Spirulina-Incorporated Biopolymer Films for Antioxidant Food Packaging

Abstract

Growing environmental concerns and the need for sustainable materials have accelerated the search for biodegradable alternatives to food packaging. Since nearly half of global plastic production is dedicated to food packaging, and less than 5% is recyclable, developing eco-friendly solutions is urgent. Biopolymeric films enriched with microalgae and cyanobacteria have emerged as promising options due to their bioactive properties. This study screened 38 film-forming formulations combining different biopolymers with varying concentrations of Spirulina (0–5%) to identify the most suitable candidates based on physical and visual characteristics. Films produced with pectin and hydroxypropylmethylcellulose (HPMC) matrices were selected for detailed characterization, including physicochemical, optical, mechanical, thermal, barrier, surface, and functional group analyses, as well as antioxidant activity. The highest elongation at break (%) was observed in the control HPMC film (16.5 ± 3.85), whereas the lowest value was recorded for the pectin film containing 1% Spirulina (2.75 ± 0.49). In parallel, the highest thickness (mm) was found in the pectin film with 5% Spirulina (0.153 ± 0.018), while the lowest thickness occurred in the HPMC film incorporating 1% biomass (0.076 ± 0.004). The incorporation of Spirulina decreased solubility and moisture content while increasing opacity. HPMC-based films demonstrated superior mechanical strength, thermal stability, barrier performance, and significantly higher antioxidant activity compared to pectin films. Antioxidant activity increased with biomass concentration, peaking at 5% (HPMC: 320.08 ± 35.7 µmol TE/g; pectin: 36.92 ± 7.63 µmol TE/g). Overall, the HPMC film containing 1% Spirulina showed the best balance of properties, including mechanical behavior and antioxidant performance, indicating strong potential for food packaging applications, particularly for protecting light-sensitive and oxidation-prone foods.

1. Introduction

Conventional plastics offer attractive features for the food industry, particularly their low cost [1], fluidity, flexibility, and profitability [2]. Food packaging is specifically designed to preserve food quality after processing and to provide protection against environmental contamination, mechanical shocks, light, and gases [3].

However, most of the plastics used in the manufacture of this packaging come from non-renewable sources such as petroleum, negatively impacting the environment, promoting pollution [4], waste generation [2], and ending up in landfills or the oceans at the end of their use. In 2018, almost 360 million tons of plastic were produced [5], of which around 47% went into food packaging, and less than 5% of the material used was recyclable [6]. In addition, around 79% of its total disposal ends up in open landfills, and it can also reach marine environments [7] and take up to 2500 years to degrade [8], affecting more than 6000 living organisms by ingesting plastic waste, thereby directly impacting the environmental balance [9]. Furthermore, microplastics and nanoplastics formed from plastic degradation can have adverse effects on human health due to their toxicity. Given the large amount of plastic waste in the environment, recent studies have detected microplastics in the bloodstream and human breast milk [8].

Society’s growing concern over the environmental risks associated with non-sustainable materials has led to the implementation of public policies in various countries to encourage recycling [10] and the production of biodegradable packaging [6]. Biopolymers, a unique class of polymers, are considered biocompatible and are synthesized from renewable resources [11]. The use of bioplastics has several advantages, especially regarding environmental impacts, reducing plastic waste accumulation, and preserving marine ecosystems, due to their biodegradability and recyclability [12]. In addition, during packaging manufacturing, there are lower levels of polluting gas emissions due to lower energy use [9,13,14]. Consequently, the use of biopolymers could be a promising alternative for our current scenario.

Several studies have explored the use of various compounds to develop films for food packaging, such as chitosan [15], microalgae, cyanobacteria [16], okra and psyllium mucilage [17,18], psyllium, locust bean, tara, durian seed, mesquite seed and cassia seed gums [6,19,20], pectin [21,22], hydroxypropylmethylcellulose [23,24], arrowroot starch [25,26] and psyllium [18,27].

In addition to being made from biodegradable materials, biopolymeric films can perform specific functions, thereby classifying them as active and/or smart packaging. Active packaging includes mechanisms that extend the shelf life of food by incorporating gas adsorption and release systems, as well as antimicrobial and antioxidant agents [28]. It also provides real-time verification of food quality by adding intelligent devices, such as data carriers (barcode and radio frequency identification labels) [19], time/temperature indicators, pH, gas, and biosensors.

Polysaccharides are commonly used in film formulation precisely because they have good film-forming properties, due to their viscosity [29] and gel-forming capacity [30], and are non-toxic and widely distributed in the environment [31]. Starch, gums, pectin, cellulose, and their derivatives are widely used to produce films and coatings [32]. Pectin, a secondary product of fruit juice and sunflower oil, is present in a variety of fruits, such as apples, peaches, and plums, and can form gels, one of the main factors enabling its use in food packaging [33]. Hydroxypropylmethylcellulose (HPMC), a natural compound derived from cellulose that has good gas barrier properties, resistance to lipids, transparency, and tastelessness [23], has been used in several studies [23,34]. Starch has been used since 1980 for the production of biodegradable matrices due to its relevant characteristics, such as low cost, film-forming capacity, and abundance in nature [35]. Psyllium is a plant of the genus Plantago, cultivated worldwide for its ability to grow in diverse extreme environments, making it an abundant and economical food source [17]. They are widely used in the food industry because of their stabilizing, thickening, structuring, and gel-forming properties, which are related to their high water-binding capacity [18]. With their distinct and wide-ranging functionalities, they have been gaining prominence in the packaging industry.

Incorporating natural antioxidants into food packaging offers numerous advantages, including extending the product’s shelf life and improving preservation. Limnospira, commonly known as Spirulina, is a blue-green, photosynthetic filamentous cyanobacterium [36] with a rich nutritional composition, including essential amino acids, polyunsaturated fatty acids, vitamins, and minerals [37]. Furthermore, several biological effects have been reported from the use of this biomass, among which stand out its antimicrobial, antioxidant, and anti-inflammatory functions [38], and it is also recognized as a Safe food by the Food and Drug Administration (FDA) [37,39,40,41]. The antioxidant and antimicrobial activity of Spirulina biomass, mainly due to the presence of phenolic compounds and pigments, is relevant for use in food products, as food is susceptible to lipid oxidation and to the presence of pathogenic or deteriorative bacteria. These compounds act by preventing lipid oxidation, reducing the formation of free radicals that can cause undesirable sensory changes in food, and extending the product’s shelf life [42].

Given the diverse properties reported for Spirulina, microalgae can also be strong candidates as thickening and emulsifying agents in food film formulations, mainly due to their protein and polysaccharide composition. Despite being an area that is still little explored [43], several studies have used Spirulina, either the biomass or its extract, to verify its behavior when incorporated into different polymeric matrices, as well as to observe the functionality of the films in relation to their bioactive properties and the possibility of application in smart films [42,44,45].

To provide new alternatives to conventional plastics and to propose packaging options with antioxidant activity, this work aimed to screen and evaluate the behavior of different polysaccharides (psyllium, arrowroot starch, pectin, and HPMC) in the formulation of films combined with integral and Spirulina biomass concentrate. Additionally, the study sought to provide relevant information on the interactions between these compounds and to characterize the films that demonstrated the best physical, visual, and compatibility properties.

2. Materials and Methods

2.1. Materials

The commercial dry organic biomass of the microalga Spirulina (Limnospira platensis) was donated by Fazenda Tamanduá and stored in a deep freezer (−40 °C). HPMC (molar weight ~90,000 Da) was purchased from Sigma-Aldrich Chemical (St. Louis, MO, USA). The pectin (derived from citrus fruits, with a degree of esterification ranging from 58% to 62%) was donated by Shandong Andre Co., Ltd., Weifang, China. The arrowroot starch was purchased by Torres Alimentos LTDA (Cidade Líder, São Paulo, Brazil). Psyllium flour was obtained from Ocean Drop Comércio LTDA (Praia Brava, Itajaí/Santa Catarina, Brazil).

2.2. Characterization of Spirulina Biomass and Biomass Concentrate

The proximate composition of the Spirulina biomass, as well as the biomass concentrate, herein designated as SpiruPower^® by our research group, was determined in triplicate according to the methodology of the Association of Official Analytical Chemists (AOAC) [46]. The difference in weight gave the moisture content after heating at 105 °C until reaching a constant weight; the ash content was obtained after incineration at 550 °C in a muffle; the fat content was determined using the Rose-Gottlieb method [46]; the protein content was analyzed using the micro-Kjeldahl method, with a nitrogen conversion factor of 6.38 [46]; pH values were determined using a digital potentiometer directly on the samples.

In addition, the phenolic compounds were extracted with methanol/water (8:2) [47] from the freeze-dried samples. The content of total phenolic compounds was determined using the Folin–Ciocalteu method, according to the procedure of Singleton & Rossi [48] and expressed as gallic acid equivalents (GAA)/100 g.

To obtain the biomass concentrate remaining after C-PC extraction from Spirulina (Figure 1A), the frozen biomass was thawed, milled in an analytical mill (IKA A11 Basic), and sieved through a 106/Tyler 150-mesh screen to ensure uniform particle size [49].

In triplicate, 8 g of the material was transferred to an Erlenmeyer flask, and 50 mL of distilled water was added. The mixture was kept at room temperature and protected from light for 1 h. Subsequently, the suspension was centrifuged at 3040× g for 15 min across four cycles. The supernatant was then separated from the resulting biomass concentrate (SpiruPower^®) according to the procedure described by Fratelli et al. [49]. SpiruPower^®, a nutrient-rich ingredient developed by our research group and incorporated into various food products since 2022 [50,51,52,53], was stored at −40 °C. Before its use as a food ingredient, the biomass concentrate was freeze-dried, milled again using the analytical mill, and sieved through a 106/Tyler 150 mesh. Because water was the sole solvent employed during C-PC extraction, SpiruPower^® remains safe for direct application as a food ingredient.

2.3. Preparation of Film Formulations

2.3.1. Psyllium Flour Films

To formulate the psyllium control films, psyllium flour was first added in different concentrations (1, 1.5, 2, and 3%) (g/mL) in distilled water heated to 80 °C. They were homogenized in a mechanical shaker (Quimis-Q250M2, Diadema, Brazil) for 10 min at 1000 rpm. After this period, glycerol in different concentrations (20, 30, and 40%) was added to the film solution and homogenized using a mechanical stirrer for 5 min at 1000 rpm. 25 mL of the solution was spread onto Petri dishes and dried in a forced-air circulation oven at 35 °C for 24 h.

After verifying the optimal concentration for the control film (2% and 3% psyllium flour with 20% glycerol), different concentrations of the biomass concentrate were added to the film solution to assess their behavior. To incorporate Spirulina biomass and SpiruPower^®, the film solution was prepared as described above and cooled to 35 °C. After cooling, Spirulina was added at different concentrations and homogenized using a mechanical stirrer at 2000 rpm for 10 min. Subsequently, 25 mL of the solution was distributed in Petri dishes and dried in an oven with forced air circulation at 35 °C for 60 h. A schematic view of the procedure is presented in Figure 1B.

2.3.2. Arrowroot Starch Films

To produce the arrowroot starch control films, a film-forming solution was prepared by dispersing 3% and 5% arrowroot starch (g/mL) in distilled water and heating it to 85 °C. The solution was homogenized using a mechanical stirrer at 1000 rpm for 10 min. The resulting film-forming solutions were added to different concentrations (20, 30, and 40%) of glycerol (mL/g of starch) and then homogenized using a mechanical stirrer for 5 min at 1000 rpm at 85 °C. After homogenization, 25 mL of the solution was spread on Petri dishes, and the films were dried in a forced-air circulation oven at 35 °C for 24 h.

After verifying the best control film (3% arrowroot starch with 20% glycerol), films were subsequently made with varying concentrations of Spirulina biomass and SpiruPower^®. Following the preparation of the film solution described above, the solution was cooled to at least 35 °C and, once this temperature was reached, the different concentrations were added and homogenized using a mechanical stirrer at 2000 rpm for 10 min. Afterwards, 25 mL of the solution was poured into Petri dishes and dried in an oven with forced air circulation at 35 °C for 48 h.

2.3.3. Pectin and HPMC Films

To produce the pectin control film, a film-forming solution (3 g/100 mL) was prepared in distilled water containing 30% glycerol and 0.3% pectin. The solution was mechanically stirred at 900 rpm (Tecnal, TE-139, Piracicaba, Brazil) at 70 °C for about 1 h to ensure homogeneity. After this period, the system temperature was reduced to 25 °C.

The HPMC control film was prepared by initially dispersing HPMC (3 g/100 mL) in hot water at 90 °C for 15 min, then hydrating at 25 °C (room temperature) for 40 min or until the solution was homogeneous. Glycerol was added as a plasticizer at 30% (w/w of HPMC).

The Spirulina biomass and SpiruPower^® were added directly to the pectin film suspensions. In the HPMC films, Spirulina biomass was incorporated in different concentrations (mass of biomass/mass of biopolymer) and homogenized. The suspensions (25 mL) were poured onto Cralplast^® 33 polystyrene plates (90 × 15 mm) and dried in an air-circulating oven (Tecnal, TE 394/2, Brazil) at 35 °C for approximately 20 h. All the films were stored at room temperature and 50% relative humidity in desiccators with saturated magnesium nitrate solutions for 2 days before being submitted for physicochemical characterization.

2.4. Characterization of HPMC and Pectin Films

Characterization was carried out on the control films and those containing Spirulina biomass under the best conditions determined by the best physical characteristics, visual appearance, and compatibility. Film thickness was determined using a portable digital micrometer (Mitutoyo Co., Kawasaki-Shi, Japan) with an accuracy of 0.001 mm. Measurements were taken at five random portions on each film, and the average thickness was calculated [54,55].

The film samples were cut to 2 cm² and weighed before and after drying in an oven at 105 °C for 24 h. The moisture content was calculated using Equation (1).

Moisture content (%) = (initial mass − final mass)/(initial mass) × 100

(1)

The water solubility of the films was determined as described by Kavoosi et al. [56], with modifications. To determine the initial dry mass, 2 cm² film samples were cut and dried at 100 ± 5 °C for 24 h. The samples were soaked in 50 mL of distilled water and, after 24 h at 23 ± 2 °C, were dried again at 100 ± 5 °C for 24 h to obtain the final dry mass. The water solubility of the film was calculated using Equation (2).

Solubility (%) = (initial mass − final mass)/(initial mass) × 100

(2)

Water vapor permeability (WVP) was determined using an adaptation of the gravimetric method (ASTM E96-92) [57]. Distilled water (2 mL) was dispensed into 2.4 cm diameter Teflon permeation cells. Circular samples were cut from each film and sealed to the cell bases. The permeation cells were placed in a 420-CLDTS climate chamber (Ethik Technologies, Vargem Grande Paulista, Brazil) at 50% relative humidity at 25 °C. The cells were weighed every hour for 34 h using an analytical balance (Gehaka AG 2000, Gehaka Co., São Paulo, Brazil). Water vapor permeability (WVP) was calculated using Equation (3) [56,58].

WVP = (m/t) × (x/(A·Δp))

(3)

where WVP is the water vapor permeability (g/m s Pa) calculated from the change in mass due to the loss of water vapor (m) through the polymeric material with width (x) and area (w) during the time interval (t) under pressure (Δp) (difference in water vapor pressure between the external and internal environment).

2.5. Optical Properties

The color of the film samples was determined using a Konica Minolta 230 CM-5 colorimeter (Minolta Camera Co., Ltd., Osaka, Japan) using the CIEl*a*b* L* (lightness), a* (red-green), and b* (yellow-blue) system. The calculation of the hue angle (h°) in degrees, considering the qualitative color attribute, was calculated according to Equation (4). The chroma index (C*), a quantitative color attribute, and the total color difference (ΔE*), were calculated according to Equations (5) and (6), respectively.

h° = tan − 1 (b*/a*)

(4)

C* = (a*² + b*²)1/2

(5)

ΔE = √((L* − L)² + (a* − a)² + (b* − b)²)

(6)

The opacity of the films was measured using a spectrophotometer, based on transparency values, following the method of Chentir et al. [59], as described in Equation (7).

Opacity = (−log(T₆₀₀))/(x)

(7)

where T₆₀₀ is the fractional transmittance at 600 nm and ‘x’ is the average film thickness (mm).

The barrier properties of films against ultraviolet (UV) and visible light were measured at wavelengths ranging from 250 to 800 nm using a UV-Vis spectrophotometer (Shimadzu 1600, Portland, OR, USA).

2.6. Thermogravimetry Analysis (TGA)

The thermal degradation profiles of the samples were obtained on a TGA Q500 (TA Instruments, Delaware, DE, USA) at a heating rate of 10 °C min⁻¹ over the temperature range 10–600 °C. The flow of nitrogen and synthetic air was maintained at 40 mL/min and 60 mL/min, respectively [60]. The weight loss (%) and the derived weight (%/°C) versus temperature of the films were obtained.

2.7. Scanning Electron Microscopy (SEM)

Film samples (6 mm²) were prepared and fixed to aluminum stubs with conductive carbon tape, then sputter-coated with a 10 nm thick gold layer using an ACE600 Sputter Coater (Leica 25 Microsystems, Wetzlar, Germany). The fractured surfaces were obtained by submerging the film samples in liquid nitrogen for 5 min and fracturing them with tweezers. The samples were mounted on aluminum stubs with the fractured surface facing upwards using conductive carbon tape, then sputter-coated with a 10 nm-thick layer of gold. The specimens were observed on a JSM 6510 microscope (Jeol, Tokyo, Japan) at 10 kV, with magnifications of the surface (1000×) and fractures (1000× and 5000×) [60].

2.8. Fourier Transform Infrared Spectroscopy (FTIR)

Fourier transform infrared spectroscopy (FTIR) measurements were obtained using a Jasco FT/IR-6200 (Tokyo, Japan) spectrometer. The spectra were recorded from 4000 to 400 cm⁻¹ at a rate of 32 scans and at a spectral resolution of 4 cm⁻¹ using an attenuated total reflectance (ATR) module [61].

2.9. Determination of Antioxidant Activity by the ABTS, DPPH, and ORAC Methods

The samples of Spirulina biomass, SpiruPower^®, pectin, and HPMC films were extracted to determine their antioxidant activity. The samples were extracted according to the method described by Fratelli et al. [49]. The ABTS radical was generated by reacting 5 mL of ABTS stock solution (7 mmol/L) with 88 μL of potassium persulfate (140 mmol/L) prepared in water, keeping the mixture in the dark and at room temperature for 16 h. This solution was diluted with ethanol to an absorbance of 0.7 ± 0.05 (734 nm) [62]. To determine the antioxidant capacity, 30 μL of the extracts prepared with methanol were added to 3 mL of the ABTS radical solution, and the absorbance was read in a spectrophotometer (734 nm) after 6 min.

Triplicates of the samples were subjected to the DPPH method, each containing 3.9 mL of DPPH solution, 0.1 mL of each extract, and 0.1 mL of distilled water. These were incubated in the dark for 1 h, and the absorbance was re-read at 515 nm, using methanol as the blank. The determination of total antioxidant activity was obtained from the Trolox standard curve in ethanol, under the same reaction conditions. The results were expressed as μmol.g sample⁻¹ of Trolox equivalents (TE) [63].

The freeze-dried samples were stirred with 20 mL of methanol for 15 min to extract the antioxidant compounds from the matrices. Samples were filtered using filter paper, and the remaining solids were washed twice with an additional 20 mL of methanol. The filtrate was concentrated using a rotary evaporator at 40 °C or lower until a total volume of 15–25 mL was obtained [47,64]. The samples were filtered using 0.22 µm cellulose acetate membranes before the experiment. To determine antioxidant activity against the peroxyl radical, the ORAC (oxygen radical absorption capacity) method was used according to Rodrigues et al. [65] using a spectrophotometer (Molecular Devices, SpectraMax^®, San Jose, CA, USA).

2.10. Statistical Analysis

Analysis of variance (ANOVA) and Tukey’s post hoc comparisons were performed at the 95% confidence level (α = 0.05). Experiments were performed in triplicate, and data are reported as mean ± standard deviation.

3. Results

3.1. Characterization of the Proximal Composition of Spirulina Biomass and Biomass Concentrate

The proximate composition, phenolic compound content, and antioxidant activity of Spirulina biomass and SpiruPower^® are shown in

Table 1. Proximal composition, phenolic compound, and antioxidant activity of Spirulina biomass and SpiruPower^® (g/100 g).

Parameter	Spirulina Biomass	SpiruPower®
Moisture	80.0 a ± 0.0	82.22 a ± 0.40
Ash (dry weight)	0.16 b ± 0.0	0.57 a ± 0.01
Lipid (dry weight)	2.8 a ± 0.01	2.39 b ± 0.18
Protein (dry weight)	57.54 b ± 0.03	66.58 a ± 0.62
Total phenolic content (EAG/100 g of sample)	146.474 b ± 0.01	358.65 a ± 0.1
ABTS (µM TE/g)	50.71 b ± 1.01	111.52 a ± 1.4
DPPH (µM TE/g)	32.41 b ± 0.01	87.05 a ± 0.02

Averages with different letters in the same line differ significantly by Tukey’s test (p ≤ 0.05).

. Other analyses of the same batch of Spirulina biomass and SpiruPower^® were conducted by the research group and published, confirming the high mineral content of these ingredients and their freedom from heavy metals [52].

3.2. Films Prepared from Arrowroot Starch and Psyllium Flour

After preparing the films, the physical characteristics and visual aspects were observed to check the quality of the material (

Table 2. Physical characteristics and visual aspects of films formulated with psyllium, arrowroot starch, HPMC, and pectin, incorporated with integral and Spirulina biomass concentrate. NE: Not evaluated; (+): Presence; (-): Absence.

Films	Malleability	Fragility	Rigidity	Adherence	Homogeneity	Brightness	Color	Images
A1	+++	-	-	-	+++	+	+
A2	+++	-	-	-	+++	++	+
A3	+++	-	-	-	++	++	++
A4	+	-	-	++	+++	+	+
A5	+	-	-	+++	+++	+	+
AR1	+++	-	+	-	++	++	+
AR2	+++	-	+	-	+	++	+
AR3	+++	-	+	-	+	++	++
AR4	+++	-	+	-	++	++	++
AR5	++	-	++	-	++	++	+++
AR6	+	+	++	-	++	++	+++
AR7	+	++	+++	-	++	++	+++
AR8	+	+++	+++	-	++	++	+++
AB1	+++	-	+	-	+	++	+
AB2	+++	-	+	-	+	+	++
AB3	+++	-	+	-	+	+	+++
B1	+++	-	+++	-	+++	++	-
B2	+++	-	+++	-	+++	++	-
B3	+	-	-	+++	+++	+	-
B4	+++	-	+	-	+++	++	-
BR1	NE	+++	-	NE	+	NE	+
BR2	NE	+++	-	NE	++	NE	++
BR3	NE	-	-	NE	+++	NE	+++
BB1	NE	+++	-	NE	+	NE	+
BB2	NE	+++	-	NE	++	NE	++
BB3	NE	-	-	NE	+++	NE	+++
C1	+++	-	-	-	+++	++	+
CR1	+++	-	-	-	++	++	+
CR2	+++	-	-	-	+	++	+
CR3	+++	-	-	-	+	++	+
CB1	+++	-	-	-	++	++	+
CB2	+++	-	-	-	++	++	++
CB3	+++	-	-	-	+	++	++
CB4	+++	-	-	-	+	++	+++
D1	+++	-	-	-	+++	++	-
DB1	+++	-	-	-	+++	++	+
DB2	+++	-	-	-	++	++	+
DB3	+++	-	-	-	++	++	++

(A1) Psyllium 1%, glycerol 20%; (A2) Psyllium 2%, glycerol 20%; (A3) Psyllium 3%, glycerol 20%; (A4) Psyllium 1.5%, glycerol 30%; (A5) Psyllium 2%, glycerol 40%; (AR1) Psyllium 3%, glycerol 20%, SpiruPower^® 1%; (AR2) Psyllium 3%, glycerol 20%, SpiruPower^® 3%; (AR3) Psyllium 3%, glycerol 20%, SpiruPower^® 5%; (AR4) Psyllium 3%, glycerol 20%, SpiruPower^® 10%; (AR5) Psyllium 3%, glycerol 20%, SpiruPower^® 15%; (AR6) Psyllium 2%, glycerol 20%, SpiruPower^® 20%; (AR7) Psyllium 2%, glycerol 20%, SpiruPower^® 30%; (AR8) Psyllium 2%, glycerol 20%, SpiruPower^® 40%; (AB1) Psyllium 3%, glycerol 20%, Spirulina biomass 1%; (AB2) Psyllium 3%, glycerol 20%, Spirulina biomass 5%; (AB3) Psyllium 3%, glycerol 20%, Spirulina biomass 5%; (B1) Arrowroot starch 5%, glycerol 20%; (B2) Arrowroot starch 5%, glycerol 30%; (B3) Arrowroot starch 5%, glycerol 40%; (B4) Arrowroot starch 3%, glycerol 20%; (BR1) Arrowroot starch 3%, glycerol 20%; SpiruPower^® 1%; (BR2) Arrowroot starch 3%, glycerol 20%; SpiruPower^® 3%; (BR3) Arrowroot starch 3%, glycerol 20%; SpiruPower^® 5%; (BB1) Arrowroot starch 3%, glycerol 20%; Spirulina biomass 1%; (BB2) Arrowroot starch 3%, glycerol 20%; Spirulina biomass 3%; (BB3): Arrowroot starch 3%, glycerol 20%; Spirulina biomass 5%; (C1) Pectin 3%, glycerol 30%; (CR1) Pectin 3%, glycerol 30%, SpiruPower^® 1%; (CR2) Pectin 3%, glycerol 30%, SpiruPower^® 3%; (CR3) Pectin 3%, glycerol 30%, SpiruPower^® 5%; (CB1) Pectin 3%, glycerol 30%, Spirulina biomass 1%; (CB2) Pectin 3%, glycerol 30%, Spirulina biomass 3%; (CB3): Pectin 3%, glycerol 30%, Spirulina biomass 5%; (CB4) Pectin 3%, glycerol 30%, Spirulina biomass 10%; (D1) HPMC 3%, glycerol 30%; (DB1) HPMC 3%, glycerol 30%, Spirulina biomass 1%; (DB2) HPMC 3%, glycerol 30%, Spirulina biomass 3%; (DB3) HPMC 3%, glycerol 30%, Spirulina biomass 5%; (-) Absence; (+) Low presence; (++) Medium presence; (+++) High presence; (NE) Not evaluated.

). The control films produced from arrowroot starch (5%) with 20% and 30% glycerol (Table 2, rows B1 and B2, respectively) had a glossy, translucent appearance and were malleable yet slightly rigid. As the concentration of plasticizer increased to 40% (Table 2, row B3), the films became more adherent, an aspect that is considerably unfavorable for this application. In general, across all these perspectives, the formulation that was most suitable and exhibited the best characteristics was the film containing 20% glycerol.

Since the starch films prepared at 5% concentration showed a certain rigidity, to incorporate the Spirulina biomass and SpiruPower^®, films were prepared with 3% arrowroot starch added to 20% glycerol (Table 2, row B4), which showed the best characteristics among the formulations. The film solutions with SpiruPower^® exhibited a greenish color and were visibly more homogenized than the Spirulina biomass, except at the 5% biomass concentration (Table 2, row BB3). Both solutions containing Spirulina biomass and SpiruPower^® adhered firmly to the Petri dish, making them impossible to remove. Samples with concentrations of 1% and 3% of integral biomass and SpiruPower^® became brittle, as seen in the BR1, BR2, BB1, and BB2 films shown in Table 2.

The psyllium films were developed using 20% glycerol and different concentrations of psyllium (1, 2, and 3%) (Table 2, rows A1, A2, and A3) to observe their behavior as the biopolymer concentration in the films varied.

Upon observation of their visual and physical characteristics, they all exhibited a yellowish color, a shine, and malleability and flexibility. However, as the psyllium concentration increased, the film became more physically resistant.

In this way, the film with 1% psyllium (Table 2, row A1) proved to be less resistant than the other concentrations, with the films with 2% and 3% (Table 2, rows A2 and A3) being the best films obtained in this regard. Also, films were produced with different concentrations of plasticizer (30% and 40%) and 1.5% and 2% psyllium (Table 2, rows A4 and A5). All the films were shiny and pliable, but they were very sticky, which is a negative aspect for use as a film. Following these evaluations, the psyllium-based films formulated with 2% and 3% polymer and 20% glycerol presented the most favorable characteristics and were therefore selected for the subsequent phase. This next step involved incorporating SpiruPower^® at levels of 20, 30, and 40% (Table 2, rows AR6, AR7, and AR8). The resulting films exhibited a glossy appearance and a dark green coloration attributable to the high biomass content. They were also characterized by increased rigidity, limited flexibility, and a tendency to fracture.

3.3. Films Prepared from HPMC and Pectin

The same concentrations of Spirulina biomass (1, 3, and 5%) and SpiruPower^® were used for pectin films. However, a 10% concentration of Spirulina biomass was also tested. Table 2 shows formulations C1, CR1, CR2, CR3, CB1, CB2, CB3 and CB4, which represent the films obtained.

All the films produced showed shine, flexibility, malleability, and resistance. The films made with Spirulina biomass (Table 2, rows CB1, CB2, CB3, and CB4) showed visibly better incorporation when compared to the SpiruPower^® (Table 2, rows CR1, CR2, and CR3). Consequently, only Spirulina biomass was used to formulate the films for further analysis. The 10% Spirulina biomass concentration (Table 2, row CB4) showed heterogeneity and a dark green color, whereas the films with concentrations of 1%, 3%, and 5% showed the best visual characteristics. Once the concentrations and type of compound to be incorporated had been established, Spirulina biomass was added to the HPMC films at 0, 1, 3, and 5% (Table 2, rows DB1, DB2, and DB3). All the films showed good flexibility and physical resistance. The HPMC films were greenish in color, gradually becoming more intense as biomass concentration increased, whereas the control film remained transparent. The pectin films, meanwhile, showed a yellowish hue in the control, which intensified as the biomass concentration increased.

Regarding the solubilization of the biomass in the polymeric matrix, the HPMC films showed visibly better incorporation than the pectin films, which showed “dots” across the entire film area, especially in the CB3 formulation (Table 2). The formulations C1, CB1, CB2, CB3, D1, DB1, DB2, and DB3 were characterized, and their optical properties, barrier properties, thermogravimetric analysis (TGA), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and antioxidant activity were determined.

3.3.1. Film Characterization

The selected films from the preliminary tests were characterized, and the results are shown in

Table 3. Mechanical and thermal properties of HPMC and pectin-based films incorporated with 0, 1, 3, and 5% Spirulina biomass.

Films		Thickness (mm)	Tensile Strength (MPa)	Elongation at Break (%)	Tonset (°C)	Tmax (°C)	R600 (%)
HPMC	Control	0.08 b ± 0.01	4.26 a ± 0.39	16.50 a ± 3.85	300.12	349.77	3.68
	Spirulina 1%	0.07 b ± 0.0	4.62 a ± 0.19	15.61 a ± 4.40	297.94	350.85	6.36
	Spirulina 3%	0.08 b ± 0.01	3.56 a ± 0.32	10.05 b ± 1.58	302.59	348.9	4.47
	Spirulina 5%	0.08 b ± 0.0	3.94 a ± 0.14	13.52 ab ± 2.30	303.66	349.38	7.37
Pectin	Control	0.11 ab ± 0.04	1.48 b ± 0.95	3.60 c ± 1.61	195.40	214.58	24.45
	Spirulina 1%	0.13 ab ± 0.01	1.22 b ± 1.03	2.75 c ± 0.49	198.02	217.44	22.09
	Spirulina 3%	0.13 ab ± 0.04	1.53 b ± 0.33	3.24 c ± 0.82	198.55	214.97	23.07
	Spirulina 5%	0.15 a ± 0.01	1.53 b ± 0.48	3.82 c ± 0.59	199.73	219.22	21.89

Averages with different letters in the same column differ significantly by Tukey’s test (p ≤ 0.05).

. The films synthesized applying HPMC and pectin as ingredients showed statistically equivalent thickness, except for the pectin sample with 5% biomass, which had a significantly greater thickness than the HPMC films.

3.3.2. Moisture Content, Water Solubility, and Water-Vapor Permeability (WVP)

All the films showed a decrease in water solubility as the biomass concentration in their formulations increased (

Table 4. Moisture properties, water solubility, and water-vapor permeability (WVP) of HPMC and pectin-based films incorporated with 0, 1, 3, and 5% Spirulina biomass.

Films		Moisture Content (%)	Water Solubility (%)	WVP (10−4 g H2O/m·h·Pa−1)
HPMC	Control	26.4 a ± 1.05	100.0 a ± 0.0	9.8 d ± 0.1
	Spirulina 1%	23.69 a ± 2.03	97.0 a ± 1.0	7.3 e ± 1.0
	Spirulina 3%	22.97 a ± 0.76	85.66 b ± 1.52	9.7 d ± 0.4
	Spirulina 5%	18.51 a ± 0.39	67.33 c ± 5.68	8.9 de ± 1.2
Pectin	Control	20.61 a ± 0.88	100.0 a ± 0.0	13.2 c ± 0.3
	Spirulina 1%	16.09 a ± 5.23	97.66 a ± 2.08	14.9 bc ± 0.3
	Spirulina 3%	17.55 a ± 1.54	87.66 b ± 6.8	16.7 ab ± 0.1
	Spirulina 5%	16.93 a ± 1.84	68.0 c ± 2.64	17.4 a ± 0.9

Averages with different letters in the same column differ significantly by Tukey’s test (p ≤ 0.05).

).

However, all the HPMC and pectin films produced in this study had significantly the same average values for the moisture parameter. Regarding water solubility, the control HPMC films and those with 1% biomass showed the highest values, while the addition of 5% resulted in the lowest. The same result can be observed for pectin films.

Concerning water vapor permeability, the HPMC films showed lower permeability than the pectin films, with a reduction in this parameter as 1% and 5% biomass was incorporated. This may be due to reduced availability of free hydrophilic groups (Figure 2) resulting from increased intermolecular interactions between biomass and HPMC in these films. In the FTIR analysis, less pronounced bands (3315 cm⁻¹) corresponding to the OH group can be observed in the HPMC films, resulting in better water properties due to increased hydrophobicity.

3.4. Optical Properties Results

The results presented in

Table 5. Color and opacity attributes of HPMC and pectin-based films incorporated with 0, 1, 3, and 5% Spirulina biomass.

Films		L*	a*	b*	C*	hº	ΔE	Opacity
HPMC	Control	90.38 a ± 0.25	−1.12 a ± 0.05	−0.56 h ± 0.05	1.26 h ± 0.05	206.13 a ± 6.29	8.01 h ± 0.14	0.56 d ± 0.0
	Spirulina 1%	80.87 c ± 0.01	−6.97 d ± 0.01	7.48 g ± 0.03	10.22 g ± 0.03	132.96 b ± 0.21	19.85 g ± 0.02	1.6 b ± 0.0
	Spirulina 3%	72.86 e ± 0.01	−13.03 e ± 0.02	17.05 f ± 0.03	21.46 d ± 0.04	127.40 c ± 0.05	33.62 d ± 0.03	1.34 c ± 0.0
	Spirulina 5%	66.51 g ± 0.01	−16.08 f ± 0.02	26.98 b ± 0.03	31.41 b ± 0.02	120.79 d ± 0.04	45.62 b ± 0.03	2.67 a ± 0.0
Pectin	Control	82.50 b ± 0.01	−1.71 b ± 0.03	18.41 d ± 0.03	18.49 e ± 0.04	95.31 e ± 0.05	27.67 f ± 0.04	0.72 c ± 0.0
	Spirulina 1%	79.41 d ± 0.01	−1.76 dbc ± 0.03	17.73 e ± 0.04	17.81 f ± 0.03	95.66 e ± 0.05	28.01 e ± 0.03	1.51 b ± 0.17
	Spirulina 3%	72.06 f ± 0.01	−1.79 c ± 0.02	26.16 c ± 0.04	26.22 c ± 0.04	93.92 e ± 0.04	38.70 c ± 0.02	2.44 a ± 0.0
	Spirulina 5%	60.55 h ± 0.01	−1.72 b ± 0.03	33.80 a ± 0.17	33.84 a ± 0.11	92.91 e ± 0.03	51.18 a ± 0.04	2.32 a ± 0.0

Averages with different letters in the same column differ significantly by Tukey’s test (p ≤ 0.05).

demonstrate that the addition of Spirulina biomass significantly affected the L*, C*, and ΔE values for all films, but only in the HPMC samples did the h° values decrease significantly.

Overall, incorporating biomass into the films decreased luminosity (L*) and increased opacity considerably, factors that enhance the concentration of microalgae in the samples, leading to the darkening of the films and making them more opaque, which is an interesting aspect for foods that require protection from light. The C* values also increased across all biomass concentrations, with only the 1% pectin sample showing a slight reduction compared to the control film. However, after adding 3% Spirulina, the chroma value increased.

In the HPMC films, adding different concentrations of Spirulina biomass reduced the h° values compared to the control, from 206.13 to 120.79–132.96, indicating a shift in coloration towards a more yellowish tone (90°). On the other hand, despite incorporating Spirulina, the pectin films did not exhibit a significant color change compared to the control, given the close h° values among the films.

The total color difference (ΔE) increased significantly in all HPMC and pectin samples, indicating that the films had colors distinguishable to the naked eye. The UV/vis light transmission rate of the films is shown in Figure 3. The pectin films showed lower light transmission (%) than the HPMC films.

The results of the rate and UV/vis light transmission of the films are shown in Figure 3. At ultraviolet wavelengths (200–350 nm), the film with the lowest transmission rate was pectin with 5% biomass. In contrast, the HPMC control film had the highest rate, given its lowest opacity among the biomass-incorporating formulations (0.56 ± 0.0%) (Figure 3B). It can therefore be inferred that the film developed from pectin with 5% Spirulina provided better protection against ultraviolet rays than the other films. Besides the pectin control films exhibiting lower L* and higher opacity values compared to the HPMC control films (Table 5), this protection could be attributed to the presence of phycobiliproteins in the Spirulina biomass. C-PC, the most representative pigment of these cyanobacteria, is a photosynthetic compound that absorbs ultraviolet light [66], a property that may have corroborated the results of this study. The results presented in Table 5 demonstrate that the addition of Spirulina biomass significantly affected the L*, C*, and ΔE values for all films, but only in the HPMC samples did it result in a significant decrease in the hº values.

3.5. Thermogravimetry Analysis (TGA)

Figure 4 illustrates the weight-loss curves (TGA) and the weight-loss derivative (DTG) obtained by TGA for films based on HPMC and pectin with 0, 1, 3, and 5% Spirulina biomass added.

This analysis shows that the films lose mass, with the first peak in the first derivative occurring at a temperature below 100 °C, which can be attributed to the loss of water from the polymer matrix. The maximum mass loss occurs at different temperatures for the HPMC and pectin films. All the films showed slight variations in maximum degradation temperature with increasing biomass (Table 3).

However, the HPMC films showed higher temperatures than the pectin films, with a difference of approximately 131 °C, whereas the HPMC film formulated with 1% biomass exhibited the highest thermal stability (350.85 °C). In addition, the endothermic peak of the HPMC films was higher than that of the pectin films, indicating slower film degradation.

3.6. Scanning Electron Microscopy (SEM)

Microstructural changes in the control films and those containing Spirulina biomass were investigated using scanning electron microscopy to obtain surface and fracture images (Figure 5 and Figure 6). SEM yields high-resolution, three-dimensional surface images and is primarily employed to elucidate the topographical attributes, surface roughness, and structural integrity of the films, thereby enabling the detection of pores, fissures, and other microstructural discontinuities [67].

3.7. Fourier Transform Infrared Spectroscopy (FTIR)

Fourier Transform Infrared Spectroscopy (FTIR) was used to determine the molecular structure of the films in this study and to evaluate the interaction between the compounds (Figure 2). The technique relies on the interaction between infrared radiation and matter, in which specific wavelengths are absorbed by functional groups, thereby inducing vibrational transitions of covalently bonded atoms. These vibrational excitations generate characteristic peaks in the infrared spectrum, enabling the identification of chemical interactions occurring within the film and providing information on its composition, as well as on interactions with additives that may influence its mechanical properties and barrier functions [67].

The spectra differ little and show a broad band at 3315 cm⁻¹, corresponding to O-H stretching, which is more pronounced in the pectin films. The OH groups correspond to the addition of glycerol and the presence of water. This band is more pronounced in the pectin films and could lead to higher film hydrophilicity, resulting in more fragile water-related properties [68]. Another band appears subtly at 2928 cm⁻¹ and is probably related to the symmetrical and asymmetrical stretching of the N-H bonds, which is more pronounced in the HPMC films, a fact that can be explained by the better incorporation of Spirulina biomass when compared to the films developed with pectin. The 1742 cm⁻¹ band present in the pectin films coincides with the C=O bonds, which are absent in the HPMC films, and at 1011 cm⁻¹, a more intense band can be observed, referring to the C-O-C bonds of the HPMC and pectin structures, showing a subtle broadening in the pectin samples compared to the HPMC films [68,69].

3.8. Determination of Antioxidant Activity by the ABTS, DPPH, and ORAC Methods

The ABTS radical-scavenging assay quantifies the antioxidant capacity of both hydrophilic and lipophilic compounds and is widely employed to evaluate the antioxidant potential of bioactive molecules and phenolic constituents, thereby determining the efficiency of antioxidants in quenching free radicals. The DPPH assay is extensively utilized to assess free-radical scavenging activity and is routinely applied in the pharmaceutical, food, and cosmetic industries. The ORAC assay, in turn, is predominantly used to determine the antioxidant capacity of food products and biological samples. This method measures the ability of antioxidants to neutralize the peroxyl radical (ROO•), a reactive oxygen species of particular relevance in oxidative processes [70].

Among the biological effects attributed to Spirulina biomass, the most widely reported in the literature is its antioxidant activity. Determining the antioxidant activity of films containing biomass is essential to assess whether this property is maintained when other ingredients are added to this complex matrix, as different interactions among the film components can either compromise or enhance it. In this study, all samples showed increased antioxidant activity with the addition of Spirulina biomass (

Table 6. Antioxidant activity by the ABTS method of HPMC and pectin-based films incorporated with 0, 1, 3, and 5% Spirulina biomass.

Samples		µmol de TE/g of Film	Samples		µmol de TE/g of Film
HPMC	Control	26.5 bA ± 6.0	Pectin	Control	0.0 bB ± 0.0
	Spirulina 1%	268.28 aA ± 17.75		Spirulina 1%	0.0 bB ± 0.0
	Spirulina 3%	284.9 aA ± 19.0		Spirulina 3%	16.81 aB ± 2.13
	Spirulina 5%	320.08 aA ± 35.74		Spirulina 5%	36.92 aB ± 7.63

Different lowercase letters on the same column represent values different from each other (p < 0.05, Tukey test), and different uppercase letters on the same line represent values different from each other (p < 0.05, Tukey test).

and

Table 7. Antioxidant activity by the ORAC method of HPMC and pectin-based films incorporated with 0, 1, 3, and 5% Spirulina biomass.

Samples		µmol de TE/g of Film	Samples		µmol de TE/g of Film
HPMC	Control	10.9 cA ± 2.2	Pectin	Control	8.21 cB ± 0.62
	Spirulina 1%	125.0 bA ± 7.44		Spirulina 1%	32.73 bB ± 8.14
	Spirulina 3%	207.9 aA ± 47.5		Spirulina 3%	19.72 aB ± 4.17
	Spirulina 5%	147.2 aA ± 25.1		Spirulina 5%	73.38 aB ± 2.35

Different lowercase letters on the same column represent values different from each other (p < 0.05, Tukey test), and different uppercase letters on the same line represent values different from each other (p < 0.05, Tukey test).

).

4. Discussion

The results section showed that the compositions that Spirulina and SpiruPower^® exhibited moisture, lipid, and ash contents were very similar (Table 1). However, the protein content of the SpiruPower^® was slightly higher than that found in the Spirulina biomass, probably because it was more concentrated than the integral biomass, due to the extraction of the C-PC. Rahim et al. [71] analyzed the chemical composition of Spirulina grown under natural conditions on a farm in Chichaoua, Morocco, and reported higher lipid values (9.25 ± 0.09) than in this study and a protein value similar to that reported in our research (53.31 ± 0.67). Still, the SpiruPower^® has a higher phenolic content and, consequently, higher antioxidant activity, as determined by both methods (Table 1). Spirulina biomass has a cell wall that contains all its constituents, including bioactive compounds such as pigments (chlorophylls, carotenoids, C-PC) and other compounds such as phenolics and flavonoids [72,73]. When we extract the C-PC, all the compounds previously surrounded by the cell wall are released and become much more available in SpiruPower^®, which is probably why the results obtained with SpiruPower^® are higher than those with the integral biomass.

Analyses of the films produced with arrowroot starch and psyllium flour (Table 2) indicated that formulations containing 2% and 3% psyllium combined with 20% glycerol exhibited the most favorable properties. These were therefore selected for the subsequent stage, in which SpiruPower^® was incorporated at 20, 30, and 40% (Table 2, rows AR6, AR7, and AR8). The resulting films displayed a glossy surface and a dark green coloration due to the high proportion of concentrated biomass. They were notably rigid, showed limited pliability, and fractured easily.

Given these outcomes, new films were prepared using 5, 10, and 15% SpiruPower^® with 3% psyllium (Table 2, rows AR3, AR4, and AR5), as this concentration produced the most suitable control film. These formulations generated films that were more flexible than those from the previous stage and lighter in color, except at 15% SpiruPower^®, which remained darker. They did not exhibit brittleness; however, the compound showed reduced solubility in the psyllium matrix. This was particularly evident in the AR4 film (Table 2), which presented a heterogeneous structure and a noticeable “striated” peripheral zone. Among these, the AR3 formulation (Table 2) offered the most satisfactory visual and physical attributes.

On the basis of these observations, additional formulations were produced containing 1% and 3% Spirulina biomass and SpiruPower^® (Table 2, rows AR1 and AR2). All films maintained a shiny appearance, flexibility, and a greenish hue. Nonetheless, incorporation of both the biomass and its concentrated form into the polymer network remained suboptimal. Films made with Spirulina biomass presented slightly better dispersion than those with SpiruPower^®, although heterogeneities were still apparent. Overall, the lowest additive concentrations (1, 3, 5, and 10%) demonstrated superior performance with respect to color uniformity and mechanical integrity compared with higher concentrations (15, 20, 30, and 40%).

From the data presented in terms of film characterization (Table 3), the highest thickness (mm) was observed in the pectin film with 5% Spirulina (0.153 ± 0.018), and the lowest thickness (mm) was observed in the HPMC film with 1% biomass (0.076 ± 0.004). The increase in pectin film thickness with 5% biomass compared to films developed with HPMC could be explained by the higher solid content resulting from Spirulina protein precipitation in the pectin film solution. The pH of the solution prepared for making the pectin film containing Spirulina was 3.2 ± 0.1, while the solution with HPMC was 6.6 ± 0.0. Given the high protein content of Spirulina biomass, the acidic pectin might have precipitated, resulting in a thicker film [74], resulting in the heterogeneity of the films produced using pectin, as well as the cracks seen in SEM analysis.

Similar behavior was observed by Ramji & Vishnuvarthanan [15], who used Spirulina biomass and nano-clay in chitosan-based nanocomposite films. All the Spirulina concentrations used (0.1–0.5 g) showed statistically equivalent values, with no interference in thickness with the addition of biomass. Regarding mechanical properties, the HPMC films showed higher tensile strength and elongation at break than the pectin films, as shown in Table 3, regardless of the Spirulina concentration added to the films. The highest elongation at break (%) was found in the control HPMC film (16.5 ± 3.85), and the lowest was observed in the pectin film with 1% Spirulina (2.75 ± 0.49). However, for the HPMC films, the control film and those containing 1% and 5% biomass showed results that were significantly equivalent, and the incorporation of 3% Spirulina differed significantly from the control and from the film containing 1% biomass. As for the films made with pectin, all showed values that were significantly equivalent, regardless of the Spirulina concentration added.

Research using Spirulina biomass polysaccharide (APP) [4] and C-PC [75] yielded films with good mechanical properties. Balti et al. [75] observed no significant change in tensile strength up to 2.5% C-PC, but at 20% Spirulina extract, they found a significant increase from 22.45 ± 1.17 to 29.65 ± 1.43 MPa. Regarding elongation at break, the films with an addition of 2.5% up to 10% showed an increase in this parameter, but from 15 to 20% of incorporated C-PC, a reduction was observed, a fact that may be related to the interactions between chitosan and Spirulina extract at high concentrations, which possibly caused a cross-linking effect, reducing the molecular mobility of the polymer matrix.

In the study by Luo et al. [76], there was a significant improvement in both tensile strength (35.26 ± 3.82 to 71.34 ± 3.05 MPa) and elongation at break (23.57 ± 4.95 to 40.78 ± 6.53%) with the incorporation of 1% APP in the films. In this way, the authors observed that increasing the polysaccharide content improved the film’s mechanical properties, due to molecular interactions between chitosan and APP, forming stronger interfacial adhesions that increased the film’s resistance.

Additionally, the study by Chentir et al. [59] reported the opposite results, showing a reduction in mechanical properties with the addition of C-PC. The tensile strength and elongation at break values of the control films and those incorporated with 12.5% C-PC decreased, respectively, from 16.14 ± 0.36 to 13.73 ± 0.12 MPa and 18.92 ± 0.09 to 14.49 ± 0.94%. The authors attributed these results to a possible reduction in the spacing between the chains and a decrease in molecular interactions between the gelatin and the added Spirulina extract.

On the other hand, considering moisture content and water solubility (Table 4), the study by Chentir et al. [59] showed that film solubility did not increase significantly with the addition of C-PC. Different findings from those reported in this work can be seen in the study by Ramji and Vishnuvarthanan [15], who found a divergent effect of adding Spirulina biomass on the water-solubility of the films. The authors found a direct, proportional relationship between solubility and the addition of Spirulina, with the control film showing the lowest solubility (10%) and the film formulated with 0.3 g of biomass showing the highest (14%).

The study by Luo et al. [4], which showed that different concentrations of polysaccharide from Spirulina biomass reduced WVP in chitosan-based films, corroborates the results of this study. In contrast, in pectin films, WVP increased with increasing biomass incorporation, suggesting an indirect relationship with solubility. This can be explained by the low compatibility of the Spirulina biomass with the pectin matrix, as well as by fractures visible in the SEM images (Figure 5).

Additionally, from the optical properties results, similar outcomes were observed using Spirulina biomass and nanoclay in chitosan nanocomposite films [15]. However, the HPMC and pectin films showed statistically equal mean values, especially when 3% and 5% biomass were added to the pectin films and 5% biomass to the HPMC film, which showed the highest opacity values.

The results of the rate and UV/vis light transmission of the films are shown in Figure 2. In the visible region (380–780 nm), the pectin films showed the lowest rates, with the 1% and 5% biomass films showing the best light barrier performance, followed by the 5% Spirulina HPMC film and the 3% biomass pectin film. These results can be related to the degree of opacity of these films (Table 5), as the color intensifies with increasing concentration of incorporated biomass, inhibiting light transmission to the film’s interior. Similar results were found in the study by Luo et al. [4], Chentir et al. [59] and Moghaddas Kia et al. [42].

Complementarily, the thermogravimetric curves (TGA) and their corresponding first-derivative profiles (DTG) obtained for the HPMC- and pectin-based films containing 0, 1, 3, and 5% Spirulina were evaluated and compared with reference values reported in the literature (Figure 4). According to Kuntzler et al. [39], Spirulina biomass exhibits an onset degradation temperature near 193 °C and a maximum degradation rate at approximately 267 °C. Based on these reference points, the thermal behavior observed in the present study suggests that all HPMC-based films provided a protective effect to the incorporated microalgal biomass, as their degradation onset and peak temperatures were consistently higher than those reported for pure Spirulina. This shift toward higher degradation temperatures indicates enhanced thermal stability, likely due to favorable physicochemical interactions between Spirulina components and the HPMC matrix, such as hydrogen bonding and polymer–protein entanglements. These interactions may reduce the mobility of thermo-labile functional groups and form a more stable composite network.

In contrast, this protective effect was not evident in the pectin-based films, which displayed degradation temperatures closer to, or in some cases lower than, the values reported for unmodified Spirulina. The weaker stabilization capacity of pectin is likely related to the acidic nature of its film-forming solution. Because Spirulina proteins exhibit pH-sensitive solubility, the acidic environment can induce protein precipitation, aggregation, or partial denaturation. Such structural modifications hinder homogeneous dispersion of the biomass and prevent the establishment of strong intermolecular interactions with the polysaccharide matrix, resulting in lower thermal resistance of the composite films.

Despite the lower thermal protection afforded by pectin, these films exhibited a considerably higher residual mass at the end of the TGA experiment. The pectin control film showed the highest residue (24.45%), approximately 17% higher than that for the HPMC film containing 5% Spirulina (7.37%). The higher char yield in pectin films may be attributed to the intrinsic chemical structure of pectin, which includes a high proportion of galacturonic acid units that favor carbonaceous residue formation during pyrolysis. Additionally, crosslinking phenomena and the presence of thermally stable ionic or esterified domains can further contribute to char retention.

Taken together, the results indicate that the selection of the polymer significantly influences the thermal properties of Spirulina-based intelligent films. When thermal protection of the incorporated biomass is desired—such as in applications requiring processing or exposure to elevated temperatures—HPMC-based formulations appear more suitable due to their ability to raise Spirulina’s degradation threshold. Conversely, when higher residual mass or char formation is advantageous, pectin-based matrices offer a clear advantage. Therefore, the choice between HPMC and pectin should be guided by the intended functional performance of the final material, taking into account both the bioactive ingredient’s thermal stability and the polymer matrix’s degradation profile.

From the SEM analysis, it can be seen that the HPMC films have a more irregular surface and contain some particle agglomerates, compared to the pectin films (Figure 5 and Figure 6). The incorporation of Spirulina biomass at higher concentrations (5%) made the HPMC film surface more irregular, with the presence of pores (Figure 6(D1)). The presence of particle agglomerates and pores can affect the water barrier properties of films and their mechanical performance [77], due to the presence of fragile points in the matrix. This result confirms the superior performance of HPMC films containing 1% Spirulina biomass, which had a more homogeneous surface with fewer particle agglomerates and no pores, compared to films at other concentrations. In addition, these films showed lower WVP values (as shown in Table 4) than those produced with 3% Spirulina biomass and the control.

Figure 5(A1,B1,D1) show cracks on the surface of the pectin films, a factor that may be linked to the higher WVP (Table 4) and lower tensile strength (Table 3) of these films compared to the HPMC films. The presence of cracks, irregularities, and pores is directly related to PVA, facilitating the diffusion of water vapor [78]. The fracture micrographs (Figure 5 and Figure 6) exhibited the presence of pores in the HPMC films (Figure 6(A1)) and more pronounced cracks in the pectin films (Figure 5(B2,C2)). In addition, Figure 5(B2,D2) and Figure 6(D2) show granulations, which may be due to the inhomogeneous incorporation of the biomass into the polymer matrix. In terms of this parameter, more homogeneous, crack-free, and pore-free films are ideal for food packaging applications, as they exhibit stronger molecular interactions within the polymer matrix, resulting in better mechanical and barrier properties.

FTIR analysis was used to assess the molecular structure of the films and the interactions between their components (Figure 2). All samples showed similar spectra, with a broad band at 3315 cm⁻¹ associated with O–H stretching, more intense in pectin films, indicating higher hydrophilicity. A subtle band at 2928 cm⁻¹, linked to N–H stretching, was more evident in HPMC films, suggesting better incorporation of Spirulina. A weak band at 2361 cm⁻¹ appeared in all samples except the pectin control and may correspond to C≡C, C≡N, or related triple-bond vibrations. The pectin films also exhibited a characteristic C=O band at 1742 cm⁻¹, which was absent in HPMC films. Additionally, both polymers showed an intense band at 1011 cm⁻¹, attributed to C–O–C bonds, with slightly broader signals in pectin samples.

In terms of biological effects, antioxidant activity is the most studied and is reported to be associated with bioactive compounds from Spirulina [79,80]. Spirulina biomass exhibits antioxidant activity, mainly due to pigments such as chlorophyll a and C-PC, which are primarily responsible for its bioactive properties [81]. In the present work, it was inferred that Spirulina-containing films showed potent antioxidant activity. HPMC films showed higher antioxidant activity compared to pectin films (Table 6 and Table 7). This was probably due to better incorporation of the Spirulina biomass into the HPMC films compared to the pectin films, as the latter showed slight greenish spots on the film samples and cracks and agglomerations in the SEM surface micrographs (Figure 5). According to the ABTS method, the HPMC films containing microalgae did not differ statistically from each other, except for the control film. As for the ORAC method, the HPMC films developed with 3% and 5% had the highest antioxidant activity values; however, there was no statistically significant difference between them, only when compared with the formulations containing 0% and 1% Spirulina.

Pectin films, using both the ABTS and ORAC methods, showed higher antioxidant activity values when incorporating 3% and 5% biomass. Still, there was also no statistically significant difference between these concentrations, only when compared to the control and the addition of 1% microalgae. To corroborate the results, the study by Balti et al. [75] reported results equivalent to the increase in antioxidant activity observed in films made with C-PC. The authors evaluated the phenolic content and antioxidant activity of the films and found that the formulation with 20% Spirulina extract showed the highest values, ranging from 1.43 mg to 21.85 mg GAE/g of film and from 4.89% to 58.77%, indicating important antioxidant properties for the formulated film.

5. Conclusions

Films produced from pectin and HPMC with added Spirulina biomass were developed in this study to check their interactions and repercussions on mechanical, physicochemical, and barrier properties. After carrying out the analyses, it was found that the HPMC films with Spirulina biomass exhibited greater antioxidant activity and better mechanical, thermal and barrier properties when compared to the pectin film samples, probably due to the better homogenization of the biomass in the polymer matrix in the films, as well as the absence of cracks verified by SEM analysis, characterizing it as a more resistant material. Regarding opacity and water solubility, all HPMC and pectin films showed reduced water solubility as biomass was added and increased opacity as the films darkened with Spirulina incorporation. Both films with biomass at its highest concentration showed good light barrier properties, a relevant aspect for application in light-sensitive food packaging. Among all the formulations, the HPMC-based film containing 1% Spirulina showed the best results for WVP, TGA, SEM, mechanical properties, and antioxidant activity, demonstrating promising potential as a food packaging material.