Introduction

Malnutrition is a public health concern caused by the deficiencies, excesses, or imbalances of vital nutrients, such as vitamins, minerals, lipids, proteins, among others (Ahmed et al., 2022; Allen and Saunders, 2023; Padhani et al., 2022), or because of the lack of an essential amino acid (EAA) (Olson et al., 2020; Scholes, 2022). Nevertheless, this problem has still not been fully explained, so it is a field of great interest for the scientific community (Ersado, 2022). Since proteins play a crucial role in various biochemical processes, protein deficiency can lead to several functional disorders (Dolganyuk et al., 2023) such as unintentional weight loss and muscle wasting, although it can be reversed with a balanced diet (Olson et al., 2020; Scholes, 2022). Currently, meat, fish, eggs, nuts, legumes, and dairy products are considered the main sources of protein in human nutrition (Pintado and Delgado-Pando, 2020; Semba et al., 2021). However, the production and transportation of animal-derived protein demands large areas of dedicated land, water, nitrogen, and fossil fuels, and their overconsumption greatly contributes to greenhouse gas emissions generating environmental impacts (Ramírez-Rodrigues et al., 2021; Semba et al., 2021). In addition, the world population has increased at an unprecedented rate, with estimates projecting it to exceed 9 billion by 2050 (United Nations, 2017). Indeed, the demand for protein is expected to double in the coming decades (Garcia et al., 2020; Sadigov, 2022; Shaghaghian et al., 2022). Therefore, there is a great necessity to find an alternative and sustainable source of protein to meet the challenges of adequate nutrition (Raja et al., 2022; Ramírez-Rodrigues et al., 2021). In that sense, Spirulina represents a potential alternative because of its composition that is rich in protein, vitamins, minerals, pigments, and bioactive compounds such as polyphenols (Guarienti et al., 2021).

Arthrospira Platensis, marketed as Spirulina, is a spiral-shaped, green-blue, photosynthetic and filamentous cyanobacterium, also called microalgae (Arahou et al., 2021; Guidi et al., 2021) that has created significant interest in several sectors, including pharmaceuticals, medicine, and food. Besides its high protein content (60–70%), it is also considered a complete and high-quality protein source because of its high digestibility, ranging between 83% and 90% (Pootthachaya et al., 2023), which is better compared to other plant sources because of the presence of mucopolysaccharide, instead of cellulose, in the cell wall (Mohammadi et al., 2024; Raczyk et al., 2022; Ragaza et al., 2020). Furthermore, from a qualitative perspective, spirulina offers a well-balanced amino acid composition (Ragaza et al., 2020), which is aligned with FAO requirements (Raczyk et al., 2022; Tessier et al., 2021). However, spirulina presents certain limitations for its application in the food industry. Spirulina’s high moisture content (Özyurt et al., 2023; Silva et al., 2023; Stramarkou et al., 2021), dark green color, stemming from pigments such as chlorophyll, phycocyanin, and carotenes, and the fishy odor imparted by acids and sulfides (Jia et al., 2024), make it unappealing to most consumers (Almeida et al., 2021; Maag et al., 2022).

Therefore, various preservation methods have been explored to overcome these challenges, and microencapsulation by spray drying is as an effective technique to protect sensitive compounds against stressing environmental factors, besides masking undesirable color and flavors of some active compounds. This process allows rapid, continuous, and scalable production of dry microspherical powder with consistent quality in a single step (Chen et al., 2021; Guarienti et al., 2021; Özyurt et al., 2023). On the other hand, although the high drying temperatures involved in the spray-drying process may induce protein denaturation (Chen et al., 2021), this technology is suitable for heat-sensitive compounds because of the brief exposure to high temperatures. In addition, the cooling effect of latent heat of evaporation may mitigate the high temperature processing conditions (Lu et al., 2021; Samborska et al., 2022). In this sense, the coating material adopted for microencapsulation by spray drying could contribute to limiting protein denaturation as well (Jiménez-González and Guerrero-Beltrán, 2021; Luo et al., 2024). Among different encapsulation agents, maltodextrin (MD) is commonly used because of its low cost and large availability. Likewise, it is a good delivery system that can prevent thermal degradation of bioactive compounds, and it has been successfully applied for phycocyanin stabilization, which is the most abundant protein in spirulina (Faieta et al., 2020).

MD is a starch-derived compound, obtained by acid and/or controlled enzymatic hydrolysis. Likewise, it can also be a whey protein isolate (Xiao et al., 2022; Zhao et al., 2022). This material is considered as a D-glucose polymer joined by α-(1,4) and α-(1,6) linkages. Moreover, it is commonly used as a drying or encapsulating agent, known for its high-water solubility, low cost, low viscosity, colorlessness, retarded crystallization, and decreased stickiness and hygroscopicity of mixture. Nevertheless, it forms stable microcapsules that maintain the integrity of sensitive flavors and fragrances under diverse environmental storage conditions (Chaudhary et al., 2024; Kosasih et al., 2023; Xiao et al., 2022). In summary, the present study focuses on the microencapsulation of spirulina by spray drying testing different processing conditions (i.e., Spirulina:MD ratio and inlet temperature) for minimizing protein denaturation and maximizing process yield, utilizing a factorial design.

Materials and Methods

Materials

Spirulina (Arthrospira platensis) biomass (SP) was obtained from the producer Intipacha Microalgas (Ica, Peru). Food-grade MD (Dextrose equivalent DE: 11.3) was bought from IFF (Peru). Bradford reagent (Lt: SLCK1933), bovine serum albumin (Lt: 0000289566), and sodium hydroxide pellets were bought from Merck.

Proximal analysis of SP

Ash (AOAC 940.26), fat (AOAC 920.177), and protein (AOAC 920.152) contents were determined for SP, while carbohydrate content was estimated by the difference method.

Elemental analysis of SP

Elemental analysis was performed using Inductively Coupled Plasma Optical Emission Spectroscopy ICP-OES (Avio 550 MAX, Perkin Elmer, USA) to evaluate the nutritional composition and ensure safety standards. The instrument was operated with a plasma gas flow rate of 8 L/min and a nebulizer flow rate of 1.7 L/min, following the protocol described by Proch and Niedzielski (2021) with little modifications.

Spray drying microencapsulation

SP and MD were mixed in different proportions (SP:MD ratio) according to a factorial design. At first, a domestic four-blade immersion blender (FPSTHB460A, Oster, China) was used at the lowest speed for 1 minute. Subsequently, the mixture, at pH: 7–7.5, was stirred at 300 rpm for 10 minutes. The aforementioned procedure was repeated three times to keep the SP in suspension and avoid agglomeration. The suspension was sprayed through the ATM3000 Spray Dryer (PIGNAT, Genas, France) at a feed flow rate of about 12 mL min⁻¹, and spray air flow pressure of 40 psi with different inlet temperatures, according to the conditions listed in Table 1. Subsequently, the spirulina microencapsulated in MD (SP-MD powder) was vacuum-sealed in embossed bags and stored in a desiccator at room temperature.

Protein quantification

Protein content was determined for each of the treatments performed. SP-MD powder (75 mg) was mixed with 5 mL of NaOH 0.5 N and shaken vigorously at 2400 rpm using a Vortex (model ZX-classic, Velp Scientifica, Italy) for 1 minute at room temperature. Subsequently, it was stirred at 300 rpm with a magnetic agitator in a water bath at boiling point for 20 minutes and afterward centrifuged at 4265 × g for 15 minutes. The supernatant obtained was used for protein determination (Papalia et al., 2019; Saxena et al., 2022). Finally, 250 µL of the solution extracted was mixed with 2.50 mL of Bradford’s reagent and shaken at 2400 rpm for 1 minute, incubated for 6 minutes at room temperature, and the absorbance of the solution was measured at 595 nm wavelength using a UV/Visible spectrophotometer (UV-1700, Shimadzu, Japan). The amount of protein was calculated based on bovine serum albumin calibration curve (concentration range of 2.5 to 40 mg L⁻¹; R²= 0.988) (Saxena et al., 2022). Subsequently, the protein content relative to dried spirulina was determined using Equation 1 and reported as gram of protein/100 g of dried spirulina.

Where DF = 0.06 is the dilution factor, %Moisture = 5% is the moisture of SP-MD powder, and dried-spirulina content refers to the amount (mg) of dried spirulina expected in the sample under consideration.

Process yield (%)

The process yield was determined as the ratio of the mass of SP-MD powder to the mass of solids contained in the feed solution. This calculation is represented in Equation 2, as described by Duran Barón et al. (2021).

Experimental design

All the experiments were carried out by triplicate, using a factorial design with two independent variables: SP:MD ratio (X₁) and temperature (X₂) (see Table 1). A one-way analysis of variance (ANOVA) was used to determine significant differences between the response variables at a confidence level of 95% (p < 0.05). In addition, the response surface methodology (RSM) enabled us to define the optimal conditions for preparing the SP-MD powder. The response variables analyzed were protein content (Y1) and process yield (Y2). The relationship between the independent variables and the response variables was modeled using a second-order polynomial equation, expressed as:

where Y represented the response variable, and β coefficients represented the effects of the factors and their interactions.

Moisture content and water activity

Moisture content (%) of SP biomass and SP-MD powders was determined thermogravimetrically using a moisture analyzer (Model QL-720A, Eurotech, Taiwan). The analysis was performed using 0.50 g of sample placed into the aluminum sample plate and heated at 105°C until constant weight was reached. Each measurement was performed in triplicate. Water activity measurement was carried out on SP and SP-MD powders at 24°C using a water activity meter (CH8853, Novasina, Switzerland) according to the method proposed by Konar et al. (2022).

Amino acid concentration

Amino acid profile was determined for both SP and the SP-MD powder. Analysis was conducted using an HPLC system (Shimadzu, Japan) equipped with a UV/VIS detector, following the method described in Francioso et al. (2017). Quantification of amino acids was made by comparison with calibration curves generated, and data were reported as milligram of amino acid per gram of dry sample.

Color evaluation

Color determination was performed using a colorimeter (CR-20, Konica Minolta, Japan), according to the method described by García et al. (2021). First, the sample holder was half-filled with SP, then the equipment was turned over ensuring that the lens pointed upward toward the plate which was placed on the top, and the measurement was performed in triplicate. Color data were collected according to the CIELAB color space, with values of L*, a*, and b* representing lightness, red-green, and yellow-blue pair, respectively. Moreover, a comparison of the SP-MD powder samples, obtained from the different treatments, and the liquid mixture before drying against the original fresh biomass was conducted using the CIELAB color difference ( calculated, as shown in equation 4. For the analysis, approximately 0.5 g of the sample was placed in the dish and compacted to ensure complete coverage of the base of the dish.

where L*₀, a*₀, and b*₀ were the color parameters of SP while L*_i, a*_i, and b*_i were the measurements of the liquid samples and all the microencapsulated treatments. Color parameters of chroma (C*) and hue (h*) were calculated using Equations 5 and 6, respectively (Faieta et al., 2020).

Fourier-transform infrared (FTIR) analysis

The identification of the functional groups in the structure and interaction between SP, MD, and the optimal treatment of SP-MD powder were performed by Fourier transform infrared spectroscopy (IR21 Prestige FTIR, Shimadzu, Japan). Previously, SP was dehydrated at 40°C for 72 hours in an oven. The spectra were obtained by scanning the region between 4000 and 400 cm⁻¹ by the attenuated total reflectance (ATR) sampling technique (Yadav et al., 2022).

Morphology, particle size, and zeta potential of microcapsules

The microstructure and surface morphology of SP-MD powder were assessed by scanning electronic spectroscopy (SEM, Quanta 650 FEG, FEI, USA), as described by (Galván-Colorado et al., 2024). Moreover, particle size and zeta potential were performed by dynamic light scattering (DLS, Brookhaven 90 plus, Holtsville, USA), using a 658 nm laser and 90^o scattering angle. Samples were diluted in deionized water at 1000 mg L⁻¹ and then sonicated for 3 minutes (Das et al., 2022). Finally, an aliquot was placed in the cell for measurement.

Release kinetic in vitro of spirulina

The study of the in vitro release assay was conducted for the optimal treatment of microencapsulated spirulina following the method described by Tuesta-Chavez et al. (2022) with some modifications. The microcapsules (0.20 g) were added to a glass beaker containing 50 mL of water and stirred continuously using an orbital shaker (Model SHO-2D, WiseShake®, Germany) at 100 rpm for 8 hours at room temperature. During the kinetic study, 2 mL of sample was withdrawn from the beaker and analyzed by UV-VIS spectrophotometry (PharmaSpec UV-1700 model, Shimadzu®, Japan) at different time intervals. After analysis, the aliquots were returned to the dissolution vessel. Spirulina released concentrations were determined at 380 nm, using a linear calibration curve (R² = 0.9949, Abs = 0.0007(C)–0.0539) within a range of 100 to 1000 mg L⁻¹. All experiments were conducted in triplicate.

Data analysis

Moisture, color (L*, a*, b*, Chroma and Hue), and protein content were determined by triplicate and expressed as the average ± S.D. (Standard deviation). These data were analyzed by ANOVA (Analysis of variance) followed by a multiple comparison (Tukey 95%) test. Optimum parameters for SP:MD and temperature were determined using the factorial design mentioned before.

Results and Discussions

Raw material

The overall composition of spirulina varies depending on the algal strain used for cultivation, environmental conditions, and seasonal factors (Alfadhly et al., 2022). In this research, SP (Arthrospira platensis) was cultivated at alkaline pH range of 8–11, temperatures between 20 and 35^oC, and salinity levels of 12–20 g L^-1 in raceway ponds or closed greenhouse systems. Under these conditions, the biomass exhibited a protein content of 66% dry weight (DW), which is consistent with values reported by Arahou et al. (2021), who found protein levels between 64 and 71% in biomass grown in the Zarrouk medium at 35°C. Likewise, de Morais et al. (2022) reported an increase in spirulina protein content up to 76% by cultivating the strain with crude glycerol as an organic carbon source. Furthermore, Bleakley and Hayes (2021) and Nunes et al. (2024) reported protein levels ranging from 70 to 76%, comparable to those found in meat and significantly higher than soybean (40%).

Regarding carbohydrate content, SP contained 16.81% DW, which aligns with the findings of Priyanka et al. (2023), who reported carbohydrate levels between 15% and 25% DW. Unlike plant-based carbohydrate, spirulina lacks cellulose and instead contains a diverse array of saccharides, including glucose, mannose, galactose, and xylose, as well as glycogen. Because of this composition, the carbohydrates present in spirulina exhibit high bioavailability and rapid digestibility, making them particularly suitable for populations such as older adults and individuals with intestinal malabsorption disorders (Alfadhly et al., 2022).

Concerning the lipid content, SP contained 9.64% DW, slightly exceeding the 7.72% reported in the USDA Food Data Central results (Taiti et al., 2023). However, previous studies have reported lipid content ranging from 5% to 10% DW (Alfadhly et al., 2022). This lipid fraction is particularly relevant because of its nutritional and functional properties, as microalgal lipids have gained significant attention in human health and nutrition. Among these, glycolipids are the most abundant and serve as a key source of polyunsaturated fatty acids (PUFAs), which are known for their anti-inflammatory properties and potential therapeutic applications (Jung et al., 2022). The lipid and carbohydrate profile of spirulina is influenced by cultivation parameters such as nutrient availability, light intensity, and pH, which can modulate their relative abundance (Rosero-Chasoy et al., 2022; Saxena et al., 2022). In particular, nitrogen limitation redirects carbon from protein synthesis to the metabolic pathways for carbohydrate or lipid production, resulting in accumulation (Saxena et al., 2022).

In addition, the ash content in SP was 7.38% DW, which is slightly lower than the previously reported values ranging from 8% to 18% DW (Ramírez-Rodrigues et al., 2021). Ash content is an indicator of the total mineral composition of a food product and plays a crucial role in determining its nutritional value (Costa et al., 2024). Among all the nutritional components found in SP, microelements are also of nutritional interest (Rubio et al., 2021). In this study, spirulina exhibited a large amount of minerals, as is shown in Table 2, with Potassium (K) (2128.49 mg kg⁻¹) being the most abundant followed by Phosphorus (P) (1165.91 mg kg⁻¹), Sodium (Na) (1106.52 mg kg⁻¹), and Sulfur (S) (918.67 mg kg⁻¹), which is consistent with the results obtained by Janda-Milczarek et al. (2023). Moreover, iron, calcium, and zinc content were 28.14, 72.07, and 1.94 mg kg⁻¹, in agreement with the results reported by Koli et al. (2022). However, spirulina can also be affected by pollution and be susceptible to accumulation of metals with high binding affinity (Wang et al. 2024). According to Rzymski et al. (2019) and USDA Foreign Agricultural Service (2018), heavy metals such as Lead (Pb), Nickel (Ni), and Cadmium (Cd) are toxic elements with established limits in spirulina: Pb < 3.00 and Cd < 3.00 mg kg⁻¹. These contaminants are of particular concern because they can cause harm during critical periods of brain development. In addition, other trace elements were analyzed, including As (< 0.1), B (< 10.00), Ba (< 0.30), Cd (< 1.0), Hg (< 0.1), Li (< 0.30), Ni (< 2.00), V (< 1.00), Al (< 7.00), and Pb (< 3.00) mg kg⁻¹ DW, consistent with findings by Rubio et al. (2021) and Al-Dhabi (2013) for commercial spirulina products intended for human consumption.

Protein quantification of spirulina microencapsulated

The protein content, in the conditions tested, decreased from 66 g per 100 g of dried spirulina (initial biomass) to a range of 21.9–34.6 g per 100 g in the SP-MD powder. These values are comparable with those reported by Batista de Oliveira et al. (2021) who found a protein content of 27 g/100g in microencapsulated Spirulina sp. LEB-18, whose initial biomass protein content was 60 g/100 g DW. In the current research, the reduction in protein content is attributed to denaturation factors, particularly the mechanical stress caused by shear forces during mixing process in the preparation, as well as the elevated inlet temperatures during the spray-drying process. These high temperatures could favor protein denaturation, through the disruption of bonds and forces that maintain the secondary, tertiary, and quaternary structures (Blanco A. and Blanco G., 2022; Butreddy et al. 2021). Haque et al. (2018) suggested that atomization and the air–water interface could introduce additional stress factors. Moreover, the combination of these factors with shear stress can synergistically amplify the destabilization of protein structures (Amaya-Farfan, 2020). In addition, a significant portion of the dried feed adhered to the spray dryer chamber walls during processing, contributing to protein losses. Hence, the results suggest that encapsulation optimization should extend beyond just the spray drying parameters but include sample preparation conditions.

As shown in Table 3, the highest protein content was T6, followed by T9. Moreover, a positive correlation was observed between the amount of MD and protein content ensuring the protective effect of the coating agent. These results are consistent with that reported by Zhang et al. (2022), who suggested that an increased solid content may act as a barrier for the biomass during processing, thereby preserving the protein portion. In addition, the best conditions to obtain the higher protein content in SP-MD powder was at 170^oC and an encapsulant ratio of 1:4. Therefore, it could be considered that the exposure to this temperature for short periods is negligible on the preservation of the phycocyanin, the most abundant protein in spirulina (Faieta et al. 2020; Özyurt et al. 2023). On the other hand, SP had a moisture content of 76.15% and a water activity of 0.85, which makes a highly perishable food, according to Silva et al. (2023) and Stramarkou et al. (2021). This disadvantage is a key challenge when handling SP, since high moisture content accelerates microbial growth and spoilage. However, the spray-drying process significantly reduced the moisture content of SP-MD powder under 8.70%, as is shown in Table 3, which is within safe microbiological limits (Özyurt et al. 2023).

Process efficiency

Regarding the process yield, a positive correlation between the amount of coating material and the process yield was observed with T8 and T9 exhibiting the highest yields, with statistically equivalent values. This can be attributed to the higher initial solid content found in these formulations (Zhang et al. 2022). Higher MD concentration would lead to an increase of glass transition temperature (Tg) of the particles, reducing droplet adhesion to the dryer chamber walls (Decker et al. 2024; Xiao et al. 2022), leading to higher spirulina content in the SP-MD powder. These treatments, along with T6, also showed higher protein content, highlighting the critical role that the solid matrix plays in maintaining protein integrity in microencapsulation. On the other hand, the temperature did not show a clear trend in influencing the process yield. However, T6 (170°C) and T9 (180°C) achieved high values for both protein content and yield, with no statistical differences, suggesting T6 for scale-production because of other benefits such as energy savings, thermal efficiency, and production costs (Tay et al. 2021).

Water activity and moisture content analysis of spirulina microencapsulated

In the current study, SP had an initial moisture content of 76.15% and a water activity of 0.85, which makes a highly perishable food according to Silva et al. (2023) and Stramarkou et al. (2021). This is a key challenge when handling SP, as high moisture content accelerates microbial growth and spoilage. However, the spray-drying process significantly reduced the moisture content of SP-MD powder under 6.70%, which is within safe microbiological limits (Özyurt et al. 2023). In addition, the water activity values ranged from 0.33 to 0.45, well below the critical threshold of 0.61, indicating that the powder was not susceptible to microbial proliferation (Barbosa-Cánovas et al., 2020). In fact, low moisture and water activity values ensure the effectiveness of spray drying, which enhances the shelf-life and stability of the spirulina. According to the moisture results, a negative correlation was observed with the temperature, indicating that as the inlet temperature increased, the moisture content in the SP-MD powder decreased. Likewise, treatments at 160°C showed a negative correlation with the amount of MD, suggesting that higher concentrations lead to the reduction in moisture content. Similar results were also reported by Tay et al. (2021).

Experimental design and model fitting

Table 4 presents the regression coefficients of the quadratic polynomial model, and the corresponding p-values obtained from the ANOVA analysis, which was used to determine the significant variables influencing the dependent variables. Moreover, the efficacy of the model was evaluated through the R² value. According to the results, X₁, X₂ (SP:MD ratio and spray drying inlet temperature, respectively), and the quadratic coefficient of X₂ significantly influence protein content (p < 0.05), whereas the R² value obtained was 0.819. In addition, the response surface plot (Figure 1A) showed that the optimal combination of temperature and SP:MD ratio—174.8°C and 1:4, respectively—resulted in the highest protein content, reaching 33.1 g/100 g dried spirulina. Regarding the responding surface plot of the yield process, Figure 1B showed that the optimal combination was 174.7°C and SP:MD ratio was 1:4. The model response indicates that higher temperatures as well as encapsulant agent amount increases the yield process. According to the ANOVA, both X₁ and X₂, as well as the quadratic effect of X₂, had a significant influence on the process yield (p < 0.05). However, the R² value was 0.461, which was lower compared to the model for residual protein content. Finally, the optimization results were integrated using a desirability function (0.83), as shown in Figure 1C, finding that the ideal conditions for both protein content and yield were found to be 174.8°C of X₁ with 1:4 of X₂. These results show the significant impact of both temperature and encapsulant/bioactive ratio on preserving protein content and maximizing process efficiency during spray drying.

Figure 1. Response surface graphs of protein content (A), process yield (B), and desirability of the nine treatments (C).

Amino acid profile comparison

SP is a well-known source of protein with its quality determined by the quantification and proportion of EAA (Alfadhly et al., 2022; Kumar et al., 2022; Markou et al., 2023). Table 5 exhibited all EAA, with a total amino acid (TAA) content of SP of 600.2 g kg⁻¹ DW, constituting 41.5% of TAA. In addition, glutamic acid was the most abundant amino acid representing approximately 15% of TAA. Other amino acids included are: aspartic acid (9.9%), leucine (8.8%), alanine (7.5%), arginine (7.2%), and valine (6.6%), which were notable because they significantly contributed to the TAA and the nutritional value of a food product. Similarly, Gentscheva et al. (2023) found that spirulina contains glutamic acid (8.15%), aspartic acid (5.34%), alanine (4.54%), leucine (4.84%), arginine (3.96%), and valine (3.34%). Therefore, based on the results, fresh spirulina whose protein content is dominated by glutamic acid has attracted a huge interest because of its high potential to develop umami ingredients (Pratama et al., 2022). On the other hand, SP-MD powder at optimal conditions preserved all amino acids with slight reduction from 600.2 to 584.44 g kg⁻¹ DW. The most abundant EAA on SP-MD powder was leucine with 11.8% of TAA, while the amino acid which presented a major reduction was aspartic acid with 34.05% of the initial content, followed by arginine (31.23%) and threonine (30.76%). Overall, it has been noticed that EAA rose from 248.90 to 298.59 g kg⁻¹ DW, and non-EAA (NEAA) was reduced from 351.3 to 285.86 g kg⁻¹ DW. Moreover, EAA represented a major percentage of TAA on SP-MD powder (51.10%) in comparison with the EAA on biomass (41.5%). In conclusion, although the protein content was reduced, the product maintained its nutritional property because of the presence of all EAA.

Color assessment

The color parameters of SP, liquid samples, and treatments of SP-MD powder are shown in Table 6. It is worth mentioning that liquid samples are divided into three groups: liquid samples 1 (LS1) is the mixture of spirulina and MD in a ratio of SP:MD equal to 1:2 which corresponded to T1, T4, and T7 according to Table 1. The luminosity parameter (L*) has significantly increased for all the samples after the drying process because of the addition of the coating material. According to the L* values of the treatments (T1–T9), a positive correlation between MD content and L* was found, while temperature did not exhibit a significant variation. A similar result was found in the study made by (Özyurt et al. (2023), where the highest whiteness values (L*) were observed with increasing concentrations of MD. This is also attributed to the capacity of protection of MD which is proved with major values of protein content (Table 3). Moreover, the greenness (−a*) and yellowness (+b*) of the SP were lower than the microencapsulated samples. In fact, the SP used was dark with greenish shades and blue with color parameters relatively like those reported by Demarco et al. (2022).

Table 6. Color parameters (CIELAB-color system) for spirulina biomass and changes in color indexes of the liquid samples and microencapsulated samples.

SP: Spirulina biomass, LS: Liquid sample refers to the mixture of spirulina and maltodextrin before it was spray dried. LS1 corresponds to T1, T4, and T7; LS2 corresponds to T2, T5, and T8; and LS3 corresponds to T3, T6, and T9.

On the other hand, it is known that values of CIELAB color difference (ΔE*) lower than 3.00 are difficult to be perceived by the human eye and are considered as the same color (García et al., 2021). In this context, the color difference of liquid samples as well as microencapsulated samples was noticeable. Regarding color parameters of liquid samples, they were also dark with little changes in color index in comparison to SP, with the LS3 sample resulting as the one with the highest color difference among the liquid samples. For the SP-MD powder, the microencapsulated samples (T1–T9) exhibited a major average color difference of 49.20, highlighting T3, T6, and T9 whose values were higher. Regarding the C* values of the treatments, they ranged 12.65 to 15.20, indicating low color saturation. The lowest values were observed in T3, T6, and T9, which correspond to treatments with the highest encapsulant ratio. However, no significant correlation was found between temperature and encapsulant ratio. Finally, h* of the treatments ranged from 154.82 to 165.28, which correspond to the cyan-green region according to Rong et al. (2024).

FTIR spectroscopy

The FTIR analysis highlights the presence of key functional groups and interactions in both the SP and the encapsulating MD, offering an insight into the chemical interactions that play a role in the encapsulation process and protein protection. Figure 2 presents the FTIR spectra corresponding to the functional groups and chemical interaction of SP, MD, and the protein encapsulated within MD. The infrared spectrum of spirulina (Figure 2, spectrum A) exhibited characteristic peaks at 3279 cm^–1, attributed to the symmetric stretching vibration of the −OH group, overlapping with N-H stretching. In addition, two bands at 2961 cm^–1 and 2920 cm^–1 were observed, corresponding to the asymmetric C-H stretching vibrations in aliphatic groups (-CH₃ and -CH₂, respectively). A notable peak at 1635 cm^–1 was identified, consistent with the stretching vibration of C=O in amide I, while bands at 1544 cm^–1 and 1239 cm^–1 were attributed to the vibrations in amide II and III, respectively. Furthermore, the stretching vibration of the C-O group was detected at 1034 cm^–1, aligning with similar results reported in the literature (Buhani et al., 2019; da Silva et al., 2019; Golmakani et al., 2024). On the other hand, in the MD spectrum (Figure 2), peaks at 3271 cm^–1 and 2920 cm^–1 were attributed to the -OH and C-H stretching vibrations, respectively. The band at 1639 cm^–1 was associated with the water–MD interaction, likely because of moisture content, a typical feature in polysaccharides (da Silva et al., 2019; Pais et al., 2021). In addition, signals at 1358 cm^–1, 1147 cm^–1, and 992 cm^–1 were linked to asymmetric -CH₂ scissoring vibrations, the asymmetric and symmetric stretching of the glycosidic bond (C-O-C), and the asymmetric stretching of C-O, respectively (Pais et al., 2021; Yu et al., 2021). The infrared spectrum of the microencapsulate (Figure 2, spectrum C) showed a slight shift in the band associated to the -OH group (from 3279 cm⁻¹ to 3285 cm⁻¹) because of the hydrogen bridge interactions that occurred between these groups with those of the MD; likewise, it was noted in the band of 1635 cm⁻¹ that shifted to 1649 cm⁻¹. Also, the presence of bands associated with amide II at 1544 cm⁻¹ and amide III at 1240 cm⁻¹ were identified, suggesting that the structural integrity of spirulina is maintained, as well as that of MD, since peaks at 1148 cm⁻¹ and 1016 cm⁻¹ were found, attributed to the vibrations of the C-O-C and C-O glycosidic bond, respectively. The results clearly show that spirulina maintains its chemical and functional characteristics, as does the encapsulant (MD), indicating that the SP remains encapsulated in the MD, generally through intermolecular Van der Waals forces and hydrogen bonds.

Figure 2. FTIR of spirulina biomass (A), maltodextrin (B), and SP-MD powder (C).

SEM and DLS analysis

In Figure 3, the micrographs show spherical microparticles of irregular sizes with the presence of cavities. This morphology could be defined as irregular shape with single core and wall (Corrêa-Filho et al., 2019; Xiao et al., 2022), primarily influenced by the presence of MD (Faieta et al., 2020). The formation of rough surfaces, dents, and concavities is attributed to the rapid and drastic evaporation of moisture during the initial stages of spray drying, as the droplets enter the drying chamber. This is followed by the subsequent cooling of the microparticles, which contributes to their final shape and surface characteristics (Norkaew et al., 2019). In this study, the surface integrity of the spray-dried samples maintained a continuous surface without visible cracks or fissures, indicating that the spray drying conditions effectively preserved the structural integrity of the microcapsules (Van et al., 2024). This is likely because of the protective role of MD as the wall material, which contributes to lower oxygen permeability, improved protection, and enhanced retention of core materials (Valková et al., 2022) These factors play a significant role in the functional properties of the microcapsules, such as their release kinetics. Nevertheless, previous studies have suggested that using MD with a higher DE value could act as a plasticizer, preventing irregular shrinkage and promoting the formation of a smoother surface (Ghani et al., 2017).

Figure 3. SEM images of optimal treatment SP-MD powder at magnifications of (A) 500×, (B) 1500×, (C) 6000×, and (D) representative particle.

Particle size mainly depends on the encapsulating agent and drying conditions (Ozdemir et al., 2021). The powder obtained in this study had an average size of 5.21 ± 0.54 µm, as measured using ImageJ software. This falls within the typical size range for food powders produced by spray drying, which generally varies between 5 and 25 µm and is classified as medium-sized by Piñón-Balderrama et al. (2020).

The hydrodynamic particle size measured by DLS was found to be 441 nm in diameter. In addition, the zeta potential was recorded at −23.7 mV, indicating that the microcapsules exhibit moderate stability over time. According to Jacob and Mullar, a zeta potential of ±30 mV is required for electrostatic stabilization, while ±20 mV is sufficient for steric stabilization (Kamble et al., 2022). However, at more alkaline pH levels, the zeta potential is expected to become more negative because of the gradual deprotonation of carboxyl and amino groups present in the encapsulated proteins (Lee et al., 2021). This increase in negative charge could enhance electrostatic repulsion between particles, reducing the tendency for aggregation and sedimentation.

In vitro release study

The release profile of spirulina from the microcapsules in an aqueous medium is shown in Figure 4. The release occurs in three distinct phases: an initial rapid release during the first 30 minutes (7.40 ± 2.23%), attributed to nonencapsulated spirulina present on the surface of the microcapsules. This behavior is similar to findings reported by Ozdemir et al. (2021), which examined the release of basil essential oil from a combined matrix of gum Arabic, whey protein isolate, and MD. The following phase is slower (between 50 and 160 minutes, 11.25 ± 0.99%), attributed to the spirulina produced by the partial microcapsule’s erosion and the final phase where the release percentage remains relatively constant (12.42 ± 0.15%), associated with the low solubility in water presented by the microparticles and is also attributed to the desorption of spirulina, produced by the erosion of microcapsules. The latter behavior is a positive parameter for the industry, since it allows the material’s incorporation in matrices with different humidity levels and also to be used in liquid products without promoting the immediate release of encapsulated spirulina (Guarienti et al., 2021).

Figure 4. Release profile of spirulina from microcapsules at pH 6 in water medium.

Conclusions

SP reported a protein content of 66 g/100 g dried spirulina based on the proximal analysis, confirming its status as a complete protein source containing all EAA. While the microencapsulated sample (SP-MD powder) presented a reduced protein content on an average of 31.65 g/100 g dried-spirulina, it still retained all EAA with a slight variation. Notably, the optimum operating conditions were 174.7^oC and an SP:MD ratio of 1:4, which resulted in higher protein content and process yield (46%). In addition, the FTIR and SEM analyses confirmed microencapsulation of spirulina within MD, validating its effectiveness as a protective coating material. Moreover, SP showed a release profile in three stages, reaching a maximum release of 12.5% at 8 hours. Further studies should focus on the optimization of biomass pretreatment before spray drying to preserve higher amounts of protein. The stability of the powder synthesized will also need to be investigated as well as its impact on food formulation from a sensory standpoint.