Zymological indicators: a new concept applied to the detection of potential spoilage yeast species associated with fruit pulps and concentrates

Abstract

In a survey of the microbial quality of raw materials used in fruit juice processing, yeast counts in fruit concentrates and pulps were found to range from <1 to 2·9×103cfu g−1. Ascomycetous yeasts were represented by 76% of the isolates while 24% were basidiomycetes. The identification of strains isolated by the simplified identification system (SIM) revealed 19 yeast species representing 12 genera. The most frequently isolated yeasts belonged to the genera Saccharomyces, Pichia, Cryptococcus, Kluyveromyces andCandida .Fatty acid yeast composition allowed the separation of contaminating yeasts into one of three major groups. Group I included yeasts without linoleic (C 18:2) and linolenic (C 18:3) fatty acids such as Saccharomyces cerevisiae. Group II comprised yeasts without C 18:3 fatty acid like Zygosaccharomyces rouxii and Torulaspora delbrueckii, and group III included yeasts with C 18:2 and C 18:3 acids that belong, among others, to one of the following yeast genera:Pichia, Candida, Kluyveromyces or Cryptococcus.Species-specific PCR primers were used for the rapid detection and identification of the most dangerous species affecting fruit concentrate stability. The simplified protocol used consisted of PCR-amplification of conserved tracts in the ITS region of the rDNA unit, thus enabling the detection of potentially dangerous flora such as Zygosaccharomyces species and T. delbrueckii in contaminated fruit concentrates. Results from PCR-typing were in full agreement with the fatty acid compositions of these species.The grouping of contaminant yeasts into three main groups showed that fatty acid composition may be used to differentiate yeasts according to their technological significance. Yeasts isolated in this work as being most dangerous to product stability belong to either group II (Z. rouxii and T. delbrueckii) or group I (Saccharomyces spp.). Group III was comprised of several species regarded as indicators of deficiencies in ‘good manufacturing practices’. Thus, each of the groups delineated may be considered to be a zymological indicator of technological significance. The conjugation of fatty acid profiles with PCR-typing methods may be used as a rapid detection system for contaminant yeasts. The fatty acid profiles provide a preliminary identification of yeasts potentially dangerous to product stability present within 48 h of isolation. Whereas the PCR-typing method is mainly used to confirm isolate identity, when required, after the initial diagnosis has been performed, over a period of 4 h.