Materials

Dry roselle (hibiscus) was purchased from Hichagol Co., Ltd., Hue, Vietnam. Acetic acid, citric acid, and sodium hydroxide were purchased from Xilong Chemical Co. Ltd. Potassium chloride and anhydrous sodium acetate were purchased from Guangdong Guanghua Sei-Tech Co. Ltd. Maltodextrin (DE: 10 - 12%) was received from Qinhuangdao Lihua Starch Co. Ltd. DPPH (2,2-diphenyl-1-picrylhydrazyl) and dimethyl sulfoxide (DMSO) were purchased from Sigma‒Aldrich.

Roselle extraction

The dry roselle flower was ground into a fine powder with a particle size of approximately 0.5 cm. An acetic acid 2% extraction solvent was prepared for the extraction process. Roselle powder was immersed in the solvent at a ratio of 4 g of roselle powder to 100 ml of extraction solvent. The mixture was incubated at 4 °C. After 24 h, the extraction mixture was collected. The solid residue after filtration was then subjected to another extraction process for 24 h. The final extracted material was stored at 4 °C for characterization.

Preparation of roselle

Four different formulas were spray-dried. Before microencapsulation, each encapsulating agent was dispersed in the roselle extracted solution and mixed until homogenization was reached. The encapsulation concentration was 1% w/v. The components of the wall materials and sample design are presented in Table 1.

Preparation of spray-dried roselle powder

The SD-1000 Eyela (Eyela, Japan) was used to spray the encapsulating mixture. Once the mixtures were made, they were fed into the spray dryer. The inlet temperatures are set at 160 °C, and the outlet temperatures are set at 80 °C. A feed flow of the solution to be dried with a spray air flow of 0.7 m/min and a pressure drop of 180 kPa was used. To maintain the homogeneity of the solution, the encapsulated solution was gently mixed via a pump. After spray drying, the powders were weighed and stored in closed plastic bags, after which they were stored at -20 °C until evaluation.

Microencapsulation particle morphology

The powders were sputter-coated with gold at 10 kV, and their microstructural characteristics were analyzed via SEM (JSM-IT100, JEOL, Japan). Different magnifications of the sample were recorded. The size and morphological structure of the encapsulated roselle powder were measured via the ImageJ program, with 30 data points collected for each sample.

Solubility

The solubility of the powder was assessed via a method outlined in the literature 14. In a concise procedure, one gram of spray-dried powder was combined with 10 millilitres of distilled water, and the mixture was stirred via a magnetic stirrer for 5 minutes. The mixture was subsequently subjected to centrifugation at 4000 revolutions per minute (rpm) for 10 minutes. The resulting supernatant was carefully transferred to a Petri dish and subsequently dried at 105 °C for 2 hours. The solubility was determined by calculating the difference in solid weight between the reconstituted solution and the original solid sample.

pH and total acidity

The total titrated acidity expressed as % citric acid was determined through acid titration, utilizing 0.1 N NaOH and phenolphthalein as indicators. The initial pH value of each sample was determined via a pH meter. Following the pH measurement, 2–3 drops of phenolphthalein indicator solution were introduced. The titration then commenced with the addition of 0.1 N NaOH until the pH reached 7, signified by a color change in the solution. Throughout the titration process, the sample in the flask was swirled continuously. The volume of NaOH solution used in the titration was carefully noted. Finally, this recorded volume was used to calculate the total titrated acidity expressed as a percentage of citric acid. The equation for titratable acidity is as follows:

where:

: normality of titrant;

: volume of titrant;

: equivalent weight of predominant acid;

: mass of sample.

Total anthocyanin content

The total anthocyanin content (TAC) of the spray-dried powder was determined following a method outlined in the literature with slight modifications15. In a concise procedure, 50 mg of the sample was accurately weighed and mixed with 1 ml of distilled water, achieving a homogenous solution through vortex mixing. Two buffer solutions, namely, 0.2 M KCl buffer (pH 1.0) and 0.1 M acetate buffer (pH 4.5), were employed to dilute the sample mixture. To assess the absorbances of the diluted samples, measurements were taken at two distinct wavelengths, 520 nm and 700 nm, via a microplate reader. The content (mg/g of dry matter) was calculated via the following equation:

= ( - ) pH 1 - ( - ) pH 4.5

: volume of diluted samples;

: 96-well plate length (0.286 cm);

: molecular weight cyanidin-3-glucoside, 449.2 g/mol; ɛ = 26,900 L/mol.

DPPH free radical scavenging activity

DPPH, which stands for 2,2-diphenyl-1-picrylhydrazyl, serves as a reagent used to measure the antioxidant activity of substances. In brief, the procedure involves the preparation of spray-dried powders mixed with DI at different concentrations (5, 10, 15, 30, and 45 mg/ml). DPPH was simultaneously dissolved in DMSO (0.1 mM). Subsequently, 20 microliters of the sample at various concentrations were added to wells in a 96-well plate, along with 180 microliters of the DPPH reagent. The reaction mixture was then incubated for 30 minutes in the dark, and the absorbance was measured at 517 nm. The percentage of DPPH-scavenging activity was calculated via the following equation:

where:

: the optical density value of the blank;

: optical density value of the test sample.

Evaluation of the antibacterial activity of the roselle powder

The antibacterial activity of the roselle powder was evaluated via the use of two representative microorganisms: () (ATCC 25922), a gram-negative bacterium, and () (ATCC 25913), a gram-positive bacterium. The minimum inhibitory concentration (MIC) was determined via a classical dilution method adapted to 96-well microtiter plates. Each assay was performed in triplicate. Serial dilutions of roselle powder were prepared in the wells, and each well was inoculated with a standardized bacterial suspension. The plates were then incubated under appropriate conditions for bacterial growth. The MIC value was defined as the lowest concentration of roselle powder that inhibited visible bacterial growth after the incubation period.

Statistical analysis

All the experiments were performed in triplicate, and the results are presented as the means ± standard deviations. Statistical analysis was conducted via one-way ANOVA for all tests, followed by Tukey’s post hoc analysis to determine significant differences between groups. A p value of less than 0.05 was considered statistically significant.

Physio-chemical profile of roselle extract

The extraction step for the dried roselle powder product was meticulously performed to isolate valuable compounds such as anthocyanins, vitamins, flavonoids, and phenols from the roselle flower. In this study, cold brew extraction was performed with 2% acetic acid utilized as the solvent, ensuring efficient extraction of these bioactive components. Initially, the properties of the extract solution were evaluated, with a focus on parameters such as pH, acid content, anthocyanins, and antioxidant capacity, before proceeding to the spray drying stage.

During the initial evaluation, the solid content in the extracted sample was determined to be 10 mg/ml. The pH and acid content of the extract were measured via titration methods, revealing an acid content value of 2.8%. The anthocyanin content in the roselle extract was 50.06 ± 2.19 mg/L, indicating the significant presence of these potent antioxidants. Additionally, the antioxidant capacity, as assessed through DPPH scavenging activity, had an IC50 value of 2.83 ± 0.03 mg/ml. The extraction process effectively evaluated the anthocyanin, antioxidant, and total titratable acidity (TTA) contents of the initial roselle extract. As anticipated, the roselle flowers presented high antioxidant capacity and high anthocyanin content. Identifying these components at the extraction stage represents a crucial initial success in the overarching goal of developing a roselle powder product with increased antioxidant and anthocyanin contents. The parameters of the extraction solution are summarized in Figure 1.

Microencapsulated-roselle powder by spray drying

After the spray drying process, the morphology of the roselle powder was evaluated. In general, microencapsulated roselle powder consists of fine particles in its dry form, despite some changes being observed among the coating and uncoating treatments. Figure 2 shows images of the spray-dried roselle powder under different blending treatments. In its uncoated form, the pure roselle powder exhibited a deep red color. Owing to the lack of coating, the use of pure extract in spray drying systems is challenging. The high-temperature drying treatment caused the sugar components in the crude roselle extract to reach the glass transition state and adhere to the drying system, resulting in only a minimal amount of pure powder remaining after the spray treatment. For particles coated with maltodextrin (MD) or xanthan gum (XG), at a 1% coating agent concentration, the roselle powder tends to be pinkish-red. The red shade is slightly lower than that of the uncoated powder because of the presence of the coating agents.

The morphology and microscopic size of the roselle powder were also evaluated via SEM. Figure 3 shows microscopy images of the different blending formulas of the roselle powder. SEM analysis of the uncoated roselle powder revealed smooth surfaces with visible bridges between the particles, indicating agglomeration (Figure 3A). In contrast, powder formulations mixed with a coating agent have uniform round particles of varying sizes depending on the content of the coating agent and the xanthan gum component. At a 1% coating agent concentration, the particle size was approximately 3–4 µm. Figure 3B shows that particles coated with maltodextrin alone have smooth surfaces with a 3.4 µm diameter (Table 2). Conversely, particles coated with xanthan gum tend to have more wrinkled surfaces. This is because xanthan gum, a large molecular weight adhesive substance, forms dynamic bonds with maltodextrin, creating high surface tension during the shaping process at the spray-dried nozzle tip. This increases the size of the spray-dried particles, but the structure rapidly shrinks when the temperature decreases, resulting in wrinkled particles with many grooves7, 16, 17, as shown in Figure 3. Owing to the relatively low coating content, the produced particles have a small diameter, corresponding to a large surface area ratio. In all SEM images of the samples in the 1% sample group, particles formed. This confirms the success of using 1% w/v encapsulation agents to produce roselle microparticles. The addition of xanthan gum effectively reduces the surface area of the particles, making the particles wink and corrupt compared with those coated with maltodextrin alone. This phenomenon has also been observed in other works with gum-derived carriers 5, 18, 19.

This increase in particle size is necessary to reduce the surface area ratio of the sample, thereby limiting the influence of temperature and environmental humidity on the extract molecules encapsulated in the carrier layer. This hypothesis can be confirmed by the significant changes in the solubility of the microencapsulated material. As the content of XG in the microencapsulated carrier increases, the solubility of the powder decreases. At the highest content of XG (RP_M8X2), the solubility of the roselle powder was only 32%, three times lower than that of the treatment with only maltodextrin. This reduction shows that by adding XG into the encapsulated component, the particles tend to be more hydrophobic, in which XG reinforces the microencapsulated wall, increasing the difficulty of penetration of water molecules into the particle.

Physio-chemical profile of the microencapsulated roselle powder

Table 3 presents the physicochemical profile of the microencapsulated roselle powders. In general, the characteristics of roselle powder differ among different carrier treatments. The pH values of spray-dried powder samples with varying concentrations of XG and maltodextrin ranged from 2.4 to 2.9. The total acidity values for RP_M, RP_M9X1, and RP_M8X2 are between 13.7% and 16.6%, showing minimal variation among formulations. This consistency suggests that the XTG concentration does not significantly impact overall acidity. Hibiscus extract, which contains anthocyanins, is used in the production of these powders. Anthocyanins, which are responsive to pH variations, exist in four forms: quinonoidal bases, flavylium cations, carbinol (pseudobase), and chalcones20. At low pH, anthocyanins are predominantly in the red flavylium cation form. As the pH increases (>5), they shift to the blue/violet quinonoidal base or colorless carbinol/chalcone forms. pH values below 5 indicate an acidic environment, preserving the red color of the anthocyanins. The total anthocyanin content in the samples obtained with maltodextrin and xanthan gum as carrier agents was 41.3 ± 1.5, 38.4 ± 0.9, and 38.0 ± 0.9 mg/g for RP_M, RP_M9X1, and RP_M8X2, respectively. The similar anthocyanin contents across formulations suggest that the XTG concentration does not significantly affect anthocyanin levels. Maltodextrin and xanthan gum provide a protective coating, enhancing anthocyanin stability by reducing susceptibility to nucleophilic attacks by water molecules. This protective mechanism is crucial for maintaining anthocyanin stability and efficacy in various applications, ensuring uniformity despite variations in carrier agents 21. The IC50 values of spray-dried powder samples with maltodextrin and xanthan gum as carrier agents were 3.3 ± 0.1, 4.0 ± 0.5, and 4.6 ± 1.1 mg/ml for RP_M, RP_M9X1, and RP_M8X2, respectively. A higher xanthan gum content in the encapsulated matrix reduced the antioxidant activity of the powder, likely because the colloidal properties of xanthan gum limit the release of the core extract. This trend was also observed in the MIC results for the gram-negative strain. Detailed data on bacterial growth inhibition are presented in Table 3.