Submitted to: World Journal of Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/11/2015
Publication Date: 2/23/2015
Publication URL: http://handle.nal.usda.gov/10113/60704
Citation: Hua, S.T., Hernlem, B.J., Yokoyama, W.H., Sarreal, S.L. 2015. Intracellular trehalose and sorbitol synergistically promoting cell viability of a biocontrol yeast Pichia anomala for aflatoxin reduction. World Journal of Microbiology and Biotechnology. doi: 10.1007/s11274-015-1824-3..
Interpretive Summary: P. anomala is a potential biocontrol agent for reduction of mycotoxin in the food chain. An important requirement for the use of biocontrol agents is the production of large quantities of the microorganisms and the development of formulations to prolong the shelf-life of the organisms. Maintaining the viability of biocontrol agents in formulated products is a great challenge. A stable liquid formulation is highly desirable for P. anomala WRL-076 because the biocontrol yeast products can be there by easily dispersed in water and delivered by spraying or dipping to the agricultural and horticultural crops and commodities. In this study we described several media compositions for supporting good growth of WRL-076 and their effects on the shelf-life of the stored yeast cells. Post growth supplementation of 5% trehalose to yeast cells at the beginning of storage was assessed for its role in promoting yeast cell viability. HPLC analysis of intracelluar trehalose and sorbitol concentrations demonstrated a synergistic effect of sorbitol and trehalose on keeping yeast cells viable after long term storage. This is the first report to show sorbitol and trehalose play important role in developing formulation for biocontrol agents.
Technical Abstract: Pichia anomala WRL-076 was discovered by a visual screening bioassay for its antagonism against Aspergillus flavus. The yeast was shown to significantly inhibit aflatoxin production and the growth of A. flavus. P. anomala is a potential biocontrol agent for reduction of mycotoxin in the food chain. Maintaining the viability of biocontrol agents in formulated products is a great challenge for commercial applications. Four media, NYG, NYGS, NYGT and NYGST are described which support good growth of yeast cells and were tested as storage formulations. Post growth supplement of 5% trehalose to NYGST resulted in 83% viable yeast cells after 12 months in cold storage. Intracellular sorbitol and trehalose concentrations were determined by HPLC analysis at the beginning of the storage and at the end of 12 month. Correlation of cell viability to both trehalose and sorbitol suggested a synergistic effect. Bonferroni (Dunn) t Test, Tukey’s Studentized Range (HSD) Test and Duncan’s Multiple Range Test, all showed that yeast cell viability in samples with both intracellular trehalose and sorbitol were significantly higher than those with either or none, at a 95% confidence level. DiBAC4(5) and CFDA-AM were used as the membrane integrity fluorescent stains to create a two-color vital staining scheme with red and green fluorescence, respectively. Yeast cells stored in formulations NYG and NYGS with no detectable trehalose, displayed mostly red fluorescence. Yeast cells in NYGST+5T showed mostly green fluorescence.