Universidade Paranaense, Umuarama, Paraná, Brazil.
Universidade Federal da Bahia, Salvador, Bahia, Brazil.
J Microbiol Methods. 2022 Jul;198:106491. doi: 10.1016/j.mimet.2022.106491. Epub 2022 May 16.
Lentinus crinitus (Basidiomycota: Polyporales) is a saprophytic fungus with biotechnological importance described more than 20 years ago. However, there are few studies on the long-term preservation of this basidiomycete. Cryopreservation is a long-term storage technique that reduces the metabolic activity of microorganisms, but its success depends on the adjustment of the freezing process, the cryoprotectants, and the protective substrates for each species. This study aimed to assess the mycelial viability and genetic stability of L. crinitus strains cryopreserved at -86 °C for two years by the wheat grain technique using different cryoprotectants and freezing methods. Three strains of L. crinitus (U9-1, U13-5, and U15-12) were subjected to different concentrations and types of cryoprotectants (dimethyl sulfoxide, glycerol, glucose, and sucrose), freezing methods such as immediate freezing from 25 to -86 °C and progressing freezing from 25 to -86 °C in a freezing container with isopropyl alcohol to control the rate of cell freezing at -1 °C min, protective substrate (wheat grain and 2% malt extract agar), and cryopreservation period (1, 6, 12, and 24 months). After thawing, samples were evaluated for mycelial viability, time to mycelial recovery, mycelial stability, and genetic stability of the fungus. All techniques achieved effective cryopreservation at -86 °C, mainly with the wheat grain technique. All cryoprotectants (3.5% glycerol, 1.5% dimethyl sulfoxide, 25% sucrose, and 5% glucose), freezing methods (immediate and gradual), and protective substrate (wheat grain and malt extract agar) were effective for cryopreservation of the three L.crinitus strains in an ultra-low temperature freezer for two years. Mycelial viability, mycelial stability, and genetic stability of the fungus were not affected after two-year cryopreservation, evidencing the robustness of the long-term cryopreservation technique and the fungus.
糙皮侧耳（担子菌门：多孔菌目）是一种具有生物技术重要性的腐生真菌，早在 20 多年前就有描述。然而，对于这种担子菌的长期保存研究甚少。冷冻保存是一种降低微生物代谢活性的长期储存技术，但它的成功取决于每种物种的冷冻过程、冷冻保护剂和保护基质的调整。本研究旨在通过麦粒技术评估在 -86°C 下冷冻保存两年的糙皮侧耳菌株的菌丝活力和遗传稳定性，使用不同的冷冻保护剂和冷冻方法。将 3 株糙皮侧耳（U9-1、U13-5 和 U15-12）分别用不同浓度和类型的冷冻保护剂（二甲亚砜、甘油、葡萄糖和蔗糖）、不同的冷冻方法（从 25°C 直接降至 -86°C 和在装有异丙醇的冷冻容器中逐渐降至 -86°C，以控制细胞在 -1°C/min 的冻结速度）、保护基质（麦粒和 2%麦芽提取物琼脂）和冷冻保存期（1、6、12 和 24 个月）进行处理。解冻后，评估样品的菌丝活力、菌丝恢复时间、菌丝稳定性和真菌遗传稳定性。所有技术都能在 -86°C 下有效地进行冷冻保存，主要是通过麦粒技术。所有冷冻保护剂（3.5%甘油、1.5%二甲亚砜、25%蔗糖和 5%葡萄糖）、冷冻方法（直接和逐渐）和保护基质（麦粒和麦芽提取物琼脂）都能有效地将 3 株糙皮侧耳菌株在超低温冰箱中冷冻保存两年。两年的冷冻保存后，真菌的菌丝活力、菌丝稳定性和遗传稳定性均未受到影响，证明了长期冷冻保存技术和真菌的稳健性。
