Changes in Spirulina’s Physical and Chemical Properties during Submerged and Solid-State Lacto-Fermentation

The aim of this study was to select a lactic acid bacteria (LAB) strain for bio-conversion of Spirulina, a cyanobacteria (“blue-green algae”), into an ingredient with a high concentration of gamma-aminobutyric acid (GABA) for human and animal nutrition. For this purpose, ten different LAB strains and two different fermentation conditions (SMF (submerged) and SSF (solid state fermentation)) were tested. In addition, the concentrations of fatty acids (FA) and biogenic amines (BA) in Spirulina samples were evaluated. It was established that Spirulina is a suitable substrate for fermentation, and the lowest pH value (4.10) was obtained in the 48 h SSF with Levilactobacillus brevis. The main FA in Spirulina were methyl palmitate, methyl linoleate and gamma-linolenic acid methyl ester. Fermentation conditions were a key factor toward glutamic acid concentration in Spirulina, and the highest concentration of GABA (2395.9 mg/kg) was found in 48 h SSF with Lacticaseibacillus paracasei samples. However, a significant correlation was found between BA and GABA concentrations, and the main BA in fermented Spirulina samples were putrescine and spermidine. Finally, the samples in which the highest GABA concentrations were found also displayed the highest content of BA. For this reason, not only the concentration of functional compounds in the end-product must be controlled, but also non-desirable substances, because both of these compounds are produced through similar metabolic pathways of the decarboxylation of amino acids.


Introduction
Currently, a healthy lifestyle is very popular, and the practice of balanced dietsincluding the consumption of functional compounds-is of great interest to both humans and animals. Gamma-aminobutyric acid (GABA) is a functional compound that can be obtained through the decarboxylation of L-glutamate by the glutamate decarboxylase intracellular enzyme [1]. It has been confirmed that GABA can be synthetized by some microorganisms, including yeasts, fungi and bacteria [2][3][4]. However, to ensure efficient synthesis of GABA, a selection of the substrate is required, which should contain its precursors, as well as the appropriate microorganisms for the decarboxylation enzymatic process. Spirulina, which belong to the Cyanobacteria class (cyanobacteria) [5][6][7] have a significant content of GABA precursors [8]. In addition, these prokaryotic microalgae are commonly used as a functional food and feed material [5,9], because of their wide range of pharmacological activities [10], including amelioration of heavy metals and pesticide toxicity [11].
Currently, the industry is turning to more sustainable technologies. For this reason, biotechnological processes are also changing to meet sustainability requirements. The fermentation process can be performed in liquid as well as in solid state conditions. Solidstate fermentation (SSF) is a microbial process occurring mostly on the surface of solid materials that have the property to absorb or contain water, with or without soluble nutrients [31,32]. Moreover, SSF is often known to reduce global costs in comparison to liquid fermentation [33]. The low water volume in SSF has a large impact on the economy of the process mainly due to smaller bioreactor size, reduced downstream processing, lower sterilization costs, etc. Furthermore, many SSF processes focus on the utilization of cheap agri-industrial byproducts as culture media [32,34]. Submerged fermentation (SMF) is a very well-known methodology in the scientific literature, while SSF occupies a very small but emerging space in biotechnology [35]. In this study, we also hypothesized that the same microorganisms can be used in both fermentation techniques, but the results will differ due to the enormous differences in processing conditions.
Taking into consideration that LAB can excrete decarboxylases, the decarboxylation process can lead to the formation of desirable (e.g., GABA formation) and/or undesirable (e.g., biogenic amines (BA)) metabolites. Most of the BA are classified as non-desirable compounds, except for beta-phenylethylamine (β-PEA), which is attributed to neurotrans-mitters [36,37]. Beta-phenylethylamine is a well-known and widespread endogenous neuroactive trace amine found throughout the central nervous system in humans [37]. This neurotransmitter modifies the release and the response to dopamine, norepinephrine, acetylcholine and GABA [38].
Moreover, during fermentation, various changes can be obtained, including that which concerns the bioconversion of lipids as well as once LAB can perform FA isomerization, hydration, dehydration and saturation in fermentable substrates [39].
The aim of this study was to select the most appropriate LAB strains for the bioconversion of Spirulina into an ingredient with a high concentration of GABA to be potentially used in human and animal nutrition.

pH Values and Color Coordinates (L*, a* and b*) in the Spirulina Samples
The pH and color coordinates of non-treated and fermented Spirulina samples are given in Supplementary File S1 (Table S1), Table 1 and Figure 1. Lactic acid bacteria strain used for fermentation × Duration of fermentation × Conditions of fermentation (submerged or solid state) a* 0.0001 b* 0.197 pH 0.486 L*-lightness; a*-redness or −a*-greenness; b*-yellowness; −b*-blueness; influence of factor or factors interaction is recognized as statistically significant when p ≤ 0.05. Significant influence of the analyzed factors or their interactions are marked in bold letters.  Among SSF samples after 48 h of fermentation, the lowest pH (4.10) was reached with Levilactobacillus brevis No. 173 strain. The increase in acidity is an indicator of the fermentation process and can be affected by many environmental and processing factors, such as LAB strains, carbon sources, type of fermentation, etc. In this respect, SSF are more effective in achieving lower pH values of the fermentable substrate when compared to SMF [40]. It was reported that the greatest changes during the fermentation of Spirulina with the Lactiplantibacillus plantarum strain are observed in the first 24 h (pH decreased from 7.3 to 5.1 and remained at the same value after 48 h) [41]. Moreover, Bao et al. [42] reported that the pH values of all fermented Spirulina samples were similar, and significant pH decreases to 4.3-5.3 were observed within the first 12 h. However, the acidification rate was the fastest when fermenting with Lactiplantibacillus. plantarum B7 strain [42]. Our study showed that the fermentation conditions (i.e., SMF or SSF) is a key factor on the final pH of the Spirulina samples (p = 0.042) ( Analysis of between-subject effects unveiled that the analyzed factors and their interactions were not significant concerning the L* coordinates of the samples (Table 1). Additionally, pH and L* coordinate values between samples presented a weak negative correlation (r = −0.277, p = 0.002). In contrast to L* coordinates, the LAB strain used for fermentation, duration of fermentation, SMF or SSF conditions, as well as all the interaction of these factors, were statistically significant with respect to a* coordinate values of Spirulina samples (Table 1).
Comparing the a* coordinate of SMF samples with the control (I), lower values were found in 14 out of 20 samples. A similar trend occurred in most of the SSF (with 24 and 48 h fermentation) samples (17 out of 20 samples) in comparison with the control (II). Regarding b* coordinate, most of the SMF samples presented lower values in comparison with the control (I), except for 24 h SMF with Levilactobacillus brevis No. 173. Opposite trends were found in b* coordinates of SSF samples, where in all cases, they were higher in comparison with the control (II). The color changes may occur because of the acidification of the substrate medium. Organic acids influence oxidation processes in fermentable substrates, leading to color changes [40]. Spirulina contains different-colored compounds, including carotenoids and C-phycocyanin [43]. In addition, β-cryptoxanthin and zeaxanthin are present in small amounts in Spirulina [44]. It was reported that the phycocyanin molecule is sensitive to environmental conditions, including pH [45,46]. As predicted, it was reported that the L* value of Spirulina has a significant correlation with pigment content [43]. Finally, our study showed that all the analyzed factors and their interactions were significant to the a* coordinate of Spirulina, and these findings led us to conclude that during the fermentation process, changes in Spirulina pigments occur.
2.2. L-Glutamic Acid (L-Glu) and Gamma-Aminobutyric Acid (GABA) Concentration in the Spirulina Samples L-Glutamic acid and gamma-aminobutyric acid concentrations of the non-treated (non-fermented) and fermented Spirulina samples are given in Supplementary File S1 (Table S2), Table 2 and Figure 2. From the comparison of glutamic acid concentration between 24 h SMF samples and control (I) samples, one may conclude that glutamic acid concentration: was lower in 3 out of 10 samples (in 24  .8%, respectively); and 1 out of 10 samples of glutamic acid concentration was similar (in 24 h SMF with Lacticaseibacillus casei No. 210). However, after 48 h of SMF, glutamic acid concentration was found to be higher in 7 out of 10 samples, and it was lower in 3 out of 10 samples when compared to the control (I).
When analyzing glutamic acid concentration SSF samples with the control (II), in most of the cases (except for in 24 and 48 h SSF with Lacticaseibacillus paracasei No. 244 samples), glutamic acid concentration increased, and the conditions of fermentation (SMF or SSF) proved to be a statistically significant factor on the glutamic acid concentration in Spirulina samples (  Despite that correlations between glutamic acid and GABA concentrations were not found, it was determined that the LAB strain used for fermentation, the conditions of fermentation (submerged or solid state) as well as the interactions between LAB strain used for fermentation and the conditions of fermentation (submerged or solid state) significantly affected GABA concentration in Spirulina samples. It was reported that LAB strains may produce glutamic acid [47]. Even though the main aim of this study was to evaluate the chemical changes in Spirulina biomass, it can be hypothesized that the correlation between glutamic acid and GABA was not found due to the characteristics of the studied LAB, for which the metabolic pathways include not only the decarboxylation of amino acids but also the production of glutamic acid. Nevertheless, further studies are needed to confirm this hypothesis.
The most common amino acids in Spirulina spp. are glutamic acid followed by leucine and aspartic acid [48]. Specific bacterial genera are involved in the production of GABA [49]. It was reported that LAB may induce the structural breakdown of cyanobacterial cell walls via hydrolysis, leading to the conversion of complex compounds [50]. Most of the glutamic acid and GABA-producing microorganisms are LAB, including species from the genera Lactococcus, Lactobacillus, Enterococcus and Streptococcus [51]. However, the production of glutamic acid and GABA can vary in relation with microorganism characteristics, and it is species-dependent [52]. In addition to these findings, our study showed that fermentation conditions (SMF or SSF) are also a very statistically significant factor, especially for GABA content in Spirulina samples.

Biogenic Amine (BA) Content in the Spirulina Samples
Biogenic amine (BA) contents in non-treated and fermented Spirulina samples are given in Supplementary File S1 (Tables S3 and S4), Table 3 and Figures 3 and 4. Table 3. Correlations between biogenic amines and gamma-aminobutyric acid (GABA) and L-glutamic acid (L-Glu) concentrations.
With respect to tryptamine (TRP) concentration, it was not found in most of the SMF samples (except for in 24   In regard to SPRMD in SMF samples, it was found that SMF decreased SPRMD concentration in Spirulina samples, on average from 3.3 to 4.9 times (in 48 h SMF with Lacticaseibacillus paracasei No. 244 and in 24 h SMF with Pediococcus pentosaceus No. 183 samples, respectively). However, opposite trends of the SPRMD concentration were found in SSF samples. Furthermore SPRMD concentrations were, on average, 7.7 times higher in SSF than in SMF samples. Additionally, all the analyzed factors and most of their interactions-except for the interaction between duration of fermentation and conditions of fermentation (submerged or solid state)-were statistically significant on SPRMD concentration in Spirulina samples (p ≤ 0.0001) ( Changes in eating habits and looking for functional compounds are often associated with the incorporation of new non-traditional food ingredients into the main diet. However, functional properties in most of these so-called "super foods" are causing concern in terms of food safety issues. For this reason, this study includes not only the evaluation of GABA but also of BA concentrations in fermented Spirulina samples due to the fact that both compounds are formed through the carboxylation of amino acids. Table 3 tabulates the correlations between BA and GABA and glutamic acid concentrations in Spirulina samples. More specifically, from these results, it was possible to unfold statistically significant correlations between TRP, PUT, CAD, HIS, TYR and SPRMD and GABA, as well as between TRP, PUT, SPRMD and SPRM and glutamic acid. TYR, HIS, PUT, CAD, SPRM and SPRMD are mainly produced by microbial decarboxylation of amino acids [53,54]. PUT is a precursor for the synthesis of SPRMD [54]. Likewise, PUT and CAD can be metabolized from ornithine and lysine, respectively [55]. TYR is associated with constricting of vascular system, and HIS is known as a vasodilator [56]. In addition to the individual toxicity of BA, Wang et al. [57] reported that the sum of primary, secondary and tertiary biogenic amines is very important. TYR causes migraines, and PUT and CAD potentiate intoxication in the presence of other BA [58]. Finally, the samples in which the highest GABA concentrations were found also presented the highest content of BA. This shows that it is important to simultaneously study the presence of functional and non-desirable compounds in the end-product, especially when both compounds (as in this case) are produced through decarboxylation pathways of amino acids. Figure 4 presents the principal component analysis (PCA) of the first two principal components (PC) and makes apparent the existence of two clusters formed by the SMF and SSF samples, respectively, and thus the existence of statistically significant differences between both type of fermentations. Our previous studies showed that during the SSF, microorganisms show more efficient capacity to excrete enzymes and to degrade fermentable substrates [59].

Fatty Acid (FA) Profile in the Spirulina Samples
Fatty acid (FA) content in non-treated and fermented Spirulina samples is given in Supplementary File S1 (Tables S5-S7) and Figure 5.
The main FA in non-treated and fermented Spirulina samples were methyl palmitate (C16:0), methyl linoleate (C18:2) and gamma-linolenic acid methyl ester (C18:3G). When investigating C16:0 content in non-treated and SMF samples, the concentration was higher in 18 out of 20 samples than that in the control (I) samples. Only in the cases of 24 and 48 h SMF with Lacticaseibacillus casei No. 210 samples was the concentration of C16:0 similar to the control (I) (on average, 42.4% from total fat content). The same investigation of C16:0 content but in the SSF samples led to discovering different trends, and the highest C16:0 concentration was obtained in 48 h SSF with Lactobacillus coryniformis No. 71 samples (on average, 60.8% from total fat content). Tests between subjects showed that the LAB strain used for fermentation, the interaction LAB strain used for fermentation * duration of fermentation and the interaction LAB strain used for fermentation * conditions of fermentation (submerged or solid state) were statistically different in terms of C16:0 concentration in Spirulina samples (Supplementary File S1, Table S6).
Regarding the content of C18:2 in Spirulina samples, the values were lower in all the SMF and SSF samples in comparison with the control (I) and control (II) samples, respectively. Tests between subjects showed that the C18:2 content in Spirulina samples was significantly affected in the following cases: LAB strain used for fermentation (p = 0.003); conditions of fermentation (SMF or SSF) (p = 0.038); LAB strain used for fermentation * duration of fermentation (p ≤ 0.0001); LAB strain used for fermentation * conditions of fermentation (SMF or SSF) (p ≤ 0.001); and LAB strain used for fermentation * duration of fermentation * conditions of fermentation (SMF or SSF) (p ≤ 0.0001) (Supplementary File S1, Table S6).

Fatty Acid (FA) Profile in the Spirulina Samples
Fatty acid (FA) content in non-treated and fermented Spirulina samples is given in Supplementary File S1 (Tables S5-S7) and Figure 5.  When comparing the content of C18:3G with the control, different trends in SMF and SSF samples were perceived. In the case of SMF, C18:3G content increased in 6 out of 20 SMF samples; C18:3G content decreased in 8 out of 20 SMF samples; and C18:3G content remained similar to the control (I) in 6 out of 20 SMF samples. In the case of SSF, C18:3G content increased in 14 out of 20 SSF samples; C18:3G content decreased in 4 out of 20 SSF samples; and C18:3G content remained similar to the control (II) in 2 out of 20 SSF samples. Tests between subjects showed that the C18:3G content in Spirulina samples was significantly affected in the following cases: LAB strain used for fermentation (p = 0.004); LAB strain used for fermentation * duration of fermentation (p ≤ 0.0001); LAB strain used for fermentation * conditions of fermentation (SMF or SSF) (p ≤ 0.001); and LAB strain used for fermentation * duration of fermentation * conditions of fermentation (SMF or SSF) (p ≤ 0.0001) (Supplementary File S1, Table S6).
Alfa-linolenic acid methyl ester (C18:3α) was only found in SSF samples, and its content ranged from 0.399 (in 48 h SSF with Liquorilactobacillus uvarum No. 245 samples) to 0.618% of total fat content (in 24 h SSF with Leuconostoc mesenteroides No. 225 samples). Tests between subjects showed that all the analyzed factors were significant regarding the concentration of C18:3α in Spirulina samples (p ≤ 0.0001) (Supplementary File S1, Table S6).
Methyl palmitoleate (C16:1), methyl stearate (C18:0), and cis, trans-9-oleic acid methyl ester (C18:1 cis, trans) contents in Spirulina samples were lower than 5% from the total fat content. In addition, the analyzed factors and their interaction proved to not be significant on C16:1 content in Spirulina samples. On the other hand, the LAB strain used for fermentation, the conditions of fermentation (SMF or SSF), and the interaction LAB strain used for fermentation * conditions of fermentation (SMF or SSF) were significant regarding C16:1 content in Spirulina samples. Likewise, the interaction LAB strain used for fermentation * duration of fermentation was significant on C18:1 cis, trans in Spirulina samples.
The proximate composition of spirulina is related to numerous factors such as the source of the cyanobacteria, the season of the year, as well as to the manufacturing technology. The lipid concentration of Arthrospira platensis can vary from ca. 5 to 10% (of the dry weight) [60]. Long-chain FA are predominant compounds in Spirulina (mainly palmitic acid and gamma-linoleic acid) [61,62]. However, other studies reported higher contents of palmitic (46%), oleic (8%) and linoleic (12%) acids in Spirulina and lower contents of gamma-linoleic acid (20%) and stearic acid (1%) [63]. One of the most significant polyunsaturated FA is gamma-linoleic acid [62,64]. In addition to the FA profile of non-treated Spirulina, it was reported that 6 days of SSF with the fungus Aspergillus niger, Spirulina spp. attained the highest concentration of linoleic acid (60.63%, from total fat content), which was significantly higher than that obtained by SSF with Lactiplantibacillus plantarum (16.93%). However, the contents of elaidic, α-linoleic, stearic and palmitic acids of Spirulina spp. were higher in SSF with Lactiplantibacillus plantarum. The desirable changes in FA profile were explained by reduction of the substrate concentration during the fermentation process, because the nutrients were used for microbial growth and secondary metabolite production [65].
Omega-6 constitutes the majority of the total Spirulina FA [66,67]. Furthermore, Spirulina contains a significant amount of palmitic acid (16:0), which represents more than 25% from the total fat content [60]. PUFA levels in Spirulina ranged from 1.5 to 2.0% of total fat [68], whereas PUFA content represented 30% of the total fat content [69]. Another study reported that the FA profile of Spirulina contains sapienic acid (2.25 mg/100 g), linoleic acid (16.7%) and γ-linolenic acid (14%) [70]. According to Liestianty et al. [71], the FA of Spirulina encompasses myristic, heptadecanoic, stearic, oleic, palmitoleic, omega-3, omega-6, linoleic and palmitic acids. According to Al-Dhabi and Valan Arasu [70], myristic, stearic and eicosadienoic acids were the predominant saturated FA in Spirulina. Spirulina is the only food source that contains large amounts of essential FA, especially γ-linolenic acid. Finally, the FA profile of Spirulina samples is highly dependent on the fermentation process; thus, by selecting the most appropriate pre-treatment conditions desirable, changes in the FA profiles may be achieved.

Conclusions
All the tested LAB strains were suitable for Spirulina fermentation, and the lowest pH value (4.10) was obtained after 48 h of SSF with Levilactobacillus brevis No. 173. Changes in the pigments of Spirulina occurred during the fermentation process, and all the analyzed factors and their interactions were significant regarding the color's Spirulina a* coordinate. The main FA in non-treated and fermented Spirulina samples were methyl palmitate (C16:0), methyl linoleate (C18:2) and gamma-linolenic (C18:3G) acid methyl esters. Likewise, changes in the FA profile of the Spirulina were detected throughout the fermentation processes. Moreover, fermentation increased glutamic acid and GABA concentrations in Spirulina samples, and the highest GABA concentration was found in 48 h SMF with Lacticaseibacillus paracasei No. 244 (286.5 mg/kg) and in 48 h SSF with Lacticaseibacillus paracasei No. 244 (2395.9 mg/kg). Furthermore, putrescine (PUT) and spermidine (SPRMD) were the main BA in fermented Spirulina samples. In addition, significant correlations were found between BA concentration and GABA and glutamic acid. Spirulina samples where the highest GABA concentrations were found also showed the highest content of BA. Such correlation underlines the importance to study not only functional compounds but also potentially undesirable substances simultaneously, especially when they are involved in similar decarboxylation pathways of the amino acids.

Spirulina and Lactic Acid Bacteria Strains Used in Experiments and Fermentation Conditions
Lyophilized Spirulina powder (Arthrospira platensis) (content per 100 g: sodium 1.1 g, total carbohydrates 30.3 g, proteins 60.6 g, calcium 151. 5 Lithuania). Before the experiment, LAB strains were incubated and multiplied in De Man, Rogosa, and Sharpe (MRS) broth culture medium (Biolife, Milano, Italy) at 30 • C under anaerobic conditions for 24 h. A total of 3 mL of fresh LAB grown in MRS broth (average cell concentration of 9.0 log 10 CFU/mL) was inoculated in 100 mL of Spirulina media (for SMF, Spirulina powder was mixed with sterilized water, in a ratio of 1:20 w/w, whereas for the SSF Spirulina/water, the ratio was 1:2 w/w)-thus giving rise to 3% (v/w) of purified LAB strain per Spirulina-water mixture.
Afterward, the algae samples were fermented under anaerobic conditions in a chamber incubator (Memmert GmbH Co. KG, Schwabach, Germany) for 24 and 48 h, at 30 • C. Nonfermented samples (mixed with sterilized water in appropriate proportions for SMF and SSF) were analyzed as a control. Before and after fermentation, the pH, color coordinates, glutamic acid, GABA, BA and FA concentrations of the samples were analyzed. The experimental design is schematized in Figure 6. w/w, whereas for the SSF Spirulina/water, the ratio was 1:2 w/w)-thus giving rise to 3% (v/w) of purified LAB strain per Spirulina-water mixture. Afterward, the algae samples were fermented under anaerobic conditions in a chamber incubator (Memmert GmbH Co. KG, Schwabach, Germany) for 24 and 48 h, at 30 °C. Non-fermented samples (mixed with sterilized water in appropriate proportions for SMF and SSF) were analyzed as a control. Before and after fermentation, the pH, color coordinates, glutamic acid, GABA, BA and FA concentrations of the samples were analyzed. The experimental design is schematized in Figure 6.

Analysis of pH and Color Coordinates (L*, a* and b*) in the Spirulina Samples
The pH of Spirulina samples was evaluated with a pH meter (Inolab 3, Hanna Instruments, Venet, Italy) by inserting the pH electrode into the algae samples. The color coordinates of the Spirulina samples were evaluated on the surface using the CIE L*a*b* system (CromaMeter CR-400, Konica Minolta, Marunouchi, Tokyo, Japan) [72]. . Method recovery ranged from 59% to 112% for GABA and from 58% to 152% for L-Glu. Method repeatability ranged from 5% to 23% for GABA and from 1% to 20% for L-Glu. The results were obtained in some rounds of experiments on different days.

Analysis of Biogenic Amine (BA) Concentration in the Spirulina Samples
Sample preparation and determination of the BAs, including tryptamine (TRP), phenylethylamine (PHE), putrescine (PUT), cadaverine (CAD), histamine (HIS), tyramine (TYR), spermidine (SPRMD) and spermine (SPRM), in Spirulina samples was conducted by following the procedure reported by Ben-Gigirey et al. [73] with some modifications. Briefly, the standard BA solutions were prepared by dissolving known amounts of each BA (including internal standard) in 20 mL of deionized water. The extraction of BA in samples (5 g) was performed by using 0.4 mol/L perchloric acid. The derivatization of sample extracts and standards was performed using dansyl chloride solution (10 mg/mL) as a reagent. The chromatographic analyses were carried out using a Varian ProStar HPLC system (Varian Corp., Palo Alto, CA, USA) with two ProStar 210 pumps, a ProStar 410 auto-sampler, a ProStar 325 UV/VIS Detector and Galaxy software (Agilent, Santa Clara, CA, USA) for data processing. For the separation of amines, a Discovery ® HS C18 column (150 × 4.6 mm, 5 µm; SupelcoTM Analytical, Bellefonte, PA, USA) was used. The eluents were ammonium acetate (A) and acetonitrile (B), and the elution program consisted of a gradient system with a 0.8 mL/min flow-rate. The detection wavelength was set to 254 nm, the oven temperature was 40 • C, and samples were injected in 20 µL aliquots. The target compounds were identified based on their retention times in comparison to their corresponding standards.

Analysis of Fatty Acid (FA) Profile in the Spirulina Samples
The extraction of lipids for fatty acids (FA) analysis was performed with chloroform/methanol (2:1 v/v), and FA methyl esters (FAME) were prepared according to Pérez-Palacios et al. [74]. The fatty acid composition of the Spirulina samples was identified using a gas chromatograph GC-2010 Plus (Shimadzu Europa GmbH, Duisburg, Germany) equipped with Mass Spectrometer GCMS-QP2010 (Shimadzu Europa GmbH, Duisburg, Germany). Separation was carried out on a Stabilwax-MS column (30 m length, 0.25 mmID, and 0.25 µm df) (Restek Corporation, Bellefonte, PA, USA). Oven temperature program started at 50 • C, then increased at a rate of 8 • C/min to 220 • C, held for 1 min at 220 • C, increased again at a rate of 20 • C/min to 240 • C and, finally, held throughout 10 min. The injector temperature was 240 • C, interface −240 • C, and ion source 240 • C. The carrier gas was helium at a flow-rate of 0.91 mL/min. The individual FAME peaks were identified by comparing their retention times with FAME standards (Merck & Co., Inc., Kenilworth, NJ, USA).

Statistical Analysis
Fermentation of the samples was performed in duplicate, and all analytical experiments were carried out in triplicate. To evaluate a potential influence of different factors (SMF or SMF conditions, duration of fermentation, type of LAB strain used for fermentation) and their interaction on Spirulina sample characteristics, the mean of values was calculated, using the statistical package SPSS for Windows (v28.0.1.0 (142), SPSS, Chicago, IL, USA), and was compared using Duncan's multiple range test with significance defined at p ≤ 0.05. A linear Pearson's correlation was used to quantify the strength of the relationship between the variables. The results were recognized as statistically significant at p ≤ 0.05.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/toxins15010075/s1, Supplementary tables with experimental results. Table S1. Changes in the pH values and color coordinates (L*, a* and b*) in the Spirulina samples. Table S2. L-Glutamic acid (L-Glu) and gamma-aminobutyric acid (GABA) concentration in the spirulina samples. Table S3. Biogenic amine (BA) content (mg/kg) in the Spirulina samples. Table  S4. Influence of the analyzed factors and their interaction on biogenic amine (BA) concentration in the Spirulina samples. Table S5. Fatty acid (FA) profile in the Spirulina samples. Table S6. Influence of the analyzed factors and their interaction on fatty acid (FA) content in the Spirulina samples. Table S7. Classification of fatty acids (FA) in the Spirulina samples.