Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Laboratory of Metabolomics, Gronostajowa 7 St, 30-387, Cracow, Poland; Jagiellonian University, Doctoral School of Exact and Natural Sciences, prof. S. Łojasiewicza 11 St. 7, 30-348, Cracow, Poland.
Polish Academy of Sciences, W. Szafer Institute of Botany, Lubicz 46, 31-512, Cracow, Poland.
Toxicon. 2024 Nov 28;251:108137. doi: 10.1016/j.toxicon.2024.108137. Epub 2024 Oct 21.
Some of the most commonly identified freshwater toxins are anatoxin-a (ATX-a), cylindrospermopsin (CYN), and microcystin-LR (MC-LR). The aim of this paper was to compare different methods of extracting and concentrating these cyanotoxins and check the impact of selected physical factors on the accumulation of biomass of Dolichospermum flos-aquae, Microcystis aeruginosa, and Raphidiopsis raciborskii. The effect of different cyanobacteria cultivation conditions on the amount of cyanotoxins synthesized showed no significant changes over time in the average concentration of all tested toxins in the medium compared to the control. Mixing cultures increases the intracellular content of ATX-a. Aerating also positively affects the concentration of MC-LR intracellularly. In order to optimize the solid phase extraction (SPE) process of toxins, the C18 phase or activated carbon was used. In general, higher toxin recoveries were achieved when using the C18 phase. The best result was achieved for ATX-a, 94% recovery with elution using methanol with 0.1% trifluoroacetic acid (TFA). For MC-LR, the best recovery was 59%, and for CYN 22%. The study evaluated the various methods to release cyanotoxins from cyanobacteria showed that: the highest ATX-a concentration (0.60 μg/mg d.w) was obtained using MilliQ water and microwave treatment for 10-15 s. For MC-LR, the highest extracted amount (6.73 μg/mg d.w) resulted from methanol treatment and boiling at 100 °C for 15 min. CYN extraction was the most effective by using MilliQ water and alternative freezing/thawing (1.54 μg/mg d.w). In conclusion, changing the optimal parameters of cyanobacterial cultivation, only slightly affects the increase in biomass accumulation and synthesis of cyanobacterial toxins. In the case of ATX, the key is the use of the TFA additive in the SPE process. No single method has been identified as the ideal approach for isolating various intracellular cyanotoxins.
一些最常见的淡水毒素是anatoxin-a (ATX-a)、cylindrospermopsin (CYN) 和 microcystin-LR (MC-LR)。本文旨在比较提取和浓缩这些蓝藻毒素的不同方法，并检查选定物理因素对大水花鱼腥藻、铜绿微囊藻和裂面鱼腥藻生物量积累的影响。不同蓝藻培养条件对合成的蓝藻毒素数量的影响表明，与对照相比，所有测试毒素在培养基中的平均浓度在时间上没有显著变化。混合培养增加了 ATX-a 的细胞内含量。曝气也对内源性 MC-LR 的浓度产生积极影响。为了优化毒素的固相萃取 (SPE) 过程，使用了 C18 相或活性炭。一般来说，使用 C18 相时，毒素的回收率更高。对于 ATX-a，甲醇洗脱回收率最高，为 94%。对于 MC-LR，最佳回收率为 59%，对于 CYN 为 22%。该研究评估了从蓝藻中释放蓝藻毒素的各种方法，结果表明：使用 MilliQ 水和微波处理 10-15 秒可获得最高的 ATX-a 浓度（0.60μg/mg d.w）。对于 MC-LR，甲醇处理和 100°C 煮沸 15 分钟可获得最高的提取量（6.73μg/mg d.w）。使用 MilliQ 水和交替冷冻/解冻（1.54μg/mg d.w）对 CYN 的提取最有效。总之，改变蓝藻培养的最佳参数，只会略微影响生物量积累和蓝藻毒素合成的增加。在 ATX 的情况下，关键是在 SPE 过程中使用 TFA 添加剂。没有一种方法被确定为分离各种细胞内蓝藻毒素的理想方法。
