Comparison of Methods for Identification of Yeasts Isolated during Spontaneous Fermentation of Freshly Crushed Pineapple Juices

Abstract

The aim of this study was to evaluate the performances of methods such as sequencing of the internal transcribed spacer (ITS) and 26S (D1/D2) regions of ribosomal DNA, RFLP analysis of the ITS region and commercial biochemical test kit for the identification of the yeasts isolated during spontaneous fermentation of fresh crushed pineapple juice. The experiments were conducted in Thailand and Australia. The yeast isolates in Thailand were identified by sequencing the ITS and 26S (D1/D2) regions of ribosomal DNA and RFLP analysis of the ITS region. The yeast isolates in Australia were identified by sequencing analysis of the two DNA regions and commercial biochemical test kit. The identification results conducted in both countries were relatively similar. Mainly, the yeast isolates could be identified by the use of 26S rDNA in combination with ITS sequencing analysis. In Thailand, approximately 80% of the yeast isolates identified by sequencing analysis of the two regions gave similar identities and included Rhodotolula mucilaginosa, Issatchenkia orientalis, Hanseniaspora uvarum, Hanseniaspora opuntiae, Pichia guilliermondii, Aureobasidium pullulans, Saccharomycodes ludwigii, Candida tropicalis, Pichia fermentans, Zygosaccharomyces bailii, Candida stellata and Erythrobasidium hasegawianum. In Australia, 86% of the yeast isolates gave similar identifications by the sequencing analysis of the two regions and included P. guilliermondii, Pichia membranifaciens, P. fermentans, H. uvarum, H. opuntiae, I. orientalis, Candida sp., Yarrowia lipolytica, Tremella globispora, R. mucilaginosa and A. pullulans. Keywords: Biochemical test kit, Fruit juices, ITS-RFLP, rDNA sequencing, Yeast diversity.