Incorporation of Agglomerated Spirulina platensis Powder in Yogurt: A Strategy for Enhancing Nutritional Quality and Bioactive Compounds

Abstract

The incorporation of Spirulina platensis has been studied as a strategy to enrich food with bioactive compounds. Recent studies have expanded the use of Spirulina in yogurts, seeking to combine its nutritional value with the practicality of functional foods. This study evaluated the physicochemical and bioactive compounds characteristics of yogurt incorporating commercial and agglomerated (with 30% maltodextrin, efficient carrier agent, in a fluidized bed) Spirulina platensis powder, at concentrations of 0.5–2.0% (w/v) prior to fermentation. This study is novel as it is the first to report the incorporation of S. platensis agglomerated in a fluidized bed into yogurt. Fermentations were carried out at 42 °C for 5 h and then the stirred yogurts were stored at 4 °C for 28 days for stabilization. All yogurts obtained achieved characteristic values according to the Brazilian Normative Instruction 46/2007 with total acidity (0.6–1.5%), pH (3.5–4.6), and viable lactic bacteria of at least 10⁶ CFU.g⁻¹, without significantly affecting the quality of the final product or the activity of lactic acid bacteria. For the nutritional composition, it was observed that the greater the amount of cyanobacteria incorporated, the higher the concentrations of proteins (4.2–5.6%) and ashes (1.3–1.8%) in the product, and for the bioactive compounds, the phenolic compounds ranged between 2.98 and 14.96 mg.100 g⁻¹ and significantly enriched the yogurt with phycocyanin (2.19–3.65 mg.100 g⁻¹), β-carotene (4.73–6.37 mg.100 g⁻¹), and chlorophyll a (12.39–13.77 mg.100 g⁻¹), for the formulations using commercial and agglomerated S. platensis powder. Agglomeration improved the stability of bioactive compounds after fermentation and stabilization processes of the yogurts. Also, it was found that the agglomerated S. platensis powder preserved a higher amount of bioactive compounds in the yogurt, which fulfills one of the main objectives of incorporating this cyanobacterium.

1. Introduction

Microalgae have been attracting increasing interest as a renewable resource for obtaining bioactive compounds, especially in the food industry. Since the 1950s, they have been studied as a promising source of proteins and are currently used in the nutritional enrichment of food and feed, in aquaculture and in the production of cosmetics [1,2]. The global market for microalgae-derived products, currently estimated at approximately USD 4.96 billion, is projected to grow at a compound annual growth rate (CAGR) of 4.8%, reaching USD 9.1 billion by 2032 [3].

The market for foods with functional and nutraceutical characteristics, with benefits to human health, is hindered by the wide variety of manufacturers and formulations, which in their vast majority do not present the same profile of benefits. Therefore, an effort is required between agencies and health professionals to regulate production guidelines, enhance the quality control, and provide correct information to consumers. This divergence is present in modern foods, mainly in the variation in the content of sugars, lipids, salts, or other additional components, such as added flavorings and preservatives, which go against the goals of consuming healthier foods [4,5].

Yogurt has been consumed globally for centuries due to its nutritional value and versatility [6,7,8]. Yogurt is rich in proteins, bioactive compounds, and essential minerals, such as B vitamins and calcium, that play key roles in bone health, metabolic processes, and neuromuscular function. It also serves as an excellent base for the incorporation of additional bioactive ingredients, including fiber, antioxidants/bioactive compounds, and plant-based proteins [9,10]. A defining feature of yogurt is its probiotic function, which distinguishes it from many other foods. The live microorganisms it contains offer health benefits for the digestive tract and immune system, making yogurt a highly suitable option for individuals pursuing a healthy lifestyle [11,12].

In this context, the incorporation of components into traditionally consumed foods increases their nutritional quality, improving their functional properties. The Spirulina sp. (Arthrospira sp.), widely used as a food source for centuries due to its high nutritional value, is commonly known as a microalga, although it is, in fact, a cyanobacterium belonging to the phylum Cyanobacteria. Among its species, Spirulina platensis (Arthrospira platensis), has been widely used as a component incorporated into foods such as biscuits, pasta, olive oil, and meat, among others [8,13,14,15].

The cyanobacterium Spirulina sp. has an excellent nutritional composition with a high content of proteins, vitamins, minerals, and antioxidant compounds, and is therefore widely used in food incorporation, due to the demand for practical and functional foods. For example, incorporating Spirulina sp. into widely consumed dairy products like yogurt presents a promising opportunity for developing functional foods that align with consumer demands, while also exploring new applications for this biomass [16,17].

There are some references to the incorporation of Spirulina sp. into yogurt, but these studies are scarce, and often the incorporation occurs after fermentation. There is limited research on the relationship that it can have with lactic acid bacteria, which can harm its probiotic characteristics. It is also necessary to verify these effects when using Spirulina biomass modified by some technique such as encapsulation or agglomeration (responsible for improving the insertion/preservation of bioactive compounds or their physical characteristics such as dispersion and homogenization) [18,19,20]. All of this is related to the optimization of the fermentation process and the guarantee of the functional capacity of the final food, highlighting the need for the involvement of both the scientific community and regulatory agencies [21,22].

Although the incorporation of cyanobacteria and microalgae into the food industry is promising, it faces significant obstacles. Issues such as biomass safety, risk of toxic compounds, high nucleic acid content, and possible allergic reactions require strict control [2,23]. In addition, undesirable sensory properties (flavor, color, and odor) and instability of bioactive compounds compromise their acceptance and economic viability. The appropriate choice of species, applying the biomass modified by some technique, such as encapsulation or agglomeration, and optimized formulation are essential to improve the palatability and functionality of the new products [20,24].

Examples of this challenge are reported by Robertson et al. [25], who developed a functional yogurt with a lipid extract of Pavlova lutheri, rich in omega-3, which increased the n-3 PUFA content and showed anti-inflammatory activity in vitro, without affecting the quality of the product. However, the low sensory acceptance indicated the need for improvements in the formulation. Similarly, Bchir et al. [26] added fresh and dried Spirulina platensis to yogurt at concentrations of 0.1%, 0.3% and 0.5% (w/v), resulting in an intensely greenish coloration, sedimentation of cyanobacteria and not good taste, aspects that negatively influenced sensory acceptance. Regarding the flavor, the scores obtained were 4.88 for the concentration of 0.5% and 5.08 for 0.1%, on a scale from 1 (I did not like it very much) to 7 (I liked it very much).

Therefore, this study aimed to explore an innovative approach to yogurt enrichment by incorporating Spirulina platensis in two forms, commercial and agglomerated with maltodextrin in a fluidized bed. The research focused on assessing the impacts of this incorporation on fermentation kinetics, physicochemical properties, and bioactive compounds content, highlighting the functional and technological advancements enabled by using agglomerated biomass in dairy product development.

2. Materials and Methods

The yogurt fermentation was divided into two stages, the exponential phase of lactic acid fermentation and the stabilization with refrigeration. During the fermentation kinetics and storage, analyses of microbial viability (lactic acid bacteria), pH, total acidity, and total sugars were performed. At the end of the 28-day storage period, physicochemical (nutritional composition) and microbiological (quantity of lactic acid bacteria) analyses were carried out on the final yogurt. The quantification of the bioactive compound content of the final yogurt was also performed. A control formulation, without the addition of Spirulina biomass, was performed to verify whether there was a noticeable change in the profile of the yogurts, either during fermentation or in the final product.

The addition of Spirulina after food fermentation presents several limitations, notably affecting its sensory acceptance and functional benefits. Its strong taste and blue-green color can negatively impact the product’s aroma, flavor, and appearance, especially at higher concentrations, despite its antioxidant benefits. Post-fermentation incorporation also reduces its interaction with fermentative microorganisms, limiting the formation of bioactive compounds and potential enhancements to fermentation kinetics. Furthermore, sensitive compounds like phycocyanin may degrade during processing, while texture and stability issues, along with possible contamination risks, include additional challenges, and for this reason, it is recommended to incorporate the cyanobacteria prior to fermentation [27,28,29,30].

2.1. Kinetics of the Fermentation Process

The first stage of the fermentation process was carried out at 42 °C for 5 h, where commercial and agglomerated cyanobacteria were incorporated, with samples collected every 0.5 h. The second stage was the storage of the yogurt in a refrigerator at 4 °C for 28 days, with samples collected every 7 days, in a laminar flow hood. Fermentations were carried out in independent duplicates (n = 2).

Initially, the milk was pasteurized at 80 °C for 10 min (Valedourado, Batch WV07, Palmeira dos Índias, Alagoas, Brazil) as recommended by Mesbah et al. [8] and Pan-Utai et al. [15]. After this process, the pasteurized milk was divided into small batches of 180 mL (containers where the fermentations were carried out), and then the milk culture for preparing fermented milk (BioRich natural yogurt type—Batch: L36595170, Hørsholm, Denmark) was added, around 3 g.L⁻¹, in order to obtain a microorganism cell concentration (inoculum) between 10⁴ and 10⁵ CFU.g⁻¹. Then, the commercial and agglomerated Spirulina platensis biomasses were added.

Commercial Spirulina platensis was obtained from the company Saúde In NATURA Alimentos Funcionais^® (Maringá, Paraná, Brazil) and the agglomerated one, produced from the same batch, was obtained in a fluidized bed (inlet air temperature of 85 °C, outlet air temperature of 25 °C, binder flow rate of 2.5 mL.min⁻¹, duration 50 min) by using an agglomeration process with maltodextrin 30% with a dextrose equivalent (DE) value between 9 and 12, while indicating the degree of hydrolysis as indicated by Carneiro et al. [31]. The composition of commercial and agglomerated Spirulina was in %: carbohydrates of 12.51 ± 0.49 and 21.45 ± 0.26; proteins of 63.90 ± 0.46 and 63.90 ± 0.46; ashes of 6.76 ± 0.02 and 8.35 ± 0.09; lipids of 8.83 ± 0.03 and 7.05 ± 0.09; and moisture of 8.00 ± 0.06 and 6.77 ± 0.05, respectively. The D₅₀ (µm) values (diameter for which 50% of the particles have a smaller size) for commercial and agglomerated Spirulina were 49.47 ± 0.21 and 170.0 ± 0.53, respectively [31].

Maltodextrin (30%) is an efficient carrier agent compared to others like gum arabic, capsul, and maltroxin, due to its strong particle-binding ability, which promotes cohesive, well-dispersed aggregates. This concentration ensures a balance between agglomeration and solubility, in addition to acting as a protective physical barrier, encapsulating bioactive compounds, and reducing their exposure to oxygen and moisture [32,33,34].

The incorporation of commercial and agglomerated cyanobacterial biomass was done in proportions of 0.5, 1.0, 1.5, and 2.0% (w/v), in relation to milk, based on work by Albuquerque et al. [20]. Visual differences between commercial and agglomerated S. platensis are shown in Figure 1.

The fermentation process was carried out in closed containers in a stirred yogurt maker (Model SF-4007, 50W, Sonifer, Yiwu, Zhejiang Province, China). Fermentation began with the stabilization of the temperature of the yogurt maker at 40–42 °C and lasted approximately 5 h, with samples collected every 0.5 h. These samples were used to analyze the exponential kinetic curve of lactic acid fermentation through the analysis of viable lactic acid bacteria (LAB) count, hydrogen potential (pH), total acidity, and total sugars.

The quantification of viable lactic acid bacteria (LAB) microorganisms used the pour plate technique on MRS Agar (Kasvi, Madrid, Spain) and was expressed in Colony Forming Units per gram (CFU.g⁻¹), as indicated by Oliveira et al. [35] and Sousa et al. [36]. The pH was measured using a digital pH meter (TECNAL TEC-5, Piracicaba, São Paulo, Brazil) previously calibrated with buffer solutions at pH 4.0 and 7.0. The amount of total sugars in the fermentations was determined using the anthrone method (Exodo Científica, Sumaré, Brazil) [37], which, after the reaction, had a spectrophotometric reading (Kasvi K17 UV/VIS 1100NM, Pinhais, Paraná, Brazil) at 625 nm.

Total acidity was determined by neutralization volumetry using titration with standardized sodium hydroxide and expressed in % [38], according to Equation (1).

$$Total acidity \left(\%\right)={\displaystyle \frac{v\ast N\ast f\ast Eq}{w\ast 10}}$$

(1)

where v = volume of sodium hydroxide solution used in the titration, in mL; N = normality of the sodium hydroxide solution (0.10 N); f = standardization factor of the sodium hydroxide solution; w = weight of the sample in grams; and eq = gram equivalent expressed in acid.

At the end of the 5 h fermentation process, the containers containing the stirred yogurt were stored in the refrigerator at 4 °C for 28 days, as indicated by Mesbah et al. [8] and Pan-Utai et al. [15] for stabilization. Finally, the yogurts were characterized physicochemically (nutritional composition) and by their quantity of bioactive compounds (antioxidants), as described in the following sections.

2.2. Physicochemical Characterization (Nutritional Composition)

For the centesimal characterization of the stirred yogurt, the following analyses were performed: moisture and ashes or dry mineral residue, according to the analytical standards of the Adolfo Lutz Institute [38], and total protein and lipid contents according to the analytical standards of the Official Method of Analysis [39]. Moisture was determined using a moisture content tester (Marconi, Piracicaba, São Paulo, Brazil, model id50, 220 v and 250 w), through a drying process at 105 °C. Total protein analysis followed the Kjeldahl method, which determined the total nitrogen content of the sample, expressed in nitrogen units multiplied by a factor of 6.25 for conversion to protein content. Lipid content corresponded to extraction with hexane in Soxhlet at 105 °C for 4 h. Ashes were obtained by incineration at temperatures between 550 and 570 °C for 3 to 4 h. Total carbohydrate content was calculated by the difference of the aforementioned analyses (moisture, protein, lipid, and ash content). All parameters were expressed as percentage in matter.

2.3. Bioactive Compounds

Bioactive compounds were analyzed as total phenolics, beta-carotene, chlorophyll a, and phycocyanin contents. They were analyzed by using the spectrophotometric method (Kasvi K17 UV/VIS 1100NM, Pinhais, Paraná, Brazil). The total phenolic content was determined by the method proposed by Waterhouse [40], using the Folin-Ciocalteu reagent, and expressed in mg of gallic acid equivalent per 100 g of sample (mg.100 g⁻¹), with reading at a wavelength of 760 nm. In brief, 0.5 mL of each extract was combined with 2.5 mL of 10% (v/v) Folin-Ciocalteu reagent and 2 mL of 4% (w/v) sodium carbonate solution in triplicate test tubes. The mixtures were vortexed thoroughly (30 s) and then incubated in the dark at room temperature (25 ± 2 °C) for 2 h to allow complete color development before spectrophotometric analysis. The beta-carotene and chlorophyll a contents were determined according to the method proposed by Nagata and Yamashita [41]. For extraction, 1 g of sample was thoroughly homogenized in a pre-chilled mortar and pestle with 10 mL of acetone: hexane (4:6 v/v) solvent mixture. The homogenate was centrifuged at 4 °C to obtain a clear supernatant for subsequent analysis. The extracts were subjected to reading in a spectrophotometer at different wavelengths (453, 505, 645, and 663 nm). The determination of the amount of phycocyanin was performed according to the methodology expressed by Silveira et al. [42], extracted with 0.1 M phosphate buffer solvent and pH 7.0. The extraction process was performed by mixing 2 g of biomass with 50 mL of solvent using a rotary shaker maintained at 30 °C. Samples were collected at 24, 48, and 72 h time points. Following centrifugation, the supernatant was analyzed spectrophotometrically, with optical density measurements recorded at 620 nm and 625 nm.

2.4. Statistical Analyses

All results for nutritional composition and bioactive compound content for all yogurt samples were analyzed by using Tukey’s test, using an online statistical calculator ASTATSA^® (https://astatsa.com/OneWay_Anova_with_TukeyHSD/ (accessed on 12 May 2025 and 25 June 2025)), and considering p < 0.05 (95% confidence level) to identify significant differences between the results obtained.

3. Results and Discussion

3.1. Kinetics of the Fermentation Process

The results obtained during the monitoring of the fermentation kinetics for the formation of lactic acid, milk sugar consumption, and growth of lactic bacteria in the stirred yogurts incorporating commercial and agglomerated Spirulina platensis are shown in Figure 2. Regarding the classical fermentation profiles, these include Deindoerfer [43] who mentions the relationship between the limiting substrate and product(s) formed, the formation of lactic acid, classified as a simple fermentation, and Gaden [44], who shows a fermentation associated with growth and who classifies this type of fermentation as product associated with growth.

Initially, when analyzing the profile of the kinetic curve of the formation of the final product, it is clear that there was an increase in the acidity of the yogurts incorporating Spirulina platensis (Figure 2A,B), observing that the acidity varied between 1.0 and 1.1%, between the control and them, as well as a decrease in the hydrogen potential (pH) (Figure 2C,D), leaving the final product with values between 4.5 and 4.6 but without a significant difference. It was noted that in approximately 4 h of fermentation, all the stirred yogurts reached the peak of acid formation. The pH indicative of the production of lactic acid is around 4.2 to 4.5, which generates an average fermentation time of approximately 4 h [8,15,45], and it agrees with the results found in this work. Normative instruction No. 46/2007 of the Ministry of Agriculture, Livestock and Food Supply—MAPA of Brazil [46] for yogurt, states that the acidity variation limit for yogurt must be between 0.6 and 1.5 total acidity (g of lactic acid.100 g⁻¹ or %) and pH between 3.5 and 4.6, in addition to a minimum number of viable lactic acid bacteria of 1.10⁶ CFU.g⁻¹ of product, with the yogurts obtained within the established limits.

In this context, the Codex Alimentarius Standard for fermented milk (CXS 243-2003) is used as an international parameter and establishes that the minimum protein content must be 2.5%, the fat content must be less than 15%, the acidity must be at least 0.6% and the amount of lactic acid bacteria—LAB of 10⁷ CFU.g⁻¹ [47]. The concentration of total sugars at the end of fermentation varied between 8 and 11 g.L⁻¹, with a small variation depending on the concentration of cyanobacteria incorporated (Figure 2E,F), with the fermentation being carried out with the agglomerated cyanobacteria closest to the control. For bacterial growth, values of around 10⁸ CFU.g⁻¹ were found for all stirred yogurts produced, in compliance with the legislation, and likewise, the concentration was required to be classified as probiotic (Figure 2G,H).

Therefore, the incorporation of commercial or agglomerated cyanobacteria biomass did not inhibit the activity of lactic acid bacteria in a way that would significantly interfere with the quality of the final product, i.e., 5 h of fermentation obtained a final pH of around 4.6 and total acidity between 1.0 and 1.1% with bacterial counts higher than 10⁸ CFU∙g⁻¹ for all stirred yogurts produced. In

Table 1. Final characteristics of the yogurts incorporating Spirulina platensis.

Sample	Total Acidity (%)	pH	Total Sugars (g.L−1)	Microbial Count * × 107 (CFU.g−1)
Control	1.031 ± 0.021 d	4.64 ± 0.02 a	8.74 ± 0.36 g	103 ± 4.24 a
Spirulina platensis commercial
0.5%	1.051 ± 0.007 d	4.66 ± 0.01 a	9.61 ± 0.16 de	103 ± 6.90 a
1.0%	1.061 ± 0.007 cd	4.63 ± 0.03 a	10.66 ± 0.33 bc	97 ± 4.24 a
1.5%	1.036 ± 0.014 d	4.64 ± 0.02 a	10.77 ± 0.16 ab	95 ± 7.07 a
2.0%	1.056 ± 0.014 cd	4.64 ± 0.01 a	11.29 ± 0.08 ab	87 ± 4.24 a
Spirulina platensis agglomerated with maltodextrin 30% (w/v)
0.5%	1.051 ± 0.007 cd	4.66 ± 0.01 a	9.32 ± 0.28 eg	99 ± 3.41 a
1.0%	1.096 ± 0.013 abc	4.63 ± 0.04 a	9.44 ± 0.38 dg	103 ± 6.90 a
1.5%	1.110 ± 0.007 a	4.63 ± 0.04 a	10.07 ± 0.16 cdf	102 ± 7.31 a
2.0%	1.092 ± 0.008 abc	4.64 ± 0.01 a	10.65 ± 0.16 abf	88 ± 8.83 a

* Microbial count is referred to lactic acid bacteria. Statistical analysis at a level of 95% confidence (p < 0.05) must be read as: different letters indicate significant differences between the samples, i.e., a, b, c, d, e, f and g are different statistically, and mixed letters, as the example ab, have no statistical difference between samples with a, b, and ab.

, final parameters for the yogurts after 28 days of stabilization and statistical analyses between them are shown. Statistical differences were observed for total acidity and total sugars in the incorporated yogurts.

This same behavior was observed in studies that incorporated commercial Spirulina platensis biomass into yogurt. For example, in the results of Yamaguchi et al. [29] and Matos et al. [48], where lactic fermentation was carried out at 42–43 °C for 4 h and both obtained a pH decreasing close to 4.5. Similarly, Pan-Utai et al. [15] produced yogurts incorporating Arthrospira (Spirulina) platensis in concentrations between 0.1 and 10% (w/v) by applying Lactobacillus bulgaricus and Streptococcus thermophillus cultures at 3% (w/v) of inoculum, fermented at 43 °C for 4 h, and obtained a final pH of 4.3. Also, Mesbah et al. [8] incorporated Arthrospira (Spirulina) platensis prior to fermentation at concentrations of 0.5–1.5% (w/v) with L. bulgaricus and S. thermophillus cultures at a ratio of 1:1 for 3% inoculum (w/v) at 42 °C and obtained a final pH of 4.6 and final sugar concentration between 6.62 and 7.26 g.L⁻¹.

In addition, Ebid et al. [49], who incorporated S. platensis at 0.2–1.0% (w/v) into yogurt, fermented with L. bulgaricus and S. thermophillus at a ratio of 1:1 for 5% inoculum (w/v) at 37 °C to pH 4.6, presented a final sugar concentration of 6.9–7.5 g.L⁻¹. In Bchir et al. [26], the production of yogurt fortified with dry and fresh Spirulina platensis, adding 0.1–0.7% (w/v) and using cultures of L. bulgaricus and S. thermophilus as inoculum in 3% (w/v), was investigated. The fermentation was conducted at 43 °C for 4 h until reaching a pH of 4.3, obtaining a final concentration of lactic bacteria between 3.65 and 4.05.10⁴ CFU.g⁻¹. Finally, Atallah et al. [50], characterizing skimmed functional yogurt enriched with whey protein concentrate, calcium caseinate, and Spirulina (1% w/v), fermented with cultures of L. bulgaricus and S. thermophilus, 3% inoculum (w/v), at 42 °C for 4 h until reaching a pH of 4.6, obtained a final concentration of lactic bacteria between 8.11 and 8.51.10¹⁰ CFU.g⁻¹.

3.2. Nutritional Composition of the Yogurts

The incorporation of cyanobacteria into dairy products can bring significant changes in the nutritional quality of the final yogurt [48,51]. The nutritional composition of the stirred yogurt samples showed that the protein and total ash content increased proportionally and more significantly with the incorporation of Spirulina sp. biomass, but with slight differences between the commercial and agglomerated biomass (

Table 2. Nutritional composition of the yogurts incorporating Spirulina platensis.

Sample	Protein (%)	Lipids (%)	Moisture (%)	Ash (%)	Carbohydrates (%)
Control	3.96 ± 0.05 d	3.13 ± 0.21 a	83.10 ± 1.30 a	1.10 ± 0.20 d	8.74 ± 0.28 c
Spirulina platensis commercial
0.5%	5.44 ± 0.26 a	2.28 ± 0.15 c	81.48 ± 1.60 a	1.31 ± 0.10 cd	9.49 ± 0.70 b
1.0%	5.74 ± 0.03 a	2.41 ± 0.15 bc	80.15 ± 1.86 a	1.52 ± 0.10 b	9.26 ± 0.50 b
1.5%	5.74 ± 0.03 a	2.52 ± 0.16 bc	80.25 ± 2.12 a	1.65 ± 0.14 a	9.86 ± 0.49 b
2.0%	5.63 ± 0.54 a	2.53 ± 0.12 bc	80.18 ± 2.52 a	1.86 ± 0.20 a	10.05 ± 0.32 ab
Spirulina platensis agglomerated with maltodextrin 30% (w/v)
0.5%	4.33 ± 0.30 c	2.42 ± 0.15 c	82.66 ± 1.10 a	1.27 ± 0.05 cd	9.68 ± 0.14 b
1.0%	4.81 ± 0.25 bc	2.64 ± 0.13 bc	81.14 ± 2.10 a	1.56 ± 0.12 ab	10.55 ± 0.16 a
1.5%	5.41 ± 0.36 a	2.70 ± 0.18 ab	80.14 ± 1.85 a	1.67 ± 0.10 a	10.43 ± 0.11 a
2.0%	5.49 ± 0.57 ab	2.79 ± 0.22 ab	80.13 ± 1.92 a	1.87 ± 0.10 a	10.36 ± 0.13 a

Statistical analysis at a level of 95% confidence (p < 0.05) must be read as: different letters indicate significant differences between the samples, i.e., a, b, c and d are different statistically, and mixed letters, as in the example ab, have no statistical difference between samples with a, b, and ab.

).

The moisture content in yogurt with or without incorporation of Spirulina was in accordance with the values found in the literature, being slightly higher than 80% [15,52]. Protein content ranged from 4.21% to 5.49% in the incorporated yogurts, compared to 3.96% in the control. Similar results were found for Atallah et al. [50] and Nazir et al. [53], with added concentrations of cyanobacteria between 1 and 3.5%, an increase in the amount of protein in the incorporated yogurt between 5.2 and 6.2% was observed, evidencing an improvement in the protein quality of the final product. The behavior for the ash content of the yogurts was similar as for proteins, having 1.27–1.87% for the incorporated yogurts and 1.1% for the control formulation. In this context, Mesbah et al. [8] and Nazir et al. [53] who incorporated 2–3% cyanobacteria biomass obtained 2.1–2.5% ash content while the control presented 1.05%.

Analyzing the lipid content, there was a slight decrease in relation to the control formulation. This behavior is similar to that found by Naziry et al. [53] and Zaid et al. [54], in which yogurts enriched with 2% Spirulina presented similar lipid contents, 0.98% and 1.02%, respectively, while the control formulation contained 1.32%. Finally, a variation between 9 and 10% of the carbohydrate content was obtained with the incorporation between 0.5 and 2.0%, presenting the same behavior found in Mesbah et al. [8] and Nazir et al. [53], who obtained a variation between 5 and 6% of the carbohydrate content for yogurt incorporated with 2–3% Spirulina. It is worth highlighting that the increase in the physical–chemical composition of the incorporated yogurt will depend on the composition of the cyanobacteria used, the type of cyanobacteria, and the cultivation conditions, among other factors [55].

3.3. Bioactive Compounds in the Incorporated Yogurts

Biological oxidizing compounds can present bioactive properties of great interest for improving the nutritional quality and biofunctional profile of foods that can exhibit antioxidant activity, which has been widely studied [53,56]. Cyanobacteria, such as S. platensis, have been studied because they have significant amounts of bioactive compounds, including, for example, antioxidants, such as phycocyanin, carotenoids, chlorophyll, and other secondary pigments. The composition of these compounds in yogurts incorporating cyanobacteria significantly increased the amount of antioxidants and inserted compounds such as phycocyanin, β-carotene, and chlorophyll, even after the fermentation process, demonstrating the stability of these bioactive compounds in the incorporated food (

Table 3. Antioxidant compounds in yogurts incorporating Spirulina platensis.

Sample	Phenolic Compounds (mg.100 g−1)	Phycocyanin (mg.100 g−1)	β-Carotene (mg.100 g−1)	Chlorophyll a (mg.100 g−1)
Control	5.58 ± 0.02 e	0.00 ± 0.00 d	0.00 ± 0.00 g	0.00 ± 0.00 e
Spirulina platensis commercial
0.5%	2.98 ± 0.02 f	2.19 ± 0.03 c	4.73 ± 0.05 f	12.39 ± 0.04 d
1.0%	5.63 ± 0.06 e	3.39 ± 0.15 a	5.09 ± 0.02 e	12.70 ± 0.01 d
1.5%	11.20 ± 0.05 c	3.57 ± 0.17 a	5.34 ± 0.06 c	13.28 ± 0.16 c
2.0%	13.62 ± 0.02 b	3.65 ± 0.13 a	5.63 ± 0.01 b	14.07 ± 0.03 a
Spirulina platensis agglomerated with maltodextrin 30% (w/v)
0.5%	11.02 ± 0.02 c	3.07 ± 0.04 b	5.06 ± 0.03 e	12.43 ± 0.08 d
1.0%	12.00 ± 0.42 c	3.12 ± 0.19 ab	5.24 ± 0.01 d	12.48 ± 0.04 d
1.5%	13.19 ± 0.32 b	3.06 ± 0.01 b	5.49 ± 0.11 c	12.60 ± 0.19 d
2.0%	14.96 ± 0.13 a	3.13 ± 0.06 b	6.37 ± 0.03 a	13.77 ± 0.12 b

Statistical analysis at a level of 95% confidence (p < 0.05) must be read as: different letters indicate significant differences between the samples, i.e., a, b, c, d, e, f, and g are different statistically, and mixed letters, as in the example ab, have no statistical difference between a, b, and ab.

).

The agglomeration of Spirulina platensis with 30% maltodextrin notably improved the stability of bioactive compounds in yogurt, as seen in the higher phenolic content compared to commercial Spirulina formulation. This enhancement is attributed to the encapsulating microstructure formed by agglomeration, which acts as a protective barrier against environmental factors, reducing compound degradation and improving functional quality [32,33,34].

Phenolic compounds are excellent antioxidants and anti-inflammatories and have cardioprotective activity. Their main group is flavonoids, which have the ability to reduce oxidative stress and inflammation in the body [57]. The addition of these components increases nutritional quality to improve health and prevent diseases. In addition to the increase in the amount of phenolic compounds in relation to the control stirred yogurt, it is interesting to note that the incorporation of agglomerated cyanobacteria preserved a higher amount of phenolic compounds, mainly at concentrations of 0.5–1.0%, after the fermentation process and constitutes a supporting rationale for using biomass in this way.

Analyzing the content of phenolic compounds, there was an increase in the quantity in relation to the control formulation, to values up to around 15 mg.100 g⁻¹ for incorporation, especially for agglomerated S. platensis, while the control formulation presented a value of 5.58 mg.100 g⁻¹. The same behavior was also found in Ebid et al. [49], who studied the impact of Spirulina platensis on antioxidant properties of incorporated yogurt with 4% Spirulina platensis obtaining 17 mg.100 g⁻¹ of phenolic compounds in comparison with the control that presented 4.59 mg.100 g⁻¹.

According to Nazir et al. [53], who studied the evaluation of the nutritional value of yogurt cream incorporating 1.5% Spirulina platensis obtained 11.37 mg.100 g⁻¹ of phenolic compounds, while the control showed 5.07 mg.100 g⁻¹. Also, Mesbah et al. [8], who studied the functional properties of yogurt fortified with 0.5% Spirulina platensis, demonstrated yogurts with 16.80 mg.100 g⁻¹ of phenolic compounds with respect to the control with 7.12 mg.100 g⁻¹.

Carotenoids are important antioxidants that contribute to the reduction of free radicals, are excellent transporters of Vitamin A, and improve immune health [58], so their addition improves the nutritional quality of yogurt. In the results, the higher the cyanobacteria biomass addition, the higher the carotenoid content in the final yogurt, with special detail in those with agglomerated cyanobacteria. Regarding the β-carotene content, there was an increase for the incorporation of 0.5–2.0% S. platensis and being absent in the control formulation.

Similarly, Mesbah et al. [8], who assessed the functional properties of yogurt fortified with Spirulina platensis and milk protein concentrate, incorporated 1.5% Spirulina platensis in the yogurt and obtained a value of 7.43 mg.100 g⁻¹ of β-carotene compared to the control that had 0.82 mg.100 g⁻¹. Also, Nazir et al. [53], who evaluated the nutritional value of yogurt incorporated with Spirulina platensis, incorporated 1.5% and obtained a content of 4.35 mg.100 g⁻¹ of β-carotene compared to the control that had 0.23 mg.100 g⁻¹.

Regarding phycocyanin, a specific pigment of cyanobacteria, which is only present in yogurt due to the addition of cyanobacteria, and which plays an important role in preserving the liver and in lipid peroxidation [29]. The phycocyanin content was present in concentration between 2.19 and 3.65 mg.100 g⁻¹, being absent in the control formulation. Although there is no official Recommended Daily Intake established specifically for phycocyanin by regulatory bodies, studies suggest that phycocyanin can be consumed up to 1 g per day as a dietary supplement [59,60].

The behavior found is similar to that in Barkallah et al. [52], who incorporated 0.25% Spirulina platensis into yogurt, obtaining 0.297 mg.100 g⁻¹ of phycocyanin in comparison and the control where it was absent. Ebid et al. [49], who studied the impact of Spirulina platensis on antioxidant properties of functional yogurt, incorporated 0.7% Spirulina platensis into yogurt, obtaining 4.73 mg.100 g⁻¹ of phycocyanin, and Pan-Utai et al. [15], who incorporated 0.5% Spirulina platensis, obtained 0.345 mg.100 g⁻¹ of phycocyanin.

Also, chlorophyll a (from photosynthetic organisms) will only be present in yogurt due to its addition through cyanobacteria, and it is associated with a high capacity to exhibit antioxidant activity via reactive oxygen species (ROS) scavenging [61,62]. Regarding the chlorophyll a content, there was a significant increase in the formulations, with a variation between 12.4 and 13.7 mg.100 g⁻¹.

In this sense, Mesbah et al. [8], who studied the functional properties of yogurt fortified with Spirulina platensis incorporated at 1.0% (w/v) the yogurt, obtained 11.32 mg.100 g⁻¹ of chlorophyll a, which was absent in the control formulation. Confirming this effect, Barkallah et al. [52] reported that fortifying yogurt with 0.25% Spirulina platensis led to a chlorophyll a concentration of 27.06 mg.100 g⁻¹, contributing to the product’s antioxidant potential.

This can be attributed to the joining of smaller particles that could promote greater encapsulation of these compounds and help their stability when incorporated into yogurt [63]. It is interesting that the incorporated cyanobacteria biomass managed to preserve a greater concentration of antioxidant compounds in the final yogurts, and this may be the result of the agglomeration of the particles that, in addition to improving the physical characteristics of the powder, also produced this secondary effect [31].

Several studies have shown that the agglomeration process is an efficient technological alternative for preserving bioactive compounds, especially those of an antioxidant nature, in dehydrated plant matrices. The technique, widely applied in the food industry, is associated with the improvement of the functional, physical–chemical, and stability properties of powders obtained by drying, contributing to greater protection against environmental factors such as oxygen, light, and temperature [64,65].

For example, Castaño Peláez et al. [64] investigated the stability of a strawberry-based powder formulation, previously obtained by spray drying and subsequently agglomerated in a fluidized bed. The samples were stored for up to 180 days at different temperatures (15, 25, and 35 °C), and the authors observed high retention rates of antioxidant activity, with values above 50% for the ABTS and DPPH methods, in addition to the preservation of 76% of the total phenolic compounds and approximately 40% of vitamin C. The results were attributed to the joint action of the carrying agents (maltodextrin and gum arabic) and the microstructure generated by the agglomeration, which contribute to the formation of a physical barrier against oxidative and thermal processes. It is also noteworthy that the best performances were obtained in the samples kept at controlled temperatures of 15 and 25 °C.

Similar results were reported by Gallón-Bedoya et al. [65], who evaluated the stability of an agglomerated powder made from Andean fruits (Physalis peruviana, Fragaria x ananassa, and Rubus glaucus) subjected to storage for 180 days. The antioxidant activity, measured by ABTS and DPPH, showed moderate losses of 22% and 36%, respectively, in the samples stored at 15 °C, evidencing the effectiveness of the microparticulate structure and the carriers used in protecting the bioactive compounds, especially phenolics and ascorbic acid, against oxidative degradation.

In line with these findings, the agglomeration process was also observed and contributed to the preservation of antioxidant activity, confirming the potential of this technique for enhancing the stability of bioactive compounds during storage. Additionally, future studies should evaluate consumer acceptance and rheological properties of the yogurt incorporating agglomerated Spirulina platensis in order to address technological and market aspects.

4. Conclusions

The integration of cyanobacterial biomass powder as an additional source of nutrients and bioactive compounds has proven to be a promising strategy to improve the nutritional profile of yogurts, expanding their functionalities and making them innovative, since they are widely consumed worldwide. The incorporation of S. platensis did not significantly alter the fermentation profile of stirred yogurt (total acidity, pH, and viable lactic bacteria), while maintaining the legal requirements for the product under all conditions studied. It was observed that the greater the amount of cyanobacteria incorporated into the yogurt, generally, the higher the concentration of proteins and ash obtained in the final product. Additionally, the antioxidant content was increased, even after the fermentation process and the stabilization period under refrigeration. The use of an agglomerated biomass resulted in greater bioactive compound stability than commercial powder. Although results are promising, challenges remain regarding Spirulina’s sensory impact and long-term bioactive stability. Future research should mainly address consumer acceptance.