Abstract: Objective: To effectively investigate yeasts involved in the spoilage of orange juice and lay the basis for the rapid detection, identification and control of yeasts in juice products. Methods: The colonial and cellular morphology of 8 yeast isolates from spoiled orange juice was observed. The 5.8S rDNA and two ribosomal internal transcribed spacers (5.8S-ITS) of yeasts were amplified with primer ITS1/ITS4. The PCR products were digested with Hha Ⅰ, Hae Ⅲ and Hinf Ⅰ, and then were sequenced using restriction fragment length polymorphism (RFLP). The D1/D2 domain of 26S rDNA was also amplified with primer NL1/NL4 and sequenced. Results: Eight yeast isolates were divided into six groups according to their colonial morphology, three groups according to cellular morphology, and four groups according to molecular analysis. RFLP analysis of 5.8S-ITS region resulted in four restriction patterns. The sequence analysis results of 5.8S-ITS region were similar with those of D1/D2. The sequence identity of eight isolates and four reference strains was as high as 99%. The phylogenetic trees constructed based on 5.8S-ITS region and D1/D2 domain were almost the same and had four branches: isolate Y1 clustered with Pichia kluyveri, Y12-3 and Y18-1 clustered with Pichia fermentans, Y22, Y23, Y26 and Y56 clustered with Meyerozyma guilliermondii, and Y47 clustered with Wickerhamomyces anomalus. Eight yeast isolates from spoiled orange juice were identified as P. kluyveri var. kluyveri, P. fermentans, M. guilliermondii and W. anomalus. M. guilliermondii is observed in spoiled orange juice for the first time. The combination of ITS-RFLP analysis, 26S rRNA gene sequencing and morphological analysis was effective for the identification of spoilage yeasts in orange juice. Therefore, rDNA analysis can allow the identification of yeast species. Colony characteristics may imply genetic differences at various species levels. Furthermore, the importance of morphological analysis cannot be ignored when yeasts are identified by molecular methods.

Key words: spoiled orange juice, yeasts, molecular identification, morphological analysis