AimsTo optimize a protective medium for freeze-dried Pichia membranifaciens and to evaluate biocontrol efficacies of agents against blue and green mould and anthracnose in citrus fruit. Methods and ResultsBased on the screening assays of saccharides and antioxidants, response surface methodology was used to optimize sucrose, sodium glutamate and skim milk to improve viability of freeze-dried Pi.membranifaciens. Biocontrol assays were conducted between fresh and freeze-dried Pi.membranifaciens against Penicillium italicum, Penicillium digitatum and Colletotrichum gloeosporioides in citrus fruit. Solving the regression equationindicated that the optimal protective medium was 606% (w/v) sucrose combined with 340% (w/v) sodium glutamate and 543% (w/v) skim milk. Pi. membranifaciens freeze-dried in the optimal protective medium showed 7680% viability, and retained biocontrol efficacy against Pe.italicum, Pe.digitatum and Co.gloeosporioides in citrus fruit. ConclusionsThe optimal protective medium showed more effective protective properties than each of the three protectants used alone. The viability of freeze-dried Pi.membranifaciens finally reached 7680%. Meanwhile, the biocontrol efficacies showed no significant difference between fresh and freeze-dried yeast against Pe.italicum, Pe.digitatum and Co.gloeosporioides in citrus fruit. Significance and Impact of the StudyThe results showed the potential value of Pi.membranifaciens CICC 32259 for commercialization.