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Chemical and Biological Technologies in Agriculture
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Characterization of caseinate-pectin complex coacervates as a carrier for delivery and controlled-release of saffron extract

Chemical and Biological Technologies in Agriculture volume 11, Article number: 118 (2024) Cite this article

Abstract

In this work, microcapsules were developed by the complex coacervation of sodium caseinate and pectin as a carrier for saffron extract. Parameters such as Zeta potential, dynamic light scattering, and microscopic techniques were investigated for their influence on the formation of these complexes. Furthermore, Fourier transform infrared (FTIR) analysis confirmed the reaction mechanism between the protein and tannic acid or saffron extract. The study revealed that core/shell and protein/polysaccharide (Pr/Ps) ratios play a role in the encapsulation efficiency (EE) and loading capacity (LC) of saffron extract, with EE and LC ranging from 48.36 to 89.38% and 1.14 to 5.55%, respectively. Thermal gravimetric analysis revealed that the degradation temperature of saffron increased significantly with microencapsulation. The use of tannic acid for hardening the microcapsules led to an increase in size from 13 μm to 27 μm. Rheological findings indicated that shear-thinning behavior in the coacervates, with cross-linking, has a minor effect on the interconnected elastic gel structures. However, cross-linking improved the microcapsules' thermal and structural properties. The increase in polymer chain length due to cross-linking and the presence of the guest molecule (saffron extract) resulted in higher rheological moduli, reflecting enhanced entanglements and correlating well with the thermal, structural, and microstructural properties of the coacervates. Kinetic release studies showed a slower release in the gastric phase compared to the intestinal phase, with the Ritger–Peppas model effectively describing saffron extract release, highlighting a dominant swelling and dissolution release mechanism. Therefore, the NaCas/HMP coacervate wall materials made saffron stable in the gastric stage and sustainably release. It in the intestinal stage, promoting excellent absorption of saffron in simulated digestion.

Graphical Abstract

Introduction

Complex coacervation is an electrostatically and entropically driven associative liquid–liquid phase separation phenomenon that may cause the formation of bulk liquid phases, or the self-assembly of hierarchical, microphase separated materials [1,2,3]. During coacervation, oppositely charged biopolymers interact through electrostatic attraction, with pH playing a major role in coacervate formation [4, 5]. Research on complex coacervates is burgeoning not only to obtain novel materials and applications in the food and pharmaceutical industries but also to gain insight into the intracellular assemblies and origin of neurodegenerative diseases [6, 7]. The promising coacervate applications in the food and pharmaceutical industries include the encapsulation of bioactive compounds, target delivery, as well as the development of new textures and functionalities of biomaterials [8, 9].

Saffron is widely recognized as the most expensive spice in the world and is often called as "red gold" or "golden spice" due to its high value. The bioactive compounds of saffron are crocin, safranal, and picrocrocin, responsible for its color, aroma, and flavor, respectively [10, 11]. Crocin or crocetin is a glycoside ester with high coloring capacity due to its high water solubility [12]. Since external conditions such as temperature, oxygen, light, pH, enzymes, ions, and additives may degrade and decrease its functionality [12,13,14,15], many researchers have investigated ways to protect it from degradation, improve its release through smart delivery, and enhance its bioavailability [16, 17]. However, various techniques and shell materials have been used to encapsulate saffron extract, including freeze-drying with modified starch, maltodextrin, chitosan, and gum Arabic [18, 19], spray drying using whey protein and pectin [20], spray drying by gelatin, gum Arabic, and maltodextrin [11, 21], emulsification by pectin-whey [17, 20], ionic gelation/complexation by chitosan-gum Arabic [22], electrospinning by zein and tragacanth [23, 24], nanoliposomal encapsulation [25], sodium alginate [26], and freeze drying and electrospinning techniques using a gelatin matrix [27]. Furthermore, complex coacervation stands out as a useful strategy among these encapsulation technologies due to easy design and preparation. It assists in better preserving unstable components, has a high loading capacity, operates at low temperatures, reduces evaporation losses of volatile principles or thermal degradation, and provides more competency in controlling the release rate of nutraceuticals [28,29,30,31] and no one evaluate the complex coacervation method in saffron extract encapsulation and its release.

The main challenge in complex coacervation is that they are highly unstable under various conditions, and chemical cross-linking is necessary to stabilize them [33]. Therefore, crosslinking can be used as an irreversible technique to enhance the mechanical qualities, high-temperature stability, ionic strength, and pH stability of coacervates [32]. General crosslinkers like formaldehyde and glutaraldehyde are toxic and hence prohibited in food and pharmaceutical applications. Instead, specific cross-linkers such as transglutaminase [33], and sodium trimetaphosphate [34] have been utilized in particular applications. Tannic acid (TA), a polyphenol rich in hydroxyl groups, can strongly bond or precipitate with proteins [31, 35], and interact with polysaccharides. Studies have reported that protein crosslinking using polyphenols at different pH levels can modify the secondary structure of proteins and improve heat stability and rheological properties [36, 37].

Rheology has been used to gain insight into the effect of guest molecules on the structure of a complex coacervate. For instance, the linear viscoelastic properties of salbutamol encapsulated by gelatin coacervates showed a significant reduction in the storage modulus, and a slight increase in loss modulus, revealing more fluidization of the material [38]. These changes in the rheological behavior of the coacervates may be vital in modeling of release processes in advanced therapeutics. While numerous studies have been focused on crosslinking proteins using TA, there has been no report on the preparation of caseinate and pectin coacervates cross-linked with TA, and their impact on coacervate properties (such as thermal and mechanical stability) and their application in saffron extract encapsulation.

Understanding the self-assembly process of the coacervate is feasible when the thermodynamic knowledge of coacervate phase behavior with the dynamics of material through the rheological experiments as well as structural properties by Fourier transform infrared (FTIR), electron microscopy, and thermal properties are properly correlated. Therefore, the structure-rheology property relationships of complex coacervates of sodium caseinate (NaCas)/high methoxyl pectin (HMP) in the encapsulation of saffron extract in the presence and absence of a cross-linker (TA) were investigated. Since the effect of pH changes (ionic charge) and protein/polysaccharide ratios has been investigated in the electrostatic interactions of NaCas/HMP [5], our focus was on the influence of crosslinker (TA) and guest molecule (saffron extract) on the complex coacervate phenomenon. Finally, a drug delivery system was employed to evaluate the in-vitro release of saffron through the simulated gastrointestinal fluids.

Materials and methods

Materials

The dried saffron stigmas were kindly provided by Qaen, Khorasan Razavi province, Iran. High methoxyl pectin (HMP, galacturonic acid content > 74%), sodium caseinate (NaCas, protein content > 97%), and tannic acid (TA) were supplied by Sigma-Aldrich Co. The other chemicals were of analytical grade from Merck Co. (Germany). Distilled water was used to prepare all aqueous solutions.

Bioactive compound extraction

The bioactive compounds of saffron were extracted using a previously published method with slight modifications [23]. The dried saffron stigmas were ground and sieved with a 0.5-mm mesh. The extraction was then performed with distilled water under continuous shaking in a dark-colored bottle at 25 °C during overnight and process completed through ultra-sonication (Iranian Ultrasonic Company, 400 W, 12 mm probe diameter) of sample at a frequency of 30 kHz with a power of 250 W for 20 min at 25 °C. Note that this process enhances the extraction efficiency [23]. The saffron aqueous extract (ASE) was filtered using Whatman filter paper (No. 42) and stored at -18 °C until further use.

Preparation of saffron extract microcapsules

Mixtures of NaCas and HMP were prepared with a mixing ratio (R = Pr/Ps) of 2:1 w/w and 4:1 w/w and a total biopolymer concentration (Ct = 0.4% w/v) at room temperature, based on our previous work [5]. Sodium azide (0.02% w/v) was added to prevent microbial growth. The complex coacervation of NaCas and HMP for ASE encapsulation in the presence and absence of a cross-linker was achieved based on previous studies with some modifications [29, 36, 39]. The ASE in the core/shell ratios (1:1, 1:2, and 1:4 w/w) was added to the mixture with stirring at 500 rpm, while the pH was adjusted to 3.0 and 3.3 for R = 2:1 w/w and 4:1 w/w, respectively, using HCl or NaOH (0.1 and 1 M) [5]. The solution temperature was instantly lowered to below 10 ± 1 °C using an ice-water bath for 30 min at 300 rpm to complete complex coacervation [39, 40].

For the microcapsules cross-linked with TA, a 10% w/v solution of TA in distilled water was prepared, and then a solution of TA at a final concentration of 0.15% v/v was added to the microcapsules for cross-linking. The microcapsules cross-linked with TA were kept for 3 h at room temperature with a stirring rate of 300 rpm to promote the formation of the microcapsules before being stored overnight at 4 ± 1 °C to allow precipitation. Finally, the microcapsules were frozen and subsequently freeze-dried (Dena Vacuum Industry Co. LTD, Tehran, Iran). Details of the encapsulated ASE in complex coacervates at different core/shell and Pr/Ps ratios in the presence and absence of TA as a cross-linker are provided in Table 1.

Table 1 The encapsulated ASE in complex coacervates samples at different core/shell and Pr/Ps ratios in the presence and absence of tannic acid
Full size table

Encapsulation efficiency and loading capacity

The amount of saffron extract in the microcapsules was determined using a UV–Vis spectrophotometer, and the saffron extract content was considered as an indication of EE. An appropriate amount of saffron extract was dissolved in deionized water as a standard solution, and the absorbance was recorded at 440 nm using a UV–Vis spectrophotometer. A standard plot was depicted, and the regression equation was applied for the EE calculation (y = 73.973x—0.0049, R2 = 0.9994). In the microcapsules, the EE was measured through the absorbance of free saffron extract in the supernatant for each sample at 440 nm. Briefly, the supernatant was separated from the microcapsules by centrifugation (10,000 rpm, 20 min). The EE and LC of the encapsulated ASE in microcapsules were determined according to the following equations [31, 41]:

$${\text{EE }}\left( \% \right) = \left( {1 - \frac{{{\text{free saffron extract}}}}{{{\text{ total saffron extract }}}}} \right) \times 100$$
(1)
$$\text{LC }\left(\text{\%}\right)=\frac{\text{ total saffron extract}-\text{free saffron extract}}{\text{ weight of produced microcapsules}}\times 100$$
(2)

where total saffron extract is the weight of total saffron extract used in each sample, and free saffron extract is the weight of unloaded saffron extract.

Zeta potential and particle size distribution analysis

The Malvern Nano Zetasizer (Malvern, United Kingdom) was used to determine the average surface electrical charge (ζ-potential) and the particle size of both non-cross-linked (CC6) and TA cross-linked (CCT6) microcapsules. The zeta potential of the diluted samples with distilled water was measured. The experiment was conducted in three replicates for each formula, and the average was reported.

Rheological measurements

Rheological measurements of the coacervates were performed using a stress-controlled rheometer (Physica MCR 301, Anton Paar, Germany) with a cone-plate geometry (CP50, angle of 2°, φ = 50 mm) and a gap value of 200 µm. Both steady-state and dynamic rheological experiments were carried out for the cross-linked and non-cross-linked NaCas/HMP complex coacervates. When the geometry was applied, approximately 200 mg of the coacervate was loaded on the Peltier plate (Viscotherm VT2, Anton Paar) at 25 °C, a solvent trap was used to minimize water evaporation during the experiments, and allowed to rest for at least 5 min before the experiment.

Flow curves of the coacervates were obtained in an upward and downward shear rate sweep 0.1–1000 s−1 25 °C. Small-amplitude oscillatory shear measurements (SAOS) were carried out in strain and frequency sweep. Strain sweep was performed to determine the linear viscoelastic region (LVR) of the NaCas/HMP complex coacervate, where dynamic G′ and G′′ are independent of strain amplitude [42]. For comprehensive investigation of the rheological properties of complex coacervates, G′, G′′, and loss tangent (tan δ) at the LVE region were determined [42]. A frequency sweep was carried out over a range of 0.1 to 100 Hz to investigate the viscoelastic behavior of coacervates at 1% strain, which was in the LVR regime under isothermal conditions at 25 °C.

Material stiffness

It was found that G′ and G′′ can be calculated at high frequency range or near the gel point as follows [43]:

$$G^{\prime}\left( \omega \right) = G_{{\infty ,\alpha }} + \left( {\sqrt {\frac{2}{\pi }} .s_{a}^{*} .{\text{cos}}\left( {\frac{\pi }{2}\alpha } \right).\omega ^{a} } \right)$$
(3)
$${G}^{{\prime}{\prime}}\left(\omega \right)=\sqrt{\frac{2}{\pi }}.{s}_{a}^{*}.\text{sin}\left(\frac{\pi }{2}\alpha \right).{\omega }^{a}$$
(4)

Here, α represents the relaxation function’s order, G∞,α stands for the equilibrium shear modulus, and \({s}_{a}^{*}\) is a parameter dependent on material strength. Within a restricted frequency range and in the gel state, G∞,α becomes negligible, and G* is defined as follows:

$${G}^{*}\cong \sqrt{\frac{2}{\pi }}.{s}_{a}^{*}.{\omega }^{a}={A}_{a}{\omega }^{a}$$
(5)

where, Aa is considered as the material stiffness parameter.

Thermal properties

The thermal characteristics of NaCas, HMP, and freeze-dried microcapsules that were cross-linked and non-cross-linked with TA were analyzed using a thermal analyzer (Bähr-Thermoanalyse GmbH, Germany). The analysis was conducted under a steady airflow and an argon atmosphere. The samples were subjected to a temperature increase from room temperature to 600 °C, with a 10 °C/min heating rate.

Structural properties determined by FTIR

To determine the FTIR spectra of NaCas, HMP, and microcapsules that were non-cross-linked and cross-linked with TA, a Shimadzu (Kyoto, Japan) FTIR spectrometer was used. The KBr pellet method was utilized in the range of 400–4000 cm−1 at approximately 23 ± 1 °C. The spectrometer was a double beam spectrometer, with a resolution of 4 cm−1.

Morphological characterization

The morphological properties of the microcapsules were evaluated using a field emission scanning microscope (FESEM, Zeiss, Germany). The samples, including non-cross-linked and TA cross-linked microcapsules, were mounted onto an amorphous glass lamella and coated with a thin layer of gold prior FESEM imaging at an accelerating voltage of 5 kV.

In-vitro release

The release of ASE was examined in simulated gastrointestinal conditions according to previous studies [44, 45]. To simulate the digestion process in the gastrointestinal tract, 100 mg of sample was dispersed in 0.5 mL phosphate buffer (pH = 7.0) introduced in a dialysis bag and put in 20 mL of simulated gastric fluid (SGF) (pH = 2.0) at 37 °C and 100 r/min. The solution analysis (1 mL) was performed at 30-min intervals until 2 h, and replaced with an equal volume of the fresh SGF. In the next stage, the dialysis bag was transferred to 20 mL of simulated intestinal fluid (SIF) (pH = 7.0). The sample analysis (1 mL) was sustained for 6 h at 30 min intervals, and it was replaced with an equal volume of fresh SIF. The ASE content was evaluated at 440 nm using a spectrophotometer (London, UK) according to the Sect. "Encapsulation efficiency and loading capacity".

To determine the release mechanism, the experimental data were fitted using different models, such as zero order (Eq. 7), first order (Eq. 8), Higuchi (Eq. 9), and Ritger Peppas (Eq. 10), which are provided as follows [12, 23]:

$${\text{C }} = {\text{ k t}}$$
(6)
$${\text{C }} = {1}{-}{\text{ e}}^{{ - {\text{kt}}}}$$
(7)
$${\text{C }} = {\text{ k t}}^{{0.{5}}}$$
(8)
$${\text{C }} = {\text{ k t}}^{{\text{n}}}$$
(9)

where C is the saffron extract concentration at time t, and k and n are the release kinetic constant, and diffusion exponent, respectively.

Statistical analysis

All the experiments were performed in triplicate unless stated otherwise, and the data were analyzed using SPSS and GraphPad Prism (Version 9.1.1). The rheological data in two replicates were analyzed using Rheoplus software (version 3.40 Anton Paar GmbH, Germany), and the graphs were plotted using Sigma Plot (version 8.0; Jandel Scientific, Corte Madera, CA, USA). One-way analysis of variance (ANOVA) was performed using a significance level of 95% (P < 0.05).

Results and discussion

Encapsulation efficiency

The encapsulation efficiency (EE, %) and loading capacity (LC, %) of different microcapsule formulas, developed through complex coacervation between NaCas and HMP at varying core/shell and protein/polysaccharide ratios, are shown in Table 2. The non-cross-linked microcapsules achieved the highest encapsulation efficiency (89.38 ± 3.07%) with core/shell and Pr/Ps ratios of 1:4 and 4:1 w/w, respectively. The TA cross-linked microcapsules had the highest EE (87.09 ± 3.09%) at the same ratios. These results indicated that the amount and ratio of biopolymer were sufficient for ASE encapsulation and confirm that the biopolymers (NaCas and HMP) effectively protect the core material. Furthermore, the highest EE was observed for the microcapsules prepared at a core/shell ratio of 1:4, demonstrating the relevance of this parameter in the encapsulation process, as also concluded by Jannasari [46] and Santos [47]. In contrast, lower EE values were observed at a core/shell ratio of 1:1, especially with the lowest Pr/Ps ratio, denoting that the amount of shell material present were not enough to encapsulate the core. Similar results have been observed in studies on vanillin/β-cyclodextrin [48], vitamin D3/tara gum [47], and lactoferrin/pectin [49].

Table 2 Encapsulation efficiency (EE, %), loading capacity (LC, %) and particle size of non-cross-linked and TA cross-linked microcapsules
Full size table

The loading capacity ranged from 1.22 to 5.5%, indicating the amount of ASE loaded into a given mass of microcapsules. Because the loading capacity largely depends on the core concentration, the highest LC was obtained for the maximum core content (0.4 g). By decreasing the core, the LC was also reduced (Table 2). The LC (%) depends on the concentration of bioactive compounds used in the system [46, 47] and therefore explains the lower values observed in our study compared to those found by other researchers. Similarly, Dehcheshmeh and Fathi [23] obtained the loading capacity (LC%) between 3.57 and 9.52% for saffron extract microcapsules, since they used a higher saffron extract concentrations for encapsulation. [46, 47]. In comparison to the EE results, the LC values at a core/shell ratio (1:1 w/w) were higher than those at other ratios (1:2 and 1:4 w/w). Therefore, there is no direct relationship between EE and LC, as many authors have stated in the literature [46, 50, 51].

Particle size

The particle size of the microcapsules is an essential analysis for its incorporation in food science because larger particles have an undesirable effect on the final texture [47]. Therefore, the particle sizes of different microcapsules in the presence and absence of cross-linkers were measured and are given in Table 2. The core concentration had an effect on the hydrodynamic diameter and varied from 6.8 to 24.8 μm. There were no significant differences in the PDI of the various microcapsules, which only showed slight changes between different samples.

In the samples without TA, the smallest particle size was accounted for CC1 by 6.2 μm, while this figure for CC6 was 14.2 μm. Similarly, this trend can be observed in the samples with TA. By increasing the core materials, the particle size was decreased, which has been similarly found for other complex coacervation systems, including vitamin C in gelatin-Arabic gum [52] and black raspberry anthocyanins in gelatin-Arabic gum [28]. This could be due to the fact that different core materials were used for encapsulation, or it may be a result of protein surface activity properties, increasing the efficiency of encapsulation and the durability of the droplets’ coalescence through the enhancement of protein concentration [53]. However, other factors can also influence the particle size [54]. The particle sizes of CC6 and CCT6 (microcapsules in presence and absence of cross-linker) was 14.23 μm with an average PDI 0.39 ± 0.14, and 24.83 μm with an average PDI 0.23 ± 0.04, respectively. Based on the sizes of the microcapsules, which are less than 1000 μm, the complex coacervates between HMP and NaCas are encapsulated with ASE-developed microcapsules.

Because the electrical charges of NaCas and HMP at pH 3.3 were 22.3 mV and -18.2 mV, respectively, the electrostatic interaction between these biopolymers, and the formation of complex coacervates is feasible according to our previous work [5]. However, the zeta-potential of the NaCas/HMP was -1.15 ± 0.28 mV close to zero, and the complexes examined had a neutral electrical charge, thereby maximizing the coacervation. On the contrary, the ASE microcapsules cross-linked and non-cross-linked with TA showed -7.88 ± 1.59 mV and -3.33 ± 0.13 mV, respectively, which indicates the repletion effect of the microcapsules, which improves its stability [55].

Rheological properties

Flow behavior

While turbidity curves elucidate the compositional aspects of the coacervation, the critical level in understanding trends in the composition of the coacervates is related to dynamic material properties, which provide insight into the molecular level interactions in the material. Thus, the steady shear rheological properties of NaCas/HMP complex coacervates were determined as a function of shear rate 0.1–1000 s−1 (Fig. 1). The shear viscosity (ηa) of both cross-linked and non-cross-linked complex coacervates decreased exponentially with an increasing shear rate, indicating shear-thinning behavior (γ = 1–1000 s−1) which may be attributed to the structural breakdown or rearrangement under shear [56]. This behavior correlated well with the power law model (R2 = 0.95) and the consistency and flow behavior indexes were K = 1.65 ± 0.15 Pa.sn and n = 0.04 ± 0.01 for cross-linked, and K = 1.19 ± 0.12 Pa.sn and n = 0.06 ± 0.01 for non-cross-linked coacervates. Similar shear-thinning properties of the complex coacervates have also been reported for canola protein isolate/chitosan [57], canola protein isolate/Arabic gum [58], BSA/pectin [59], and rice bran protein/flax seed gum coacervate [42]. However, a local maximum in the shear viscosity at very low shear rates (γ < 1 s−1) was observed, which can be attributed to non-steady state conditions [60]. This apparent shear thickening at low shear rates can be completely attributed to non-equilibrium measurements of viscosity, which have been observed for other polymer solutions such as xanthan and flaxseed gum [61]. Cross-linked coacervates showed higher viscosity at all shear rates due to increased electrostatic interactions between polymers [42, 49, 57]. However, it has been observed that excessive charge and electrostatic repulsion significantly impact the apparent viscosity, with the most pronounced electrostatic interaction occurring at electroneutrality (pHopt and R = 4:1)[42, 62, 63]; the effect of cross-linking as a guest molecule did not consider. Cross-linking also increased polymer chain length and side chains, enhancing viscosity. Furthermore, the upward and downward curves of the cross-linked coacervates were closely superimposed, indicating the time-independent rheological behavior. Time-independent rheological behavior was observed in the cross-linked coacervates, while time-dependent behavior was noted in the non-cross-linked coacervates, especially at low shear rates.

Fig. 1
figure 1

Steady state shear rheological properties of complex coacervates of NaCas/HMP as affected by cross-linker (tannic acid) as a function of shear rate 0.1–1000 s−1. Downward curves are plotted in red color. Lines are depicted to easy follow by eye

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SAOS measurements

The effect of cross-linking on the dynamic rheological properties of NaCas/HMP coacervates over a strain range of 0.1–100% under a frequency of 1.0 Hz was investigated. A typical strain sweep of NaCas/HMP cross-linked and non-cross-linked coacervates is shown in Fig. 2. The linear viscoelastic region (LVR) was determined according to the strain sweep data in which G′ and G′′ were independent of strain. Although there was only a negligible difference in the G′ value between cross-linked and non-cross-linked complex coacervates, the G′ was greater than G′′ in the LVR region [42]. This behavior showed the interconnected gel-like network structures with mainly elastic behavior [49]. The values of G', G", G* and tan δ in the LVE region are also given in Table 3. The storage modulus in the LVE range (G'LVE), which reveals the structural strength of NaCas/HMP coacervates, was not statistically influenced by cross-linking (p < 0.05). On the other hand, the effect of cross-linking on the network structure of coacervate was small. The loss tangent (tan δ) as an indication of the physical behavior of the coacervates was reduced from 0.36 to 0.34 by cross-linking with TA which showed viscoelastic behavior of the coacervate [64]. Similar electrostatic interactions have also been observed for bovine serum albumin (BSA)/pectin [59], β-lactoglobulin/pectin [65], NaCas/gum tragacanth [66], WPI/RBP system [67], RBP/flaxseed gum [42], WPI/flaxseed gum [62], and whey protein/HMP[49]. In all of these works, the carbohydrate polymers have shown the synergistic effect on the formation and strength of a physical gel.

Fig. 2
figure 2

Effect of tannic acid on the storage (G', filled symbols) and loss moduli (G", blank symbols) of NaCas/HMP complex coacervates (25 °C, frequency = 1 Hz)

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Table 3 Structural strength (G' LVE), loss modulus (G"LVE), loss-tangent (tan δ LVE) in the LVR region for NaCas/HMP coacervates as determined by strain sweep (25°C, f = 1 Hz)*
Full size table

Frequency sweep

Mechanical spectra of the NaCas/HMP as affected by tannic acid are provided in Fig. 3. Both moduli (G′ and G′′) improved with increasing in frequency, and the G′ was greater than G′′ without any crossover point, which indicated highly interconnected gel-like network structure [42, 62]. However, the G′ increased through crosslinking with tannic acid, and there was a small difference in G′ between cross-linked and non-cross-linked coacervates. Furthermore, the storage modulus of the non-cross-linked coacervates was superimposed with cross-linked coacervates at high frequencies (> 5 Hz). It has been found that pH changes weakened the electrostatic interactions between polymers and decreased the moduli [1, 42, 62, 68]. In contrast, increasing in the polymer chain length caused an increase in moduli due to the increased propensity for entanglements, up to an asymptotic limit for an infinitely long polymer chain [69]. Similarly, varying complex coacervates of β-lactoglobulin/pectin [65], NaCas/tragacanth [66], BSA/pectin [59], rice bran protein/flax seed gum [42], and whey protein/pectin [70] have shown the gel-like network structure of the coacervates, whereas some researchers have found vice versa and explained the liquid-like behavior such as whey protein/gum Arabic [63], and β-lactoglobulin/lactoferrin [2].

Fig. 3
figure 3

Effect of cross-linker (tannic acid) on the mechanical properties of NaCas/HMP coacervates at 25 oC, strain 1%. Cross-linked sample (circle) and non-cross-linked (square), G' (filled symbols) and G'' (blank symbols)

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The complex viscosity (η*) was decreased linearly by frequency until frequency of 5 Hz (shear-thinning behavior) and then improved along with increasing the frequency. This behavior is mainly driven from the high linear viscoelastic limit (1%) which was used in frequency sweep measurements. In addition, G' and G″ showed low dependency at the frequency range < 5 Hz, which means the formation of a strong network structure of the NaCas/HMP coacervates. Accordingly, the elastic behavior of the NaCas/HMP coacervates stems from the electrostatic interactions between the proteins and polysaccharide chains, which is consistent with previous works [42, 49, 65].

The network strength of the NaCas/HMP coacervates was determined using the material stiffness factor (Aa) according to the Friedrich and Heyman model [43]. The log–log plot of G* and frequency in the range of 0.1–5 Hz was depicted to determine the Aa. Since, the storage modulus of the NaCas/HMP coacervates is lower than 1 Pa, the material stiffness of the NaCas/HMP cross-linked and non-cross-linked coacervates was much lower than that of the rice bran protein/flaxseed gum (2450 Pa.rad−α.sα)[42], and whey protein/HMP (20,960 Pa.rad−α.sα)[49] complex coacervates (Table 4). The value of α, as the order of relaxation function parameter used to identify the extension of network, indicates a higher number of interactions when its lower value [71]. Consequently, it can be assumed that the NaCas/HMP cross-linked coacervates have more electrostatic interactions than that of whey protein/HMP and rice bran protein/flaxseed gum coacervates. Moreover, the low dependency on frequency indicates the development of a strong network structure within the NaCas/HMP coacervate.

Table 4 Synergistic/non-synergistic interactions of NaCas/HMP complex coacervate in the LVE range and material stiffness parameter (Aa) as affected by crosslinking with tannic acid
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Thermal characteristics of the microcapsules

Thermal degradation of NaCas, HMP, freeze-dried non-cross-linked (CC6), and TA cross-linked microcapsules (CCT6) are shown in Fig. 4. All samples experienced a 5% decrease in mass at approximately 200 °C due to the evaporative loss of both bound and free water [72]. However, NaCas and HMP exhibited a significant decline in mass between 210 °C and 550 °C on their TGA curves, whereas the microcapsules experienced a monotonous decline. The HMP experienced a sharp decrease in mass between 200 °C and 280 °C due to the decomposition of the carboxyl group and the dehydration of the saccharide ring [73]. The decomposition of the NaCas from 210 °C to 550 °C was due to the thermal brakeage of the protein backbone [56].

Fig. 4
figure 4

Thermal gravimetric analysis (TGA) of sodium caseinate, pectin and microcapsules cross-linked and non-cross-linked with tannic acid from 20 to 550 °C

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It was also found that there is a slight change in the mass loss between TA-cross-linked and non-cross-linked microcapsules. Interactions between NaCas and TA were thought to have replaced the water molecules of the microcapsules via hydrogen bonding, and non-covalent, and covalent bonding, resulting in a higher affinity between these biopolymers. Therefore, the TA-cross-linked microcapsules showed more resistance to thermal degradation and lower weight loss than the non-cross-linked microcapsules. Similar results have also been found for the gelatin/Arabic gum cross-linked by TA [39], and for the gelatin/HMP coacervates cross-linked using TA [36]. Consequently, microcapsules along with TA led to the complexes with improved thermal properties, which is in agreement with previous works on TA [74]. These results confirm previous FTIR analysis finding that the interaction between TA and the biopolymer matrix creates a stable chemical bond.

FTIR features of the microcapsules

FTIR spectra were utilized to reveal changes in the band shifts resulting from the interactions occurring within the microcapsules. FTIR spectra for TA, NaCas, HMP, freeze-dried saffron extract, and microcapsules that are non-cross-linked and cross-linked with TA are given in Fig. 5a, b. The characteristic peaks of NaCas were identified at 3348 cm−1, 2925 cm−1, 1651 cm−1, and 1539 cm−1. The hydrogen bond region (3200–3400 cm−1) primarily corresponds to the stretching vibration of O–H, while the hydrophobic band relates to C–H stretching. The amide I band (1750—1600 cm−1) primarily involves the C–O stretching vibration, and the amide II band (1550 -1510 cm−1) primarily relates to the C–N tension and the bending vibration of N–H [75,76,77]. The HMP spectrum displayed characteristic peaks typical of carbohydrates, including a broad and strong peak at 3315 cm−1, and 2943 cm−1, corresponding to O–H and C–H stretching vibrations, which are consistent with prior research findings [78]. Peaks at 1751 cm−1 and 1647 cm−1 signify esterified carbonyl stretching (C–O) and carboxylic acid (COOH) bands, respectively. In addition, C–O–C bending vibration and hydroxyl stretching vibration were evident between 1000 cm−1 and 1200 cm−1 [77,78,79].

Fig. 5
figure 5

FTIR spectra of (a) sodium caseinate, pectin and their coacervates (b) saffron, tannic acid, non-cross-linked, and cross-linked microcapsules with tannic acid

Full size image

TA exhibited a wider band within the 3600–3000 cm−1 range as a result of the –OH stretching. An approximately 1723 cm−1 band indicated the carboxylic carbonyl group's presence. The band found at 1602 indicated the presence of –C=C– in the aromatic ring, while the band at 1448 cm−1 indicated the deformation of –C–C– of the phenolic group, and the band at 1315 cm−1 was related to the phenol group. The band at 1180 cm−1 was related to C–H, and the vibration bands at 1100–1000 cm−1 were due to C–H and C–O deformation. The bands between 800 and 600 cm−1 were related to C-H bonds in the benzene ring, and all the bands are in line with the previous works on the TA characterization [36, 80, 81].

The peak stretch frequency of saffron was between 3200 and 3400 cm−1 and corresponded to the O–H bond of hydroxyl groups involved in hydrogen bonding and was related to the functional groups of alcohols and phenolic compounds [26]. The peak at 2923 cm−1 corresponded to C–H stretching, while the two peaks at 1706 and 1654 cm−1 were attributed to the carbonyl (C=O) group found in esters, ketones, and aldehydes. The peaks at 1074, 1228, and 1363 cm−1 were attributed to sugars and polysaccharides, respectively [23, 26].

FTIR spectra revealed significant structural differences due to microcapsule crosslinking with TA. FTIR spectra of TA cross-linked microcapsules showed shifts from 3402 to 3394 cm−1 related to hydrogen bonding formed between NaCas/HMP coacervate and TA, which is in accordance with previous works [31, 36, 39]. The changes at 1645 cm−1 to 1654 cm−1 (Amide I, related to C = O stretching/hydrogen bonding) in microcapsules owing to cross-linking with TA. A spectra change was seen in the region at 2920 cm−1 in microcapsules to 2931 cm−1 after cross-linking with TA. Furthermore, a change at 1525 cm−1 (Amide II) in microcapsules to 1533 cm−1 was found, which is related to cross-linking. Moreover, peak 1232 cm−1 (Amid III) in microcapsules changed to 1226 cm−1 after crosslinking with TA. In addition, the bands 1456 cm−1 and 1340 cm−1 were shifted to 1450 and 1336 cm−1 owing to crosslinking, respectively. The new band at 1725 cm−1 is attributed to the carboxyl carbonyl group of TA in the cross-linked microcapsules. The overlap of the spectra shows that saffron is completely encapsulated in the NaCas-HMP coacervate.

Microstructural properties of the microcapsules

The FESEM microstructures of the non-cross-linked and TA cross-linked microcapsules are shown in Fig. 6. A-H. The non-cross-linked and cross-linked microcapsules illustrated a wide range of particle sizes. Furthermore, the dried microcapsules had an irregular and uneven exterior surface with roughness and cracks. This implies that the drying of the microcapsules changed their surface characteristics. The cracking on the surface can be attributed to the stresses induced by shrinkage during the drying process, which may be due to the polymer network breaking [82]. It has also been observed that TA cross-linked microcapsules had a uniform and compact shell structure with smoother external surfaces, which is advantageous for enhancing the stability of microcapsules. FESEM images have shown that employing TA for cross-linking in gelatin/gum Arabic microcapsules reduced surface roughness by transforming the microcapsules' shell structure into a less porous structure [83]. Our results are in line with FTIR and particle size findings, indicating a strong interaction between NaCas, HMP, and TA within the system. The interaction between amino acids in the NaCas chain and the phenol group in TA leads to the formation of larger particles and a significant increase in size and shape variation [84].

Fig. 6
figure 6

FESEM micrographs of the non-cross-linked microcapsules (A–D); and cross-linked microcapsules by tannic acid (E–H). All the experiment were prepared at 15 kV

Full size image

Saffron extract release

The gastrointestinal tract contains massive amounts of enzymes that degrade the active substances, resulting in low bioavailability [56]. Therefore, the encapsulated structures should not only provide protection from gastric digestion but should also be able to release their active compounds for biological activity [24]. Thus, the saffron extract release profile under both gastric and intestinal conditions as a function of time is provided in Fig. 7. There was a weak initial release during the first 30 min, which can be attributed to the fraction of saffron extract adhered weakly onto the surface of the complexes. The microcapsules, whether cross-linked with TA or not, did not display burst release, indicating excellent control over release properties. The initial slope of the release profile of saffron alone was faster than that of the non-cross-linked and cross-linked microcapsules, which can be attributed to the increase in shell thickness and thus reduction in number of pores and complete coverage around the core, which diminished the diffusion rate. Therefore, the burst-release phenomenon on crocin depends on the surface and shell properties of the microcapsules, which have been reported similar results in previous works [17, 22,23,24].

Fig. 7
figure 7

Cumulative release of saffron in simulated gastric and intestine fluids for freeze-dried saffron extract, resulting in the complete release of saffron in SGF and SIF, non-cross-linked and cross-linked microcapsules with tannic acid, resulting in the prevention of saffron release in stomach conditions

Full size image

In comparison with non-cross-linked and TA cross-linked microcapsules, the 45% of saffron was detected in the SGF release medium and another 55% was detected in the SIF release medium. However, only about 6%–8% of saffron in two microcapsules, whether cross-linked with TA or not, was released in the SGF release medium, and the about 70% of saffron extract was detected in the SIF release medium. Consequently, the release rate in the intestinal phase was higher than that of the gastric phase, which exhibited the better control of the saffron extract release during the simulated digestion. Similarly, the release rates of peppermint essential oil from β-lactoglubolin/alginate complexes (84.4%) [50], cinnamon essential oil from aloe vera mucilage/gelatin (68%) [44], allyl-isothiocyanate from gelatin/Arabic gum cross-linked with TA (46%) [85], and vitamin D from GE/cress seed gum (~ 100%) [46] have been published. No significant difference was observed between non-cross-linked and TA cross-linked microcapsules, which is consistent with previous studies [31, 86]. According to our findings, the NaCas/HMP complexes cross-linked with TA can be applied for saffron encapsulation, and it is a suitable delivery system for bioactive compounds with a targeted release in the gastrointestinal tract.

Modeling and mechanism of sustained release

Release modeling has a great effect on the design of encapsulation systems and approaches a desirable release rate. Therefore, the sustained release of saffron extract during in-vitro digestion was described by some mathematical models that can provide the kinetics and mechanism of saffron extract release, and the model parameters are summarized in Table 5. Although the Higuchi model has the lowest fit (low coefficient of determination = R2(, the Ritger-Peppas model has the highest coefficient for all the samples at gastric, intestinal, and whole release. The low R2 means that the release of saffron extract from the microcapsules is not only dependent on concentration, but the release mechanism is mainly governed by diffusion and swelling mechanisms [12]. The root means square error (RMSE) and R2 are used to assess the quality of fit. The best model is selected on the basis of the highest R2 and lowest RMSE, which is defined as R2/RMSE. A RMSE value of < 10 indicates an excellent fit, while an index > 30 suggests a weak fit. An RMSE values in the range of 10–20 and 20–30 indicate good and relatively good fits, respectively [87].

Table 5 Kinetics release parameters of saffron extract from tannic acid cross-linked microcapsules obtained from different models *
Full size table

According to Table 5, the best model for SGF, SIF, and the whole of the gastrointestinal tract was Ritger-Peppas. As previously reported, this model is commonly used to describe the release of a solute from a polymeric matrix [88]. In the Ritger-Peppas model, the n value, the diffusion exponent, exhibits the release mechanism: when n < 0.43 the release from the core is derived by the Fickian diffusion mechanism (case I), In case II, n > 0.85 is indicated for the dissolution phenomenon or swelling-controlled system, and 0.43 < n < 0.85 is governed by the combination of two mechanisms including diffusion and swelling release [89, 90]. In Case II, where release is independent of time and the diffusion rate is faster than the relaxation rate, release is controlled by microcapsule shell relaxation [88, 91]. Since the electrostatic interactions are governed between HMP and sodium caseinate, the hydration of the microcapsules in aqueous media destroys these interactions, which partially deteriorate the microcapsules shells. Consequently, the diffusion of saffron extract was occurred as a partial weakness of the microcapsules.

Conclusion

Saffron is a medical plant with applications in the food and pharmaceutical industries, but its bioactive compounds are sensitive to external factors. Encapsulation using polymers like sodium caseinate and pectin can protect these compounds. Our study found that a core/shell ratio of 1:4 w/w and Pr/Ps 4:1 w/w resulted in the highest encapsulation efficiency. The microcapsules improved the thermal stability of saffron extract and protected it from gastrointestinal conditions. The rheological study showed that the coacervates have shear-thinning behavior. Cross-linking slightly affected the gel structures but improved the thermal and structural properties of the microcapsules. Thermal gravimetric analysis results revealed that tannic acid crosslinked microcapsules could be relatively more heat resistant. The sustained release of saffron extract was confirmed, and the Ritger–Peppas model was a suitable model for describing the release kinetics. The encapsulation mechanism illustrated that hydrophobic interaction, hydrogen bonding, and electrostatic interaction controlled the formation of saffron extract complexes. Our results illustrated that the NaCas/HMP coacervates properties can be improved using tannic acid and applied to prepare microcapsules for the food and pharmaceutical industries.

Availability of data and materials

No datasets were generated or analyzed during the current study.

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Acknowledgements

I would like to express my very great appreciation to Dr. Haghighi asl and Dr. Rafe for his valuable and constructive suggestions during the planning and development of this research work.

Funding

The authors would like to thank Semnan University gratefully for providing the financial support of this project.

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Authors and Affiliations

  1. Faculty of Chemical, Petroleum and Gas Engineering, Semnan University, P. O. Box: 35195-363, Semnan, Iran

    Faezeh Ardestani & Ali Haghighi Asl

  2. Department of Food Physics, Research Institute of Food Science and Technology (RIFST), P. O. Box: 91775-1163, Mashhad, Iran

    Ali Rafe

Authors
  1. Faezeh Ardestani
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  2. Ali Haghighi Asl
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  3. Ali Rafe
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Contributions

F. A.: Data curation; investigation; methodology; writing-original draft. A.H.A.: conceptualization; supervision; validation; writing-review and editing, A.R.: conceptualization; supervision; validation; visualization; writing-review and editing. All authors read and approved the final manuscript.

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Correspondence to Ali Haghighi Asl or Ali Rafe.

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I, Faezeh Ardestani give my consent for the submitted manuscript to be published in the Chemical and Biological Technologies in Agriculture.

II, Ali Haghighi asl give my consent for the submitted manuscript to be published in the Chemical and Biological Technologies in Agriculture.

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Ardestani, F., Haghighi Asl, A. & Rafe, A. Characterization of caseinate-pectin complex coacervates as a carrier for delivery and controlled-release of saffron extract. Chem. Biol. Technol. Agric. 11, 118 (2024). https://doi.org/10.1186/s40538-024-00647-0

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