Development of a new protocol for freeze-drying preservation of Pseudoalteromonas nigrifaciens and its protective effect on other marine bacteria

Background: Freeze-drying is known as one of the best methods to preserve bacterial strains. Protectant is the key factor affecting the survival rate of freeze-dried strains. In addition, salinity, bacterial suspension concentration, drying time, and other factors can also affect the survival rate of strains to varying degrees. At present, there are relatively few studies on freeze-drying preservation of marine bacteria. In the present study, we performed the freeze-drying protectant screening and optimized the preservation conditions for Pseudoalteromonas nigrifaciens, which is widely distributed in marine environment. The protective effects of the screened protectants were verified by 18 other marine bacterial strains. Results: The results indicated that the combination of 5.0% (w/v) lactose, 5.0% (w/v) mannitol, 5.0% (w/v) trehalose, 10.0% (w/v) skim milk powder, 0.5% (w/v) ascorbic acid and 0.5% (w/v) gelatin was the best choice for the preservation of P. nigrifaciens. The suggested salinity and concentration of initial cell suspension were 10 g/L NaCl and 1.0 × 109 CFU/mL, respectively. Furthermore, stationary-phase cells were the best choice for the freeze-drying process. The highest survival rate of P. nigrifaciens reached 52.8% when using 5­10% (w/v) skim milk as rehydration medium. Moreover, the other 18 marine strains belonging to Pseudoalteromonas, Vibrio, Photobacterium, Planomicrobium, Edwardsiella, Enterococcus, Bacillus, and Saccharomyces were freezedried under the abovementioned conditions. Their survival rates were 2.3­95.1%. Conclusion: Collectively, our results supported that the protectant mixture and parameters were beneficial for lyophilization of marine bacteria

Comparison of thris-EDTA and threhalose on DNA quality under different storage conditions "medico-legal view"

DNA storage is important to ensure integrity of DNA sample and maintain its availability while investigations. The best known condition for storage of DNA samples is by using Tris-EDT [TE]; as preservative agent, stored at -80[degree sign]C. A potential alternative to TE is trehalose which could stabilize any biological molecule at room temperature [RT]. Assessment of the optimal storage conditions which maintains quality of blood DNA samples with economical advantage. A case-control study using 8 groups of human blood DNA stored at 2 different temperatures [-80 [degree sign]C,RT] and preserved by using TE and trehalose. The effectiveness of storage conditions were tested at certain intervals [at set-up then after 3 and 6 month] using PCR assay of 18s ribosomal gene to evaluate DNA quality. DNA was assessed by running yield gels. PCR success rate were 43.8% and 62.8% using TE and trehalose respectively. After 6 months, PCR success rate were 25% for TE and 62.5% for trehalose [p<0.05]. The relative risk [RR] of poor quality associated with using trehalose is 0.4. Trehalose serves as an alternative to expensive freezer storage. It has a DNA protective effect which helps in preservation even trace DNA while judicial proceedings continue

Purification and characterization of trehalose-6-phosphate synthase from Saccharomycopsis fibuligera A11.

Mutant A11, a mutant of Saccharomycopsis fibuligera Sdu with low acid and neutral trehalase was found to accumulate over 18% (w/w) trehalose from starch in its cells. In this study, trehalose-6-phosphate synthase (Tps1) was purified to homogeneity from this mutant, with a 30-fold increase in the specific enzyme activity, as compared to the concentrated cell-free extract, from initial cells. The molecular mass of the purified enzyme as determined by SDS-PAGE was 66 kDa. The optimum pH and temperature of the purified enzyme were 6.6 and 37 degrees C, respectively. The enzyme was activated by Ca2+, K+ and Mg2+, with K+ showing the highest activation at 35 mM. On the other hand, Mn2+, Cu2+, Fe3+, Hg2+ and Co2+ inhibited the enzyme. The enzyme was also strongly inhibited by protease inhibitors such as iodoacetic acid, EDTA and PMSF.