Yeast species associated with honey: different identification methods

Abstract

In the present study, three different methods were used to identify yeast isolated from Trás-os-Montes, Portuguese honey. A total of 24 isolates were identified using a partial sequence of the 26S rRNA gene (rDNA), restriction patterns generated from the region spanning the internal transcribed spacers (ITS1 and ITS2) of the 5.8S rRNA gene and the API 20C AUX kit. Nine different yeast species were identified representing six different genera. Among the isolated honey samples, Rhodotorula mucilaginosa, Candida magnoliae and Zygosaccharomyces mellis were the predominant species. Partial sequence of the 26S rDNA yielded the best results in terms of correct identification, followed by-5.8S-ITS analysis. The commercial identification kit API 20C AUX was able to correctly identify only 58% of the isolates. Two new 5.8S-ITS profiles were described, corresponding to Trichosporon mucoides and Candida sorbosivorans. RESUMENEn este estudio, se han utilizado tres métodos para identificar levaduras aisladas de la miel de Trás-os-Montes, Portugal. Se han identificado un total de 24 aislados usando una secuencia parcial del gen rRNA 26S (rDNA), los patrones de restricción generados de la región de los espaciadores transcritos internos (ITS1 e ITS2) del gen rRNA 5.8S (rDNA) y el kit API 20C AUX. Fueron identificadas nueve especies distintas de levaduras que representan seis géneros distintos. Entre las muestras aisladas de la miel, Rhodotorula mucilaginosa, Candida magnoliae y Zygosaccharomyces mellis eran las especies predominantes. La secuencia parcial del rDNA 26S rindió los mejores resultados para la correcta identificación, seguida por el análisis del 5.8S-ITS. El kit comercial de identificación API 20C AUX. sólo identificó correctamente el 58% de los aislados. Se describen dos perfiles nuevos de 5.8S-ITS, correspondiendo a Trichosporon mucoides y Candida sorbosivorans.

Effect of Storage on Microbiological Quality of Honey

“…Many authors indicated physicochemical properties of honey, which should inhibit or even destroy the microorganisms (1,2,8,10,15,22). Several experiments showed a very strong antibacterial activity of honey against different microorganisms, including human and animal pathogens (3)(4)(5). On the other hand, under certain conditions, such as leaky containers, accessibility of oxygen and water, or high temperature of storage, some microorganisms may survive or even multiply (7,9,15).…”

“…Honey may be contaminated with different microorganisms. In general, the spore forming anaerobes and aerobes are reported as predominant microflora of honey, but several others genera of microorganisms were also detected (1,3,4,8,13,19,20). The primary sources of microorganisms in honey may be pollen, digestive tract of honey bees, soil, water, air, and nectar.…”

Natural fruits, flowers, honey, and honeybees harborHelicobacter pylori‐positive yeasts

“…53 Amplification was performed with 500-1000 ng yeast DNA as a template and initial denaturation at 94°C for 5 minutes, followed by 35 cycles of denaturation at 94°C for 1min, annealing at 55°C for 1 minute and extension at 72°C for 2 minutes, and final extension at 72°C for 10 minutes. 53 Amplification was performed with 500-1000 ng yeast DNA as a template and initial denaturation at 94°C for 5 minutes, followed by 35 cycles of denaturation at 94°C for 1min, annealing at 55°C for 1 minute and extension at 72°C for 2 minutes, and final extension at 72°C for 10 minutes.…”

“…52 Primers used to amplify the ITS region were ITS1(5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5-TCCTCCGCTTATTGATATGC-3′). 53 Amplification was performed with 500-1000 ng yeast DNA as a template and initial denaturation at 94°C for 5 minutes, followed by 35 cycles of denaturation at 94°C for 1min, annealing at 55°C for 1 minute and extension at 72°C for 2 minutes, and final extension at 72°C for 10 minutes. PCR products were electrophoresed, using 1% agarose gel in Tris/Borate/EDTA (TBE, 0.5 X), and digested without further purification, using restriction endonucleases Hha I, Hae III, and Hinf I (Promega, USA and Bioron, Germany).…”

“…Amplification of D1/D2 region of 26S rDNA from 29 yeasts was carried out, using primers NL1 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 (5′-GGTCCGTGTTTCAAGACGC-3′). 53 PCR was performed with 500-1000 ng of yeast DNA and initial denaturation at 94°C for 1min, followed by 35 cycles of denaturation at 94°C for 1min, annealing at 50°C for 1 minutes, and extension at 72°C for 2 minutes with final extension of 72°C for 5 minutes. The PCR products were electrophoresed on a 1% agarose gel, and their size was determined as 600 bp, based on comparison with molecular ladder (50-1500 bp) (CinnaGen, Iran).…”