | −14.9 | 28 | 22.84 ± 0.16b |
| Casein–inulin complex with the chrysin concentration of 0.02 mg mL−1 at pH 8 | 382.3 | −12.3 | 27.7 | – |
| Sample | Particle size (nm) | Zeta potential (mV) | Polydispersity Index (%) | Antioxidant activity (%) |
|---|---|---|---|---|
| Pure chrysin† | 7.50 ± 0.63e | |||
| Casein + Chrysin† | 230.9 | −14.3 | 27.6 | 14.36 ± 0.19d |
| Casein–chitosan complex with chrysin† | 256.1 | −11.1 | 24.0 | 17.54 ± 1.09c |
| Casein–gum arabic complex with chrysin† | 237.4 | −11.1 | 28.8 | 25.16 ± 0.10a |
| Casein–inulin complex with chrysin† | 225.9 | −14.9 | 28 | 22.84 ± 0.16b |
| Casein–inulin complex with the chrysin concentration of 0.02 mg mL−1 at pH 8 | 382.3 | −12.3 | 27.7 | – |
The concentration of casein was 0.5% (w/v), chrysin was 0.01 mg mL−1 and polysaccharide was 1% (w/v) at pH 7 in all the samples, unless mentioned otherwise. Results are expressed as mean ± standard deviation from triplicate experiments. Values with different letters (a–e) represent significant differences for the samples (P < 0.05).
Particle size distribution, zeta potential, Polydispersity Index (PDI) and antioxidant activity of encapsulated chrysin
| Sample | Particle size (nm) | Zeta potential (mV) | Polydispersity Index (%) | Antioxidant activity (%) |
|---|---|---|---|---|
| Pure chrysin† | 7.50 ± 0.63e | |||
| Casein + Chrysin† | 230.9 | −14.3 | 27.6 | 14.36 ± 0.19d |
| Casein–chitosan complex with chrysin† | 256.1 | −11.1 | 24.0 | 17.54 ± 1.09c |
| Casein–gum arabic complex with chrysin† | 237.4 | −11.1 | 28.8 | 25.16 ± 0.10a |
| Casein–inulin complex with chrysin† | 225.9 | −14.9 | 28 | 22.84 ± 0.16b |
| Casein–inulin complex with the chrysin concentration of 0.02 mg mL−1 at pH 8 | 382.3 | −12.3 | 27.7 | – |
| Sample | Particle size (nm) | Zeta potential (mV) | Polydispersity Index (%) | Antioxidant activity (%) |
|---|---|---|---|---|
| Pure chrysin† | 7.50 ± 0.63e | |||
| Casein + Chrysin† | 230.9 | −14.3 | 27.6 | 14.36 ± 0.19d |
| Casein–chitosan complex with chrysin† | 256.1 | −11.1 | 24.0 | 17.54 ± 1.09c |
| Casein–gum arabic complex with chrysin† | 237.4 | −11.1 | 28.8 | 25.16 ± 0.10a |
| Casein–inulin complex with chrysin† | 225.9 | −14.9 | 28 | 22.84 ± 0.16b |
| Casein–inulin complex with the chrysin concentration of 0.02 mg mL−1 at pH 8 | 382.3 | −12.3 | 27.7 | – |
The concentration of casein was 0.5% (w/v), chrysin was 0.01 mg mL−1 and polysaccharide was 1% (w/v) at pH 7 in all the samples, unless mentioned otherwise. Results are expressed as mean ± standard deviation from triplicate experiments. Values with different letters (a–e) represent significant differences for the samples (P < 0.05).
Chrysin release profile
The release of chrysin from its encapsulated complexes was studied at different time intervals up to 48 h in the pH range of 3–10. Different buffers were used to emulate foods with different pH: citrate buffer (pH 3, 4 and 5), phosphate buffer (pH 6, 7 and 8) and bicarbonate buffer (pH 9 and 10). The encapsulated chrysin was resuspended in suitable buffers in 1:10 ratio and kept on a magnetic stirrer. Samples were taken at regular intervals and centrifuged at 10 000 r.p.m. for 20 min. The amount of chrysin released from the complex was determined by measuring the content of chrysin in the supernatant using a UV–Visible spectrophotometer at 316 nm using eqn 1.
Results and discussion
Effect of casein concentration
Casein is considered a suitable carrier for hydrophobic flavonoids such as chrysin, because the flavonoids can interact with proteins in the hydrophobic core of casein particles via electrostatic attraction, hydrogen bond and van der Waals force (Wang & Zhao, 2022). The EE was studied at different concentrations of sodium caseinate. The highest EE was observed with 0.5% (w/v) of sodium caseinate. The EE decreased significantly (P < 0.05) (Fig. 1) upon further increase in the sodium caseinate concentration. Similar results were obtained for the encapsulation of quercetin in reassembled casein particles (Ghatak & Iyyaswami, 2019). The decrease in EE with an increase in sodium caseinate concentration can be attributed to casein's tendency to form aggregates at higher concentrations, which reduces its capability to encapsulate (Ye & Harte, 2013).
Effect of different concentrations of sodium caseinate on the EE of chrysin. Results are expressed as mean ± standard deviation of triplicate experiments. Letters (a–e) represent significant differences among different polysaccharides.
Effect of polysaccharide concentration
Under controlled conditions, many polysaccharides with their terminal reducing sugars can form covalent bonds with free amino groups on the surface proteins of casein, and their imbalanced interactions (non-neutralised charges) yield novel functional conjugates for food applications (Abd El-Salam & El-Shibiny, 2020; Cortés-Morales et al., 2021). From the experimental results, it was observed that the addition of gum arabic, inulin and chitosan showed the highest EE in that order (Fig. 2), and therefore were chosen for studying the effect of their concentration in the range of 0.1% to 1% (w/v). Although a few other polysaccharides also showed good EE, gum arabic, inulin and chitosan were chosen for further studies because, apart from their top performance, they possess negative, neutral and positive charges, respectively, which enables a comprehensive study of the interaction between sodium caseinate and polysaccharides with distinct charges.
Screening and effect of concentrations of different polysaccharides on the encapsulation of chrysin with casein micelles. Results are expressed as mean ± standard deviation of triplicate experiments. Different letters (a–e in lower case) represent significant differences in EE at different concentrations of polysaccharides, and letters (A–C in upper case) represent significant differences in EE among different polysaccharides at the same concentration.
A significant decrease in the EE was observed with increasing polysaccharide concentration for all three polysaccharides till a casein-to-polysaccharide ratio of 1:1, at which the EE reduced; the EE was found to again increase significantly till 1:2 (casein:polysaccharide). At the casein-to-polysaccharide ratio of 1:1, the EE was low due to the formation of emulsions instead of sediments, while at other ratios, stable sediments were formed. The statistically significant differences in EE at different concentrations of polysaccharides and across individual polysaccharides are shown in Fig. 2.
Chitosan's interaction with casein increased beyond 1:1 casein: polysaccharide, due to the charge stabilisation between the cationic chitosan and the anionic nature of casein at pH 6.8. At low concentrations, chitosan is said to precipitate casein due to bridging flocculation. A low chitosan concentration is insufficient to fully cover the casein surface due to reduced surface electrostatic potential which in turn increases the collision rate between the casein molecules, and the chitosan-free patches on the casein surface allow the chitosan molecules to attach simultaneously to more than one casein particle leading to aggregation (Ding et al., 2019). As the concentration of chitosan increases, more casein particles are independently covered by chitosan. This indicates that the electrostatic interactions are likely the major driving force for spontaneous chitosan–casein complexation, while other forces such as hydrogen bonding and hydrophobic interactions could play a minimal role (Celli et al., 2018).
Higher encapsulation was achieved with lower concentrations of gum arabic with casein to gum arabic ratio of 1:0.2. Higher ratios of gum arabic might have been unfavourable for encapsulation because of the negative charge offered by casein and gum arabic at pH 6.8.
The highest EE was observed at a casein-to-inulin ratio of 1:2. Since inulin is a neutral polysaccharide, it does not show much electrostatic interaction with the casein particles (Ye, 2008). The interaction is mainly due to hydrogen bond formation and hydrophobic interactions along with dipole–dipole and charge–dipole interactions. According to Esmaeilnejad Moghadam et al. (2019) and Schaller-Povolny & Smith (2002), three types of proteins in caseins are capable of interaction with inulin but with different bonds: inulin-α-casein involves in an electrostatic band, β-casein due its strong hydrophobic property forms a hydrophobic band with inulin, while with k-casein–inulin forms a covalent bond complex. Casein–inulin complex can be deemed to have contributed to the highest encapsulation of chrysin due to the formation of stable complexes by the neutrally charged inulin with negatively charged casein particles at pH 6.8–7. Moreover, chrysin at this pH is mostly charge-neutral, as its pKa is 6.74. This uncharged form of chrysin could also have contributed towards efficient encapsulation in casein–inulin complex, as uncharged forms of the target analyte have been reported to have maximised EE (Xing & Chen, 2013).
Effect of pH and chrysin concentration
Interaction between a polysaccharide and a protein during the preparation process is influenced by pH (Cortés-Morales et al., 2021). Studies were performed for casein systems with inulin (1% w/v) and gum arabic (0.1% w/v) at different pH. The significant differences in the EE with respect to different pH and within a given pH for both gum arabic and inulin have been depicted in Fig. 3a. The highest EE was observed at pH 8 followed by 7 for both inulin and gum arabic. The higher EE at pH 7 and 8 could be because of casein's tendency to loosen at higher pH values due to electrostatic repulsions which can help the entry of hydrophobic chrysin into the casein core. Moreover, the presence of polysaccharides helps in masking the charges between the casein molecules preventing large-scale aggregation of casein at basic pH, possibly leading to an overall environment for efficient encapsulation (Bian & Plank, 2012; Vasina, 2016). It can be noted that inulin showed significantly better EE with change in the pH values, as compared to gum arabic.
(a) Effect of pH on EE of chrysin in casein–gum arabic and casein–inulin complexes; (b) Effect of chrysin concentration on EE of chrysin in casein–inulin complex. Results are expressed as mean ± standard deviation of triplicate experiments. Different letters (a–d in lower case) represent significant differences in EE at different pH for a polysaccharide and letters (A–B in upper case) represent the significant difference in EE among the two polysaccharides at a single pH.
Chrysin concentration was studied in the range of 0.005 to 0.05 mg mL−1. A significantly increasing trend of EE was observed till a chrysin concentration of 0.02 mg mL−1 and showed a significant decrease thereafter with higher chrysin concentrations (Fig. 3b). This decrease could mainly be because of the shortage of casein–polysaccharide complex for the encapsulation of higher amounts of chrysin. At higher concentrations of chrysin, most of the hydrophobic core of casein is saturated by chrysin, leading to the unavailability of newer sites on casein for further interaction.
Characterisation of the encapsulated chrysin
Chromatographic analysis of the encapsulated chrysin
The retention time of standard chrysin was found to be around 5.27 min, of the loaded chrysin in casein–chrysin complex was 5.27 min, and 5.26 min in casein–inulin complex (Figure S1), confirming the successful formation of casein–inulin–chrysin complex and the intactness of chrysin throughout the encapsulation process and even after reverse extraction. From the area of the elution peaks, the EE for casein–chrysin complex was found to be 88.18 ± 4.19% and for casein–inulin–chrysin complex was 98.01 ± 1.54%.
Particle characterisation
A hydrodynamic diameter of 382.3 nm (with an accuracy better than ±2% on NIST traceable standards) and zeta potential value of −12.3 mV with a Polydispersity Index (PDI) of 27.7% (accuracy of ±0.5%) were observed for casein–inulin–chrysin complex (Table 1). The average hydrodynamic diameter of empty casein complexes was reported to be 182 nm (Ghatak & Iyyaswami, 2019). The increase in the hydrodynamic diameter of the complexes indicates successful incorporation of chrysin molecules within casein and casein–inulin complex.
It was observed from Table 1 that the zeta potential values (−10 to −15) of different encapsulated chrysin samples have favourable surface charges to retain the colloidal stability of the particles without agglomerations. The obtained zeta potential values of the samples are also comparable to the values obtained by Nosrati et al. (2018) and Sabzichi et al. (2017), where chrysin encapsulated in bovine serum albumin (BSA) and nanolipid carriers showed zeta potential values of −11 mV.
The PDI values between 24% and 28% for different samples indicate the homogeneity of the particles with an acceptable size distribution. The PDI values obtained are comparable to those obtained by Kim et al. (2019) where chrysin encapsulated with phospholipid matrices gave PDI values between 20% and 30%.
Antioxidant activity
The results (Table 1) demonstrate a higher free radical scavenging activity of encapsulated chrysin than pure chrysin. The enhanced antioxidant activity could be attributed to the enhanced solubility and dissolution rate caused due to encapsulation. Encapsulation with casein can synergistically increase oxygen-scavenging ability due to the release of casein peptides along with chrysin (Rao et al., 2016; Wu et al., 2021). The results obtained here are in agreement with that of Chakraborty et al. (2010), where chrysin encapsulated in β-cyclodextrin showed better antioxidant activity than standard chrysin. Although both gum arabic and inulin have inherent antioxidant activities (Karimi et al., 2015; Mirghani et al., 2018), gum arabic encapsulated chrysin showed slightly higher antioxidant activity possibly due to its film formation over casein and its stabilising effect (Rao et al., 2016), leading to a more controlled release of chrysin from the encapsulated complex.
Chrysin release profile
The rate at which chrysin is released from the encapsulated complex is a significant factor in the formulation and optimisation of products with required release rate properties. From Fig. 4, it can be observed that encapsulated complex is stable over a wide range of pH. The release of chrysin is slow, with 8.8 ± 0.42, 6.0 ± 0.2, 38.7 ± 0.37, 50.7 ± 1.5, 58.0 ± 0.09, 54.2 ± 0.25, 33.0 ± 1.10 and 62.0 ± 0.20% release at pH 3, 4, 5, 6, 7, 8, 9 and 10, respectively, even after 48 h, which is a desirable property for its various applications. It can also be observed from Fig. 4 that chrysin release from the casein–inulin complex is slower and stabler than that from casein–chrysin at different pH. Casein loses its emulsifying properties at its pI (pH 4.6) and begins to flocculate, but the addition of polysaccharides to caseins could enhance the emulsion stability due to the formation of an extended network and changing of the viscoelastic properties (Vasina, 2016). Nosrati et al. (2018) found that 20% of the chrysin loaded in BSA was released within the first 10 h during in vitro drug release studies and 35% in 96 h, showing a more controlled release. Siddhardha et al. (2020) observed an initial burst of chrysin release from chitosan matrix within 2 h followed by a steadier release thereafter until the 6th hour.
Release profile of chrysin at different pH from (a) casein–chrysin complex; (b) casein–inulin complex. Results are expressed as mean ± standard deviation of triplicate experiments.
Casein shows a pH-dependent behaviour, that is, at alkaline pH it exhibits an anionic charge density due to the deprotonation of the amino acids. This negative charge increases with an increase in pH and leads to increased electrostatic repulsions within casein and loosening of the complex, which results in increased chrysin release rates at higher pH values (Bian & Plank, 2012; Ye & Harte, 2013). The presence of a neutral polysaccharide like inulin helps in reducing the electrostatic interactions between casein proteins by masking the charges (Vasina, 2016; Cortés-Morales et al., 2021). The chrysin encapsulated in casein–inulin complex most likely had a stronger interaction and was more tightly bound and therefore more stable as compared to casein–chrysin complex.
Chrysin might also have been involved in the interaction of the two macromolecules and, based on steric hindrance and the available sites in or on the casein–polysaccharide complex, could allocate itself in the core, or the surface, or may be uniformly distributed in the complex (Cortés-Morales et al., 2021). The hydrophobic unsubstituted B ring in chrysin is likely to place itself in the hydrophobic core of the complex, while the A ring of the chrysin might interact with the polysaccharides (Bilia et al., 2014). The interaction of chrysin with the casein–polysaccharide complex and its placement might play a role in how quickly it is released from its encapsulating matrix.
Casein is an easily digestible protein, and it does not interfere with the nutritional or functional properties of the encapsulated flavonoids, making it suitable for applications in food industry (Ye & Harte, 2013). Casein–polysaccharide complexes are known to retain their physical integrity and promote controlled release during the intestinal phase of human digestion ensuring bioavailability of the encapsulated molecule (Ding et al., 2019).
Conclusion
The study finds that both casein and casein–polysaccharide complexes act as suitable encapsulating materials for chrysin. The addition of polysaccharides particularly chitosan, gum arabic and inulin increased the encapsulation of chrysin, as can be seen in Fig. 2. EE as high as 90.71 ± 0.68% with casein and 98.23 ± 0.22% with casein–inulin complex were attained. The encapsulated chrysin was able to form a stable homogenous solution and also showed higher antioxidant activity as evidenced by the results in Table 1. Chrysin encapsulated in casein–inulin complex showed slower and steadier release at different pH as compared to chrysin encapsulated in only casein. The percentage release of chrysin from the chrysin–casein–inulin complex was 8.8 ± 0.42, 6.0 ± 0.2, 38.7 ± 0.37, 50.7 ± 1.5, 58.0 ± 0.09, 54.2 ± 0.25, 33.0 ± 1.10 and 62.0 ± 0.20% at pH 3, 4, 5, 6, 7, 8, 9 and 10, respectively, after 48 h (Fig. 4). The data indicate that encapsulation has successfully overcome the limitation of rapid release of chrysin and that the encapsulated chrysin can be amenable for food applications at a wide pH range. Moreover, both casein and inulin are natural, biodegradable, non-toxic and individually already in use in various food applications, which makes the chrysin–casein–inulin complex developed here relevant for various potential food applications including nutraceuticals and probiotics.
Acknowledgments
The authors would like to thank the National Institute of Technology Karnataka, Surathkal for providing the necessary infrastructure and support for carrying out this work.
Author contributions
Kruthika Parappa: Data curation (equal); formal analysis (equal); investigation (equal); methodology (equal). Prajna Rao Krishnapura: Data curation (equal); methodology (equal); project administration (equal); supervision (equal); writing – original draft (equal). Regupathi Iyyaswami: Conceptualization (lead); methodology (lead); resources (lead); supervision (lead); writing – review and editing (equal). Prasanna D. Belur: Formal analysis (equal); project administration (equal); resources (equal); supervision (equal); writing – review and editing (equal).
Funding
This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors.
Conflict of interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Ethical statement
Ethics approval was not required for this research.
Data availability statement
The authors confirm that the data supporting the findings of this study are available within the article.
References
This reference offers comprehensive insights into the sources and properties of chrysin, providing detailed information. A profound understanding can be gained regarding the correlation between its properties and human health.
This reference provides methodology for the encapsulation process of quercetin within reassembled casein particles. This research serves as a foundational basis for conducting the current study, as chrysin shares the same flavonoid class as quercetin.
This reference provides knowledge about the antioxidant activity of compounds that are complexed or encapsulated with casein. These also provide insights into the properties of these compounds, highlighting their potential as antioxidants when incorporated into casein-based systems.
This reference offers comprehensive insights into the sources and properties of chrysin, providing detailed information. A profound understanding can be gained regarding the correlation between its properties and human health.
