A New Method for Olive Oil Screening Using Multivariate Analysis of Proton NMR Spectra

A new NMR-based method for the discrimination of olive oils of any grade from seed oils and mixtures thereof was developed with the aim of allowing the verification of olive oil authenticity. Ten seed oils and seven monovarietal and blended extra virgin olive oils were utilized to develop a principal component analysis (PCA) based analysis of ¹H NMR spectra to rapidly and accurately determine the authenticity of olive oils. Another twenty-eight olive oils were utilized to test the principal component analysis (PCA) based analysis. Detection of seed oil adulteration levels as low as 5% v/v has been shown using simple one-dimensional proton spectra obtained using a 400 MHz NMR spectrometer equipped with a room temperature inverse probe. The combination of simple sample preparation, rapid sample analysis, novel processing parameters, and easily interpreted results, makes this method an easily accessible tool for olive oil fraud detection by substitution or dilution compared to other methods already published.     

Activation of Kraft Lignin by an Enzymatic Treatment with a Versatile Peroxidase from Bjerkandera sp. R1

Enzymatic lignin activation may be an environmentally friendly alternative to the use of chemicals in the production of wood fibers composites. Most studies on enzymatic activation of lignin for improving the adhesion of lignocellulosic products have been carried out using laccases. In this work, the use of a versatile peroxidase (VP) from the white-rot fungus Bjerkandera sp. (anamorph R1) for activating Kraft lignin was studied. The effect of enzyme dosage, incubation time, and H₂O₂ addition profile on lignin activation was evaluated by quantifying the phenoxy radicals formed using electron paramagnetic resonance (EPR) spectroscopy. Two alternative enzymatic systems based on the use of VP (a two-stage and an enzymatic cascade system) were also assayed. At optimal conditions (dose of 15 U?g^?1 and continuous addition of H₂O₂ (5.24 μmol?h^?1) during 1 h) the content of phenoxy radicals was doubled as compared with an untreated control. Moreover, using the two-stage VP system, a lignin activation similar to that found at optimal conditions could be reached in a shorter time.     

Determination of betaines in vegetable oils by capillary electrophoresis tandem mass spectrometry--application to the detection of olive oil adulteration with seed oils

A CE–tandem mass spectrometry (MS²) methodology enabling the simultaneous determination of betaines (glycine betaine, trigonelline, proline betaine and total content of carnitines) in vegetable oils was developed. Betaines were derivatized with butanol previous to their baseline separation in 10 min using a 0.1 M formic acid buffer at pH 2.0. Ion trap conditions were optimized in order to maximize the selectivity and sensitivity. Analytical characteristics of the proposed method were established by evaluating its selectivity, linearity, precision (RSDs ranged from 4.8 to 10.7% for corrected peak areas) and accuracy by means of recovery studies (from 80 to 99%) and LODs and LOQs at 0.1 ppb level. The method was applied for the determination of the selected betaines in seed oils and extra virgin olive oils. MS² experiments provided the fingerprint fragmentation for the betaines identified in vegetable oils. In extra virgin olive oils, carnitines were not detected, making it possible to propose them as a feasible novel marker for the detection of adulterations of olive oils. Application of the developed method for the analysis of different mixtures of extra virgin olive oil with seed oil (between 2 and 10%) enabled the detection and quantitation of the total content of carnitines. The results obtained show the high potential of the developed method for the authentication and quality control of olive oils.     

Multiresidue analysis of three groups of pesticides in washing waters from olive processing by solid-phase extraction-gas chromatography with electron capture and thermionic specific detection

A multiresidue solid phase extraction-gas chromatography (SPE-GC) method is developed for the analysis of 28 organochlorine (OCPs), organophosphorus (OPPs) and organonitrogen (ONPs) pesticides at ng l^− 1 levels in washing water from olive processing. Pesticides are separated from the water matrix, containing estimable amounts of olive oil, by eluting them from a C₁₈ SPE cartridge using dichloromethane. The target compounds are determined in the final extract by gas chromatography using thermionic specific (TSD) and electron capture (ECD) detection. Recoveries range from 79% to 129% in spiked washing water samples and detection limits are between 1 and 4 ng l^− 1. The method was successfully applied to determine OCPs, OPPs and ONPs in 25 samples of water collected directly in the inlet and outlet of the washing devices of three olive mills at different locations in Jaén (Spain). The most frequently encountered pesticide residues were terbuthylazine, simazine and diuron. In some of the water samples coming from the inlet of the washing devices, terbuthylazine and diuron measured concentrations were above the concentration limits scheduled by law.     

Simple Extraction and Rapid Quantitative Analysis of Isoflavones in Soybean Seeds

A simple extraction and rapid HPLC method, suitable to separate and quantify the isoflavonoids in soybean seeds, are proposed and discussed. The method has been applied to the separation and quantification of seed isoflavonoids in five naturally grown soy cultivars; the total amount of isoflavones ranged from 1.83 g kg^–1 to 11.88 g kg^–1 of fresh weight. The detection limits (200 g/kg^–1), evaluated for genistein and daidzein, are presented together with a list of the soy polyphenols analysed by HPLC-DAD and HPLC-MS.     

Oleocanthal Quantification Using 1H NMR Spectroscopy and Polyphenols HPLC Analysis of Olive Oil from the Bianchera/Belica Cultivar

The cultivar Bianchera is an autochthonous variety from the eastern part of northern Italy, but it is also cultivated in the Slovenian and Croatian peninsula of Istria where it is named Belica (Slovenia) and Bjelica (Croatia). The properties of oleocanthal, a natural anti-inflammatory ibuprofen-like compound found in commercial monocultivar extra virgin olive oils, were determined by means of both quantitative ¹H NMR (qNMR) and HPLC analyses, where qNMR was identified as a rapid and reliable method for determining the oleocanthal content. The total phenolic content (TPC) was determined by means of the Folin–Ciocalteau method and the major phenols present in the olive oils were also quantified by means of HPLC analyses. All these analyses confirmed that the cultivar Bianchera was very rich in polyphenols and satisfied the health claim provided by the EU Commission Regulation on the polyphenols content of olive oils and their beneficial effects on human health.     

Pulse-radiolysis studies on 4-pyridinemethanol and 4-pyridinecarboxaldehyde in aqueous solutions: dimer radical anion formation in the case of 4-pyridinecarboxaldehyde

Reactions of e _aq ^– , H atom and OH radicals with 4-pyridinemethanol (4-PM) and 4-pyridinecarboxaldehyde (4-PCA) have been studied at various pH values using the pulse radiolysis technique. Reaction of e _aq ^– with 4-PM and 4-PCA leads to the formation of pyridinyl and ketyl radicals of 4-PM and 4-PCA, respectively. Ketyl radicals formed from 4-PCA react with the parent molecule to give a dimeric radical species. At pH 7, the equilibrium constant for the dimer formation was determined to be 13 500 M^-1. At pH 13 also dimer radical formation was observed. Reaction of e _aq ^– with 4-PM was found to give highly reducing pyridinyl radicals. Reaction of OH radicals with 4-PM gives a mixture of species,viz., OH adducts and radicals formed by H-atom abstraction from the –CH₂OH moiety. Radicals formed by H-atom abstraction reaction from 4-PM were found to be reducing in nature. O^– radicals were found to react with 4-PM exclusively by H-abstraction pathway.     

Rapidly differentiating grape seeds from different sources based on characteristic fingerprints using direct analysis in real time coupled with time‐of‐flight mass spectrometry combined with chemometrics

The seeds of grapevine (Vitis vinifera) are a byproduct of wine production. To examine the potential value of grape seeds, grape seeds from seven sources were subjected to fingerprinting using direct analysis in real time coupled with time‐of‐flight mass spectrometry combined with chemometrics. Firstly, we listed all reported components (56 components) from grape seeds and calculated the precise m/z values of the deprotonated ions [M–H]^–. Secondly, the experimental conditions were systematically optimized based on the peak areas of total ion chromatograms of the samples. Thirdly, the seven grape seed samples were examined using the optimized method. Information about 20 grape seed components was utilized to represent characteristic fingerprints. Finally, hierarchical clustering analysis and principal component analysis were performed to analyze the data. Grape seeds from seven different sources were classified into two clusters; hierarchical clustering analysis and principal component analysis yielded similar results. The results of this study lay the foundation for appropriate utilization and exploitation of grape seed samples. Due to the absence of complicated sample preparation methods and chromatographic separation, the method developed in this study represents one of the simplest and least time‐consuming methods for grape seed fingerprinting.     

Reactivity of certain hydrogen donors and olefins with respect to α,α-dichloroalkyl radicals

The rate constants of hydrogen splitting from hydrogen donors (DH) by the ClCH₂(CH₂)₃CCl₂ radicals (R¹) were determined by the method of competitive kinetics in the temperature range of 413–433 K. As a competing reaction with splitting of hydrogen from the DH, the isomerization of radicals R¹ into ClCH(CH₂)₃CCl₂H radicals with 1,5-migration of hydrogen was chosen, for which log k (sec^–1)=9.343 –10.56/2,303RT (kcal/mole) was calculated. It was found that the relative reactivities are similar of R¹ and (CH₃)₃CO radicals (R⁴) in splitting hydrogen from HD. This makes it possible to predict the values of analogous constants for radicals R¹ from known rate constants of splitting hydrogen from various DH by radicals R⁴. The rate constants of the addition of Cl(CH₂)₂Cl₂ radicals (R²) to amethylstyrene and methyl methacrylate were determined. Taking into account the data in [7], where the same constants were determined for styrene, methyl acrylate, acrylonitrile and vinyl methyl ketone, the existence of a linear dependence was shown of the logarithm of the rate constant of the addition of radicals R² to olefins on the polar properties of the above-enumerated olefins.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 8, pp. 1811–1816, August, 1990.The authors wish to express their gratitude to T. T. Vasil'eva for providing the reference samples of 1,5,5-trichloropentane and 1,1,5,5-tetrachloropentane.     

Thermal Shift Assay as a Tool to Evaluate the Release of Breakdown Peptides from Cowpea β-Vignin during Seed Germination

The present work aimed to characterize the molecular relationships between structure and function of the seed storage protein β-vignin, the vicilin storage protein of cowpea (Vigna unguiculata, l. Walp) seeds. The molecular characterization of β-vignin was carried out firstly by assessing its thermal stability, under different conditions of pH and ionic strength, by thermal shift assay (TSA) using SYPRO Orange fluorescent dye. Secondly, its aggregation propensity was evaluated using a combination of chromatographic and electrophoretic techniques. Two forms of β-vignin were considered: the native form purified from mature quiescent seeds, and a stable breakdown intermediate of 27 kDa produced while seeds germinate. TSA is a useful tool for determining and following over time the structural changes that occur to the protein during germination. The main result was the molecular characterization of the 27 kDa intermediate breakdown polypeptide, which, to the best of our knowledge, has never been described before. β-vignin seems to retain its trimeric conformation despite the evident degradation of its polypeptides.     

Chemical,Thermal and Antioxidant Properties of Lignins Solubilized during Soda/AQ Pulping of Orange and Olive Tree Pruning Residues

Some agroforestry residues such as orange and olive tree pruning have been extensively evaluated for their valorization due to its high carbohydrates content. However, lignin-enriched residues generated during carbohydrates valorization are normally incinerated to produce energy. In order to find alternative high added-value applications for these lignins, a depth characterization of them is required. In this study, lignins isolated from the black liquors produced during soda/anthraquinone (soda/AQ) pulping of orange and olive tree pruning residues were analyzed by analytical standard methods and Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance (solid state ¹³C NMR and 2D NMR) and size exclusion chromatography (SEC). Thermal analysis (thermogravimetric analysis (TGA), differential scanning calorimetry (DSC)) and antioxidant capacity (Trolox equivalent antioxidant capacity) were also evaluated. Both lignins showed a high OH phenolic content as consequence of a wide breakdown of β-aryl ether linkages. This extensive degradation yielded lignins with low molecular weights and polydispersity values. Moreover, both lignins exhibited an enrichment of syringyl units together with different native as well as soda/AQ lignin derived units. Based on these chemical properties, orange and olive lignins showed relatively high thermal stability and good antioxidant activities. These results make them potential additives to enhance the thermo-oxidation stability of synthetic polymers.     

Determination of oleic acid in sunflower seeds by infrared spectroscopy and multivariate calibration method

A method for the determination of oleic acid in sunflower seeds is proposed. One hundred samples of sunflower seeds were analyzed by near-infrared diffuse reflectance spectroscopy (NIRDRS). The direct measures were realized in ground and sifted seeds. The PLS multivariate calibration model was obtained using first derivate absorbance values as response matrix, while the oleic acid concentration matrix was obtained analyzing the seed samples by gas chromatography with a flame ionization detector (GC-FID). The NIRDRS-PLS model was validated externally using unknown samples of sunflower seeds. The accuracy and precision of the method was evaluated using GC as reference method. The following figures of merit (FOM) were obtained: LOD = 3.4% (w/w); LOQ = 11.3% (w/w); SEN = 8 × 10⁻⁵; SEL = 0.15; analytical sensibility (γ) = 1.5 and linear range (LR) = 18.1-89.2% (w/w). This method is useful for the fast determination of oleic acid in sunflower seeds and for quality control of raw materials.     

Determination of styrene in olive oil by an automatic purge-and-trap system coupled to gas chromatography-mass spectrometry

Summary A new gas chromatographic method using an automatic purge-and-trap system coupled to a GC with mass selective detection to analyze styrene at the parts-per-trillions (ng kg^–1) level is described. The method shows a good sensitivity and the detection limit is 10 ng kg^–1 with a relative standard deviation (RSD) of 4.7% for 164 ng kg^–1 styrene in olive oil. This analytical method has been successfully applied to the analysis of styrene in extra-virgin olive oil from the European market.     

Determination of edible oil parameters by near infrared spectrometry

A chemometric method has been developed for the determination of acidity and peroxide index in edible oils of different types and origins by using near infrared spectroscopy (NIR) measurements. Different methods for selecting the calibration set, after an hierarchical cluster analysis, were applied. After discrimination of olive oils from maize, seed and sunflower, the prediction capabilities of partial least squares (PLS) multivariate calibration of NIR data were evaluated. Several preprocessing alternatives (first derivative, multiplicative scatter correction, vector normalization, constant offset elimination, mean centering and standard normal variate) were investigated by using the root mean square error of validation (RMSEV) and prediction (RMSEP), as control parameters. Under the best conditions studied, the validation set provides RMSEP values of 0.034 and 0.037% (w/w) for acidity in (I) olive oil group and (II) sunflower, seed and maize oils group. RMSEP values for peroxide in both sample groups, expressed as mequiv. O₂ kg⁻¹, were, respectively 1.87 and 0.79. The limit of detection of the methodology developed was 0.03% for acidity in both groups of edible oils (I and II), and 0.9 and 0.8 mequiv. O₂ kg⁻¹ for peroxide in the olive oil and other edible oils groups, respectively. In fact, the methodology developed is proposed for direct acidity quantification and for the screening of peroxide index in edible oils, requiring less than 30 s per sample without any previous treatment.     

Migration of Ethylene Terephthalate Oligomers from Roasting Bags into Olive Oil

Summary A high-performance liquid-chromatographic method with UV detection (HPLC–UV) has been developed for quantification of ethylene terephthalate oligomers in olive oil, from which they were extracted with acetonitrile. Oligomers, from monomers (M₁) to pentamers (M₅), were jointly and/or individually identified by liquid chromatography with mass spectrometry (electron-impact mass spectrometry (EIMS) low- and high-resolution) and were quantified by HPLC–UV using an acetonitrile solution of the major oligomer (the trimer M₃) as standard. For M₃ recovery was 98.9%, the detection limit was 60 g L^–2, and method precision was 2.03% (RSD). Migration of oligomers M₁–M₅ into 50 mL olive oil sealed in each of two brands of 10 cm × 10 cm poly(ethylene terephthalate) roasting bag was evaluated under two sets of conditions that approached but remained below the limit at which the bag material became physically deformed – heating for 7 min at 850 W in a microwave oven, or for 60 min at 200 °C in a conventional oven. Total migration was approximately 2.7 mg dm^–2 under the former conditions and 3.5–4.1 mg dm^–2 under the latter.Presented at the International Symposium on Separation and Characteristics of Natural and Synthetic Macromolecules, Amsterdam, The Netherlands, February 5–7, 2003     

Comparative Assessment of Phytoconstituents,Antioxidant Activity and Chemical Analysis of Different Parts of Milk Thistle Silybum marianum L.

Silybum marianum L. is a therapeutic plant belonging to the family Asteraceae, which has exhibited silymarin, a principal component used to cure various physiochemical disorders. The study appraised the phytochemical analysis, antioxidant activity and chemical analysis of an extract from the seed, stem and leaves. Qualitative and quantitative phytochemical analysis was evaluated by the Folin–Ciocalteu reagent method and aluminum chloride colorimetric method, respectively. While the antioxidant activity was determined by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and acetate buffer in ferric chloride (FRAP) assay, respectively, the chemical profile was evaluated by Gas Chromatography-Mass Spectrometry (GC-MS) assay. The study outcomes identified that alkaloids, glycosides, flavonoids, terpenoids, steroids and catcholic tannins were present in seed, stem and leaves extracts except for saponins and Gallic tannins. Whereas, phenols were absent only in seed extract. Quantitative analysis revealed the presence of phenols and flavonoids in appreciable amounts of 21.79 (GAE/g), 129.66 (QE/g) and 17.29 (GAE/g), 114.29 (QE/g) from the leaves and stem extract, respectively. Similarly, all extracts expressed reasonable DPPH inhibition (IC₅₀) and FRAP reducing power such as 75.98, 72.39 and 63.21% and 46.60, 51.40 and 41.30 mmol/g from the seeds, stem and leaves extract, respectively. Additionally, chemical analysis revealed the existence of 6, 8 and 9 chemical compounds from the seeds, stem and leaves extract, respectively, corresponding to 99.95, 99.96 and 98.89% of the whole extract. The chemical compound, Dibutyl phthalate was reported from all extracts while, Hexadecanoic acid, methyl ester and Silane, (1,1-dimethylethyl), dimethyl (phenylmethoxy) were reported only from the seed and leaves extract. Moreover, Methyl stearate was also a major compound reported from all extracts except for seed extract. It is demonstrable that extracts from different parts of S. marianum possess significant antioxidant activity, as well as valuable chemical compounds accountable for therapeutic effects that might be incorporated as an alternative to synthetic chemical agents.     

Resistant Starch Contents of Starch Isolated from Black Longan Seeds

A large quantity of longan fruits (Dimocarpus longan Lour.) produced annually are processed into many products, one of which is black longan, from which the dried, dark-brown meat has been used medicinally in traditional medicine, while the starch-containing seeds are discarded. In this study, starch samples (BLGSs) were isolated from seeds of black longan fruits prepared using varied conditions. The in vitro digestibility was determined in comparison with those extracted from fresh (FLGS) and dried (DLGS) seeds. Scanning electron microscopy (SEM), X-ray diffraction (XRD), differential scanning calorimetry (DSC), and Fourier transform infrared (FTIR) spectroscopy were employed to evaluate the starch properties. The results showed that the yields of FLGS, DLGS, and BLGSs were 20%, 23%, and 16–22% w/w, respectively. SEM images showed starch granules of mixed shapes, with sizes up to 15 µm in all samples. XRD patterns confirmed an A-type crystallinity for FLGS and DLGS, with strong refraction peaks at 2θ = 15°, 17°, 18°, and 23°, while BLGSs also showed detectable peaks at 2θ = 10° and 21°, which suggested V-type structures. Thermal properties corroborated the changes by showing increases in peak gelatinization temperature (T_p) and enthalpy energy (ΔH) in BLGSs. The paste viscosity of BLGSs (5% w/w) decreased by 20–58% from that of FLGS. The FTIR peak ratio at 1045/1022 and 1022/995 cm⁻¹ also indicated an increase in ordered structure in BLGSs compared to FLGS. The significant increase in the amounts of slowly digestible starch (SDS) and resistant starch (RS) in BLGSs compared to FLGS, especially at a prolonged incubation time of 20 (4.2×) and 30 days (4.1×), was proposed to be due to the heat-induced formation of starch inclusion with other components inside the seed during the black longan production process. Thus, black longan seed could be a new source of starch, with increased RS content, for potential use in the food and related industries.     

Pulse radiolysis of 3-pyridine methanol and 3-pyridine carboxaldehyde in aqueous solutions

Reactions of e_aq^-, H-atom and OH radicals with 3-pyridine methanol (3-PM) and 3-pyridine carboxaldehyde (3-PCA) have been studied at various pHs using pulse radiolysis technique. e_aq^- was found to be highly reactive with both 3-PM and 3-PCA (k approx. 10¹⁰ dm³ mol¹ s^-1). Semi-reduced species formed in both cases were strongly reducing in nature. In the case of 3-PM, electron addition leads to the formation of pyridinyl radicals whereas in the case of 3-PCA, PyCHOH type radicals are formed. At pH 6.8, H-atom reaction with 3-PCA also gives semi-reduced species (PyCHOH), whereas at pH 1, H-atoms add to the ring. (CH₃)₂˙COH radicals were found to transfer electron to 3-PCA at all the pH values tested and by making use of changes in the absorption spectra, pK_a values of the semi-reduced species were determined to be 4.5 and 10.6. OH radicals were found to undergo addition reaction with 3-PCA, whereas in the case of 3-PM they reacted by H-abstraction as well as addition reaction. By following the yield of methylviologen radical cation formed by electron transfer reaction, it was estimated that approx. 50% of OH radicals react with 3-PM by H-atom abstraction at pH 6.8, giving reducing radicals, whereas at pH 3.2, where 3-PM is in the protonated form, the same is only about 10%. At pH 13, O^-˙ radical anions were found to react exclusively by H-atom abstraction. Reaction of SO₄^-˙ radicals with 3-PCA was found to give a species identical to the one formed by one electron reduction of nicotinic acid at acidic pH values.     

Protein, Oil, and Isoflavone Contents in Lipoxygenase- and Kunitz Trypsin Inhibitor-Deficient Soybean Seeds

Two traditional soybean cultivars, CAC-1 and Garimpo, two backcrossed-derived isolines lacking lipoxygenase (LOX^?KTI⁺), and two backcrossed-derived isolines lacking lipoxygenase and Kunitz trypsin inhibitor (LOX^?KTI^?) in the seeds were grown in São Gotardo and Paracatu, Brazil, and their protein, oil, and isoflavone contents were assessed. The loss of LOX or the combined loss of LOX and KTI brought about no significant changes in seed oil content. Significant decreases in seed protein content were observed in isolines of the CAC-1 genetic background, but not in isolines of the Garimpo background. Total isoflavone contents in LOX^?KTI⁺ and LOX^?KTI^? isolines were higher than those in the normal parental cultivars. Genetic eliminations also changed isoflavone composition. These results suggest that the genetic removal LOX and KTI may be an efficient approach to improve nutritional and nutraceutical values of soybean seeds, however, performances of null lines should be evaluated for undesirable modifications in seed composition.     

Free Radicals in the Photolysis and Radiolysis of Polymers: IV. Radicals in γ- and UV-Irradiated Wood and Lignin

Free radicals formed in the radiolysis and photolysis of wood and lignin were studied using X-band and D-band EPR measurements. It was found that singlet spectra at g 2, which appeared upon the low-temperature (77 K) - and UV irradiation of wood and lignin, or singlets detected in a posteffect on heating the irradiated samples belong to radicals having conjugated carbon–carbon bonds. Formyl radicals in -irradiated wood and peroxide radicals in - and UV-irradiated wood were detected for the first time using EPR spectra. The radiation-chemical and quantum yields of radical formation reactions were determined. In wood at 77 K, G _R 3.2 1/100 eV and _R 2 × 10^–3.