Detection of Hazelnut and Almond Adulteration in Olive Oil: An Approach by qPCR.

Virgin olive oil (VOO), characterized by its unique aroma, flavor, and health benefits, is subject to adulteration with the addition of oils obtained from other edible species. The consumption of adulterated olive oil with nut species, such as hazelnut or almond, leads to health and safety issues for consumers, due to their high allergenic potential. To detect almond and hazelnut in olive oil, several amplification systems have been analyzed by qPCR assay with a SYBR Green post-PCR melting curve analysis. The systems selected were Cora1F2/R2 and Madl, targeting the genes coding the allergenic protein Cor a 1 (hazelnut) and Pru av 1 (almond), respectively. These primers revealed adequate specificity for each of the targeted species. In addition, the result obtained demonstrated that this methodology can be used to detect olive oil adulteration with up to 5% of hazelnut or almond oil by a single qPCR assay, and with a level as low as 2.5% by a nested-qPCR assay. Thus, the present research has shown that the SYBR-based qPCR assay can be a rapid, precise, and accurate method to detect adulteration in olive oil.

Microencapsulation of a Commercial Food-Grade Protease by Spray Drying in Cross-Linked Chitosan Particles.

In this study, the use of spray-drying technology for encapsulating Flavourzyme® (protease-peptidase complex) was evaluated to overcome the limitations (low encapsulation efficiency and no large-scale production) of other encapsulation processes. To the best of our knowledge, spray drying has not been applied previously for the immobilization of this enzyme. Firstly, bovine serum albumin (BSA), as a model protein, was encapsulated by spray drying in chitosan and tripolyphoshate (TPP) cross-linked-chitosan shell matrices. The results showed that the chitosan-TPP microcapsules provided a high encapsulation efficiency and better protein stability compared to the non-crosslinked chitosan microcapsules. The effect of enzyme concentration and drying temperature were tested during the spray drying of Flavourzyme®. In this regard, an activity yield of 88.0% and encapsulation efficiency of 78.6% were obtained with a concentration of 0.1% (v/v) and an inlet temperature of 130 °C. Flavourzyme®-loaded chitosan microcapsules were also characterized in terms of their size and morphology using scanning electron microscopy and laser diffractometry.

Rational design of mitochondria targeted thiabendazole-based Ir(III) biscyclometalated complexes for a multimodal photodynamic therapy of cancer.

Despite their outstanding properties as potential photosensitizers for photodynamic therapy (PDT), Ir(III) biscyclometalated complexes need both further developments to overcome remaining limitations and in-depth investigations into their mechanisms of action to reach clinic application in the treatment of cancer. This work describes the synthesis of a family of Ir(III) complexes of general formula [Ir(C^N)2(N^N')]Cl (N^N' = thiabendazole-based ligands; C^N = ppy (2-phenylpyridinate) (Series A), or dfppy (2-(2,4-difluorophenyl)pyridinate) (Series B)) and their evaluation as potential PDT agents. These complexes are partially soluble in water and exhibit cytotoxic activity in the absence of light irradiation versus several cancer cell lines. Furthermore, the cytotoxic activity of derivatives of Series A is enhanced upon irradiation, particularly for complexes [1a]Cl and [3a]Cl, which show phototoxicity indexes (PI) above 20. Endocytosis was established as the uptake mechanism for [1a]Cl and [3a]Cl in prostate cancer cells by flow cytometry. These derivatives mainly accumulate in the mitochondria as shown by colocalization confocal microscopy experiments. Presumably, [1a]Cl and [3a]Cl induce death on cancer cells under irradiation through apoptosis triggered by a multimodal mechanism of action, which likely involves damage over mitochondrial DNA and mitochondrial membrane depolarization. Both processes seem to be the result of photocatalytic oxidation processes.

Development and optimization of an efficient qPCR system for olive authentication in edible oils.

The applicability of qPCR in olive-oil authentication depends on the DNA obtained from the oils and the amplification primers. Therefore, four olive-specific amplification systems based on the trnL gene were designed (A-, B-, C- and D-trnL systems). The qPCR conditions, primer concentration and annealing temperature, were optimized. The systems were tested for efficiency and sensitivity to select the most suitable for olive oil authentication. The selected system (D-trnL) demonstrated specificity toward olive in contrast to other oleaginous species (canola, soybean, sunflower, maize, peanut and coconut) and showed high sensitivity in a broad linear dynamic range (LOD and LOQ: 500ng - 0.0625pg). This qPCR system enabled detection, with high sensitivity and specificity, of olive DNA isolated from oils processed in different ways, establishing it as an efficient method for the authentication of olive oil regardless of its category.

Novel qPCR systems for olive (Olea europaea L.) authentication in oils and food.

The traceability of olive oil is an unresolved issue that remains a challenge. In this field, DNA-based techniques are very powerful tools for discrimination that are less negatively influenced by environmental conditions than other techniques. More specifically, quantitative real time PCR (qPCR) achieves a high degree of sensitivity, although the DNA that it can directly isolate from these oils presents drawbacks. Our study reports the analysis of eight systems, in order to determine their suitability for olive detection in oil and oil-derived foodstuffs. The eight systems were analyzed on the basis of their sensitivity and specificity in the qPCR assay, their relative sensitivity to olive DNA detection and DNA mixtures, their sensitivity and specificity to olive in vegetable oils and the detection of olive in commercial products. The results show that the PetN-PsbM system, designed in this study, is a suitable and reliable technique in relation to olive oil and olive ingredients in both food authentication and food safety processes.

Development of a method to recovery and amplification DNA by real-time PCR from commercial vegetable oils.

This study describes the design of a suitable DNA isolation method from commercial vegetable oils for the application of DNA markers for food safety and traceability. Firstly, a comparative study was made of eight methods for the recovery of high quality DNA from olive, sunflower and palm oils, and a CTAB-based method was selected. In order to optimize this method, the effect of the organic compounds and several components in the lysis buffer and the lysis and precipitation time were evaluated. For the purpose of overcoming the limitations detected in spectrophotometric and PCR DNA yield evaluations, the performance of the extraction protocols during the optimization processes was evaluated using qPCR. The suggested DNA extraction optimized is less time consuming than other conventional DNA extraction methods, uses a reduced oil volume and is cheaper than available commercial kits. Additionally, the applicability of this method has been successfully assayed in ten commercial vegetable oils and derivatives.

Pea detection in food and feed samples by a real-time PCR method based on a specific legumin gene that allows diversity analysis.

Real-time polymerase chain reaction is currently being used for the identification and quantification of plant and animal species as well as microorganisms in food or feed samples based on the amplification of specific sequences of low copy genes. We report here the development of a new real-time PCR method for the detection and quantification of the pea (Pisum sativum) based on the amplification of a specific region of the legS gene. The specificity was evaluated in a wide range of plant species (51 varieties of Pisum sp., and 32 other plant species and varieties taxonomically related or nonrelated). The method allows the detection and quantification of as low as 21.6 pg of DNA, which corresponds to 5 haploid genome copies. The system has been shown to be sensitive, reproducible and 100% specific for the rapid detection and quantification of pea DNA in processed food and feed samples, being therefore suitable for high-throughput analysis.