José Antonio Gabaldón

ORAC-fluorescein assay to determine the oxygen radical absorbance capacity of resveratrol complexed in cyclodextrins.

The effect of the complexation of resveratrol with hydroxypropyl-beta-cyclodextrins (HP-beta-CDs) on the antioxidant capacity of the polyphenol is studied for the first time by means of the oxygen radical absorbance capacity (ORAC) method, using fluorescein (FL) as the fluorescent probe. The method is validated through its linearity, precision, and accuracy for measuring the ORAC of resveratrol in the absence or presence of cyclodextrins (CDs). The complexation of resveratrol in CDs increased the net area under the FL decay curve (net AUC) of resveratrol up to its saturation level, at which the polyphenol showed almost double the antioxidant activity it shows in the absence of CDs. The complexation constant ( K c) between resveratrol and HP-beta-CDs was calculated by linear regression of the phase solubility diagram ( K c = 18048 M (-1)). The antioxidant activity of resveratrol was dependent on the complexed resveratrol because CDs acts as a controlled dosage reservoir that protects resveratrol against rapid oxidation by free radicals. In this way, its antioxidant activity is prolonged and only reaches its maximum when all the resveratrol is complexed.

Comparative study of different methods to measure antioxidant activity of resveratrol in the presence of cyclodextrins

The antioxidant activity of resveratrol in the absence and presence of increasing concentrations of HP-β-CDs was determined using three different methods: ORAC, ABTS and DPPH. The three methods were validated and compared for their linearity, precision and accuracy in measuring resveratrol antioxidant activity. The results indicated that the most sensitive method is the ORAC assay, which can measure the lowest resveratrol concentration (0.15-2 μM) with the highest precision. In the presence of increasing concentrations of HP-β-CDs, the antioxidant activity of resveratrol was seen to increase when it was measured by the ORAC and ABTS assays. However, no increase was observed when the DPPH assay was used. With the ORAC assay, the antioxidant activity increased until all the resveratrol had been included in HP-β-CDs (0.4 mM CDs), whereas in the case of ABTS assay the plateau in antioxidant activity was reached after 2 mM HP-β-CDs, suggesting that the CDs interferences in the measurement method. When the DPPH assay was used, no effect was observed when increasing concentrations of HP-β-CDs, indicating that in a methanolic medium resveratrol is free. Therefore, so this method cannot be used to measure the effect of resveratrol complexation with CDs on its antioxidant activity.

Detection of sulphathiazole in honey samples using a lateral flow immunoassay

A lateral flow immunoassay (LFIA) was developed in the competitive reaction format and applied to test sulphathiazole (STZ) residues in honey samples. To prepare the assay test, a hapten conjugate and goat antirabbit antiserum as capture and control reagent, respectively, were dispensed on nitrocellulose membrane. Polyclonal antiserum against sulphathiazole was conjugated to colloidal gold nanoparticles and used as the detection reagent. The visual limit of detection (cut-off value) of the sulphathiazole LFIA was 15ng/g, reaching qualitative results within 10min. The assay was evaluated with STZ spiked honey samples from different geographical origins (n=25). The results were in good agreement with those obtained from liquid chromatography separation and mass spectroscopy detection (LC-MS), indicating that the LFIA test might be used as a qualitative method for the determination of sulphathiazole residues without expensive equipment. The test was also highly specific, showing no cross-reactivity to other chemically similar antibiotics. To our knowledge, this is the only work where a development of LFIA tests for the detection of sulphathiazole residues is performed.

Use of cyclodextrins to recover catechin and epicatechin from red grape pomace

The capacity of cyclodextrins (CDs) to extract phenolic compounds from grape pomace was evaluated and compared with that of ethanol/water or aqueous extraction. The extraction method (stirring and ultrasound), temperature and time were also studied. Total phenolic compounds (TPC) and antioxidant activity were measured, and HPLC analysis was used to identify the phenolic compounds. The extracts obtained using the ethanol/water mixture presented the highest TPC content and antioxidant activity, followed by those obtained using CD solutions. The addition of CDs to the extractant agent had a selective effect on the extraction of catechin and epicatechin. The yield of catechin and epicatechin by using aqueous solutions of CDs was similar to that obtained using organic solvents as ethanol.

Detection of chemical residues in tangerine juices by a duplex immunoassay

A rapid duplex ELISA for the simultaneous determination of two of the most widely used organophosphorous insecticides in tangerine juices is described. To accomplish this aim, two individual enzyme-linked immunosorbent assays for chlorpyrifos and fenthion pesticides were integrated into one ELISA test. The strategy uses 96-well plates with specific wells coated with the corresponding haptenized conjugate. The optimized duplex ELISA was accomplished within 40 min achieving a detection limit of 0.20±0.04 µg/L and 0.50±0.06 µg/L, for chlorpyrifos and fenthion, respectively in tangerine juice samples. The determination of residues of both pesticides was carried out by simple sample dilution, without any extra sample clean-up procedure. Results of testing precision, stability, and selectivity demonstrated that the assay provided reliable analytical performances for the simultaneous determination of residues of chlorpyrifos and fenthion in fruit juice samples below the established European maximum residue limits (MRL). In addition, the accuracy and reliability of this duplex bioanalytical method is demonstrated by analyzing blind spiked juice samples and the results, correlated well with those achieved using a well-established GC/MS method (recoveries between 95% and 106%).

Detection of mandarin in orange juice by single-nucleotide polymorphism qPCR assay

A dual-probe real time PCR (qPCR) DNA-based analysis was devised for the identification of mandarin in orange juice. A single nucleotide polymorphism at the trnL-trnF intergenic region of the chloroplast chromosome was confirmed in nine orange (Citrus sinensis) and thirteen commercial varieties of mandarin, including Citrus reticulata and Citrus unshiu species and a mandarin × tangelo hybrid. Two short minor-groove binding fluorescent probes targeting the polymorphic sequence were used in the dual-probe qPCR, which allowed the detection of both species in single-tube reactions. The similarity of PCR efficiencies allowed a simple estimation of the ratio mandarin/orange in the juice samples, which correlated to the measured difference of threshold cycle values for both probes. The limit of detection of the assay was 5% of mandarin in orange juice, both when the juice was freshly prepared (not from concentrate) or reconstituted from concentrate, which would allow the detection of fraudulently added mandarin juice. The possible use of the dual-probe system for quantitative measurements was also tested on fruit juice mixtures. qPCR data obtained from samples containing equal amounts of mandarin and orange juice revealed that the mandarin target copy number was approximately 2.6-fold higher than in orange juice. The use of a matrix-adapted control as calibrator to compensate the resulting C(T) bias allowed accurate quantitative measurements to be obtained.

Fluorimetric determination of sulphathiazole in honey by means the formation of CDs inclusion complexes

The inclusion complex of sulphathiazole in β-cyclodextrin has been investigated. A 1:2 stoichiometry of the complex was established and formation constants K2 (42.83 ± 3.27 M(-1)) and K1 (4.98 ± 0.36 M(-1)) were calculated by using the changes produced on the native fluorescence of the drug, when included on the hydrophobic cyclodextrin cavity. An enhancement in the fluorescence emission of sulphathiazole and protection of the drug against photochemical reactions has been attained upon inclusion. In solutions of β-CD dual emission (458 nm) was noticed in STZ. Formation of the inclusion complex of STZ should result in dual emission, which is due to a twisted intramolecular charge transfer band (TICT). A fluorimetric method for the determination of sulphathiazole has been proposed and applied in honey without sample treatment. The optimized fluorimetric method showed detection and quantitation limits of 9.74 ng/g and 32.48 ng/g, respectively. Selectivity is high, showing no cross-reactivity to other chemically related antibiotics. The results obtained for blind honey samples (mean recovery 97%), were in good agreement with those obtained by liquid chromatography separation and mass spectrometry detection (LC-MS) (mean recovery 102%), showing that the proposed method might be used for the determination of sulphathiazole residues without expensive equipment.

Microbiological and Sensory Quality of Fresh Ready-to-Eat Artichoke Hearts Packaged under Modified Atmosphere

In recent years the sales of minimally processed vegetables have grown exponentially as a result of changes in consumer habits. The availability of artichoke buds as a ready-to-eat product would be, therefore, highly advantageous. However, minimally processed artichoke hearts are difficult to preserve because of their rapid browning and the proliferation of naturally occurring microorganisms. We developed artichoke hearts prepared as ready-to-eat products that maintain the characteristics of the fresh product. The microbiological stability, sensory qualities, and shelf life of the processed artichoke hearts were determined. During the shelf life, Salmonella, Listeria monocytogenes , and Escherichia coli counts were below the limits legally established by European regulations for minimally processed vegetables. The pH played an important role in microbial growth. Artichoke hearts had lower microbial counts in experiments conducted at pH 4.1 than in experiments conducted at pH 4.4, although the recommended threshold value for total plate count (7 log CFU/g) was not exceeded in either case. Sensory parameters were affected by the microorganisms, and artichoke products at lower pH had better sensory qualities. Vacuum impregnation techniques, modified atmosphere packaging, and low storage temperature were very effective for increasing the shelf life of minimally processed artichokes. The average shelf life was approximately 12 to 15 days.