PCR-fingerprinting and RAPD approaches for tracing the source of yeast contamination in a carbonated orange juice production chain

Abstract

Aims: To investigate the sort and the origin of the contamination of a packed fruit juice. Methods and Results: Fifty-eight yeast isolates were collected in a survey of two different visits to a carbonated orange juice factory. In each visit, samples were collected, six times, from seven points in the production chain. For each visit, no significant differences were observed among the yeast average values obtained in the control points considered. The random amplified polymorphic DNA (RAPD) with primer P24 and the PCR-fingerprinting with the microsatellites primers (GTG) 5 and (GAC) 5 were used, in order to discriminate the isolates, rendering 29 composite profiles; the most frequent one (24/58) was profile c, which included the yeast isolates from the final product and strains isolated before and after the pasteurization of the juice. These contaminant strains were identified as Pichia galeiformis by sequence analysis of D1/D2 26S rRNA gene. Conclusions:The results obtained point to an inefficient pasteurization of the juice related to the fouling of the heat-transfer surfaces of the plate-type exchanger. Significance and Impact of the study: The combination of PCR-fingerprinting and RAPD assays showed to be very useful in tracking the route of contamination in a carbonated juice production chain.

Identification of yeasts of dairy origin by amplified ribosomal DNA restriction analysis (ARDRA)

“…The methods described for yeasts are based on sequences of the small (18S) and large (28S) rDNA subunit and three internal transcribed spacer regions (ITS1, ITS2, IGS) harbouring the 5.8S gene between ITS 1 and ITS 2 (White, Bruns, Lee, & Taylor, 1990). Several primer pairs have been used for yeast identification on species level: NS1-ITS2 (Dlauchy, Tornai-Lehoczki, & Peter 1999;Redzepovic, Orlic, Sikora, Majdak, & Pretorius 2002;Vasdinyei & Deak, 2003), ITS1-ITS4 (Clemente-Jimenez, Mingorance-Cazorla, Martinez-Rodriguez, Las Heras-Vazquez, & Rodriguez-Vico 2004;Deak, Chen, & Beuchat 2000;Esteve-Zarzoso, Belloch, Uruburu, & Querol 1999;Pina et al, 2005;Sipiczki, 2004;de Souza Liberal, Silva Filho, Morais, Simoes, & Morais, 2005), and CNL12-CNS1 (Romero et al, 2005). Another approach for yeast identification was recently published by Gente et al (2007); the authors developed species-specific primers for the detection of Clavispora lusitaniae, D. hansenii var.…”

“…An extensive study on Geotrichum classification was published recently by Gente, Sohier, Coton, Duhamel, and Gueguen (2006). Molecular approaches for yeast identification on species level such as PCR-fingerprinting and random amplified polymorphic DNA (RAPD) analysis have been successfully used in recent years (Barszczewski & Robak, 2004;Pina et al, 2005). Amplified ribosomal DNA restriction analysis (ARDRA) is widely used for bacteria and was established for the identification of cheese smear bacteria (Brevibacterium spp., Corynebacterium spp., Arthrobacter spp., Microbacterium spp., and Staphylococcus spp.…”

Genetic diversity of Fusarium solani f.sp. cucurbitae the causal agent of crown and root rot of watermelon in Tunisia using ISSR markers

“…A total of six ISSR primers were evaluated for their capacity to produce polymorphic, scored and reproducible DNA fingerprint patterns. The primers used were two dinucleotide, and four trinucleotide repeats with or without 5' anchors: 5'DVD (CT) 7C (Mahuku et al, 2002), 5'YHY (GT) 7G, 5'DDB (CCA) 5 , 5' (GAC) 5, 5' (GTG) 5 (Pina et al, 2005) (Tib Molbiol, Berlin). Each PCR reaction contained 1 PCR buffer, 2.5 mM MgCl2, 100 mM each dNTPs, 0.4 mM of each primer, 0.5 U DNA Taq polymerase (Dominion MBL, Cordoba), and 0.5-5 ng template DNA were quantified spectrophotometrically.…”

RAPD Analysis of Salt-tolerant Yeasts from Contaminated Seasoned Pickled Plums and Their Growth Inhibition Using Food Additives

“…On the other hand, the identification and typing of microbes using gene analysis techniques is becoming more common. In the food industry, these techniques are applied to detect yeasts in wine (Comi et al, 2000;Guillamon et al, 1998;Pramateftaki et al, 2000;Querol et al, 1992), cheeses (Andrighetto et al, 2000;Romano et al, 1996;Suzzi et al, 2000), sausages (Cocolin et al, 2006), carbonated orange juice (Pina et al, 2005), and other food (Foschino et al, 2004 1-b). The DNA fraƒÊment patterns were classified into four groups.…”