BACKGROUND: Salep is a highly valued food product, susceptible to adulteration with less costly additives such as starch. The aim of this study is to develop a real-time polymerase chain reaction (PCR) method for the detection and quantification of authentic salep using primers designed from the nr-ITS2 region. RESULTS: The designed primers were shown to amplify selectively with salep DNA and cloning/sequencing of end-point PCR products obtained with authentic salep (Orchideceae) confirmed amplimers to be copies of the intended target region. Detection of salep DNA using the designed primer set was possible at levels as low as 20 pg. After initial evaluation of the real-time PCR method with binary DNA mixtures of salep and cornflour, quantification of salep was conducted in binary mixtures of potato starch (a more likely adulterant) and salep, showing that quantification within the range 25%-100% could be achieved. The real-time assay was performed using the 18S rDNA region co-amplified as an internal standard and evaluated using the Ct method. The calibration curves obtained as a result of real-time PCR runs showed linearity, with R 2 values above 99%. CONCLUSION: A real-time PCR method using SybreGreen intercalating dye was established for salep detection and quantification based on the specific amplification of salep DNA. Method validation was performed utilizing blind and commercial samples.
Cyclodextrin glycosyltransferase (CGTase) enzyme is used industrially to obtain cyclodextrins. In this study, a newly isolated alkaliphilic CGTase producing Bacillus sp. SD5 was used as the source organism for the gene encoding for CGTase. The gene was amplified and sequenced in preparation for cloning into Pichia pastoris for methanol‐induced expression and extracellular secretion. Three strategies were employed in designing the amplification primers for obtaining the fragments to be used in transformation. Hence, three P. pastoris X33 clones were obtained: N2 (with natural gene sequence), S3 (also encoding the c‐myc epitope and polyhistidine tail), and M5 (encoding the polyhistidine tail). Extracellular CGTase activity was monitored throughout fermentation for each clone. The molecular weights of the enzymes were in the range of 85–90 kDa and all three clones showed maximum CGTase activity upon 24 h of induced fermentation. The CGTase enzyme obtained from the M5 clone had Km and Vmax values 13.59 mg/mL and 1153 µmol/(mg min), respectively. The enzyme was purified and displayed optimum activity at 50°C and pH 6 and pH 9. HPLC analysis of reaction products following 10 min of incubation in a reaction solution containing 4% starch showed 852.5 mg/L β‐cyclodextrin and 89.6 mg/L γ‐cyclodextrin being produced.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.