D-Trehalose

32200250

Freeze-drying technology has been widely considered for decades as a suitable technique to preserve microorganisms. However, protective agents must be added prior to freeze drying to improve the survival and storage stability of the bacteria. The objective of our study was to evaluate the effect of a new protectant medium containing sucrose (10 %), trehalose (10 %), skimmed milk (10 %) and antioxidants on the viability of gut bacteria under different storage conditions. Two strains were tested, Escherichia coli and Akkermansia muciniphila, as examples of facultative aerobic and anaerobic bacteria, respectively. We studied the cell viability and bacterial morphology in 5 fecal samples in the presence and absence of this protectant medium using plating technique, flow cytometry and scanning electron microscopy. The results of bacterial viability assessed by plating method showed that the protectant medium yielded higher survival rates for both strains whatever the storage conditions (85-93 %) compared to normal saline solution (0.36-37.50 %). It also showed its effectiveness on fecal samples, where bacterial viability after freeze-drying was 89.47 ± 7.63 % and 84.01 ± 7.44 %, as evidenced by flow cytometry analysis and plating method. However unprotected samples showed the lowest cell viability at 19.01 ± 12.88 % and 13.23 ± 9.56 %, as measured by flow cytometry and plating method. In addition, bacterial size and shape were conserved in the protectant medium. In contrast, storage without protectant medium severely damaged bacterial morphology. In conclusion, our study is the first to use morphological features as well as culture-dependant and culture-independent tests to evaluate the effectiveness of a new protectant medium.

Bacterial viability; Freeze-drying; Gut microbiota; Preservation; Protectant medium; Survival.

A new protectant medium preserving bacterial viability after freeze drying

Sara Bellali 1, Jacques Bou Khalil 2, Anthony Fontanini 2, Didier Raoult 1, Jean-Christophe Lagier 3

2020 Jun

32191747

Harmonia axyridis is a major bio-control agent of pests in agriculture and forest ecosystems. It is also a globally important invasive insect species. To test whether dark elytra colour is associated with greater cold hardiness, we compared the survival rate of prolonged cold exposure in both yellow and black colour morphs of female and male H. axyridis. We determined the trehalose and glycogen content, trehalase activity, and the dynamics of genes associated with the trehalose metabolic pathway. Yellow forms predominated before winter began, however black forms increased from 11.15 to 30.46% after overwintering. There was no significant difference in trehalose content between the females and males during overwintering. Glycogen content in over-wintering yellow females and black males increased significantly, while it decreased in black females. Soluble trehalase activity increased significantly in all the insects except black females. Membrane-bound trehalase activity increased in black males, and decreased in black females. Trehalose and glycogen content and trehalase activity were regulated by differential expression of TRE and TPS genes. Female beetles weighed more than males and survived in low temperatures for longer periods of time, regardless of elytra colour, suggesting that mass is a stronger predictor of overwintering survival rather than colour morph. Our results provide a guide for comparing cold resistance in insects and a theoretical basis for cold storage of H. axyridis for use as natural enemies of pests in biological control programs.

Effect of long-term cold storage on trehalose metabolism of pre-wintering Harmonia axyridis adults and changes in morphological diversity before and after wintering

Boping Zeng 1, Shasha Wang 2, Yan Li 2, Zhongjiu Xiao 1, Min Zhou 2, Shigui Wang 2, Daowei Zhang 1

2020 Mar 19;