Selection and characterization of DNA aptamers for highly selective recognition of the major allergen of olive pollen Ole e 1.

In this study, single-stranded DNA aptamers with binding affinity to Ole e 1, the major allergen of olive pollen, were selected using systematic evolution of ligands by exponential enrichment (SELEX) method. Binding of the aptamers was firstly established by enzyme-linked oligonucleotide assay (ELONA) and aptaprecipitation assays. Additionally, aptamer-modified monolithic capillary chromatography was used in order to evaluate the recognition of this allergenic protein against other non-target proteins. The results indicated that AptOle1#6 was the aptamer that provided the highest affinity for Ole e 1. The selected aptamer showed good selective recognition of this protein, being not able to retain other non-target proteins (HSA, cyt c, and other pollen protein such as Ole e 9). The feasibility of the affinity monolithic column was demonstrated by selective recognition of Ole e 1 in an allergy skin test. The stability and reproducibility of this monolithic column was suitable, with relative standard deviations (RSDs) in retention times and peak area values of 7.8 and 9.3%, respectively (column-to-column reproducibility). This is the first study that describes the design of an efficient DNA aptamer for this relevant allergen.

Olive leaf extracts for shelf life extension of salmon burgers.

In this work, the effect of the addition of olive leaf extracts on the quality of vacuum-packed salmon burgers stored at 4 ℃ during 16 days has been studied. Olive leaf extract and its hydrolysate were initially characterized and then incorporated to salmon burgers. A shelf life study was conducted in three different batches (control, olive leaf extract, and hydrolyzed olive leaf extract burgers). Among the chemical indices determined, total volatile base nitrogen values were lower in hydrolyzed olive leaf extract and olive leaf extract burgers than in control samples. Lipid oxidation was lower in salmon burger with olive leaf extract. Salmon mince treated with hydrolyzed olive leaf extract showed lower microbial counts during the whole study, which extended the shelf life of the fish product. Therefore, the potential of olive leaf extracts to preserve salmon burgers during cold storage has been demonstrated.

Proteomic fingerprinting of mistletoe (Viscum album L.) via combinatorial peptide ligand libraries and mass spectrometry analysis.

Combinatorial peptide ligand libraries (CPLLs), coupled to mass spectrometry (MS) analysis, have been used to investigate in depth the proteome of Viscum album L. (VA), commonly named European mistletoe, in order to provide a first proteomic fingerprinting. For this purpose, the proteins were captured via CPLLs at two different pH values (acidic and neutral). A total of 648 non-redundant proteins were identified by using two different databases. The two pH values, chosen for bead incubations, have contributed to increment the capture ability: 56% and 31% of CPLLs species were respectively recognized at pH7.2 and at pH2.2. Finally the biological function of identified proteins was evaluated in order to understand their role on human health and the potential benefits of mistletoe extracts in medicine. SIGNIFICANCE: Viscum album L. (VA) extracts are recently used as supporting medicine for cancer therapy, improving patients' survival and increasing their quality of life in medicine. These anticancer effects are investigated and they are probably due to mistletoe's capability to favor tumor cell's death and to modulate the immune system. Although the increasing interest in VA medical benefits, the role of its components in human health remains unclear. In order to exploit this aspect, it is important to comprehensively study proteins present in Viscum album L. (VA) extracts. Nevertheless, since plant proteomics analysis is in most cases handicapped by the presence of high-abundance proteins masking the detection of the low-abundance ones, it is important to overcome this challenge. In this sense, combinatorial peptide ligand libraries (CPLLs) have been used to reduce the dynamic protein concentration range to enable the identification of a higher amount of proteins than employing conventional methods. In this work, a total of 648 non-redundant proteins were identified: 56% and 31% of CPLLs species were respectively recognized at pH7.2 and at pH2.2. This deep proteome identification was useful to investigate the biological functions of proteins in order to evaluate their potential role in human health.

According to the CPLL proteome sheriffs, not all aperitifs are created equal!

Combinatorial peptide ligand libraries (CPLLs) have been adopted for investigating the proteome of a popular aperitif in Northern Italy, called "Amaro Branzi", stated to be an infusion of a secret herbal mixture, of which some ingredients are declared on the label, namely Angelica officinalis, Gentiana lutea and orange peel, sweetened by a final addition of honey. In order to assess the genuineness of this commercial liqueur, we have prepared extracts of the three vegetable ingredients, assessed their proteomes, and compared them to the one found in the aperitif. The amaro's proteome was identified via prior capture with CPLLs at two different pH values (2.2 and 4.8). Via mass spectrometry analysis of the recovered fractions, after elution of the captured populations in 4% boiling SDS, we could confirm the presence of the following: six proteins originating from honey, 11 from orange peels, 29 from G. lutea and 46 from A. officinalis (including shared species), plus 33 species which could not be attributed to the other secret ingredients, due to paucity of genomic data on plant proteins, for a total of 93 unique gene products (merging shared proteins). This fully confirmed the genuineness of the product. Considering that most of these species could be present in trace amounts, undetectable by conventional techniques, the CPLL methodology, due to its ability to enhance the signal of trace components up to 3 to 4 orders of magnitude, could represent a powerful tool for investigating the genuineness and natural origin of commercial beverages in order to protect consumers from adulterated products.

Tannin analysis of chestnut bark samples (Castanea sativa Mill.) by HPLC-DAD-MS.

In the present investigation, an HPLC-DAD/ESI-MS method for the complete analysis of tannins and other phenolic compounds of different commercial chestnut bark samples was developed. A total of seven compounds (vescalin, castalin, gallic acid, vescalagin, 1-O-galloyl castalagin, castalagin and ellagic acid) were separated and quantified, being 1-O-galloyl castalagin tentatively identified and found for the first time in chestnut bark samples. Thus, this method provided information regarding the composition and quality of chestnut bark samples, which is required since these samples are commercialised due to their biochemical properties as ingredients of food supplements.

Chemical analysis and antioxidant activity of the essential oils of three Piperaceae species growing in the central region of Cuba.

The present study describes the phytochemical profile and antioxidant activity of the essential oils of three Piperaceae species collected in the central region of Cuba. The essential oils of Piper aduncum, P. auritum and P. umbellatum leaves, obtained by hydrodistillation, were analyzed by gas chromatography-mass spectrometry. The main components of P. aduncum oil were piperitone (34%), camphor (17.1%), camphene (10.9%), 1,8-cineol (8.7%) and viridiflorol (7.4%), whereas that of P. auritum and P. umbellatum was safrole (71.8 and 26.4%, respectively). The antioxidant properties of the essential oils were also evaluated using several assays for radical scavenging ability (DPPH test and reducing power) and inhibition of lipid oxidation (ferric thiocyanate method and evaluation against Cucurbita seed oil by peroxide, thiobarbituric acid and p-anisidine methods). P. auritum showed the strongest antioxidant activity among the Piper species investigated, but lower than those of butylated hydroxyanisol and propyl gallate.

Chemical composition, antioxidant properties and antimicrobial activity of the essential oil of Murraya paniculata leaves from the mountains of Central Cuba.

The essential oil of Murraya paniculata L leaves from the mountains of the Central Region of Cuba, obtained by hydrodistillation, was analyzed by gas chromatography-mass spectrometry. Eighteen compounds, accounting for 95.1% of the oil were identified. The major component was beta-caryophyllene (ca. 30%). The antioxidant activity of essential oil was evaluated against Cucurbita seed oil by peroxide, thiobarbituric acid and p-anisidine methods. The essential oil showed stronger antioxidant activity than that of butylated hydroxyanisole and butylated hydroxytoluene, but lower than that of propyl gallate. Moreover, this antioxidant activity was supported by the complementary antioxidant assay in the linoleic acid system and 2, 2'-diphenyl-1-picrylhydrazyl. The essential oil also showed good to moderate inhibitory effects against Klebsiellapneumoniae and Bacillus subtilis.

Capillary electrophoresis of free fatty acids by indirect ultraviolet detection: application to the classification of vegetable oils according to their botanical origin.

A method for the determination of fatty acids in vegetable oils by capillary electrophoresis with indirect UV-vis detection has been developed. The separation of fatty acids was optimized in terms of Brij surfactant nature and concentration and organic modifier (2-propanol) percentage. The optimal background electrolyte consisted of 10 mM p-hydroxybenzoate, 5 mM Tris at pH 8.8, 80 mM Brij 98, 40% acetonitrile, and 10% 2-propanol. Under these conditions, vegetable oils from five botanical origins (avocado, corn, extra virgin olive, hazelnut, and soybean) were analyzed and the fatty acid contents established. Linear discriminant analysis (LDA) models were constructed using fatty acid peak areas as predictors. An excellent resolution among all category pairs was obtained, and all samples were correctly classified with assignment probabilities of >95%.

Classification of pumpkin seed oils according to their species and genetic variety by attenuated total reflection Fourier-transform infrared spectroscopy.

Attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR), followed by multivariate treatment of the spectral data, was used to classify seed oils of the genus Cucurbita (pumpkins) according to their species as C. maxima, C. pepo, and C. moschata. Also, C. moschata seed oils were classified according to their genetic variety as RG, Inivit C-88, and Inivit C-2000. Up to 23 wavelength regions were selected on the spectra, each region corresponding to a peak or shoulder. The normalized absorbance peak areas within these regions were used as predictors. Using linear discriminant analysis (LDA), an excellent resolution among all categories concerning both Cucurbita species and C. moschata varieties was achieved. The proposed method was straightforward and quick and can be easily implemented. Quality control of pumpkin seed oils is important because Cucurbita species and genetic variety are both related to the pharmaceutical properties of the oils.

Methacrylate ester-based monolithic columns for nano-LC separation of tocopherols in vegetable oils.

The separation and determination of tocopherols (Ts) in vegetable oils by nano-LC chromatography with UV-vis detection using lauryl methacrylate ester-based monolithic columns has been developed. The separation of Ts was optimized in terms of mobile phase composition on the basis of the best compromise among efficiency, resolution and analysis time. Using a mobile phase composed of ACN/methanol/water, an excellent resolution between Ts was achieved within 18 min. The LODs were lower than 0.26 µg/mL, being repeatability values of retention time and peak area below 0.15 and 3.1%, respectively. The method was applied to the quantification of Ts and tocotrienols present in several vegetable oils from different botanical origins.

Fast separation and determination of sterols in vegetable oils by ultraperformance liquid chromatography with atmospheric pressure chemical ionization mass spectrometry detection.

A method for the determination of sterols in vegetable oils by ultraperformance liquid chromatography (UPLC) with atmospheric pressure chemical ionization mass spectrometry detection has been developed. The separation of sterols was optimized in terms of mobile phase composition, column temperature and flow rate. The optimal conditions were achieved using an Acquity UPLC BEH C18 column (50 x 2.1 mm, 1.7 microm) with a mobile phase consistent of acetonitrile/water (0.01% acetic acid) using a linear gradient, at a flow rate of 0.8 mL min(-1) and column temperature of 10 degrees C, giving a total analysis time below 5 min. The determination was performed in selective ion recording mode. The limits of detection were in all cases below 0.07 microg mL(-1), with relative standard deviation values of retention times and peak areas below 0.4 and 5%, respectively. The content of main sterols present in several vegetable oils with different botanical origins was also established.

Determination of tocopherols and tocotrienols in vegetable oils by nanoliquid chromatography with ultraviolet-visible detection using a silica monolithic column.

A method for the determination of tocopherols and tocotrienols in vegetable oils by nanoliquid chromatography with UV-vis detection has been developed. The separation of tocopherols was optimized in terms of mobile phase composition on the basis of the best compromise between efficiency, resolution, and analysis time. The optimal conditions were achieved using a C18 silica monolithic column (150 mm x 0.1 mm) with an isocratic elution of acetonitrile/methanol/water (acidified with 0.2% acetic acid) at a flow rate of 0.5 microL min(-1), giving a total analysis time below 18 min. The method has been applied to the quantification of tocopherols and tocotrienols present in several vegetable oils with different botanical origins.

Classification of extra virgin olive oils produced at La Comunitat Valenciana according to their genetic variety using sterol profiles established by high-performance liquid chromatography with mass spectrometry detection.

A method to classify extra virgin olive oils (EVOOs) according to their genetic variety using sterol profiles obtained by high-performance liquid chromatography (HPLC) with mass spectrometry (MS) detection has been developed. Sterol extracts were chromatographed on a dC18 Atlantis column (100x3 mm, 3 microm) with a gradient of acetonitrile/water (0.01% acetic acid) at a flow rate of 1.0 mL min(-1) and positive-ion mode MS detection. Using linear discriminant analysis of the HPLC-MS data (extracted ion chromatograms), EVOO samples belonging to six genetic varieties cultivated at La Comunitat Valenciana, Spain (Arbequina, Borriolenca, Canetera, Farga, Picual, and Serrana), were correctly classified with an excellent resolution among all of the categories.

Rapid evaluation of oxidized fatty acid concentration in virgin olive oils using metal oxide semiconductor sensors and multiple linear regression.

This works aims to set up a rapid and nondestructive method to evaluate the advanced oxidation of virgin olive oils (VOOs). An electronic nose based on an array of six metal oxide semiconductor sensors was used, jointly with multiple linear regression (MLR), to predict the oxidized fatty acid (OFA) concentration in VOO samples characterized by different oxidative status. An MLR model constructed using five predictors was able to predict OFA concentration with an average validation error of 9%.

Prediction of the genetic variety of extra virgin olive oils produced at La Comunitat Valenciana, Spain, by fourier transform infrared spectroscopy.

Fourier transform infrared spectroscopy (FTIR), followed by multivariate treatment of the spectral data, was used to classify extra virgin olive oils (EVOOs) according to their genetic variety. EVOO samples corresponding to seven different genetic varieties (Arbequina, Borriolenca, Canetera, Farga, Hojiblanca, Picual, and Serrana) were analyzed. The wavelength scale of the FTIR spectra of the oils was divided into 20 regions. The normalized absorbance peak areas within these regions were used as predictor variables. Classification of the EVOO samples according to their genetic variety was achieved by linear discriminant analysis (LDA). A good resolution among all categories was achieved using a LDA model constructed with only nine predictor variables. With this LDA model, 88% of the EVOOs were correctly classified, with assignment probabilities higher than 95%. This method is helpful for olive oil producers because it provides useful information related to the genetic variety of EVOOs, which is required by European Community regulations.

Study of chemical changes produced in virgin olive oils with different phenolic contents during an accelerated storage treatment.

Chemical changes produced in an extra virgin olive oil sample in the presence (EVOO) and absence (EVOOP) of its phenolic fraction during an accelerated storage treatment at 60 degrees C up to 7 weeks were studied. Modifications in phenol content, as well as changes in several quality parameters (free acidity, peroxide value, UV absorbance, fatty acid composition, oxidative stability index, and tocopherol content) were also evaluated under the same storage conditions and compared to those of the same sample deprived of phenolic compounds. When the phenolic extract of the EVOO was studied, a decrease of the antioxidants first present in the sample and an increase of the oxidized products were observed. In addition, oxidation seemed to produce the transformation of such phenolic compounds as secoiridoids and the appearance of oxidized forms of them. These latter compounds could be used as molecular markers of the lack of extra virgin olive oil freshness.

Rapid determination of sterols in vegetable oils by CEC using methacrylate ester-based monolithic columns.

A method for the determination of sterols in vegetable oils by CEC with UV-Vis detection, using methacrylate ester-based monolithic columns, has been developed. To prepare the columns, polymerization mixtures containing monomers of different hydrophobicities were tried. The influence of composition of polymerization mixture was optimized in terms of porogenic solvent, monomers/porogens and monomer/crosslinker ratios. The composition of the mobile phase was also studied. The optimum monolith was obtained with lauryl methacrylate monomer at 60:40% (wt:wt) lauryl methacrylate/ethylene dimethacrylate ratio and 60 wt% porogens with 20 wt% of 1,4-butanediol (12 wt% 1,4-butanediol in the polymerization mixture). Excellent resolution between sterols was achieved in less than 7 min with an 85:10:5 v/v/v ACN-2-propanol-water buffer containing 5 mM Tris at pH 8.0. The limits of detection were lower than 0.04 mM, and inter-day and column-to-column reproducibilities at 0.75 mM were better than 6.2%. The method was applied to the determination of sterols in vegetable oils with different botanical origins and to detect olive oil adulteration with sunflower and soybean oils.

Determination of tocopherols in vegetable oils by CEC using methacrylate ester-based monolithic columns.

The separation and determination of tocopherols (Ts) in vegetable oils by CEC using methacrylate ester-based monolithic columns has been developed. The effects of pore size of the monolithic columns were studied, and the composition of mobile phase was optimized. The optimal pore size of the monolith was obtained with 12 wt% 1,4-butanediol in the polymerization mixture. Excellent resolution between tocopherols was achieved within 10 min analysis time with a 99:1 v/v MeOH-aqueous buffer containing 5 mM tris(hydroxymethyl)aminomethane at pH 8.0. The LODs were lower than 2.3 microg/mL, and interday and column-to-column reproducibilities at 25 microg/mL were better than 5.6%. Using a 93:7 v/v MeOH-aqueous buffer, both tocopherols and tocotrienols (T(3)s) of grapeseed and palm oils were resolved. Application to the detection of olive oil adulteration with low-cost edible oils was demonstrated.