**3. Conclusions**

The results support the hypothesis that the Llayta biomass analysed does not contain MC-LR or BMAA toxins, and it can be considered a safe ingredient for human consumption.

#### **4. Materials and Methods**

To evaluate whether Llayta is a safe ingredient for human consumption, we evaluated the presence of target cyanotoxin genes in the genomes of Llayta (dry biomass) and four strains of cyanobacteria (LLC-10, LLA-15, CAQ-15, LCHI-10) isolated from various water bodies in northern Chile. Additionally, the presence of microcystin-LR (MC-LR) and BMAA was evaluated by HPLC-MS in the dried biomass of Llayta. Genomic DNA was extracted and purified from all isolates and Llayta (dry biomass) using the PureLink Genomic DNA Mini Kit (Invitrogen, USA), according to the manufacturer's instructions. Table 1 shows the target genes (*mcy*A, *mcy*E*, cyr*J, *ana*C*, sxt*A and *sxt*G) and the primers used for PCR. The cyanobacterial strains *Aphanizomenon gracile* LMECYA-040, *Anabaena* sp. LEGE X-002, *Cylindrospermopsis raciborskii* LEGE 97047 and *Microcystis aeruginosa* LEGE 91339 were used as positive control for saxitoxin, anatoxin, cylindrospermopsin and microcystin, respectively, and obtained from Blue Biotechnology and Ecotoxicology Culture Collection (LEGE Culture Collection) of CIIMAR/University of Porto.

**Table 1.** Primers used to show the presence/absence in Llayta samples of target genes required for toxin biosynthesis.


After molecular screening of the samples, the dry Llayta biomass was also examined by liquid chromatography coupled with mass spectrometry (LC-MS) in order to confirm the presence of MC-LR and BMAA. One gram of Llayta (dry biomass) was macerated with 8 mL of methanol 75% in liquid nitrogen. After two hours, 12 mL MeOH 75% was added and the methanolic suspension (20 mL) was sonicated (5 times, 60 s, 60 Hz) on ice to extract the intracellular toxins. After centrifugation (4000× *g* for 2 min, at 4 ◦C), the supernatant was recovered and concentrated in a rotary evaporator. This residue was suspended in 20 mL MeOH 75%, sonicated, centrifuged and evaporated as before. The final residue was dissolved in 50% (v/v) methanol and analysed by LC-MS to detect microcystin. To detect the presence of BMAA by LC-ESI-MS/MS, the resulting pellet from the maceration of Llayta was subjected to acid hydrolysis dissolving it in 1 mL 6M HCL at 110 ◦C for 24 h [21].

The LC-MS system used to identify and quantify MC-LR in Llayta was a Liquid Phase Chromatograph Finnigan Surveyor (Thermo Scientific, San Jose, CA, USA), coupled with a spectrometry detector (MS Mass LCQ FleetTM ion trap), with electrospray interface (ESI), including a Surveyor LC pump, a Surveyor auto sampler and a Surveyor photoelectric diode array detector (PDA). The program used for data acquisition and processing was XcaliburTM version 2 (Thermo Scientific, San Jose, CA, USA). The mass spectrometer was operated in full scan mode. The capillary voltage and tube lens were maintained at 22 and 120 kV, respectively; the spray voltage was 4.5 kV. Nitrogen was used as a sheath and auxiliary gas. The sheath gas flow rate was set at 80 (arbitrary units) and the auxiliary gas at 10. The capillary temperature was held at 350 ◦C. Helium was used as a collision gas in the ion trap at a pressure of 3 bar. Separation was achieved on C18 Hypersil Gold column (100 × 4.6 mm I.D., 5 μm, Thermo Scientific, Waltham, MA, USA) kept at 25 ◦C, with a flow rate of 0.7 mL/min. The injected volume was 10 μL in loop partial mode. Samples were injected in positive polarity mode, in Full scan (270–2000 m/z). The standards and samples were injected in duplicate and a blank and two standards of different concentration were introduced at each set of 6 samples. The standard solution of MC-LR was purchased from DHI LAB Products (Hørsholm, Denmark, Batch nº MCLR-110), with a concentration of 11.026 μg/mL. The system was calibrated using seven dilutions of the standard solution of MC-LR (between 8.5 and 180 μg/L) diluted in 50% acetonitrile (ACN). A gradient elution was used with mobile phase A (ACN) and B (water), both acidified with 0.1% formic acid (55% A and 45% B at 0 min, 90% A and 10% B at 12 min, 100% A at 12.5 min, 100% A at 15 min, 45% A and 55% B at 15.01 and 25 min). Under these conditions the MC-LR retention time (RT) was 7.21 min and the LOD and LOQ were 5.7 μg/L and 8.5 μg/L, respectively. Samples were analysed using the mass-to-charge ratio (m/z) transition of 995 > 599, at 35 eV collision energy. The MC-LR transition was monitored for 1 microscan time. The precursor ion (m/z 995) and MC-LR reference fragment ions with m/z values of 375, 553, 599, 866 and 977 were monitored in the MS/MS mode, in order to validate the presence of the toxin. To assess the presence/absence of other microcystin variants, precursor ions of [DMAdda5] MC-LR (m/z 981), [ADMAdda5] MC-LR (m/z 1009) and [D-Asp3, ADMAdda5] MC-LR (m/z 1023) were searched in the MS/MS obtained spectra. Diagnostic ions at m/z 553 [Mdha-Ala-Leu-MeAspArg + H+] and m/z 627 [Arg-ADMAdda-Glu + H+] were also scanned, as well as DMAdda (m/z 121.06) and ADMAdda (m/z 163.08) specific fragment ions. The qualitative analysis of BMAA in Llayta hydrolysates was done by LC-ESI-MS/MS as described above with the following modifications: the capillary voltage and tube lens were maintained at 33 and 115kV/a, respectively; the spray voltage was 5 kV; nitrogen was used as a sheath and auxiliary gas at a flow rate of 60 (arbitrary units) and the auxiliary gas at 20; the column used was a HILIC (100 × 4.6 mm I.D., 2.6 μm, Phenomenex, USA) kept at 40 ◦C, with a flow rate of 0.5 mL/min; samples were injected in positive polarity mode, in full scan (50–500 m/z); the standard solution was a mixture of BMAA and L-2, 4-Diaminobutyric acid dihydrochloride (DAB) from Fluca (Batch nº 0001418988), with a concentration of 0.145 μg/g; a gradient elution was used with mobile phases A (MeOH) and B (water), both acidified with 0.1% formic acid (90% A until 10 min, 60% A until 20 min, 50% A until 26 min), returning the start conditions and equilibrating 10 min and finally, samples were analysed using the mass-to-charge ratio (m/z) of 119 at 20 eV collision energy.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6651/12/6/382/s1, Figure S1. Extracted Ion Chromatogram and spectrum of Blank solution (methanol 50% + 0.1% Formic acid), Figure S2. Extracted Ion Chromatogram and Full MS2 spectrum of MC-LR Standard (20 ppb) at 3.01 min, Figure S3. Extracted Ion Chromatogram and Full MS2 spectra of Llayta extract at *m*/*z* 995.50, Figure S4. Extracted Ion Chromatogram and Full MS<sup>2</sup> spectra of Llayta extract at *m*/*z* 981, Figure S5. Extracted Ion Chromatogram and Full MS<sup>2</sup> spectra of Llayta extract at *m*/*z* 1009, Figure S6. Extracted Ion Chromatogram and MS2 spectra of Llayta extract at *m*/*z* 1023, Figure S7. Extracted Ion Chromatogram and MS<sup>2</sup> spectrum of Llayta extract at *m*/*z* 121.06, Figure S8. Extracted Ion Chromatogram and MS2 spectrum of Llayta extract at *m*/*z* 163.07.

**Author Contributions:** Conceptualization, A.G. and V.V.; methodology, A.G., J.A., R.C.-B., V.V., formal analysis, A.G., J.A., R.C.B, F.O.; investigation, all authors.; resources, A.G., B.G.-S., V.V.; writing—original draft preparation, A.G. writing—review and editing, all authors; funding acquisition, A.G., B.G.-S., V.V. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by MINEDUC-UA, Universidad de Antofagasta, Project [project code ANT 1755, 2018]; Proyecto Semillero de Investigación, Universidad de Antofagasta [SI-5305, 2018]; CeBiB, CONICYT-Chile [FB-0001, 2018]; and the CIIMAR [FCT Project UIDB/04423/2020 and UIDP/04423/2020 and by the Atlantic Interreg Project—EnhanceMicroAlgae—High added-value industrial opportunities for microalgae in the Atlantic Area (EAPA\_338/2016)].

**Acknowledgments:** Special thanks to CIIMAR members from the Laboratory Blue Biotechnology and Ecotoxicology.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
