Autochthonous yeasts associated with pineapple wine fermentation and chemical changes during fermentation process

Abstract

The main goal of this study is to undertake investigation of the yeasts associated with spontaneous pineapple juice fermentation to find specific yeasts, which could be applied as starter cultures for quality pineapple wine fermentations. Initially, the indigenous yeasts associated with pineapple fruits and with freshly crushed pineapple juices allowing natural fermentation to occur were investigated. The indigenous yeasts associated with pineapple fruits at various stages of maturity throughout cultivation in the fields of Thailand including damaged fruits were examined for their populations and species of yeasts, performing the isolation of yeast by rinsing and enriching methods. The yeast isolates were identified by the combination of cultural and three molecular methods, PCR-RFLP analysis, sequencing analysis of the 26S (D1/D2) and ITS region of ribosomal DNA, and ID 32 C system. With rinsing method, the results showed that the population of yeast on pineapple surfaces increased in parallel with maturity development of pineapple fruits. Aureobasidium pullulans was found as a common yeast on intact fruits skin at every cultivation stage, whereas only fermentative yeast, Pichia guilliermondii, was found as the prevalent species on mature, intact fruits. On damaged fruits, only Zygosaccharomyces bailii was found. For the enrichment methods, P. guilliermondii and Hanseniaspora uvarum were consistently found on intact pineapple skins at every cultivation stages. On damaged fruits, Z. bailii was still found as the main species. Not many yeast species were additionally found on pineapple skins, even when isolated by enrichment cultures. Apart from unidentified yeast, there were just four yeast species, Candida tropicalis, Candida sp., Erythrobasidium hasegawianum and Saccharomycodes ludwigii, which were found on a few occasions. For the investigation of the yeasts in spontaneous fermentation, pineapple samples from Thailand and Australia were studied to investigate the influence of climate and region on the yeasts associated with the fermentation systems. The yeasts associated with the spontaneous fermentations were determined by cultural isolation and PCR-DGGE analysis. Based on the data obtained from both methods, H. uvarum and P. guilliermondii were the main species similarly isolated from the fermentation systems of freshly crushed pineapple juice in all samples. P. guilliermondii was present as dominant species during the early stage of the fermentation, then H. uvarum became more prevalent until the final day of fermentation. Their populations increased from initial approximately 5 to 8 log cfu ml-1 through to the end of fermentation. Ethanol generated in the system of these natural fermentations was varied between 1 to 4 % (v/v). The yeast isolates obtained from this study were selected to investigation of their alcoholic fermentation characteristics if it generated alcohol higher than 5% (v/v). Based on the fermentation characteristics, S’codes ludwigii (Sl) was selected since it could produce the highest amount of ethanol content relative to the other yeast isolates. In addition, it showed the other fermentation characteristics, which were similar to a commercial S. cerevisiae. H. uvarum1 (Hu1) was also selected since it could generate 2-phenylethyl acetate, which is reported as a volatile compound giving rose and flowery odors. Therefore, these two yeast isolates and the commercial S. cerevisiae were applied as mixed starter cultures for pineapple wine fermentation. It was found that the mixed cultures of S’codes ludwigii (Sl) and H. uvarum1 (Hu1) had positive interaction. S’codes ludwigii (Sl) could play role as the main yeasts to produce the ethanol and complete the fermentation same as S. cerevisiae. In addition, it could extend the survival of H. uvarum1 (Hu1) during the fermentation allowing H. uvarum1 (Hu1) to carry out the fermentation and produce the 2-phenylethyl acetate in pineapple wine.