3D-printed soy protein and microalga films: A sustainable approach with antioxidant functionality

Abstract

This study investigated the optimization and fabrication of soy protein isolate (SPI)–green microalga (MA) 3D-printed films. For optimizing 3D printing, the effects of MA concentration, nozzle size (0.52–0.81 mm), and speed(10–20 mm/s) were examined. The printed films were then dried, and color, mechanical properties, water vapor permeability, structure, and antioxidant activity were analyzed. All the formulations showed shear-thinning behavior and rapid recovery. The concentration of 3 % MA, nozzle size of 0.72 mm, and printing speed of 20 mm/s were selected as the optimized conditions for the best 3D printability. Compared with the control, their elongation at break decreased by more than 16 %, while puncture strength increased by over 12 %, and tensile strength rose by more than 40 %. Water vapor permeability decreased by more than 40 % with the addition of MA. The microstructure images and secondary structure confirmed the formation of a less porous and stronger gel network with an increase in MA concentration from 0 to 5 % (w/w). The antioxidant properties of SPI films also increased two-fold with the addition of MA. These findings highlight that the 3D-printed edible films with antioxidant properties could be used as an eco-friendly and nutritious alternative to petroleum-based films in food packaging.

Graphical abstract

Highlights

• •Soy protein isolate–Chlorella Vulgaris films were fabricated via a 3D food printer.
• •The optimal printing was achieved using nozzle size of 0.72 mm and speed of 20 mm/s.
• •Microalga incorporation improved gel strength and puncture resistance.
• •The microalga-added films became less permeable to water vapor.
• •The microalga-loaded films showed significant antioxidant properties.

1. Introduction

Packaging applications use art, science, and technology to ensure quality and cost-effective delivery of goods with consumer acceptability (Priyadarshi et al., 2023). In the food industry, packaging is the most important factor in preserving product safety and freshness during storage (Priyadarshi et al., 2023; Kadirvel et al., 2025). Conventional packaging materials, such as plastics, while they are inexpensive and effective, contribute to environmental and health concerns (Gupta et al., 2017). This caused a growing demand for sustainable and biodegradable alternatives (Kadirvel et al., 2025). Recent advances in this field include active packaging, which incorporates functional components that extend shelf life and preserve nutritional quality (Onyeaka and Nwabor, 2022). Bio-based films are a type of active packaging that can be used as a promising solution in packaging systems (Elafify et al., 2024). Edible films have emerged as thin, continuous layers of edible materials with sustainability and eco-friendly properties, which are placed on or between food components to inhibit the migration of the food's moisture, gases, and aromas (Cooper and Costa, 2021; Hellebois et al., 2020; Dalla Rosa, 2019). Recently, edible films were produced using Chlorella vulgaris, a single-cell green microalga belonging to the Chlorophyta family and has been widely used as human food and supplement for over 30 years in the United States (Wang et al., 2024a; Zheng et al., 2024). It is classified as Generally Recognized as Safe (GRAS) by the U.S. Food and Drug Administration (FDA) (Zheng et al., 2024). C. vulgaris is a highly nutritious food with a composition based on dry mass of approximately 62 % protein, 13 % fat, and 14 % carbohydrates, antioxidants, vitamins (folic acid, vitamin C, B-complex, biotin, B₁, D, α-tocopherol, menadione), minerals (Fe, K, Na, Ca, P, Mg), pigments (lutein, β-carotene, astaxanthin, chlorophyll-a and -b, canthaxanthin), polysaccharides, and other bioactive compounds (Abdel-Latif et al., 2022). The food-grade and nutritional properties make C. vulgaris a good candidate for improving the functionality of polymer-based edible films (Abdel-Latif et al., 2022).

These biofilms for food applications possess suitable mechanical strength, barrier properties, and UV shielding. Current film fabrication methods (i.e., casting) are limited due to poor thickness control, small size, high material use, and increased amount of waste, restricting industrial applications (Jeya Jeevahan et al., 2020). To overcome these challenges, recent advances have explored three-dimensional food printing (3DFOODP) as a more accurate and flexible alternative to traditional approaches (Ahmadzadeh et al., 2023). 3DFOODP is an advanced technique that uses computer science, engineering, food science, and culinary arts to create visually appealing and structurally complex structures (Truong-Le et al., 2025; Ahmadzadeh et al., 2025). Therefore, this tool provides the opportunity to design customized packaging for different products (Arshad et al., 2025). However, for successful printing with high resolutions, optimizing formulation and printing parameters are essential (Balasubramanian et al., 2019).

Chlorella vulgaris was chosen because it is rich in natural biopolymers and provides antimicrobial and antioxidant properties that can improve biofilm functionality (Wang et al., 2024a; Zheng et al., 2024). Soy protein isolate (SPI) was selected for its excellent film-forming ability, strong mechanical properties (Truong-Le et al., 2025), and compatibility with microalgal biomass. Traditional methods like extrusion and film-casting are fast and scalable (Kumar et al., 2025), but often result in uneven films, excess waste, slow drying, and high costs, so there's a clear need for a more precise and flexible approach, and the novel 3DFOODP method offers a promising solution with unique potential for customized edible packaging and personalized food designs (Truong-Le et al., 2025). 3DFOODP has been recently used for fabricating protein- (Ahmadzadeh et al., 2023), and starch-based (Truong-Le et al., 2025) films where phenolic compounds were added to improve film structure through forming crosslinking with proteins. For example, SPI enriched with polyphenol-rich grape seed or green tea extracts (Ahmadzadeh et al., 2023) and corn starch-gelatin with hawthorn berry extract (Leaw et al., 2021) have been employed for 3D printing. However, no studies have optimized printing parameters or investigated the use of whole dry green microalga (C. vulgaris) to enhance film printability and uniformity. Previous work only investigated the algal phenolic extracts (Karabulut and Goksen, 2025) without using 3DFOODP, and the effect of the whole microalgae on modifying protein-based film structure and mechanical properties remains unclear. This study is the first report to utilize and optimize 3D printing to produce soy protein–microalga edible films with high antioxidant activity.

Thus, to find the effect of MA content on edible films, different formulations of soy protein gels with MA (0, 1, 3, and 5 % w/w) and glycerol as plasticizer were formulated. The rheological properties of the gels were evaluated. The effect of nozzle size and printing speed during printing was optimized. Then, 3D printability, mechanical properties, water vapor properties, microstructure, total phenolic content, and antioxidant activity of the 3D-printed dried films were evaluated. We hypothesized that the addition of MA into SPI would increase the antioxidant and mechanical properties of edible films obtained with high precision and flexibility through 3DFOODP. This work introduces an edible film with suitable mechanical properties and structure for food packaging applications and provides insight into developing edible ink by using 3D printing technology as a tool to prepare functional packaging materials with uniform thickness. It also addresses current limitations in food packaging and biomaterial applications and advances the field of sustainable 3D-printed biomaterials.

2. Materials and methods

2.1. Materials

Whole grain soybeans (line MO-S17–17168) were kindly supplied by Fisher Delta Research Center at the University of Missouri (MO, USA). Chlorella vulgaris powder, containing 52 % protein, 12 % carbohydrates, 14 % lipids, 6 % ash, and other minor components based on a dry basis, was bought from NY Spice (NY, USA) and used as is without further treatments. Glycerol (CAS No. 56-81-5, >99 % purity), sodium hydroxide (CAS No. 1310-73-2, 497-19-8, ≥97 % purity), and hexanes (CAS No. 92112-69-1, 99.9 % purity) were purchased from ThermoFisher Scientific (MI, USA). Ethanol (CAS No. 64-17-5, 100 % purity) was sourced from Decon Labs (Pennsylvania, USA). All other chemicals were of analytical grade. Ultra-purified deionized water for all analyses was prepared using a PURELAB flex water purification system (Veolia Water Solutions and Technologies, IL, USA).

2.2. Extraction of soy proteins

Soy flour was defatted by mixing with n-hexane in a 1:2 (w/v) ratio for 3 h. The mixture was then vacuum filtered, defatted again, and dried overnight under a hood. The dried flour was stored in an airtight container at 4 °C. To extract proteins, the defatted soy flour (passed through a 60-mesh sieve) was mixed with deionized water in 1:9 (w/v) ratio. The pH of the mixture was adjusted to 9.5 using 3N NaOH. This mixture was stirred for 3 h and centrifuged at 3000×g for 15 min to remove solid residues. The remaining solids were subjected to one more extraction cycle to maximize protein recovery. Next, the supernatants were pooled and adjusted to pH 4.5 to precipitate the proteins. The samples were stored overnight at 4 °C, and then centrifuged at 7000×g for 75 min at 4 °C. The resultant protein was washed with slightly acidic water (pH 4.5), mixed with distilled water adjusted to pH 7.0, and lyophilized at −43 °C and 5.6 Pa (Labconco, MO, USA) for 48 h. The dried protein isolate with 84.7 ± 1.9 % (w/w) protein content was stored in an airtight container at 4 °C (Barekat and Ubeyitogullari, 2025a).

2.3. Formation of SPI-microalga gels

For the formulation of the SPI–microalga gels, the method of Ahmadzadeh et al. (2023) was used with some modifications. An aqueous suspension of 11 % SPI and microalgae powder (0 %, 1 %, 3 %, and 5 %, w/w) were mixed continuously at room temperature (23 °C) for 2 h (Table 1). The concentration of 7 % (w/w) cased gel network began to lose water and due to the syneresis, the 5 % (w/w) was selected as the maximum concentration for MA. Then, glycerol (3.3 %, w/w based on SPI) was added as a plasticizer and degassed under vacuum. The pH of the solution was set to 8.5 using 1 M NaOH solution. The final solutions were heated in a water bath to 85 °C for 30 min to form gels (Ahmadzadeh et al., 2023).

Table 1.

Composition of SPI-microalga gels used for 3D printing.

Sample	Soy protein (%w/w)	Microalga (%w/w)	Glycerol (%w/w)
SPI	11	0	3.3
SPI-MA1	11	1	3.3
SPI-MA3	11	3	3.3
SPI-MA5	11	5	3.3

The samples were labeled as SPI, SPI-MA1, SPI-MA3, and SPI-MA5. “SPI” refers to the soy protein isolate gels, while “SPI-MA” indicates gels prepared from soy protein isolate and green microalga. The numbers (1, 3, and 5) showed the concentration (% w/w) of microalga.

2.4. Rheological properties

The rheological properties of the SPI, SPI-MA1, SPI-MA3, and SPI-MA5 gels were tested using a modular compact rheometer (MCR 302e, Anton Paar, Graz, Austria) with a 50 mm parallel plate setup (PP50). All measurements were done at a 1 mm gap and at 23 °C, which matches the 3D printing conditions (Barekat and Ubeyitogullari, 2025b).

2.4.1. Viscosity measurement

To measure viscosity, the samples were exposed to a gradually increasing shear rate (from 0.01 to 100 s⁻¹), and the resulting shear stress was recorded (Ahmadzadeh et al., 2025). The resulting data were fitted to the power-law model to obtain consistency (K) and flow behavior index (n) (Eq. (1)).

η = Kγⁿ-1 Eq. 1

where η, K, γ, and n are the viscosity (Pa.s), the consistency (Pa.s), the shear rate (s⁻¹), and the power-law index (Ahmadzadeh and Ubeyitogullari, 2022).

2.4.2. Dynamic rheological tests

A strain sweep test was performed at 1 Hz, with strain values ranging from 0.01 % to 100 %, to determine the linear viscoelastic region using the same equipment described above. A frequency sweep test was conducted at a constant stress of 1 Pa and a range of 0.1–100 Hz, to measure the storage modulus (G′) and loss modulus (G''). To prevent the gels from drying, silicone oil (Alfa Aesar, MA, USA) was used to cover the samples (Barekat and Ubeyitogullari, 2025b).

2.4.3. Three-step recovery tests

These tests were used to mimic the printing process and check how the 3D-print can recover its structure. The shear rate was set to 1 s⁻¹ for 3 min, increased to 100 s⁻¹ for 2 min, and then returned to 1 s⁻¹ for another 3 min (Barekat and Ubeyitogullari, 2025b).

2.5. 3D printing of films

The developed gels (SPI, SPI-MA1, SPI-MA3, and SPI-MA5) were loaded into the 10 mL extruder of an Allevi 2 Bioprinter (Allevi, Inc., PA, USA) for 3D printing. The SPI and SPI-MA inks were printed using nozzle diameters of 0.52, 0.64, 0.72, and 0.81 mm. The extrusion pressure was set between 0.020 and 0.062 MPa for optimal printing. The nozzle height was kept the same at 0.2 mm, and samples were printed on 10 cm × 10 cm plastic sheets. The printed films were square in shape (5 cm × 5 cm) with a thickness of 0.025 cm. They were dried using an oven for 4 h at 50 °C and relative humidity controlled (45 % RH) by a magnesium nitrate saturated salt solution. After drying, the films were stored between wax paper sheets in a chamber at 25 °C and salt-controlled relative humidity for 24 h before further testing (Ahmadzadeh et al., 2023). The moisture content of these films was 12 ± 0.5 % (w/w). Finally, the 3D-printed films were labeled as SPI-F, SPI-MA1-F, SPI-MA3-F, and SPI-MA5-F. “SPI-F” refers to the soy protein isolate film, while “SPI-MA-F″ indicates films formulated from SPI and green microalga. The numbers (1, 3, and 5) represent the concentration (% w/w) of microalgae (Fig. 1).

Fig. 1.

Schematic diagram of the 3D printing process.

2.6. 3D printability

3D printability was evaluated by comparing the printed samples to the original digital 3D model. All four sides and the thickness of the printed cubic films were measured using ImageJ software (National Institutes of Health, MD, USA) with a scale bar applied after printing.

2.7. Film thickness

The film thickness was measured using a digital caliper (Mitutoyo Corporation, Absolute Digimatic Caliper, Japan) with an accuracy of 0.01 mm. Measurements were taken at 10 randomly selected spots on each film, and the average thickness was reported (Ahmadzadeh et al., 2023).

2.8. Color

The color of 3D-printed films after drying was measured using a MINOLTA CR-300 colorimeter (Konica Minolta, NJ, USA) with diffuse illumination and 0° viewing geometry. The Lab color space was used, where L∗ indicates lightness, a∗ represents the red-green axis (positive values for redness, negative for greenness), and b∗ represents the yellow-blue axis (positive values for yellowness, negative for blueness). The colorimeter was calibrated using device's white standard calibration plate with values of L∗ = 97.12, a∗ = +5.25, and b∗ = −3.49 before measurements (Ahmadzadeh et al., 2023). The total color difference (ΔE∗) was calculated to detect the change in color between MA-F and SPI-F (Eq. (2)):

ΔE∗ = √(ΔL∗)²+(Δa∗)²+(Δb∗)² Eq. 2

ΔE∗ indicates the overall color difference between two objects; a higher ΔE∗ value represents a more perceptible difference to the human eye (Khashayar et al., 2014).

2.9. Mechanical properties

The tensile and puncture strength of the 3D-printed films were measured using a TA-XT2i Texture Analyzer with Exponent software (Stable Micro Systems, Ltd., Godalming, Surrey, UK) (Ahmadzadeh et al., 2023; Azevedo et al., 2017).

2.9.1. Tensile Strength (TS) and Elongation at Break (EB)

Film strips (40 mm × 15 mm) were clamped to the texture analyzer using film extension grips, with an initial grip separation of 20 mm. The analyzer pulled the grips apart at a speed of 2 mm/s until a 20 mm extension was obtained, and the TS and EB were recorded.

2.9.2. Puncture Strength (PS)

For PS testing, film samples were mounted using the TA-108S film-testing fixture. A 2 mm diameter probe (TA-52) pierced the film at a speed of 100 mm/min. The force needed to puncture the film was measured and expressed as puncture strength (N/mm). To account for variations in film thickness, puncture strength was normalized by dividing the force by the film thickness.

2.10. Water Vapor Permeability (WVP)

WVP was measured following the ASTM E96/E96M-10 standard method with some modifications. Calcium chloride (0.5 g) was used in 20 mL vials (2.2 cm diameter, 5.5 cm height). Films were cut to 2 cm² squares and sealed over vial openings. The vials were then placed in a humidity control chamber at 23 °C and 50 % relative humidity controlled using a saturated magnesium nitrate solution. The vials were weighed at set times over 24 h, and the WVP was calculated using the following equation (Ahmadzadeh et al., 2023):

$$\text{WVP}=\frac{\text{weigth}\text{difference}\left(\mathrm{g}\right)\times \text{film}\text{thickness}\left(\mathrm{m}\right)}{\text{exposed}\text{area}\left(m^2\right)\times \mathrm{t}\text{ime}\left(\mathrm{s}\right)\times \text{pressure}\text{difference}\left(\text{Pa}\right)}$$ Eq. 3

2.11. Microstructure

The microstructure of the freeze-dried 3D-printed films was examined using a scanning electron microscope (SEM; FEI Nova Nanolab 200 Dual-Beam system, FEI Company, OR, USA), equipped with a 30 kV field emission gun (FEG) and a 30 kV focused ion beam (FIB). For imaging, both surface and cross-sectional areas of the samples were carefully sliced and coated with a thin layer of gold using an EMITECH SC7620 sputter coater (MA, USA). SEM images were taken at an accelerating voltage of 15.0 kV and a current of 10 mA, with magnifications of×1000 and × 4000 to visualize the microstructure (Ahmadzadeh et al., 2023; Barekat and Ubeyitogullari, 2025b).

2.12. Fourier-Transform Infrared (FTIR) spectroscopy

The functional groups present in 3D-printed films were identified using FTIR spectroscopy (IRAffinity-1S, Shimadzu, Kyoto, Japan) with a Quest attenuated total reflectance (ATR) accessory. Spectra were recorded at the range of 400–4000 cm⁻¹ with a resolution of 8 cm⁻¹ and a total of 64 scans. To analyze overlapping peaks, Gaussian peak fitting was applied using OriginPro software (OriginLab Corporation, MA, USA) (Barekat and Ubeyitogullari, 2025b).

2.13. Total phenolic content

Phenolic compounds were extracted from films (5 mg) using 3 mL of 70 % ethanol for 24 h. A 0.3 mL portion of the extract was mixed with 2.5 mL of 10 % (v/v) Folin–Ciocalteu reagent, followed by the addition of 2 mL of 7.5 % (w/v) sodium carbonate solution (Kaur S and Ubeyitogullari, 2024). The mixture was incubated at 50 °C for 5 min. The absorbance was then recorded at 760 nm using a UV–vis spectrophotometer (Milton Roy Spectronic 1201, PA, USA). A standard curve was created using gallic acid solutions (0–1000 mg/L; R² = 0.99), and the results were expressed as micrograms of gallic acid equivalent per gram of film (μg GAE/g film).

2.14. Antioxidant activity

2.14.1. DPPH free radical scavenging assay

To measure antioxidant properties, 25 mg of film was soaked in 3 mL of 70 % ethanol and left overnight for extraction. Briefly, 0.3 mL of the film samples was mixed with 1 mL of 0.1 mM ethanolic DPPH solution. After incubating in the dark at 23 °C for 30 min, the absorbance was recorded at 517 nm, and results were expressed as % of DPPH radical scavenging (Barekat et al., 2023).

2.14.2. ABTS assay

ABTS radicals were prepared by mixing 7 mM ABTS with 2.45 mM potassium persulfate in a 1:1 (v/v) ratio and storing the mixture in the dark at 23 °C for 16 h. Ethanol was then added to the ABTS solution to adjust the absorbance to 0.70 ± 0.02 at 734 nm. A 40 μL of film or gallic acid standard solution (0–0.001 mg/mL) was added to 3960 μL of the ABTS solution. After incubating in the dark for 6 min, the absorbance was recorded at 734 nm. Results were expressed as μg Trolox equivalent per gram of film (μg TE/g dried film) (Kaur S and Ubeyitogullari, 2024).

2.15. Statistical analysis

All experiments were performed in triplicate, and the results are reported as mean ± standard deviation. Data was analyzed using Statistical Analysis System software (SAS, version 9.4, SAS Institute Inc., Cary, NC, USA). Statistical significance was considered at P < 0.05.

3. Results and discussion

3.1. Rheological properties

The rheological behavior of film inks indicates their suitability for 3D printing, especially during extrusion, recovery, and stability (Barekat and Ubeyitogullari, 2025a). The viscosity measurements showed that all samples’ viscosities decreased with increasing shear rate (Fig. 2a), showing shear-thinning behaviors. The flow behavior index (n; 0.19^b, 0.33^a, 0.25^b, 0.35^a) and consistency index (K; 20.68^c, 24.93_^b, 35.09^a, 35.14^a Pa·sⁿ) with R² ≥ 0.99, were determined for SPI, SPI-MA1, SPI-MA3, and SPI-MA5, respectively, where the means within the same category that do not share a common superscript letter are significantly different (P < 0.05). The consistency index (K) increased from 20.68 to 35.14 Pa·sⁿ with increasing the MA concentration, resulting in more viscous inks. Shear-thinning is necessary for continuous ink flow during extrusion to prevent clogging (Barekat and Ubeyitogullari, 2025b). In 3DFOODP, printable material should be strong enough to support the layer-by-layer deposited structure but easily flow through a nozzle with a smaller diameter during the extrusion (Ahmadzadeh et al., 2023). SPI showed the lowest viscosity, while SPI-MA5 showed the highest viscosity. As the concentration of MA was increased from 1 % to 5 % (w/w), the slope reduced, indicating a higher viscosity. Similarly, Ahmadzadeh et al. (2023) reported that the SPI inks (11 %, w/w) exhibited shear-thinning behavior. In their study, the addition of grape seed or green tea extracts decreased the viscosity due to disrupted protein–protein interactions. Polyphenols, as multidentate ligands, can bind to multiple sites on protein chains, causing them to coil and form more compact structures, which leads to reduced viscosity (Ahmadzadeh et al., 2023). However, in the current study, the addition of MA increased the viscosity. Other studies also have shown that processed microalgae and Spirulina platensis suspensions (Wang et al., 2022, 2023), when added to SPI, can improve the viscosity and gel network formation and interactions. The polysaccharides and proteins within the microalgae powder interact with the SPI network. For Spirulina, food processing can dissolve cell wall polysaccharides, which then fill the existing SPI network. This increased physical interactions and formed a denser and more compact structure in the SPI-microalgae hydrogel (Wang et al., 2022, 2023).

Fig. 2.

Rheological properties of the gels: (a) apparent viscosities of gels as a function of shear rate; (b) frequency sweeps; and (c) recovery tests. The samples were labeled as SPI, SPI-MA1, SPI-MA3, and SPI-MA5, corresponding to 11 % (w/w) soy protein gel and soy protein gel containing 1 %, 3 %, and 5 % (w/w) microalga, respectively.

The G′ is a measure of elastic solid-like behavior, that is, the resistance to elastic deformation of the sample, while the G″ is the viscous response, which is the ratio of stress to strain under vibration conditions. When G′ is greater than G″, the material behaves primarily as a solid, and vice versa for a viscous fluid, exhibiting a slight frequency dependence (Wang et al., 2024b). In all gel samples, G′ was much higher than G″, showing an elastic-dominant behavior. The bioinks showed pseudoplastic behavior, with G′ remaining higher than G″ across all frequencies (Fig 2b), confirming their gel-like nature. A higher G′ value mainly shows stronger mechanical strength, and the ink can hold its shape after printing (Liu and Zhong, 2013; Liu et al., 2017). It was also found that both G′ and G″ increased significantly (P < 0.05) as the MA concentration in PPG increased. The increase in both G′ and G″ might be due to the formation of a stronger and more rigid gel structure.

The recovery time provides the time it takes for the extruded material to regain the initial viscosity, which can be evaluated by altering the shear rate using a rheometer. Greater printing fidelity could be achieved with materials that naturally recover quickly (Xie, 2023). For all samples, viscosity dropped sharply after 204 s and then rose after 318 s (Fig. 2c). This simulates the ink flowing through the nozzle and confirms the shear-thinning behavior (Sahu et al., 2021). When the shear or force was removed, like the ink being deposited, the gels began to recover their initial viscosity. All the SPI and SPI-MA samples showed good recovery, quickly returning to their viscosity (Fig. 2c).

3.2. 3D printability

As shown in Fig. 3, the concentration of MA and the nozzle size had a great impact on 3D printability. By increasing the concentration of MA from 1 to 5 % (w/w), the color became darker (as expected), and the amount of printed sample increased. It is clear in Table 2 that by increasing the MA concentration, the thickness of the films increased. Ahmadzadeh et al. (2023) reported that adding grape seed (GS) or green tea (GT) extracts to SPI films significantly increased film thickness, with GS having a greater impact than GT. They explained this increase by the interaction between high molecular weight phenolic compounds in the extracts and SPI molecules, mainly through hydrogen bonding and hydrophobic interactions. These interactions changed the film structure, resulting in thicker films (Ahmadzadeh et al., 2023). However, in the current study, by evaluating and optimizing the printing parameters, a similar thickness to the digital model was achieved, even by adding MA (Table 2).

Fig. 3.

Images of 3D-printed films at printing speeds of 10 and 20 mm/s using nozzle sizes of 0.52, 0.64, 0.72, and 0.81 mm. The film (F) samples were labeled as SPI-F, SPI-MA1-F, SPI-MA3-F, and SPI-MA5-F, corresponding to 11 % (w/w) soy protein gel and soy protein gel containing 1 %, 3 %, and 5 % (w/w) microalga, respectively. All the images have the same scale bar as given in the first image.

Table 2.

The dimensions of the digital 3D model and the 3D-printed films at different printing speeds of 10 and 20 mm/s and nozzle sizes of 0.52–0.81 mm. The film (F) samples were labeled as SPI-F, SPI-MA1-F, SPI-MA3-F, and SPI-MA5-F, corresponding to 11 % (w/w) soy protein gel and soy protein gel containing 1 %, 3 %, and 5 % (w/w) microalga, respectively.

Sample	Nozzle size (mm)	Printing speed (mm/s)	Length (cm)	Area (cm2)	Thickness (mm)
Model			5.00	25.00	0.25
SPI-F	0.64	10	–	–	0.15f±0.00
	0.64	20	–	–	0.15fg ± 0.02
	0.72	10	4.90a±0.15	24.49abc±0.58	0.27d ± 0.00
	0.72	20	4.89a±0.05	24.83abc±0.67	0.25de ± 0.01
	0.81	10	5.03a±0.05	25.67a±0.38	0.52a±0.02
	0.81	20	5.09a±0.08	26.20a±0.30	0.57a±0.07
SPI-MA1-F	0.64	10	4.80a±0.03	–	0.12fg ± 0.04
	0.64	20	4.94a±0.15	25.41a±0.54	0.16f±0.02
	0.72	10	4.89a±0.09	24.15c±0.15	0.26de ± 0.13
	0.72	20	4.99a±0.16	25.05b ± 0.00	0.25de ± 0.04
	0.81	10	4.85a±0.09	23.17c±0.38	0.46b ± 0.01
	0.81	20	5.20a±0.19	25.67a±0.84	0.47b ± 0.04
SPI-MA3-F	0.64	10	4.98a±0.10	–	0.14g ± 0.00
	0.64	20	-	–	0.14fg ± 0.05
	0.72	10	4.84a±0.05	24.07c±0.50	0.25de ± 0.01
	0.72	20	4.97a±0.06	24.91bc±0.39	0.25e±0.00
	0.81	10	4.97a±0.11	24.66bc±0.53	0.42b ± 0.01
	0.81	20	5.12a±0.16	24.70bc±0.49	0.44bc±0.06
SPI-MA5-F	0.64	10	–	–	–
	0.64	20	–	–	0.17f±0.05
	0.72	10	4.92a±0.05	23.74c±0.15	0.27de ± 0.03
	0.72	20	4.92 a±0.06	24.33bc±0.25	0.28d ± 0.02
	0.81	10	4.85a±0.06	24.16bc±0.63	0.40bc±0.03
	0.81	20	4.88 a±0.05	23.99c±0.14	0.39c±0.01

∗Means in the same column that do not share a common superscript letter are significantly different (P < 0.05).

Nozzle diameter is the other main factor affecting the printing process and should be optimized for 3D structure accuracy (Tian et al., 2024). Nozzle size significantly affected the 3D printability of films, where with the lowest size (0.52 mm), printing was not possible, and after printing the first layer, the nozzle clogged, and the printing was not completed. SPI-F, SPI-MA1-F, and SPI-MA3-F were printable for the first layer using a nozzle size of 0.64 mm. For SPI-MA5-F and SPI-MA3-F at a speed of 20 mm/s, printing stopped due to nozzle clogging. This might be attributed to the increased flow behavior of gels; as MA increased, viscosity also increased (Fig. 2andFig. 3), requiring higher pressure for extrusion. For successful 3D printing, layers must be deposited one-by-one. When the flow stops even for a second, the printing of bioink gets interrupted as described by Barekat and Ubeyitogullari (2025a) for sorghum and soy gels. This is the main reason why nozzle size strongly affects printability (Karabulut and Goksen, 2025).

Printing speed, which combines extrusion and filling speeds, is key to evaluating material behavior in 3D printing (Tian et al., 2024). At the higher speed of 20 mm/s, the flow rate for creating the shape was faster, and printing became easier. However, the speed should not be too high, as the flow needs sufficient time to form the printed shape and support the next layer. It has been explained in several studies that high extrusion with low filling speeds can cause excess material extrusion, making shape accuracy difficult (Barekat and Ubeyitogullari, 2025a; Tian et al., 2024; Dankar et al., 2018). Conversely, low extrusion and high filling speeds may cause filament discontinuities, risking print collapse (Barekat and Ubeyitogullari, 2025a; Tian et al., 2024; Dankar et al., 2018). This might be the reason why the sample with a 0.52 mm nozzle size printed better at a speed of 20 mm/s. While at a 0.64 mm nozzle size, printing was not successful, as the flow was too fast and there was not enough time for the printed ink to remain close enough to form the square shape. With nozzle sizes of 0.72 and 0.81 mm, the final layer was successfully printed, demonstrating the suitability of these sizes. This was probably because of the high ink flow at these nozzle sizes, and so the importance of printing speed was not significant. However, increasing the speed of printing ensures shorter print time. So, it is preferred to have a higher printing speed, as printing speed and printing time are inversely proportional (Tian et al., 2024). Barrios-Rodríguez et al. (2024) also reported that for the 3D printing of rice protein, print speed alone did not significantly affect printability (P > 0.05). However, its interaction with layer height and nozzle diameter influenced sample weight, print time, and mass flow (Barrios-Rodríguez et al., 2024). Between 0.72 mm and 0.81 mm, the smaller nozzle size was better as it showed a closer thickness to the digital model. At the nozzle size of 0.81 mm, the high amount of ink released resulted in an increased thickness of the printed object (>0.39 mm). This was more than 56 % of the thickness of the digital model. So, by considering Fig. 3 and Table 2, the concentration of 3 % MA, nozzle size of 0.72 mm, and printing speed of 20 mm/s were selected as optimized conditions in view of 3D printability.

The effects of processing parameters, such as nozzle diameter and nozzle movement speed, and their combination on print quality also depend on the bioink type, its flow behavior, and final geometry (Barrios-Rodríguez et al., 2024; Yang et al., 2018; Hao et al., 2010). In our previous study, the optimum condition for 3D printing of the SPI layer of the final 4-layered sorghum protein gel– SPI gel (11 %w/w) in cubic shape, was a nozzle size of 0.52 mm and a speed of 10 mm/s. In that study, the effect of gel type (sorghum or SPI) used for printing the first layer was evaluated. When the sorghum gel was the first layer for printing, the 0.52 mm nozzle size created an accurate layer with well-defined edges and corners of the structure. The speed of 10 mm/s was suitable for drying of the sorghum layer and holding the shape for the SPI layer. This shows that the final shape, bioink type, and 3D structures can all influence the printability (Barekat and Ubeyitogullari, 2025a).

3.3. Color

Color and transparency are known as one of the most important attributes of both edible and non-edible packages that greatly influence their consumer acceptability and product stability (Dey et al., 2022; Dakhili et al., 2025). The color of the SPI film was significantly different from the SPI-MA in films, as expected (Fig. 4). SPI-F had the highest L∗ value (82.40) and a small negative a∗ value (−2.44) with a low b∗ value (11.51). These showed that SPI was light in color and looked light yellow. Ahmadzadeh et al. (2023) similarly reported values of L∗ = 90.83, a∗ = 3.19, and b∗ = 10.67 for a 11 % SPI film. The L∗ value in their study decreased significantly with adding grape seed or green tea extracts. They noted that the addition of extracts in film can scatter light, contributing to this darkening effect (Ahmadzadeh et al., 2023).

Fig. 4.

The color and appearance of dried 3D-printed films. The film (F) samples were labeled as SPI-F, SPI-MA1-F, SPI-MA3-F, and SPI-MA5-F, corresponding to 11 % (w/w) soy protein gel and soy protein gel containing 1 %, 3 %, and 5 % (w/w) microalga, respectively.

SPI-MA-F samples all had much larger negative a∗ values (−40.56, −45.89, and −51.89), resulting in a visually green color (Table 3). At the same time, their L∗ values became lower (50.70, 29.21, and 20.21), so the color became darker from 1 % MA to 5 % MA. The b∗ values for these samples (70.24, 74.32, and 77.32) were all positive and high, which shows that the green colors also had a yellow tone. The ΔE∗ values were 39.27, 60.10, and 71.63, showing significant differences in color between the samples. Mondal et al. (2022) evaluated the color of ultrasound-assisted extracts from de-oiled algae biomass residue. The color of the extract was L∗ = 72.62 ± 0.04, a∗ = −14.1 ± 0.01, and b∗ = 59.29 ± 0.05. The authors explained that the negative value to the green color came from chlorophyll, while the high positive b∗ value indicated a yellow hue due to carotenoids (β-carotene) pigments in green algae (Mondal et al., 2022). Carissimi et al. (2018) developed biodegradable 4 % cassava starch films blended with 0–2.0 % (w/w) extracts from Heterochlorella luteoviridis and Dunaliella tertiolecta. Microalgae addition produced darker, less transparent films with lower L∗ and higher opacity, effectively blocking UV and visible light (Carissimi et al., 2018). Fabra et al. (2017) found that adding 400 mg/L Nannocloropsis gaditana to 2 % (w/w) corn starch films reduced transparency and gave a green hue. While this limits some food-packaging uses, the films fully block UVB and UVC radiation, protecting light-sensitive components in foods (Fabra et al., 2017). Dakhili et al. (2025) also explained that the algae addition greatly improves films’ UV and visible light protection but reduces transparency. These trade-off limits are used for visibility-critical products, but the benefits of packaging include light and oxygen shielding (Dakhili et al., 2025).

Table 3.

Color parameters of the 3D-printed films. The film (F) samples were labeled as SPI-F, SPI-MA1-F, SPI-MA3-F, and SPI-MA5-F, corresponding to 11 % (w/w) soy protein gel and soy protein gel containing 1 %, 3 %, and 5 % (w/w) microalga, respectively.

Sample	L∗	a∗	b∗	ΔE
SPI-F	92.40a±1.54	−2.44d ± 0.04	11.51d ± 1.98	–
SPI-MA1-F	50.70b ± 3.09	−40.56c±1.34	70.24c±0.70	39.27c±2.41
SPI-MA3-F	29.21c±2.23	−45.89b ± 0.76	74.32b ± 1.09	60.10b ± 1.50
SPI-MA5-F	20.21d ± 0.54	−51.89a±1.00	77.32a±0.98	71.63a±0.87

∗Means in the same column that do not share a common superscript letter are significantly different (P < 0.05).

3.4. Mechanical properties

Mechanical flexibility and strength are important for the fabrication of food packaging systems (Ahmadzadeh et al., 2023). EB values showed a notable decrease in flexibility with increasing MA concentrations in SPI films (Table 4). The SPI-film exhibited the highest EB (122.93 %), suggesting a relatively ductile network structure. Addition of 1 % and 3 % MA reduced EB to 103.21 % and 99.51 %, respectively, while 5 % MA caused a reduction to 70 %. This decrease showed that MA components might be due to crosslinking and the formation of a more rigid matrix. In contrast, TS exhibited an increase with MA addition. The SPI-F showed a TS of 6.42 MPa, whereas SPI-MA1-F, SPI-MA3-F, and SPI-MA5-F reached 9.11 MPa, 11.71 MPa, and 13.77 MPa, respectively. This trend might be due to enhanced intermolecular interactions, such as hydrogen bonding between MA polysaccharides and soy protein chains. These interactions may lead to a denser, more continuous network, resulting in a higher TS (Ahmadzadeh et al., 2023; Dakhili et al., 2025). Similarly, PS values increased with MA concentration, from 6.33 N in the SPI-F to 8.01 N at SPI-MA5-F. Increased PS is advantageous for applications where resistance to mechanical damage is required, especially for packaging and handling products. The mechanical parameters of SPI-F were in good agreement with the results reported in a previous study for 3D-printed soy protein films (Ahmadzadeh et al., 2023; Dey et al., 2022). Ahmadzadeh et al. (2023) reported values for SPI films of 117.93 % EB, 4.62 ± 0.61 MPa TS, and 58.63 N PS (), which are similar to the present results (Table 4).

Table 4.

Tensile strength (TS), elongation at break (EB), and puncture strength (PS) of the 3D-printed films. The film (F) samples were labeled as SPI-F, SPI-MA1-F, SPI-MA3-F, and SPI-MA5-F, corresponding to 11 % (w/w) soy protein gel and soy protein gel containing 1 %, 3 %, and 5 % (w/w) microalga, respectively.

Sample	EB (%)	TS (MPa)	PS (N)
SPI-F	122.93a ± 9.90	6.42d ± 0.31	6.33c ± 0.22
SPI-MA1-F	103.21b ± 8.22	9.11c ± 1.12	7.21b ± 0.20
SPI-MA3-F	99.51b ± 10.01	11.71b ± 0.20	7.31b ± 0.8
SPI-MA5-F	70.00c±8.22	13.77a ± 1.12	8.01a ± 1.20

∗Means in the same column that do not share a common superscript letter are significantly different (P < 0.05).

Dakhili et al. (2025) explained that the addition of microalgae to biodegradable films such as cassava starch and proteins increased film flexibility and hydrophilicity, whereas it enhanced TS but reduced extensibility. These effects depend on the microalgae type and concentration. This provided the possibility to tailor edible film properties for specific applications (Dakhili et al., 2025). Other studies also confirm that adding different types and concentrations of microalgae altered film mechanical properties. In rice bran protein films (5.0 %), increasing red algae/gelatin blend caused up to 3.0 % enhanced TS but reduced EB (Shin et al., 2011). In persimmon peel (PP) films with red algae, the best formulation was achieved with a 4 % PP/1 % red algae composite, resulting in a TS of 7.31 MPa, EB of 8.16 %, and WVP of 4.99 ng m/m²·s·Pa. However, increasing the algae content to 2.0 % reduced TS to 3.32 MPa, while EB increased significantly (Jo et al., 2014). Kontogianni et al. (2022) formed edible whey protein concentrate (WPC) films containing Spirulina and found that higher Spirulina concentration increased TS, but reduced EB compared to the control. This effect is attributed to strong interactions between algae components (proteins, lipids, etc.) and WVP functional groups, producing a more rigid film structure (Kontogianni et al., 2022). All these previous studies used the casting method to form the films, with no reports on 3D-printed microalgae-based films.

3.5. WVP properties

WVP is a critical indicator of film's ability to act as a moisture barrier (Ahmadzadeh et al., 2023). The measured WVP values for the SPI and the SPI-MA films are presented in Table 5. The SPI film exhibited the highest WVP (7.20 × 10⁻¹⁰ g m⁻¹·s⁻¹·Pa⁻¹), indicating a relatively high permeability to water vapor (Ahmadzadeh et al., 2023). Addition of MA into the SPI film resulted in an increasing reduction in WVP: 5.01 × 10⁻¹⁰ for 1 % MA, 4.93 × 10⁻¹⁰ for 3 % MA, and 4.80 × 10⁻¹⁰ for 5 % MA. The observed decrease in WVP with increasing MA content might be due to structural and compositional changes within the film matrix. MA contains polysaccharides and phenolics capable of forming additional hydrogen bonds with soy proteins, resulting in a more compact, crosslinked, and less porous structure (Jo et al., 2014). This is in accordance with the results obtained for the microstructure of films, where the porosity of films decreased with increasing the concentration of MA in a film formulation. Also, these changes are likely due to the structure of the microalgal components, protein interactions and polysaccharides form a denser, more rigid network, which makes the film stronger and better at blocking moisture, but less flexible. Ahmadzadeh et al. (2023) reported the SPI film had a WVP of 9.57 × 10⁻⁶ g m⁻¹ s⁻¹ Pa⁻¹, where adding grape seed or green tea extracts reduced WVP. The 1 % extracts reduced WVP by 53 % (GS) and 43 % (GT), and 5 % extracts by around 60 % (GS) and 56 % (GT) (Ahmadzadeh et al., 2023), due to interactions between SPI and the phenolics in extracts (Jo et al., 2014). Additionally, an improvement in moisture barrier performance was observed with changes in mechanical properties. As alga content increased, TS and PS increased, while EB decreased. Similar correlations between increased rigidity and enhanced barrier properties have been reported in previous studies for algae-starch films (Dakhili et al., 2025; Carissimi et al., 2018).

Table 5.

Water vapor permeability (WVP) of the 3D-printed SPI films. The film (F) samples were labeled as SPI-F, SPI-MA1-F, SPI-MA3-F, and SPI-MA5-F, corresponding to 11 % (w/w) soy protein gel and soy protein gel containing 1 %, 3 %, and 5 % (w/w) microalga, respectively.

Sample	WVP ( × 10-10 g m−1 s−1 Pa−1)
SPI-F	7.20a ± 0.90
SPI-MA1-F	5.01b ± 0.12
SPI-MA3-F	4.93c ±0.01
SPI-MA5-F	4.80c ±0.12

∗Means in the same column that do not share a common superscript letter are significantly different (P < 0.05).

3.6. Microstructure

SEM images of films from the surface and cross-section of the 3D-printed samples are shown in Fig. 5. The 3D-printed films showed a uniform surface without any cracks and large bubbles, indicating a high accuracy and rigidity of the 3D printing process and drying of films. At the lower magnification, the accuracy of 3D printing was confirmed as all layers were printed, and no gap was found between the layers. The thickness of films was aligned with the data reported in Table 2. At higher magnification ( × 4000), the microstructure was accessible with more details, where the porosity and the gel network were obviously shown. By adding and increasing the MA in the film's formulation, the porosity was reduced, which showed the formation of a stronger gel network and was aligned with the results obtained for rheological properties, mechanical properties, and WVP. MA might have bound to the hydrophobic side chains of protein, resulting in chain entanglement between nearby molecules, leading to more compact microstructures of the SPI films (Ahmadzadeh et al., 2023). Previously, Mikus et al. (2021) and Ahmadzadeh et al. (2023) also reported that the SPI film (5 % and 11 % w/w, respectively) had a homogeneous and smooth surface.

Fig. 5.

SEM images taken from surfaces and cross-sections of the dried 3D-printed films. The film (F) samples were labeled as SPI-F, SPI-MA1-F, SPI-MA3-F, and SPI-MA5-F, corresponding to 11 % (w/w) soy protein gel and soy protein gel containing 1 %, 3 %, and 5 % (w/w) microalga, respectively.

3.7. FTIR spectra

FTIR spectroscopy was used to understand the secondary structure and molecular interactions within the SPI and MA 3D-printed films. As shown in Fig. 6, all samples exhibited major absorbance bands between 2000 and 1000 cm⁻¹. The 900–1200 cm⁻¹ broad bands of C–O–C and C–O stretching vibrations are found in polysaccharides, indicating the presence of carbohydrate structures from MA, and phosphate-related signals from MA biomolecules (Augusto et al., 2024).

Fig. 6.

FTIR spectra of 3D-printed dried films. The film(F) samples were labeled as SPI-F, SPI-MA1-F, SPI-MA3-F, and SPI-MA5-F, corresponding to 11 % (w/w) soy protein gel and soy protein gel containing 1 %, 3 %, and 5 % (w/w) microalga, respectively.

The broad band in the 2900–3500 cm⁻¹ region is linked to O–H stretching vibrations of hydroxyl groups and bound water, N–H stretching of amide A in proteins, and C–H stretching of aliphatic –CH₂/–CH₃ groups. The intensity of this band increased with the addition of MA. This might be due to the presence of MA's polysaccharides rich in hydroxyl groups, as well as additional aliphatic structures from lipids and pigments (Abdel-Latif et al., 2022). Additionally, by increasing the concentration of MA in film formulation, from 1 % w/w to 5 % w/w, the absorbance of bands increased (Fig. 6), which might show the interaction of SPI and MA.

FTIR is a widely used technique for evaluating the structure of proteins by the analysis of the amide I and amide II regions. The amide I band (1700–1600 cm⁻¹) arises from the C=O stretching vibrations. The amide II band (around 1550 cm⁻¹) is associated with N–H bending and C–N stretching vibrations. The amide I region is using for identifying secondary structures, including α-helices (1650–1660 cm⁻¹), β-sheets (1660–1690 cm⁻¹, 1630–1638 cm⁻¹, 1620–1630 cm⁻¹), intermolecular β-sheet aggregates (1690–1695 cm⁻¹, 1610–1620 cm⁻¹), and random coils (1640–1648 cm⁻¹). The α-helix is a right-handed coiled structure, and it is stabilized by hydrogen bonding between the carbonyl (C=O) and amide (N–H) groups along the polypeptide backbone. However, β-sheets with a sheet-like arrangement are stabilized by hydrogen bonds that form between neighboring strands (Barekat and Ubeyitogullari, 2025a, 2025b).

Previous studies have also shown that the protein secondary structures might be related to the mechanical properties of protein-based films (Htut et al., 2021). The combination of original FTIR spectra and the deconvoluted spectra of the amide I band (1700–1600 cm−1) was used for curve fitting (Fig. 7a). Detailed analysis of the deconvoluted spectra showed changes in percentages of α-helix, random coil. and β-aggregate (Fig. 7b). The addition of MA into SPI increased both α-helix and β-sheet/β-aggregate contents, accompanied by a reduction in random coil structures. The small rise in β-turn was also observed, which might show local chain rearrangements of the protein network (Barekat and Ubeyitogullari, 2025a). The hydroxyl-rich polysaccharides present in MA might interact with SPI via hydrogen bonding and electrostatic forces, stabilizing existing secondary structures. This might cause the folding of flexible coil regions into α-helices and β-sheets. Also, MA contains 52 % protein, and protein-protein interactions (e.g., hydrogen bonding, electrostatic interactions, hydrophobic interactions, and van der Waals forces) might affect this transition. The combined increase in α-helix, β-sheet/β-aggregate and β-turns with the reduction in random coil might confirm this possibility that the MA addition produces a denser and more ordered film network. Dionne et al. (2017) and Htut et al. (2021) reported that the higher β-sheet and aggregate content are linked to higher stiffness but lower stretchability, while higher unordered content is linked to lower stiffness but higher stretchability.

Fig. 7.

(a) The peak-fitted FTIR spectra of samples in the range of 1600–1700 cm⁻¹; (b) the secondary structural components. The film (F) samples were labeled as SPI-F, SPI-MA1-F, SPI-MA3-F, and SPI-MA5-F, corresponding to 11 % (w/w) soy protein gel and soy protein gel containing 1 %, 3 %, and 5 % (w/w) microalga, respectively.

3.8. TPC and antioxidant properties

The antioxidant properties and TPC of films are given in Table 6. The SPI-F exhibited a TPC of 908.38 μg GAE/g. The addition of MA into the film matrix significantly increased TPC values, with the highest value observed at 5 % microalgae (1920 μg GAE/g). This was more than 2 times the SPI-F's TPC. This increase might be attributed to the rich phenolic profile of C. vulgaris, which contains various bioactive compounds such as flavonoids, phenolic acids (gallic acid), β-carotene, and other antioxidants (Mondal et al., 2022; Zainal-Abidin et al., 2024).

Table 6.

Total phenolic content (TPC), DPPH, and ABTS antioxidant activities of the 3D-printed films. The film (F) samples were labeled as SPI-F, SPI-MA1-F, SPI-MA3-F, and SPI-MA5-F, corresponding to 11 % (w/w) soy protein gel and soy protein gel containing 1 %, 3 %, and 5 % (w/w) microalga, respectively.

Sample	TPC (μg GAE/g dried film)	DPPH (%)	ABTS (μg TE/g dried film)
SPI-F	908.38d ± 4.9	22.34d ± 1.3	2020d ± 30.67
SPI-MA1-F	1509c ±23.12	31.54c ± 3.5	4200c ± 44.30
SPI-MA3-F	1640b ± 14.21	37.76b ± 1.8	5356b ± 45.03
SPI-MA5-F	1920a ±16.54	42.34a ± 1.9	6478a ± 24.87

∗Means in the same column that do not share a common superscript letter are significantly different (P < 0.05).

The DPPH scavenging activity increased from 22.34 % for SPI-F to 42.34 % for SPI-MA5-F. The ABTS assay showed the antioxidant properties by measuring the ability to quench the ABTS•+ radicals (Barekat et al., 2023). Similar to DPPH, ABTS scavenging activity increased with increasing MA content, rose from 2020 μg TE/g in the SPI to 6478 μg TE/g at 5 % MA concentration. The antioxidant properties of MA have been reported in several studies (Mondal et al., 2022; Zainal-Abidin et al., 2024; Ferdous et al., 2023). Fassi Fihri et al. (2024) reported that C. vulgaris extracts obtained using a deep eutectic solvent exhibited inhibitions ranging from 20.59 % to 90.49 %, indicating notable antioxidant potential (Fassi Fihri et al., 2024). The higher ABTS values further demonstrate enhanced radical scavenging potential associated with phenolics. This trend for antioxidant properties aligned with the increase in TPC, confirming that phenolic compounds in MA contributed significantly to the antioxidant capacity. Kontogianni et al. (2022) reported that the control whey protein film resulted in a radical scavenging activity of 26.41 ± 3.45. In comparison, films containing Spirulina showed activities that were approximately 1.34, 1.09, 1.10, and 1.13 times higher than the control, at Spirulina concentrations of 2, 4, 6, and 8 %, respectively (Kontogianni et al., 2022). Mondal et al. (2022) also reported that increasing the concentration of crude MA extract from 0 to 28 % in biodegradable chitosan-based films increased TPC more than 38 times and antioxidant properties more than 48 % (Mondal et al., 2022).

4. Conclusions

This study optimized the 3D printing conditions for soy protein isolate-C. Vulgaris edible films, and evaluated their mechanical, structural, and antioxidant properties. The optimal printing, with the highest similarity to the digital design, was achieved using a nozzle size of 0.72 mm and a printing speed of 20 mm/s. All film gels showed shear-thinning behavior and high recovery (51–65 %). The addition of MA changed the appearance and chemomechanical properties of the SPI films. The SPI-MA-F were less transparent, with improved mechanical and WVP properties compared to SPI-F. Microalgae incorporation improved gel strength and puncture resistance at the expense of flexibility. The addition of MA, by altering the microstructure, made the films less permeable to water vapor due to reduced porosity in the films. In addition, the phenolic content and antioxidant properties of edible films with 3 % MA increased by 44 %, and 41–62 %, respectively. Overall, this study describes an approach utilizing sustainable food-grade materials for fabricating edible films to overcome the limitations of traditional film fabrication methods. It allows precise layer-by-layer deposition, reducing material waste and enabling formulations with lower additive content while achieving the desired film properties. Furthermore, it provides novel ink for 3D printing of eco-friendly and active packaging materials.

Author contribution statement

Sorour Barekat: Methodology, Validation, Formal Analysis, Writing – Original Draft– Review & Editing, Visualization. Buse Dogan: Methodology, Writing – Review & Editing. Sibel Uzuner: Methodology, Writing – Review & Editing. Ali Ubeyitogullari: Conceptualization, Methodology, Supervision, Validation, Writing – Review & Editing, Project Administration, Funding Acquisition, Resources.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.