Microencapsulation of Black Carrot Anthocyanins by Conventional and Three-Nozzle Spray-Drying: Effect on Storage Stability and Bioaccessibility

1. Introduction

Consumers’ changing habits have led to a notable rise in demand for functional meals, which are seen as wholesome. This has also increased interest in replacing synthetic dyes with natural colorants, mainly because synthetic colorants have been restricted by official regulations in the EU and the USA due to their potential adverse effects on human health [1,2].

In this regard, the black carrot (Daucus carota L.) is a potential source of natural colors. It is also a good source of minerals, vitamins, and polyphenols, such as anthocyanins. Its composition includes approximately 88% water, 1% protein, 8% carbohydrate, 0.14% fat, and 2.5% fiber [3,4,5]. The black carrot stands out for its attractive bluish-purple color and high levels of anthocyanins [6,7]. These compounds produce a bright red color under acidic conditions. Black carrots are used as a nutraceutical ingredient in various food matrices, including fruit juices, soft drinks, yogurt, jellies, and confectionery [6,8,9]. Consequently, the use of black carrot extract as a natural colorant may also provide health benefits and enhance the oxidative stability of foods [6,8]. The main anthocyanins are cyanidin derivatives; among them, 64- 77% are acylated anthocyanins. A significant property of cyanidins is their antioxidant activity, which plays an important role in the prevention of neuronal and cardiovascular diseases, cancer, and diabetes, among others [3,4,6,10,11,12,13,14,15,16].

Black carrots are reported to contain up to 350 mg of anthocyanins per 100 g of fresh weight. For comparison, total anthocyanin concentrations are approximately 113 mg/100 g in blueberries and 48 mg/100 g in raspberries [12]. The structure of anthocyanins is affected by pH, temperature, water activity, exposure to light and oxygen, enzymatic activity, the presence of co-pigments, self-association, metallic ions, ascorbic acid, sugar, and their degradation products [4,15,16,17,18,19]. Stability is an important consideration when using anthocyanins as antioxidants. Foods containing anthocyanins are thermally processed before consumption, and this processing can significantly influence the anthocyanin content of the final product [4,18,19].

The first challenge for anthocyanin (cyanidin)-based ingredients is protecting the actives from environmental conditions. The second challenge is preserving antioxidant capacity and bioaccessibility until they are ingested by consumers. These aspects can be addressed using microencapsulation to protect cyanidins [2,20,21,22] and/or to control cyanidin release at a specific site or time [23]. Encapsulation by spray-drying has been described as a technique in which an active compound is introduced into or entrapped within a polymeric wall to protect it from environmental conditions, interactions with other food components, and exposure to the gastrointestinal tract, or to control the specific conditions (time and/or site) of active compound release [21].

Spray-drying is a process that converts a feed solution into a dry powder in a single, continuous step. The feed solution is atomized into fine droplets within a hot drying medium. Upon contact with the drying medium, water evaporates rapidly, producing a powder almost instantly [24]. SD equipment commonly uses a conventional two-way nozzle, which allows the spray-drying of a single feed solution and is therefore limited to feed solutions containing compatible materials (conventional SD) [24]. By contrast, using a different nozzle, such as a three-way nozzle, during SD may be a suitable strategy for forming an outer layer coating microparticles, since the solution sprayed by the external peripheral nozzle covers the solution sprayed by the central nozzle during the atomization process, generating an outer layer [25,26,27,28].

Various encapsulating agents have been used to encapsulate black carrot anthocyanins by spray drying (maltodextrins, proteins, and others). Among the biopolymers available, maltodextrins with different dextrose equivalents (DE) are widely used as encapsulating agents in spray-drying because they have high water solubility and low viscosity, allowing an infeed solution with high solid content; in addition, they have a bland flavor and produce colorless solutions [20]. For outer layers, sodium alginate has been reported as the most used polymer [26,27]. SA is a controlled-release polymer used as an intestinal trigger. It is an anionic biopolymer that imparts high viscosity and low solids content to infeed solutions. Moreover, it is important to determine how digestion affects polyphenols, as this will influence their bioaccessibility [4,13,29]. Accordingly, the potential health-promoting effects of black carrot anthocyanins depend on their processing, stability during storage, and gastrointestinal tract absorption [4,29]. Some research has shown that microencapsulation can increase the bioaccessibility of anthocyanins by up to 20% compared with free extracts or juices. However, the specific impact of multilayer systems formed with a three-fluid nozzle on the stability of black carrot anthocyanins remains to be thoroughly evaluated [2,15,16,29].

Therefore, the aim of this study was to develop and optimize spray-dried microencapsulation systems for black carrot juice anthocyanins and to compare the effects of conventional and three-nozzle spray-drying on encapsulation efficiency, storage stability, and in vitro bioaccessibility.

2. Materials and Methods

2.1. Raw Material

Black carrot (Daucus carota L.) was cultivated at the Agricultural Research Institute of Chile (INIA) in Los Tilos, Santiago, Chile. A random sample of black carrots was selected from the field, in accordance with the experimental design.

Encapsulating agents: maltodextrin (Prinal, Santiago, Chile) and sodium alginate (Alginatos Chile S.A., Paine, Chile).

2.2. Black Carrot Juice

2.2.1. Preparation of BCJ

A pool of black carrots was washed with a cold-water spray and ground in a blender (Thermomix, Vorwek, Germany) to obtain a juice according to Vergara et al. [2]. Briefly, the pH was adjusted to 3 with a citric acid solution to inactivate oxidation, and black carrot juice was then extracted by pressing. The juice was centrifuged, and the supernatant was concentrated in a rotary evaporator R-100 (Büchi, Flawil, Switzerland) at 50 °C until reaching 65 °Brix. BCJ was then stored at -20 °C until analysis.

2.2.2. Characterization of Black Carrot and BCJ

The black carrot (raw material) and BCJ were characterized by moisture, measured with an infrared moisture analyzer (PMB202, ADAM, Maidstone Road, UK), and soluble solids (°Brix), measured with a refractometer (HI 96801, Hanna Instruments, Rhode Island, USA).

AT content: Total anthocyanins were determined by the pH-differential method [30], as described by Lee et al., using spectrophotometry. This method includes measurements at pH 1 and 4.5, with absorbance at 510 and 700 nm. Anthocyanin content was quantified by mixing 200 µL of the sample with 800 µL of buffer at pH 1 or pH 4.5. The mixture was filtered through a 0.45 µm syringe filter, and measurements were taken.

AT Profile: The anthocyanin profile and quantification were performed by HPLC-DAD [31], using HPLC equipment with a photodiode-array detector (Jasco interface LC-NetII/ADC 7059-J012A with a diode array detector MD-4010, quaternary pump PU-4180-LPG, column oven CO-4060, and an autosampler AS-4050 with cooling system TC-4000-1 (Jasco, Tokyo, Japan)) and a C18 column (PerkinElmer Universal LC 250 x 4.6mm, particle size 5 µm). The samples (20 μL) were injected. The mobile phases, A (0.2% v/v TFA in water), B (0.2% v/v TFA in acetonitrile), and C (0.2% v/v TFA in methanol), were used under the following conditions: initial, 7% B and C; linear change to 40 min to 13% B and 13% C. The column oven temperature was 40°C. Cyanidins were detected at 520 nm. Quantification was carried out using cyanidin-3-O-glucoside as the standard. All analyses were carried out in triplicate.

Antioxidant activity: Determined using the FRAP method, as described by Benzie and Strain [32]. All analyses were carried out in triplicate.

2.3. Microencapsulation of BCJ by Spray-Drying

2.3.1. Conventional Spray-Drying

Statistical design: Encapsulation of BCJ with MD (BCJ-MD) was performed using a mini-spray dryer B-290 (Büchi, Flawil, Switzerland) with a conventional two-fluid nozzle. The experimental conditions were set according to a Central Composite design (12 runs) with a BCJ/MD ratio (1:0.5–1:2) and an inlet air temperature (120-180°C). The drying conditions were airflow of 600 L/h, atomization pressure of 20 psi, and feeding rates of 0.12 mL/min and 1.2 mL/min for the inner and outer infeeds, respectively. The response variable was encapsulation efficiency (EE) of AT, calculated according to Equation (1). Response surface methodology (RSM) was applied to optimize EE, aiming to maximize it. Data were fitted to a second-order regression model that included linear, quadratic, and cross-product terms for inlet air temperature and the BCJ/MD ratio.

$$\mathrm{E}\mathrm{E}(\mathrm{\%})={\displaystyle \frac{Total AT-surface AT}{total AT experimental}}\times 100$$

(1)

2.3.2. Three-Fluid Nozzle Spray-Drying

The encapsulation of BCJ with MD was optimized using an experimental design with a two-fluid nozzle (conventional spray drying). Then, microparticles of BCJ-MD-SA were prepared under optimized BCJ-MD conditions using a three-fluid nozzle, following the method of Cáceres et al. [26]. The preparation of the inner and outer infeed and the spray-drying conditions were as follows.

Inner infeed: MD was dissolved in distilled water, then mixed with the AT and homogenized at 20,000 rpm for 2 min using a Polytron PT-2100 (Kinematica AG, Luzern, Switzerland).

Outer infeed: SA was dispersed in distilled water under magnetic stirring for 2 h. The inner: outer infeed ratio was 1:10 to prepare the outer layer, following Cáceres et al. [27].

Spray-drying: To prepare microparticles with an outer layer of BCJ-MD-SA, the inner and outer infeeds were delivered via a mini-spray using a three-fluid nozzle. Drying conditions were airflow of 600 L/h, atomization pressure of 20 psi, feeding rates of 0.12 mL/min and 1.2 mL/min for the inner and outer infeeds, respectively, and an inlet air temperature of 150 °C. Microparticles were stored at –20 °C until analysis.

2.3.3. Characterization of BCJ-MD and BCJ-MD-SA Microparticles

2.3.3.1. Encapsulation Efficiency of AT

Experimental total anthocyanin determination: Microparticles (200 mg) were dispersed in 2 mL of methanol: acetic acid: water (50:8:42 v/v/v) and vortexed for 1 min. They were then ultrasonicated twice for 10 min and finally centrifuged at 112,000 g for 5 min. The anthocyanin content was determined by the pH-differential method and HPLC-DAD.

Superficial anthocyanin determination: Microparticles (100 mg) were dispersed in 2 mL of ethanol: methanol (1:1), vortexed for 1 min, then centrifuged at 112,000 g for 5 min. Anthocyanin content was determined by the pH-differential method and by HPLC-DAD.

2.3.3.2. Moisture Content, Water Activity, and Morphology

The moisture content was measured with an infrared moisture analyzer (PMC50, Radwag, USA). Water activity (aw) was measured using the dewpoint method (Hygrolab 2, Rotronic, USA; 20 ± 0.3 °C).

2.3.3.3. Morphology

The outer structures of the microparticles were examined by scanning electron microscopy (SEM). The powder was coated with gold/palladium using a Varian PS 10E vacuum evaporator and then analyzed with a LEO 1420VP SEM (LEO Electron Microscopy Ltd., Cambridge, UK) operating at 20 kV. The images were then collected digitally using EDS 7424 software (Oxford Instruments, Oxford, UK).

2.4. The Storage Stability of BCJ Microparticles

The BCJ microparticles obtained under optimal conditions by spray-drying BCJ-MD and BCJ-MD-SA (100 mg) were transferred to clear glass vials (16 × 100 mm) and stored at 60 ± 1 °C in a forced-air oven (UFE 500, Memmert, Schwabach, Germany) with controlled temperature and in the absence of light (in triplicate). The progression of AT degradation was monitored by collecting seven samples at specific time intervals. Anthocyanin contents were quantified by HPLC-DAD, and the anthocyanin degradation rate constant was determined. To determine the kinetic degradation of anthocyanin, the study was conducted over a 6-month period.

Kinetic analysis. The data were best fit by a first-order kinetic model according to Equation (2):

LnC= LnC₀−kt

(2)

where C₀ is the initial concentration of anthocyanin (mg/g), C is the anthocyanin concentration at time t, k is the anthocyanins degradation rate constant, and t is the storage time. The degradation rate constants (k) and correlation coefficient were obtained from the slope of a plot of the natural log of the percentage retention of anthocyanin versus time for first-order kinetics at each studied temperature (60 °C).

2.5. In Vitro Gastrointestinal Digestion

An in vitro method was performed as described by Aravena et al. [33], with minor modifications. First, for oral digestion, artificial saliva (9 mL) was added to each flask containing a sample (about 0.5 g of microparticles). The artificial saliva contained 14.4 mM sodium bicarbonate, 21.1 mM potassium chloride, 1.59 mM calcium chloride, and 0.2 mM magnesium chloride. The pH was adjusted to 7 with 1 M HCl. On the day of analysis, 60 α-amylase units per milliliter of buffer were added. Samples were incubated at 37 °C at 185 rpm in a thermostatic bath (Zhicheng ZHWY-110X30).

For gastric digestion, after the oral stage, the pH of the samples was adjusted to 2.0 with 1 M HCl, and 36 mL of a pepsin solution (25 mg/mL in 0.02 M HCl) was added. Samples were incubated for 2 h at 37 °C with stirring at 130 rpm.

Finally, the gut digestion stage was performed by first adjusting the samples’ pH to 6.0 with NaHCO3 (1 M), then adding 0.25 mL of an artificial gut solution per mL of sample, containing pancreatin (2 g/L) and bile salts (12 g/L) dissolved in NaHCO3 (0.1 M). The samples were incubated for 2 h at 37 °C with shaking at 45 rpm. Each digestion product was transferred to 50 mL Falcon tubes, and the pH was adjusted to 3 [33]. The tubes were then centrifuged for 10 min at 5000 rpm to recover the liquid fraction. Next, the liquid digestion product was centrifuged at 12,000 g before anthocyanin analysis. Anthocyanin bioaccessibility was calculated according to equation (3):

$$Bioaccessibility(BA)\left[\mathrm{\%}\right]={\displaystyle \frac{{AT}_d\times V_T}{{AT}_m\times G}}\times 100$$

(3)

where ${AT}_d$ is the anthocyanin in the liquid phase after digestion, representing the fraction released from the microparticle matrix; V_T is the final volume after digestion; ${AT}_m$ is the anthocyanin in the microparticles before digestion; and G is the amount of sample analyzed.

2.6. Statistical Analysis.

Differences among particle systems for each parameter were analyzed using ANOVA and Tukey’s test. Linear regression (95% confidence limits) was used to determine reaction order and the cyanidin release constant in studies of particle stability during storage and under simulated digestion. To determine statistical differences in release rates across temperature and release medium, ANOVA was performed. All statistical analyses were conducted using Statgraphics Plus 7.0.

3. Results

3.1. Characterization of Black Carrot and BCJ

The physical and chemical characteristics of black carrot (raw material) and BCJ are presented in Table 1, which also lists the concentrations of individual anthocyanins. Figure 1 shows the chromatogram of black carrot.

The HPLC profile of AT from black carrot showed cyanidin derivatives, including non-acylated and acylated forms with hydroxycinnamic acids (sinapic, ferulic, and coumaric acid) [34,35,36]. The main peak identified by HPLC corresponds to Cyanidin-3-xylosyl(feruloylglucosyl)galactoside, accounting for approximately 83% of the total AT, as was reported by other authors [36,37]. Cyanidin-3-xylosyl-glucosyl-galactoside and cyanidin-3-xylosylgalactoside correspond to peaks 1 and 2, respectively (non-acylated cyanidin derivatives), and acylated anthocyanins correspond to peaks 3, 4, and 5. The non-acylated anthocyanins are 5.2%, while the acylated anthocyanins are 94.8% in raw black carrot (see Table 1 and Figure 1).

3.2. Optimization of Microencapsulation of BCJ

Table 2 shows the experimental runs and their response variable (EE of AT) for the SD microencapsulation of BCJ using MD as encapsulating agent. The EE of AT ranged from 50% to 98%. According to the ANOVA, EE was significantly affected by the linear and quadratic effects of both inlet air temperature and the BCJ:MD ratio (p < 0.05).

The equation describing the effect of the independent variables on the EE of AT is the following:

EE (%) = -235,714 + 50,8446*X₂ + 3,57029*X₁ - 9,04638*X₂^2 - 0,0255556*X₁*X₂ - 0,0114596*X₁^2

(4)

The Response Surface Methodology plot is shown in Figure 2. When the BCJ:MD ratio increases, and when the temperature is intermediate within the studied range, the EE of AT increases (red zone in the plot). This quadratic model explained over 98% of the variability (R² = 98.6% and R²ajustado = 97.3%).

The optimal SD conditions were 150 °C (inlet air temperature) with a BCJ:MD ratio of 1:2. These conditions were used to produce microparticles under optimal conditions for conventional SD (BCJ-MD) and three-nozzle SD (BCJ-MD-SA).

3.3. Characterization of the BJC-MD and BJC-MD-SA Systems Obtained Under Optimal Conditions

Table 3 shows the physical and chemical characteristics of the BCJ-MD and BCJ-MD-SA systems obtained under optimal conditions. It is possible to indicate that the micro-particles obtained under optimal conditions are comparable since there are no significant differences in their water activity or in AT content.

Figure 3 (a and b) shows SEM images of BCJ-MD and BCJ-MD-SA microparticles produced under optimal conditions by spray-drying conventional and using a three-fluid nozzle, respectively.

3.4. Stability of BCJ, BCJ-MD, and BCJ-MD-SA Microparticles During Storage at 60°C

Figure 4 (a) shows the evolution of AT retention over time (hours). To evaluate the effects of storage temperature, the initial total AT content of encapsulated black carrot anthocyanins at 0 h was set to 100%. The stability and retention of encapsulated black carrot anthocyanins were monitored for 4,750 hours (198 days) using AT non-encapsulated (BCJ), conventional SD (BCJ-MD), and three-fluid nozzle SD (BCJ-MD-SA).

Figure 4(b) shows the fit to first-order kinetics (k_obs and r²) for BCJ (non-encapsulated), BCJ-MD, and BCJ-MD-SA microparticles, respectively. The AT retention data were best fit by a first-order kinetic model (Equation 2). The degradation rate constants (k_obs) and correlation coefficient (r²) were obtained from the slope of a plot of the natural log of the percentage retention of anthocyanin versus time at 60 °C.

All systems follow first-order kinetics that depend on the concentration of the anthocyanin compound. The AT degradation rate constant (k_obs) was 26.7×10^3 h^-1 (r² 96.9 %) for free AT (non-encapsulated) BCJ, and 2.0×10^4 h^-1 (r² 96.7 %) and 1.0×10^4 h^-1 (r² 94.8 %) for BCJ-MD and BCJ-MD-SA microparticles, respectively.

3.5. In Vitro Bioaccessibility of Anthocyanins from BCJ, BCJ-MD, and BCJ-MD-SA Microparticles

Table 5 presents the final bioaccessibility (BA) (%) of AT after in vitro digestion of BCJ (non-encapsulated) and of BCJ-MD and BCJ-MD-SA microparticles. Table 5 shows BA for the total AT (calculated by the pH-differential method) and BA for individual AT, determined by HPLC-DAD. The BCJ-MD microparticles reach the highest final BA of AT, and for individual ATs, a BA greater than 100% is observed, which is related to the deacetylation of AT.

4. Discussions

4.1. Characterization of Black Carrot and BCJ

Moisture levels, soluble solids, antioxidant activity, and AT content are comparable with the reported ranges for black carrots [8,34,35] (see Table 1). The AT content in black carrot is consistent with the literature, ranging from 1000 to 1750 mg/kg fresh weight [15,34,35]. The BCJ reached 65 °Brix, and its AT content and antioxidant activity were significantly higher than those of raw black carrot due to the juice-concentration process. These parameters fall within the typical range for a concentrated juice produced by evaporating a water extract.

The HPLC profile of anthocyanins from black carrots shows a predominance of acylated forms (94.8%), with the Cyanidin-3-xylosyl(feruloylglucosyl)galactoside being the majority component (~83%). Similar results have already been reported [10,34,36,37]. According to the literature, acylated anthocyanins from black carrot are up to six times more stable and resistant to hydration and pH changes than non-acylated anthocyanins [6].

4.2. Microencapsulation of BCJ

This study employed a composite central design, optimized using RSM), to identify the optimal conditions for microencapsulating BCJ with MD via spray-drying (SD). The independent variables for EE of AT were the inlet air temperature (a process variable) and the BCJ:MD ratio (a formulation variable). RSM was applied to maximize the response variable, that is, to obtain EE values for carrot and blackberry anthocyanins close to 100%.

The EE of AT ranged from 50% to 98%. The ANOVA for the EE of anthocyanins showed that the EE of ATs was significantly affected by the linear and quadratic forms of the inlet air temperature and the BCJ:MD ratio (p<0.05). This model explained over 98% of the variability (R2 = 98.6% and R2 adjusted = 97.3%), with residuals below 4.0. The lack-of-fit was not significant, indicating that the mathematical model fits the experimental data well.

For the BCJ:MD ratio, the EE of AT rose with increasing MD content, likely due to rapid dry-crust formation on droplet surfaces. In contrast, inlet air temperature exerted only a minor effect on the EE of AT [2,38].

The optimal spray-drying conditions were 150 °C with a BCJ:MD ratio of 1:2 for both conventional SD processes.

The same spray-drying conditions were applied to the three-nozzle SD processes, applying SA to the outer layer.

4.3. Characterization of the BJC-MD and BJC-MD-SA Systems Obtained Under Optimal Conditions

The moisture, a_w, and AT content were within the range reported for polyphenols or anthocyanin microparticles obtained by spray-drying with MD [22,25,29,30].

The EE of a microparticle depends on achieving high retention of the core material within the encapsulation wall material and minimizing its presence on the microcapsule surface. The EE of microcaparticles produced with MD and MD–SA is shown in Table 3. The EE for all treatments exceeds 90%, indicating that the black carrot anthocyanin encapsulation process was successful. The EE in both systems (BCJ-MD and BCJ-MD-SA) is higher than previously reported values for purple potato juice (86%) [2] and blueberries (74–85%) using maltodextrin (MD) [39]. This high performance suggests strong hydrogen-bonding interactions between the hydroxyl groups of the AT and the MD matrix [2,22,24,39]. However, recent studies indicate that EE can be further optimized through polymer synergy. Thus, combining MD with sericin proteins [15] or soy isolates has been shown to improve not only EE but also the total recovery of AT after drying by forming a denser protective crust that limits diffusion of the core to the surface. In this study, adding SA via a three-fluid nozzle maintained a high EE (> 93%), comparable to the complex systems of gum Arabic and inulin (97.8%) used in myrtle berries [40].

The external morphology observed by SEM images of BCJ-MD and BCJ-MD-SA was produced under optimal conditions; the particles are spherical, with irregular surfaces that tend to agglomerate. The indentations observed on particles during spray-drying result from particle shrinkage, which can occur at both high and low inlet air temperatures. At higher temperatures, rapid water evaporation and increased internal pressure cause shrinkage, whereas at lower temperatures, slower water diffusion leads to longer shrinkage times [41]. Similar external morphologies have been observed in particles of anthocyanin pigment encapsulated by conventional SD with MD, including those from black carrot [15,41], maqui juice [39], and purple potato juice [2].

4.4. Stability of BCJ Microparticles During Storage

All systems follow first-order kinetics that depend on the concentration of the anthocyanin compound, driven by oxidation, cleavage, or heating-induced oxidation [43]. In both graphs, the protective effect of microencapsulation is evident. Both microparticle systems significantly reduced the AT degradation rate constant (26.7×10^3 h^-1) compared with BCJ (26.7×10^3 h^-1). No significant differences in the degradation rate constants of anthocyanins were observed between systems with and without an outer layer, suggesting that MD is the primary stabilizing factor via hydrogen-bond interactions. The same order has been reported for AT degradation in microparticle systems from black carrot extract [41], maqui juice [39], and purple potato juice [2], where microencapsulation techniques significantly improved the stability of AT and phenolic compounds. Encapsulation enhanced AT stability through MD–AT interactions, minimizing damage to active AT caused by adverse storage conditions and thereby prolonging shelf life while maintaining AT retention and color

4.5. In Vitro Bioaccessibility of Anthocyanins

Bioaccessibility is the amount of a compound released from the matrix following simulated in vitro digestion (oral, gastric, and intestinal phases) [42]. Microencapsulation of black carrot anthocyanins with MD effectively protected them against simulated gastric conditions, minimizing chemical degradation in the gut environment (see Table 5). The final BA of total AT was higher for BCJ-MD (~88%) than for BCJ-MD-SA (~44%) and for BCJ (non-encapsulated) (~57%). On the other hand, encapsulation appears to protect the AT during digestion against environmental conditions (especially pH) when MD is used as the encapsulating agent. The final BA of the BCJ and BCJ-MD systems was higher than that reported by Vergara et al. (2020) [2] for free and encapsulated purple potato juice and by Fredes et al. (2018) [39] for free and encapsulated maqui juice.

A critical finding was the marked difference in final BA: 88.4% for BCJ-MD compared with a drastic 44.1% for BCJ-MD-SA. While MD alone enabled efficient, protected release in the intestine, the external alginate layer appeared to overly restrict release or fail to protect the core during the transition from gastric to intestinal pH. In contrast, systems that incorporate proteins along with MD have shown AT bioaccessibility from black carrot exceeding 91% [15], suggesting that proteins may be more effective carriers than alginate for gastrointestinal delivery of AT.

When examining the achieved BA of individual anthocyanins, it is evident that for non-encapsulated BCJ and BCJ-MD microparticles, the final BA of individual non-acylated forms can reach 100%. This is explained by deacetylation during ingestion and intestinal transit of acetylated anthocyanins, which converts them into their non-acylated forms (see Table 5, Cyanidin-3-xylosyl-glucosyl-galactoside and Cyanidin-3-xylosyl-galactoside). This aligns with observations by Carrillo et al. [16], who report that intestinal conditions can cause the cleavage of Cyanidin-3-xylosyl(feruloylglucosyl)galactoside (the main anthocyanin of black carrot) to Cyanidin-3-xylosyl-glucosyl-galactoside. Additionally, HPLC data confirmed deacetylation during intestinal digestion; the complex acylated forms of black carrot degraded into simpler non-acylated cyanidins due to neutral pH and enzymatic activity. This phenomenon is crucial because, although the BA of the complex forms decreases, the availability of simpler forms may influence subsequent colonic absorption. The determining factor was pH and its impact on bioaccessibility. While anthocyanins are stable under acidic conditions, a shift to a neutral pH in the intestine promotes their transformation or degradation.

5. Conclusion

Microencapsulation by spray-drying with maltodextrin as the encapsulating agent is a highly effective technique for stabilizing anthocyanins from black carrot. Adding an outer sodium alginate layer via a three-fluid nozzle did not confer additional thermal stability and drastically reduced the bioaccessibility of the active compounds. The in vitro digestion study showed that MD has the potential to improve microcapsule stability during passage through simulated oral, gastric, and intestinal conditions, thereby leading to high bioaccessibility. Therefore, a better strategy is to use MD alone to impart stability to the AT for formulating healthy ingredients based on black carrots.