Microencapsulation of Betacyanin from Dragon Fruit (Hylocereus polyrhizus) Peel by Foam-Mat Drying for Natural Food Colorant Application

2.1 Materials

Dragon fruits were procured from local farmers in Bantul Regency, Yogyakarta, Indonesia. The peels were carefully separated from the fruit flesh and cleaned to remove any green outer portions. The cleaned peels were thoroughly washed and gently dried using paper towels to remove surface moisture. The cleaned dragon fruit peel is shown in Figure 1. The drying process was conducted in two stages: initially, the peels were dehydrated using a food dehydrator at 50℃ for 24 hours, followed by a secondary drying phase at 70℃ for 4 hours to ensure complete removal of residual moisture. Once dried, the peels were ground into a fine powder using a grinding machine. The ground powder was subsequently sieved through a 40-mesh sieve to achieve uniform particle size. The resulting dragon fruit peel powder was stored in sealed aluminium-plastic packaging with silica gel to prevent moisture absorption. Storage of the powder in an aluminum plastic bag can preserve its quality, thereby extending its shelf life [14]. The packaged powder was refrigerated at 8 - 10℃ to preserve its stability and quality until further use. Storage of the powder at temperatures between 8 - 10℃ can inhibit the degradation of bioactive compounds [11].

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                                        (a)                                  

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(b)

Figure 1. Cleaned dragon fruit peel (a) outer fruit peel and (b) inner fruit peel

2.2 Optimization of ultrasound-assisted extraction

The extraction of dragon fruit peel powder was performed using an ultrasound-assisted extraction (UAE) method. The powder was extracted with ethanol at varying concentrations, solid-to-solvent ratios, and temperatures, as determined by the experimental design. The extraction process was performed for 10 minutes at 80% amplitude with a duty cycle of 1/s. After extraction, the mixture was centrifuged at 4000 rpm for 15 minutes to separate the supernatant. The supernatant was filtered through Whatman No.1 filter paper to remove residual solids. The resulting extract was transferred to glass bottles, covered with aluminum foil to prevent light-induced degradation, and stored at 8-10℃ in a refrigerator until further analysis.

A Box-Behnken Design (BBD) was employed to optimize the UAE process. This design used three levels (-1: low, 0: medium, 1: high) and three independent variables: temperature (X₁), solvent concentration (X₂), solid-to-solvent ratio (X₃), with total betacyanin content and color intensity as the response variables (Table 1). The experimental setup consisted of 15 runs, including three replicates at the central point to estimate experimental variability. Data analysis was conducted using Minitab software (Minitab Ltd, Brandon Curt, UK). The significance of the variables and their interactions, as well as the model's goodness of fit, were evaluated using analysis of variance (ANOVA). A second-order polynomial (Eq. (1)) was applied to predict the responses as a function of the independent variables.

$\begin{aligned} y=\beta_0+\sum_{i=1}^k \beta_i X_i & +\sum_{i=1}^k \beta_{i i} X_i^2 +\sum_{i=1}^k \sum_{j=1, j \neq i}^k \beta_{i j} X_i X_j\end{aligned}$     (1)

The response variable y represents the response variable (e.g., total betacyanin or color intensity), $\beta_0$is the intercept, $\beta_i$ are linear coefficients, $\beta_ii$ are quadratic coefficients, and $\beta_ij$ are interaction coefficients. $X i$ and $X j$ are the independent variables, $k$ is the number of factors (in this case, 3), and $\varepsilon$ accounts for random error. The coefficients were estimated using the least squares regression method.

The response surface equation generated from the model was used to perform multi-response optimization (MRO) to identify the optimal extraction conditions for the two responses: total betacyanin content and color intensity. This approach allowed the simultaneous optimization of multiple factors and responses, leveraging methodologies successfully demonstrated in prior studies [15].

Table 1. Selected factors and their level

2.3 Foaming agent screening

A total of 100 mL of dragon fruit peel extract was mixed with 5% w/v maltodextrin and homogenized using an Ultra-Turrax T50 homogenizer (IKA, Staufen, Germany) for 3.5 minutes at 10,000 rpm. Subsequently, 1% w/v of a commercial foaming agent was added to the solution. The foaming agents evaluated included: a mixture of monoglyceride, diglyceride, and polyglycerol ester of fatty acid (SP); a combination of monoglyceride, polyglycerol ester, and sorbitan monostearate (TBM); and mono- and diglycerides derived from animal and vegetable fats and fatty acids (Ovalet) (PT. Gunacipta Multirasa, Banten, Indonesia). The solution was then further homogenized at 14,000 rpm for 15 minutes. The resulting foam was analyzed for its foaming properties, including foam expansion, density, and stability. The addition of maltodextrin in the range of 2-10% can provide good foam stability [16]. Meanwhile, the use of a 1% foaming agent is capable of producing optimal foam [17].

2.4 Microencapsulated with foam-mat drying

Dragon fruit peel extract was combined with 5% w/v maltodextrin, which served as a foam stabilizer, and homogenized using an Ultra-Turrax T50 homogenizer (IKA, Staufen, Germany) for 3.5 minutes at 10,000 rpm. Subsequently, 1% w/v of a foaming agent was added, and the mixture was homogenized again at 14,000 rpm for 15 minutes. The resulting foam was evenly spread onto trays and dried in a vacuum dryer (Memmert, Eagle, USA) at 60℃ for six hours. A temperature of 60℃ is considered safe for prolonged drying without compromising the bioactive components of the material [18]. Once dried, the foam sheet was ground into a fine powder, referred to as foam-mat dried powder. The powder was then sealed in airtight bags and stored in a refrigerator at 8-10℃ to maintain stability.

2.5 Microencapsulated with spray drying

Spray drying was performed following a reference method with modifications [19]. Dragon fruit peel extract was mixed with 5% w/v maltodextrin as a foam stabilizer and homogenized for 3.5 minutes at 10,000 rpm. Subsequently, 1% w/v of a foaming agent was added, and the solution was homogenized again at 14,000 rpm for 15 minutes. The prepared solution was then processed using a lab-scale spray dryer (BUCHI Labortechnik AG 9230 B-290, Switzerland). During spray drying, the airflow rate was maintained at 5 m³/min, the feed rate at 1 mL/min, and the inlet temperature at 100℃. A temperature of 100℃ is considered optimal for spray dryer treatment to preserve bioactive components [19]. The resulting microencapsulated powder was collected and stored in airtight containers at 8-10℃ until further analysis.

2.6 Moisture analysis

The moisture content of the samples was determined using a moisture analyzer [20]. Approximately 1-2 g of each sample was placed on the analyzer's plate, and the moisture content was measured by heating the sample at 105℃ for 8 hours until a constant weight was achieved. The sample was then reweighed, and the moisture content was calculated as the percentage difference between the initial and final weights.

2.7 Color analysis

Color analysis was performed using a Chroma meter (Model CR-310, Konica Minolta, Inc., Tokyo, Japan) [21]. The analysis measured three color parameters: L*, representing lightness, a*, indicating the green-to-red spectrum, and b*, representing the blue-to-yellow spectrum. Chroma, which specifies the color intensity of the sample, was calculated from the a* and b* parameters using Eq. (2).

Chroma $=\sqrt{a^2}+b^2$     (2)

2.8 Foam expansion

Foam expansion (FE) was determined following the reference method [17]. The volume of the solution was measured using a graduated cylinder both before ($V i$, cm³) and immediately after ($V f$, cm³) the foaming process, and foam expansion was calculated using Eq. (3).

$\mathrm{FE}(\%)=\frac{V_f-V_i}{V_f} \times 100$     (3)

2.9 Foam density

Foam density (FD) was determined following the reference method [17]. The volume of the solution before the foaming process (V_i, cm³) was measured using a graduated cylinder. After the foaming process, the generated foam was collected using a scoop, transferred into a pre-weighed container, and weighed to determine its mass (m, mg). Foam density was calculated using Eq. (4).

$\operatorname{FD}\left(\frac{g}{c m^3}\right)=\frac{m}{V_i}$    (4)

2.10 Foam stability

Foam stability (FS) was determined using a modified reference method [17]. A transparent graduated cylinder containing 50 mL of the foamed solution was maintained at room temperature (28 ± 2℃) for one hour to assess foam stability. The foam volume after a 15-minute interval (V_t, cm³) was compared to the initial foam volume (V_i, cm³) to calculate foam stability using Eq. (5).

Foam stability $(\%)=\frac{V_t}{V_i} \times 100$     (5)

2.11 Total betacyanin content

The total betacyanin content was determined following the reference method with modifications [22]. Two grams of dragon fruit peel powder were extracted under the optimized conditions. The extraction results were centrifuged at 4000 rpm for 15 minutes to obtain the supernatant, which was then filtered using Whatman No.1 filter paper. The supernatant was placed into a cuvette, and the absorbance at the maximum wavelength for betacyanin (λ = 538 nm) was measured using a UV-Vis spectrophotometer (Genesys 10, Thermo Scientific Co.). The total betacyanin content was calculated using Eq. (6).

Total betacyanin content $\left(\frac{m g}{g}\right)=\frac{A(M W) V(D F)}{\varepsilon L}$     (6)

where, A is the absorbance at 538 nm, MW is the molecular weight of betanin (550 g/mol), V is the volume of extract, DF is the dilution factor, ɛ is the mean molar absorptivity (60,000 mol/L·cm), and L is the path length (1 cm). The results were expressed as mg of betacyanin per gram of dry matter.

2.12 Encapsulation efficiency

Encapsulation efficiency was expressed as the percentage ratio of the betacyanin content within the microcapsules (W_f) to the initial betacyanin content in the extract (W_i) [21]. The encapsulation efficiency was calculated using Eq. (7) and expressed in percentages.

Encapsulation efficiency $(\%)=\frac{W_f}{W_i} \times 100$     (7)

2.13 Solubility of microcapsules

The solubility of microcapsules was determined following a reference study [23] with modifications. One gram of microcapsules was mixed with 25 mL of purified water and stirred for 5 minutes using a mechanical stirrer. The solution was then transferred to a centrifuge tube and centrifuged at 4000 rpm for 10 minutes at 25℃. The supernatant was collected and transferred to pre-weighed Petri dishes, which were dried for 5 hours in an oven at 105℃. The solubility percentage, representing the ratio of the dried supernatant weight (W_initial) to the initial weight of the supernatant before drying (W_final), was calculated using Eq. (8).

Solubility (%) $=\frac{W_{final}}{W_{initial}} \times 100$    (8)