The challenge of feeding the growing world population and the pressure upon the global natural resources are forcing the food industry in developing alternative strategies to meat production, whose growing demand is ultimately not sustainable [World Health Organization (WHO), 2016]. Moreover, the consumption of functional foods and dietary supplements containing protein is an important and growing source of nutrition in diets of the consumers. Consumers use these products in their diet to achieve a healthy nutrition lifestyle (Kuesten and Hu, 2020). Apart from the source of energy (4 kcal/g protein) and essential amino acids, dietary proteins contain different sequences known as bioactive peptides (Sánchez and Vázquez, 2017) having the amino acid sequences with specific positive impacts on health. These are generally obtained by the proteolytic cleavage of a larger protein molecule through microbial fermentation, enzymatic activities, or during food processing (Patil et al., 2020). Protein hydrolyzates and peptides from natural resources can be used as “functional foods” and “nutraceuticals” on the basis of their bioactivity or as technological components due to their functional properties. The functional products and nutraceuticals may contain the whole hydrolyzate and/or isolated and purified peptides (Lafarga et al., 2016). Because the bioactivity and functionality of peptides depend on their amino acid composition, sequence, and molecular mass (Lassoued et al., 2015), peptides with varying effects might be derived from a single hydrolyzate.

Different microbes and algae, containing more than 30% protein in their biomasses, can provide an optimal balance of essential amino acids, moreover, requiring limited inputs, arable land, and water compared to plant-based protein production (Ritala et al., 2017).

Microbial protein sources, generally defined as single-cell protein (SCP), have high potential for the food and feed industry of developed and developing countries (Chama, 2019). Amidst SCP, Cyanobacteria, also known as blue-green algae, are a phylum of microorganisms related to bacteria but capable of performing photosynthesis; therefore, these are commonly included into the group of microalgae. Only few species of microalgae, among which Arthrospira platensis, Chlorella vulgaris, Dunaliella salina, and Parietochloris incise, are currently authorized for human consumption (Rizwan et al., 2018). Spirulina, which is the trade name referring to Cyanobacteria A. platensis and Arthrospira maxima, is one of the most well-known and worldwide cultivated microalgae (Lafarga et al., 2020). Spirulina is characterized by a very high protein content, which is around 60% on a dry weight basis and can reach up to 70% depending on cultivation conditions (Lafarga et al., 2020). The most relevant proteins in Spirulina are phycobiliproteins, water-soluble pigments that can be classified depending on their composition and chromophores content, with phycocyanins, phycoerythrins, and allophycocyanins being the main ones (Noreña-Caro and Benton, 2018). Phycobiliproteins have also been associated with several potential positive health effects, proving to be excellent sources of bioactive peptides with potential application in the functional foods industry as antihypertensive, antidiabetic, antiobesity, and antioxidant ingredients among other positive bioactivities assessed both in vitro and in vivo using animal models (Samarakoon and Jeon, 2012; Ovando et al., 2018; Lafarga et al., 2021). Aside from protein, Spirulina is also valued for the presence of several minerals, including potassium, calcium, magnesium, selenium, iron, zinc, and vitamins especially those belonging to the B group (Carcea et al., 2015; Lafarga et al., 2020).

Notwithstanding all the positive qualities Spirulina offers, some drawbacks, such as unusual color and flavor, limit its use as food ingredient (Chama, 2019). Moreover, the presence of cell wall, which, for many microalgae, is recalcitrant and not digestible by monogastric animals, limits the efficient extraction of intracellular components and prevents nutrients utilization, posing as the main nutritional issue (Coelho et al., 2020).

The aim of this study was to evaluate the effect of the combination of microbial and enzymatic treatments on the nutritional and functional features of Spirulina. Fermentation with selected strains of Lactiplantibacillus plantarum T0A10 already showing promising results in terms of bioactive peptides (Rizzello et al., 2017) and enzyme-assisted extraction method using Alcalase^® as proposed by Verdasco-Martín et al. (2019) were evaluated. Besides, aiming at evaluating the effect of drying on the nutritional quality, the above treatments were performed on wet, dried at low temperature, and lyophilized Spirulina biomasses. Indeed, different drying treatments are currently used at the industrial level to reduce the amount of water in Spirulina biomass, with some nutritional concerns related to the nutrient loss due to high temperature or removal of soluble compounds (Desmorieux and Hernandez, 2004). An integrated approach, including chromatographic and electrophoresis analysis, was used to evaluate the effect of the different bioprocessing options on the degree of proteolysis of Spirulina protein, which, in turn, may affect the digestibility and the release of potentially bioactive compounds.

Nowadays, the nutritional composition – particularly rich in proteins, minerals, vitamins, and essential fatty acids along with the evolving awareness of diet influence on the state of health and the pressing need of sustainable food sources – is the main reason to its fast-emerging applications as food supplement and ingredient. Microalgae feasibility as food ingredients or source of nutrients and/or bioactive compounds and their health effects have been widely studied (Barros de Medeiros et al., 2021). Indeed, as bioactive peptides are generated from proteins and microalgae are protein-rich feedstocks, the potential utilization of microalgae as a source for peptides has gained increased interest during the past decade (Lafarga et al., 2021). Currently, only few species of microalgae are grown, produced, and used on a commercial scale, and among all, Arthrospira platensis is one of the most used (Barros de Medeiros et al., 2021). Overall, the market value of algae products, which in 2018 amounted to 2.6 billion US$, is forecast to reach over 3.45 billion US$ by 2025 (Statista, 2019).

Fresh Spirulina, in its natural state, is made of regularly wrapped spiral multicellular filaments, which are well compartmentalized with a lot of proteins partitioned within the cells. However, the cell wall is made of multiple layers, the first of which containing β-1,2-glucan (Vernès et al., 2019; Chen et al., 2020), that might limit the bioaccessibility of such acclaimed components.

In this framework, the present study aimed at optimizing a biotechnological protocol able to favor the proteolysis of the Spirulina proteins, which may increase the functional (e.g., bioactive compounds) and nutritional (e.g., protein digestibility) features, by comparing and combining two methods recently explored by the literature: treatment with Alcalase^®, having endopeptidase activity; and fermentation with a selected strain of LAB. Among bioactive compounds used as food ingredients, peptides are one of the most studied (Fernandes et al., 2019). Although peptides can be released using several treatments, the use of commercial proteases is the most common strategy because of the high specificity of proteins and the possibility to control the process (Lafarga et al., 2021). Among other enzymatic preparations, Alcalase^® was found to increase the extraction yield of hydrophilic compounds by 90% compared to the simple solvent extraction (Verdasco-Martín et al., 2019). Moreover, the Alcalase^® treatment contributed to the production of bioactive peptides, which can positively impact human and animal metabolism and health, as extensively reported by authors (Tacias-Pascacio et al., 2020; Lafarga et al., 2021). On the other hand, microbial fermentation has widely been reported as a promising tool for the release of bioactive peptides with Lactobacillus species being the predominantly used (Patil et al., 2020). Fermentation with selected strains of L. plantarum was attempted by de Marco Castro et al. (2019) and Niccolai et al. (2019) to increase in vitro antioxidant properties. Nevertheless, because the functionality of protein hydrolyzates may differ between strains due to the presence of different proteolytic systems in microorganisms (Patil et al., 2020), the specific L. plantarum T0A10 strain, previously selected to release bioactive compounds (Rizzello et al., 2017), was used in this study.

Because of its high-water content and, therefore, high perishability, drying processes are necessary to preserve Spirulina, although often associated with loss of quality compared to the fresh product, especially if conducted at high temperatures (Desmorieux and Hernandez, 2004). Although spray drying is preferred at industrial level, small Spirulina producers generally dry the produced biomass using convective drying processes. Other strategies such freeze-drying have been evaluated but are not yet as relevant because considered a time- and energy-consuming manufacturing process (Lafarga et al., 2021). Nevertheless, freeze drying is considered as the best way to preserve the high-value compounds (Soni et al., 2017; Noreña-Caro and Benton, 2018), and several freeze-dryers and continuous freeze-drying tunnels have already been used for the food industry (Pisano et al., 2019). An ad hoc optimization of the parameters may help resolve scale-up issues by reducing cost and operation time (Butreddy et al., 2020).

In this study, wet (80% moisture), dried (12% moisture), and freeze-dried (5% moisture) Spirulina were used and subjected to the enzymatic treatments and or microbial fermentation.

The analysis of the three different types of Spirulina biomass showed remarkable lower contents of peptides and FAA in dried compared to WB. The process used for drying Spirulina after harvesting includes biomass filtration and pressing to drain excess water, extrusion in 1- to 2-mm thin layers or rods (“spaghetti” shape), and drying at relatively low temperature. A large part of the soluble compounds like amino acids and peptides are therefore lost, explaining the gap between their content in wet and dried biomasses (Table 1). Moreover, the decrease of protein (10–20%) during drying was previously reported (Oliveira et al., 2009) with losses directly proportional to the process temperature (Desmorieux and Hernandez, 2004). Overall, drying treatments are also responsible for an alteration of the biomass, filaments unfold, and begin to fragment; nevertheless, the cells remain intact (Vernès et al., 2019).

On the contrary, increases of peptides and amino acids (up to 180 mg/g combined) were observed in lyophilized compared to WB. In this case, Spirulina was stored at circa−20°C after harvesting, until the freeze-drying. Low temperature storage is considered as the optimal approach for cryopreservation of organisms, yet it relies upon their ability of being cooled avoiding cryo-injury induced by physical and chemical changes associated with freezing. Kapoore et al. (2019) observed that storage at −15°C, especially if for prolonged time (up to 4 months), leads to a rapid reduction in viability of C. vulgaris due to damage associated with ice crystal development and colligative wound due to excessive concentrations of solutes (Mazur, 1965). The concentration of solutes can affect the permeability of the membrane and, as a result, large quantities of low MW solutes dialyze, in quantity roughly proportional to the percentage of damaged cells (Mazur, 1965). It is therefore possible that the freeze-storage and freeze-drying processes led to the lysis, or at least damage, of the cells, causing the release of peptides and amino acids. Indeed, the freezing and drying steps of the lyophilization process may cause protein unfolding and alteration of the protein structure due to the dehydration stress and ice-liquid interfacial stress (Butreddy et al., 2020).

Spirulina biomass is normally cultivated in medium varying for their osmotic, temperature, and salt concentrations, mostly being highly alkaline (Lafarga et al., 2021); indeed, the pH of WB exceeded 9, preventing both enzymatic and microbial activity. Thus, a solution of food grade sodium acetate was added to all samples to decrease the pH to circa 6.6, and it remained stable throughout the treatments except when L. plantarum was inoculated. The high pH during cultivation also prevents the growth of other microbial species into the substrate (Soni et al., 2017). Indeed, except for mesophilic aerobic bacteria, none of the microbial groups investigated (yeasts, molds, and Enterobacteriaceae) were found in raw and treated biomasses. However, the incubation at 30°C for 24 h with Alcalase^® allowed an increase of total mesophilic bacteria, probably due to the higher availability of nutritional compounds. As expected, when inoculated, LAB exceeded a cell density of 9 log10 cfu/g, leading to the synthesis of high concentrations of organic acids.

A more comprehensive overview of what the protein fraction underwent to during the treatments was provided by electrophoresis. The protein fraction in Spirulina mainly consists of phycobiliproteins, chromophore-bearing, highly fluorescent, and water-soluble protein components of the photosynthetic complexes of cyanobacteria. They are classified into two groups on the basis of their colors: red for phycoerythrin and blue for phycocyanin. The latter includes C-phycocyanin (C-PC), R-phycocyanin (R-PC), and allophycocyanin (APC) (Kuddus et al., 2013). APC and C-PC often occur as a trimer, hexamers, and other oligomers, and C-PC constitutes up to 20% of the dry weight of Spirulina (Chaiklahan et al., 2011; Kuddus et al., 2013). SDS-PAGE results showed an intense proteolysis determined by Alcalase^®, involving mainly the α- and β-subunits of phycobiliproteins, having MW slightly lower than 20 kDa (Chaiklahan et al., 2011). Because of the enzymatic treatment, as well as to the proteolytic system of L. plantarum T0A10, the other major change in the protein profile concerned proteins with MW over 100 kDa, which faded considerably in biomasses singly and combined treated with Alcalase^® and microbial strain. Moreover, lyophilized biomass was characterized by much weaker bands, compared to WB, with the loss of polypeptides between 15 and 20 kDa (Figure 2). It is hypothesized that the precipitation of some proteins followed the salt concentration increase due to the freeze-drying. With water removal, ions and proteins compete for water molecules, leading to protein-protein interactions, which become stronger than protein-water interaction, resulting in aggregation and precipitation (Wingfield, 2016).

Because mono- and bi-dimensional electrophoresis only deemed proteins within the range 10–200 kDa, the effect of the treatments on peptides below 10 kDa and amino acids was evaluated through chromatographic techniques. The chromatographic analysis based on hydrophobic interactions highlighted significant differences in the peptide fraction among samples. Although the total number of detected peaks decreased with bioprocessing (compared to wet and lyophilized biomasses), the total area was overall subjected to significant increases. Indeed, values 70, 36, 30, and 50% higher than their corresponding untreated samples were found for wet, dried, and lyophilized biomasses treated with Alcalase^® and L. plantarum T0A10, and lyophilized biomasses treated with Alcalase^®, respectively (Supplementary Table 2), suggesting substantial degradation of higher MW proteins, as confirmed by the SDS-PAGE and 2DE. As a matter of fact, because of the wide range of amino acids that it can recognize, the hydrolysis catalyzed by Alcalase^® has a strong tendency to give hydrolyzates with many peptides of small size (Tacias-Pascacio et al., 2020). Whereas, the contribution of the complex system of proteases and peptidases own by LAB is well known (Kieliszek et al., 2021).

A further confirmation, of what observed so far, was provided by analyzing the single FAA profiles. Their content in wet Spirulina was in accordance with that reported elsewhere (Mišurcová et al., 2014; Bashir et al., 2016). Spirulina contains lysine and sulfur containing amino acids (further enhanced by Alcalase^® and fermentation), which are generally limiting in cereals; thus, it can be an excellent choice for supplementation of cereals-based products. Whereas in wet and lyophilized biomasses, A and AF were the treatments that led to the highest releases of FAA, linearly with what reported for peptides and proteins, in dried Spirulina, Alcalase^® allowed the most relevant increases, up to 24-fold higher than the initial concentration (Figure 3). Notwithstanding, it is worth of notice that the amount of each FAA in dried biomass, due to drying process explained above was one to 21 times lower than WB, with many amino acids close to 0 mg/g.

The effect of the treatments on protein digestibility was evaluated by mimicking the gastrointestinal digestion with an in vitro multi-step enzymatic assay. Digestibility of WB was similar to that previously reported (Kose et al., 2017) and substantial increases were found in lyophilized Spirulina, as consequence of the release of peptides and FAA. Overall, it was found that Alcalase^® and fermentation (alone or combined) led to IVPD increases proportional to the proteolysis degree, compared with their respective untreated samples.

Besides the increased digestibility of the protein fraction, proteolysis, carried out by Alcalase^® or LAB, can result in the formation of bioactive peptides having any number of functions (Rizzello et al., 2016b; Tacias-Pascacio et al., 2020). Moreover, bioactive peptides derived from Spirulina were already characterized for antibacterial, antiallergic, antihypertensive, antitumor, and immunomodulatory properties (Ovando et al., 2018; Lafarga et al., 2021). Hence, all samples were tested for their ability to inhibit microbial growth and scavenge radicals. In general, biomass suspensions had high antioxidant activity, suggesting a higher contribution of the protein component of the matrix, to the antioxidant properties observed. Alcalase^® and fermentation were the treatments that led to the highest increases of the radical scavenging activity, obtaining similar results to those reported by Niccolai et al. (2019) when Spirulina was fermented with a L. plantarum strain.

One of the main concerns of the food industry is ensuring food safety during shelf life, preventing contamination by pathogenic and spoilage microorganisms, and the use of natural antimicrobic instead of synthetic ones is becoming the preferred option. In our study, 100% inhibition of P. roqueforti growth was achieved when lyophilized biomass enzymatically treated was used. As a matter of fact, antimicrobial activity of protein hydrolyzates obtained with Alcalase^® was reported (Tacias-Pascacio et al., 2020). On the other hand, only fermented Spirulina (F, AF, and SF) was able to inhibit food pathogen E. coli, but most of all, only in wet and lyophilized Spirulina. Much more subtle differences were observed in dried biomasses, most likely because the compounds responsible for such activity derive from the hydrolysis of soluble proteins lost during the drying process. However, further studies are necessary to evaluate a higher inhibition spectrum against fungi and food pathogenic bacteria (including gram positive bacteria), as well as the compounds responsible for such activity.

Data collected from the characterization of Spirulina biomasses were subjected to PCA as showed in Figure 5. The first and second factors explained, respectively, the 57.35 and 19.51% of the total variance. Except for dried samples, the first factor separated bioprocessed (A, F, and AF) from raw samples, whereas the second factor clearly distinguished wet and lyophilized biomasses. Bioprocessed samples of wet and lyophilized biomasses were characterized by the lower protein content to which corresponded higher concentration of peptides and FAA with potentially bioactive properties. On the contrary, more subtle changes were apported by enzymatic treatment and fermentation, when performed on dried biomasses, hence the concentration in the I quarter of the PCA plane (Figure 5).

This study aimed at assessing the effect of desiccation and biotechnological treatments on the Spirulina protein profile. Overall, aiming at stabilizing the biomass, by reducing the moisture content, the lyophilization proved to be the most promising method due to its effect on the structure of Spirulina, which led to the release of amino acids and small peptides in substantial amount. On the other end, exsiccation at low temperature, because of the filtering and pressing process, resulted in the loss of most of the soluble compounds originally characterizing Spirulina biomasses. Hence, more thought should be put when considering the drying method to apply and, although lyophilization entails expenses seldom accepted at industrial level, the cost-benefit ratio represents the needle of the scale.

Moreover, to enhance the bioaccessibility of peptides and amino acids, while releasing potentially bioactive compounds showing antioxidant and antimicrobial activities, the combination of Alcalase^® and fermentation with selected L. plantarum T0A10 was found to be the most appropriate approach. The release of bioactive peptides outside the gastro-intestinal tract is of major significance because they not only confer nutritional advantages to the body once ingested but also to the food itself, preventing or delaying its spoilage and shelf life.

Overall, freeze-dried Spirulina biomass treated with Alcalase^® and fermented with selected L. plantarum T0A10 showed scavenging activity toward ABTS radical (up to 60%) and promising antimicrobial activity against fungal spoilage P. roqueforti and food pathogen E. coli. Therefore, on the basis of the results of this work, the selected treatments will be the topic of further studies involving the identification of the compounds responsible for such activities and the use of bioprocessed Spirulina in the development of functional foods.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

MV was responsible for formal analysis, data curation, investigation, and writing the original draft. CD was responsible for formal analysis and data curation. CR was responsible for resources and funding acquisition and for manuscript revision. EP was responsible for the conceptualization of the study, investigation, validation, writing, reviewing, and editing. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.