Autochthonous yeasts associated with mature pineapple fruits, freshly crushed juice and their ferments; and the chemical changes during natural fermentation
Introduction
The pineapple variety “Smooth cayanne” (Ananas comosus (L.) Merr.) is a significant economic farm-plant that is widely grown in every part of Thailand, one of the largest exporters of pineapple fruits, and some pineapple products, in the world. Recently, interest in developing pineapple wine, one of the new pineapple products, has increased as a potential product for exportation. Pineapple can be used to produce fruit wine (also known as country wines) since it contains a unique fruity flavour and has sufficient nutrients (including nitrogen) for yeast growth and fermentation, giving acceptable wine characters to the resultant (typically dry) white wine (Callens and De Smet, 1991, Ruengrongpanya, 1996, Ayogu, 1999). Moreover, its juice can be easily extracted, yielding up to 55% juice by weight, and so giving a good and easily obtained yield (Salvi and Rajput, 1995). Finally, the extracted juice is easy to filter and decontaminate, somewhat similar to the nature of grape juice, and thus is amenable to large scale commercial production. Therefore, pineapple has a high appeal for tropical fruit wine making and has been used for the successful production of fruit wines (Ayogu, 1999), with commercial production in Hawaii and Japan. Overall, the process of pineapple wine making is similar to grape wine. However, the important art or technology specific to pineapple wine manufacture, that is required to improve and standardize the quality, consistency and varieties of pineapple wine, is still very much at the initial stage of development.
The alcoholic fermentation of fruit (e.g. grape) juice into wine, is carried out by a complex and temporally dynamic interplay between the changing communities of microorganisms involving bacteria and principally yeasts. Indeed, the growth and metabolic activity of yeasts generate, in addition to ethanol, many of the secondary metabolites that significantly impact on the basic chemical structure and individuality of the wine flavour and aroma. As such they are some of the key determinants of pineapple wine quality, consistency and economic value. These wine properties depend in part upon the species and strains of yeasts that develop during the fermentation (Fleet, 2003). Typically, the non-Saccharomyces autochthonous yeasts dominate the ferment for the first few days before Saccharomyces takes over with rising ethanol concentrations (Bisson and Kunkee, 1993). However, during the initial few days of non-Saccharomyces domination, as well as later with the residual ethanol-tolerant surviving yeasts, a lot of the important chemistry that affects the taste and aroma of the wine is performed, exemplifying the importance of the indigenous fruit flora to the resultant wine.
Autochthonous yeasts are reported to be wild yeasts that produce the unique flavours and exceptional quality when used for traditional wine fermentation (Fleet et al., 2002). To commence the development of pineapple wine making technology, the study of natural yeast ecology from the spontaneous fermentation of freshly crushed pineapple juice should provide fundamental knowledge of ubiquitous or regional autochthonous yeasts that are associated with pineapple wine fermentation. These yeasts could then be selected and developed to be species-specific starter cultures by further investigating their functions in fermentations and roles in pineapple wine flavours.
Therefore, the main goal of this study was to determine the autochthonous yeasts and their function in the natural fermentation system of pineapple juice. However, since climatic and regional variations, as well as fruit varieties and agricultural practices, can influence the yeasts associated with fruits including pineapples, then pineapples grown in two well separated locations in both Thailand and Australia were subjected to investigation. The yeast communities on the fruit skin, and in the extracted juice and resultant spontaneous fermentations, were determined by culture isolation and then compared to each other and reference species by PCR-denaturing gradient gel electrophoresis (PCR–DGGE) analysis. The yeast isolates obtained throughout the study were identified by three methods. Two molecular methods, that is the sequence analysis of the ITS1-5.8S rRNA-ITS2 region and that of the D1/D2 regions of the 26S rDNA were used as the main diagnostic methods for examination of yeast isolates from both countries, leading to a molecular operational taxonomic unit (MOTU) based classification. Homology searching against known taxonomic species was used to ascribe species level identification to MOTU classifications. In addition, RFLP analysis of the ITS1-5.8S rRNA–ITS2 region amplicons was used to distinguish the yeast isolates found in Thailand, whilst the commercial ID 32 C system was used instead of RFLP analysis for the examination of yeasts isolated in Australia, leading to a direct species classification. The relationship among the chemical changes, ethanol, organic acids, sugars and dynamic yeast species and the population levels were also investigated to characterize the potential role and limiting factors of the autochthonous yeasts in the fermentation systems.
Section snippets
Pineapple sources
Pineapple samples of the “Smooth cayanne” cultivar (Ananas comosus (L.) Merr.) were collected from two growing regions of Thailand; one located in the Prachuapkirikhun province (Western Thailand) and the other in Rayong province (Eastern Thailand). In the first region, called TH1 hereafter, pineapple samples at commercial harvest stages were aseptically collected from the field where they were grown without agrochemical applications. The pineapple sample consisted of a composite of batches
Results
In this study, the populations and identification of yeast species associated with freshly crushed pineapple juice that was allowed to undergo natural fermentation for 6 days are reported. The yeasts associated with the fermentations were determined by culture isolation and morphology, biochemical tests and by DNA sequence homology and similarity analysis including PCR–DGGE profiling based analysis. The populations of individual species were counted, and the levels of alcohol, sugars and
Discussion
In the spontaneous fermentation of pineapple juice evaluated here, both the culture plating and PCR–DGGE evaluation methods support that H. uvarum and P. guilliermondii were the main yeast species isolated from freshly crushed pineapple juice ferments from Thailand and Australia. This is consistent with the pattern of yeasts present on the maturing fruit skin (at TH1), where P. guilliermondii was a common species on mature pineapple fruits and easily isolated by rinsing as well as enrichment
Conclusions
From this study, some new information has been added. It was known that there was less diversity of yeast species associated with the pineapple fruits, freshly crushed juices and their ferment ecosystems. H. uvarum and P. guilliermondii were the main species isolated from the freshly crushed pineapple juice and natural ferments from Thailand and Australia. Thus, these two yeasts did not appear to depend on climatic and regional traits but rather maybe ubiquitous. In the natural fermentation
Acknowledgements
We are grateful to Food Science, School of Chemical Sciences and Engineering, The University of New South Wales, Sydney, NSW for providing research facilities when conducting research in Australia. We are also grateful to Miss Padchara Sawatdiruk and Miss Patcharee Pattharathadakiat for their helpful assistance and cooperation. Additionally, we are thankful to Royal Thai Government and CU Graduate School Thesis Grant, Chulalongkorn University for financial support, and the Publication
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