Detektion och kvantifiering av mögel på spannmål med realtids PCR

Tillväxt av mögel och jäst är ett problem vid lagring av spannmål ensilage och andra fodermedel. Traditionella metoder för identifiering och kvantifiering av sådana mikrosvampar är tidskrävande och mödosamma. Det finns därför ett behov för att utveckla nya, snabba och pålitliga förfaranden för identifiering och kvantifiering. Vi har utvecklat en molekylär metod som kan används för kvantifiering av mögel och jäst som växer på spannmål eller andra fodermedel. Förfarandet är baserat på direkt isolering av mikroorganismernas DNA från spannmål och kvantifiering av organismspecifika gener med realtids-PCR. Metoden utprovades i ett projekt för odling av mycelväxade svamp (Rhizopus oligosporus) och jäst (Saccharomyces cerevisiae) på korn. En enkel DNA-extraktion var effektiv för mögel, jäst och även mjölksyrabakterier. Koncentrationen av organismspecifika gener (determinerade i realtids PCR) korrelerade med ergosterolhalten (som indikerar svamptillväxt) i R. oligosporus ren kultur och med antal kolonibildande enheter av jäst och mjölksyrabakterier. Detektionen var mycket specifik för de testade organism, och ingen korsdetektion upptäcktes. Förfarandet är snabbt och pålitligt med möjlighetet till analys av 50 prover under två dagar. Metoden kan antagligen användas för detektion och kvantifiering av även andra mögelarter på andra sorts spannmål. Man kan även identifiera specifika gener, t.ex. gener som är involverade i mykotoxinproduktionen

Selection and identification of lactic acid bacteria that inhibit yeast contaminants isolated from fermented plant beverages

In order to investigate yeast contamination in finished products of fermented plant beverages (FPBs), 27 FPBs samples were collected from northern Thailand. Nine samples from finished products werecontaminated with yeast and 36 yeast isolates were isolated and identified to the genera level by conventional methods. These included 12 isolates of Rhodotorula sp., 9 isolates of Pichia sp., 9 isolates of Hansenula sp.,3 isolates of Saccharomyces sp. and 3 isolates of Candida sp. Rhodotorula sp. was chosen to use as a target organism for the primary screening of lactic acid bacteria (LAB) with antiyeast activity, using a dual cultureoverlay assay. Fifteen of the 72 LAB cultures isolated from Thai fermented foods and the FPBs produced a strong inhibition against the Rhodotorula sp. Ten of these also had a broad antiyeast spectra (at least 5genera inhibited). Three of the isolates that gave the best inhibition (DW1, 3 and 4) were identified as Lactobacillus plantarum strains based on conventional identification methods

Rhizopus oligosporus and yeast co-cultivation during barley tempeh fermentation-Nutritional impact and real-time PCR quantification of fungal growth dynamics

Barley tempeh was produced by fermenting barley kernels with Rhizopus oligosporus. The potential of the yeasts Saccharomyces cerevisiae (three strains), S. boulardii (one strain), Pichia anomala (one strain) and Kluyveromyces lactis (one strain) to grow together with R. oligosporus during barley tempeh fermentation was evaluated. All yeast strains grew during the fermentation and even during cold storage of tempeh (P < 0.01). The growth of yeasts slightly increased the ergosterol contents, but did not influence amino acid contents and compositions, and did not reduce phytate contents. Slight increases of vitamins B-6 and niacinamide, and slight decreases of B, and biotin were observed. Quantification of fungal growth is difficult during mixed species fermentations because ergosterol is found in all fungal species, and colony-forming-unit (cfu) estimations are not reliable for R. oligosporus and other sporulating fungi. Therefore, we developed a quantitative real-time PCR method for individually quantifying S. cerevisiae and R. oligosporus growth in barley tempeh. The PCR results were highly correlated with the ergosterol content of R. oligosporus and with the number of cfu of S. cerevisiae. Thus, real-time PCR is a rapid and selective method to quantify yeasts and R. oligosporus during mixed species fermentation of inhomogenous substrate such as barley tempeh

Genome and physiology of the ascomycete filamentous fungus Xeromyces bisporus, the most xerophilic organism isolated to date

Xeromyces bisporus can grow on sugary substrates down to 0.61, an extremely low water activity. Its genome size is approximately 22Mb. Gene clusters encoding for secondary metabolites were conspicuously absent; secondary metabolites were not detected experimentally. Thus, in its dry' but nutrient-rich environment, X.bisporus appears to have relinquished abilities for combative interactions. Elements to sense/signal osmotic stress, e.g. HogA pathway, were present in X.bisporus. However, transcriptomes at optimal (approximate to 0.89) versus low a(w) (0.68) revealed differential expression of only a few stress-related genes; among these, certain (not all) steps for glycerol synthesis were upregulated. Xeromyces bisporus increased glycerol production during hypo- and hyper-osmotic stress, and much of its wet weight comprised water and rinsable solutes; leaked solutes may form a protective slime. Xeromyces bisporus and other food-borne moulds increased membrane fatty acid saturation as water activity decreased. Such modifications did not appear to be transcriptionally regulated in X.bisporus; however, genes modulating sterols, phospholipids and the cell wall were differentially expressed. Xeromyces bisporus was previously proposed to be a chaophile', preferring solutes that disorder biomolecular structures. Both X.bisporus and the closely related xerophile, Xerochrysium xerophilum, with low membrane unsaturation indices, could represent a phylogenetic cluster of chaophiles'

Genome and physiology of the ascomycete filamentous fungus Xeromyces bisporus, the most xerophilic organism isolated to date

Xeromyces bisporus can grow on sugary substrates down to 0.61, an extremely low water activity. Its genome size is approximately 22Mb. Gene clusters encoding for secondary metabolites were conspicuously absent; secondary metabolites were not detected experimentally. Thus, in its dry' but nutrient-rich environment, X.bisporus appears to have relinquished abilities for combative interactions. Elements to sense/signal osmotic stress, e.g. HogA pathway, were present in X.bisporus. However, transcriptomes at optimal (approximate to 0.89) versus low a(w) (0.68) revealed differential expression of only a few stress-related genes; among these, certain (not all) steps for glycerol synthesis were upregulated. Xeromyces bisporus increased glycerol production during hypo- and hyper-osmotic stress, and much of its wet weight comprised water and rinsable solutes; leaked solutes may form a protective slime. Xeromyces bisporus and other food-borne moulds increased membrane fatty acid saturation as water activity decreased. Such modifications did not appear to be transcriptionally regulated in X.bisporus; however, genes modulating sterols, phospholipids and the cell wall were differentially expressed. Xeromyces bisporus was previously proposed to be a chaophile', preferring solutes that disorder biomolecular structures. Both X.bisporus and the closely related xerophile, Xerochrysium xerophilum, with low membrane unsaturation indices, could represent a phylogenetic cluster of chaophiles'