Using <Superscript>1</Superscript>H and <Superscript>13</Superscript>C NMR techniques and artificial neural networks to detect the adulteration of olive oil with hazelnut oil

The lack of any official analytical method to detect the adulteration of olive oil with a low percentage of hazelnut oil is explained by the similarities in the chemical compositions of both kinds of oils. To counter this problem, an artificial neural network based on ¹H-NMR and ¹³C-NMR data has been developed to detect olive oil adulteration, and the results from this ANN are presented here. A training set consisting of hazelnut oils, pure olive oils, and olive oils blended with 2–20% hazelnut oils was used to design and train a multilayer perceptron with 100% correct classifications. This mathematical model was also validated using an external validation set of blend samples (3–15%) and genuine samples. The detection limit of the model was around 8%.     

A study of the evolution of the physicochemical and structural characteristics of olive and sunflower oils after heating at frying temperatures

The evolution of the density, viscosity, oil/water interfacial tension and structure of vegetable oils after heating at frying temperatures was studied to explore the possibility of reusing waste vegetable oils as solid agglomerants for different purposes. Commercial olive and sunflower oils were heated at 150 and 225 °C in the time interval of 1–15 days to achieve a wide range of alteration degrees. Structural changes in the oils were monitored by FT-IR and ¹H NMR. Significant variations occur in the physicochemical and structural characteristics of these oils, which may affect their agglomeration capability, when they were heated at frying temperatures for periods above two days. Under these conditions, the viscosity of the oils increased very quickly, whereas their unsaturation degree decreased noticeably. Decreases in the relative intensities of the NMR spectra signals of the unsaturation-related protons were observed, those corresponding to the diallyl protons occurring much faster. Of the two vegetable oils studied, sunflower oil was found to be more sensitive to thermal treatment, undergoing greater changes in its properties, especially in viscosity, which may show a marked increase.     

A facile NMR method for the quantification of total,free and esterified sterols in virgin olive oil

This study describes a simple analytical technique based on NMR spectroscopy for the determination of free and esterified sterols in olive oil. Free sterols were determined by ³¹P NMR upon derivatization of free sterols with a phosphorous reagent, whereas total (free + esterified) sterols were obtained from ¹H NMR. The present NMR method was validated by performing a comparison study with the reference method of gas chromatography. The agreement between NMR and GC methods was evaluated by using the Bland and Altman statistical analysis. The distribution of the data points in the bias plot showed that ∼96% of the measurements of total, free and esterified sterols in 24 extra virgin olive oil samples from various regions of Greece were within the limits of agreement of the two methods.     

NMR and statistical study of olive oils from Lazio: A geographical,ecological and agronomic characterization

NMR and statistical procedures were used to analyse olive oils obtained from trees grown in different areas of Lazio, an Italian region, under different irrigation conditions. In order to obtain information on “real” commercial olive oils and to study the effects of some agronomical and ecological factors on the olive oil composition, we studied commercial multi-varietal olive oils, all produced in well-characterized areas of Lazio. ¹H and ¹³C NMR techniques, coupled to a suitable multivariate statistical procedure, were used to analyse 72 multi-varietal extra virgin and PDO (Protected Denomination of Origin) olive oils harvested in 2003, from the northern area, the centre and the southern area of Lazio. The intensity of selected ¹H and ¹³C NMR variables were submitted to three different statistical methods, namely, analysis of variance (ANOVA), principal component analysis (PCA) and linear discriminant analysis (LDA). ¹H and ¹³C NMR spectroscopy allowed us to obtain a good chemical characterization of the samples, giving information on major and minor compounds with an experimental error exactly the same and always extremely low for all the analyzed components. As a result of the statistical analysis, olive oils from the same geographical areas were well grouped. Since the amounts of some minor volatile components, such as aldehydes, terpenes and squalene, as well as, the content of β-sitosterol, the most important sterol present in olive oils, are sensitive to the pedoclimatic conditions, the intensity of the corresponding NMR signals turned out to be the most discriminating factors in the geographic classification. Moreover, the NMR and statistical protocol allowed us to investigate the roles of irrigation and altitude on the olive oil composition: the contents of oleic and saturated fatty acids turned out to be strongly influenced by the irrigation practice, whereas the content of volatile compounds was sensitive to the altitude of the olive trees. As a result of our study, olive oils were well grouped according to the irrigation practice as well as to the altitude at which olive trees were grown.     

Discrimination of olive oil adulterated with vegetable oils using dielectric spectroscopy

The study focused on application of dielectric spectroscopy to identify the adulteration of olive oil. The dielectric properties of binary mixture of oils were investigated in the frequency range of 101 Hz–1 MHz. A partial least squares (PLS) model was developed and used to verify the concentrations of the adulterant. Furthermore, the principal component analysis (PCA) was used to classify olive oil sample as distinct from other adulterants based on their dielectric spectra. The results showed that the dielectric spectra of binary mixture of olive oil spiked with other oils increased with increasing concentration of soy, corn, canola, sesame, and perilla oils from 0% to 100% (w/w) over the measured frequency range. PLS calibration model showed a good prediction capability for the concentrations of the adulterant. For olive oil adulterated with soy oil, the results showed that the RMS was 0.053, sd(RMS), 0.017 and Q² value was 0.967. PCA classification plots for all oil samples showed clear performance in the differentiation for the different concentrations of the adulterant. Each of the oil samples could be easily grouped in different clusters using dielectric spectra. From the results obtained in this research, dielectric spectroscopy could be used to discriminate the olive oil adulterated with the different types of the oils at levels of adulteration below 5%.     

Classification of Brazilian honeys by physical and chemical analytical methods and low field nuclear magnetic resonance (LF H NMR)

This study evaluated the possibility of differentiating the botanical origin of honeys using Low Field Nuclear Magnetic Resonance (LF ¹H NMR). Eighty samples of honey from 8 different botanical sources (eucalyptus, “assa-lipto”, oranges, Barbados cherry, cashew tree, “assa-peixe”, “cipó-uva” and polyfloral) were analyzed. A close correlation (p < 0.01) was established between the LF ¹H NMR analysis and physical and chemical measurements, including water content, water activity, pH and color. Bi-exponential fitting of the transverse relaxation (T₂) data revealed two water populations in all samples, T₂₁ and T₂₂, corresponding to relaxation times of 0.6–1.8 ms and 2.3–5.4 ms respectively. The observed differences in the relaxation times suggest that these were influenced by the differences in botanical origins. Good linear correlations were observed between the T₂ and T₂₁ parameters and the physical and chemical data. This study demonstrated that LF ¹H NMR can be a viable technique for use in classifying honeys by their botanical origin.     

Compositional study of different soybean (Glycine max L.) varieties by H NMR spectroscopy,chromatographic and spectrometric techniques

Compositional study of different soybean varieties was performed by use of ¹H nuclear magnetic resonance (NMR) spectroscopy, chromatographic and spectrometric technique. Compositions of amino acids, fatty acids, sugars, elements, and isoflavones in two glyphosate-tolerant soybean varieties and three Chinese conventional soybean varieties were studied by ¹H NMR spectroscopy, gas chromatography-mass spectrometry, high performance liquid chromatography and inductively coupled plasma-atomic emission spectrometry, respectively. Principal component analysis suggested that glyphosate-tolerant and conventional soybeans had different compositional profile characteristics of amino acids and fatty acids. Then, the contents of some typical soybean components involving main nutrients and antinutrients were compared. As key nutrients of glyphosate-tolerant soybeans, the content of crude protein increased at 8.9–40%, while the contents of α-tocopherol and γ-tocopherol decreased at 12–64%. As antinutrients of glyphosate-tolerant soybeans, the content of tannin decreased at 32–51%, while the content of raffinose increased at 63–197%, compared with conventional soybeans. Systematical study of compositional profile characteristics provided an effective method for discriminating different soybean varieties and useful reference values for soybean consumption.     

Determination of herbicide residues in olive oil by gas chromatography–tandem mass spectrometry

This paper reports a method for the analysis in olive oil of multiresidues of the herbicides of low-medium polarity most widely used by Andalusian olive growers. The method, which uses gas chromatography/tandem mass spectrometry (GC–MS/MS), was developed within the framework of Project CAO00-005, which spanned the period from 2000 to 2004. The results obtained for more than 3000 samples of virgin olive oil and organic olive oil analyzed over such a period are reported. Samples were extracted with an acetonitrile/n-hexane mixture and cleaned up by passage through Florisil columns prior to analysis. A linear determination range for the herbicides from 1 to 500 μg kg⁻¹ and a correlation coefficient better than 0.996 were achieved. The reproducibility, as relative standard deviation, was quite acceptable (8–11%), and so were herbicide recoveries (90–102%). The proposed method has been transferred to both public and private laboratories in the Andalusian region.     

Antioxidant and antiacetylcholinesterase activities of essential oils from Cymbopogon schoenanthus L. Spreng. Determination of chemical composition by GC–mass spectrometry and C NMR

Cymbopogon schoenanthus L. Spreng, is an aromatic herb consumed in salads and used to prepare traditional meat recipes in Tunisia. The chemical composition, antioxidant activities and acetylcholinesterase inhibitory properties of the essential oils from fresh leaves, dried leaves and roots collected from three different locations in southern Tunisia, were evaluated. Essential oils were analysed by GC–mass spectrometry and ¹³C NMR. The major components were limonene (10.5–27.3%), β-phellandrene (8.2–16.3%), δ-terpinene (4.3–21.2%) and α-terpineol (6.8–11.0%). Antioxidant activity was measured by DPPH assay. The results ranged from 36.0% to 73.8% (2 μl of essential oil per mL of test solution).     

PRELIMINARY EVALUATION OF THE APPLICATION OF THE FTIR SPECTROSCOPY TO CONTROL THE GEOGRAPHIC ORIGIN AND QUALITY OF VIRGIN OLIVE OILS

ABSTRACT

A rapid Fourier transform infrared (FTIR) attenuated total reflectance spectroscopic method was applied to determine qualitative parameters such as free fatty acid (FFA) content and the peroxide value (POV) in virgin olive oils. Calibration models were constructed using partial least squares regression on a large number of virgin olive oil samples. The best results (R² = 0.955, root mean square error in cross validation [RMSECV] = 0.15) to evaluate FFA content expressed in oleic acid % (w/w) were obtained considering a calibration range from 0.2 to 9.2% of FFA relative to 190 samples. For POV determination, the result obtained, built on 80 olive oil samples with a calibration range from 11.1 to 49.7 meq O₂/kg of oil, was not satisfactory (R² = 0.855, RMSECV = 3.96). We also investigated the capability of FTIR spectroscopy, in combination with multivariate analysis, to distinguish virgin olive oils based on geographic origin. The spectra of 84 monovarietal virgin olive oil samples from eight Italian regions were collected and elaborated by principal component analysis (PCA), considering the fingerprint region. The results were satisfactory and could successfully discriminate the majority of samples coming from the Emilia Romagna, Sardinian and Sicilian regions. Moreover, the explained variance from this PCA was higher than 96%.

PRACTICAL APPLICATIONS

The verification of the declared origin or the determination of the origin of an unidentified virgin olive oil is a challenging problem. In this work, we have studied the applicability of Fourier transform infrared coupled with multivariate statistical analysis to discriminate the geographic origin of virgin olive oil samples from different Italian regions.

     

Detection of irradiated beef by nuclear magnetic resonance lipid profiling combined with chemometric techniques

The combination of ¹H NMR lipid profiling with multivariate analysis was applied to differentiate irradiated and non-irradiated beef. Two pattern recognition chemometric procedures, stepwise linear discriminant analysis (sLDA) and artificial neural networks (ANNs), provided a successful discrimination between the groups investigated. sLDA allowed the classification of 100% of the samples into irradiated or non-irradiated beef groups; the same result was obtained by ANNs using the 1 kGy irradiation dose as discriminant value suggested by the network. Furthermore, sLDA allowed the classification of 81.9% of the beef samples according to the irradiation dose (0, 2.5, 4.5 and 8 kGy). ¹H NMR lipid profiling, coupled with multivariate analysis may be considered a suitable and promising screening tool for the rapid detection of irradiated meat in official control of food.     

Study of the suitability of HRMAS NMR for metabolic profiling of tomatoes: Application to tissue differentiation and fruit ripening

The feasibility of HRMAS NMR as an efficient technique for metabolic studies of tomato fruit and tomato tissues is described. A good-taste tomato variety from Almería (Spain) (commercial name Zayno) was chosen for this study. Fruits of three different ripening stages (green, turning and red) were selected, based on their external appearance. NMR data of whole fruits were obtained through ¹H HRMAS NMR spectra of tomato purée. Flesh, peel and seeds from mature red fruits were separately analysed. ¹H HRMAS NMR spectra showed resolutions similar to that of solution ¹H NMR with the advantages of minimal sample manipulation and possibility of analysing polar and non-polar compounds simultaneously. Seeds could be clearly distinguished by visual comparison of the spectra. Tryacylglycerols, in which linoleic (47%) and oleic (28%) acyl chains preponderate, were the major components of seeds. By contrast, signals of fructose, glucose, citric acid, and the amino acids GABA, glutamine and glutamate dominated the spectra of flesh, peel and tomato purée. Principal components analysis, performed on the covariance matrix, revealed a clear separation between peel and flesh. Apart form the presence of the cutin biopolymer, peel showed a specific accumulation of sugar and sugar moieties related to pectin degradation, as well as glycosylated metabolites. Chemometric analysis also allowed the metabolic trajectory from green to red mature stages to be visualised. However, sample heterogeneity in the analysis of the whole fruit proved to be an important issue. The source of such heterogeneity was assigned to the presence of tissues of different hardness in the small amount of sample used. The presence of two clusters of samples at the turning stage indicated that the levels of certain metabolites could diverge significantly in fruits with almost identical appearance.     

High-power gradient diffusion NMR spectroscopy for the rapid assessment of extra-virgin olive oil adulteration

A high gradient diffusion NMR spectroscopy was applied to measure diffusion coefficients (D) of a number of extra-virgin olive, seed, and nut oils in order to ascertain the suitability of this rapid and direct method for discrimination of adulterated olive oils. Minimum adulteration levels that could be detected by changes in D were 10% for sunflower (SuO) and soybean oil (SoO), and 30% for hazelnut (HO) and peanut oil (PO). Qualitative and quantitative prediction of adulteration was achieved by discriminant analysis (DA). The highest prediction accuracy (98–100%) was observed only when two DA models were concomitantly used for sample classification. The first DA model provided recognition of high adulterated EVOO with more than 20% of SuO or SoO, and 30% with PO, whilst the second model could differentiate EVOO adulterated with 10% of SuO or SoO, and more than 30% of HO. The validation test performed with an independent set of randomly adulterated EVOO samples gave 100% classification success. The high accuracy levels together with minimal requirements of sample preparation, and short analyses time, prove the high-power gradient diffusion NMR spectroscopy as an ideal method for rapid screening of adulteration in valuable olive oils.     

Rapid quantitation of curcumin in turmeric via NMR and LC–tandem mass spectrometry

A rapid, sensitive and accurate ¹H NMR method has been developed for the quantitation of curcumin isolated from Curcuma longa rhizome (turmeric) extract, the results of which were compared with a validated LC–MS/MS method. The relative standard deviations of the methods were found to be 2.49% and 3.48% for the ¹H NMR and LC–MS/MS methods, respectively. The correlation coefficients were 0.998 for ¹H NMR and 0.995 for LC–MS/MS assay in the calibration range. The recoveries at 5 mg mL⁻¹ and 50 μg mL⁻¹ concentrations averaged to 99–101% for both techniques, respectively. The uncertainty of the measurement of curcumin via ¹H NMR spectroscopy was determined to be 5.80% while in LC–ESI-MS/MS method was 7.38%.     

Monitoring olive oil oxidation under thermal and UV stress through synchronous fluorescence spectroscopy and classical assays

Synchronous fluorescence (SyF) spectra combined with principal component analysis (PCA) is proven to be a useful tool for monitoring olive oil deterioration under UV irradiation at 80 °C. Spectra acquired in the range 300–500 nm during 12 h accelerated oxidation stress using a surface per volume ratio of 0.95 cm² ml⁻¹ reveal five different classes after PCA. Parallel monitoring of lipid oxidation parameters peroxide value (PV), anisidine value (AV) shows that after 12 h, TOTOX value increases 10-fold, 19-fold and almost 38-fold for extra virgin olive, olive and olive-pomace oil, respectively.     

Determination of the overall migration from silicone baking moulds into simulants and food using 1H-NMR techniques

Different silicone baking moulds (37 samples) were characterized with respect to potential migrating substances using ¹H-NMR, RP-HPLC–UV/ELSD and GC techniques. In all cases cyclic organosiloxane oligomers with the formula [Si(CH₃)₂–O] _n were identified (n = 6 … 50). Additionally, linear, partly hydroxyl-terminated organosiloxanes HO–[Si(CH₃)₂–O] _n –H (n = 7 … 20) were found in 13 samples. No substances other than siloxanes could be detected, meaning the migrants mainly consist of organopolysiloxanes. Based on this knowledge, a ¹H-NMR quantification method for siloxanes was established for the analysis of both simulants and foodstuffs. Validation of the ¹H-NMR method gave suitable performance characteristics: limit of detection 8.7 mg kg^–1 oil, coefficient of variation 7.8% (at a level of 1.0 mg kg^–1 food). Migration studies were carried out with simulants (olive oil, isooctane, ethanol (95%), Tenax) as well as preparation of different cakes. From the 1st to 10th experiment, siloxane migration into cakes only slightly decreased, with a significant dependence on fat content. Migration never exceeded a level of 21 mg kg^–1 (3 mg dm^–2) and was, therefore, well below the overall migration limit of 60 mg kg^–1 (10 mg dm^–2). However, migration behaviour into simulants differed completely from these results.     

A NMR metabolomics study of the ripening process of the Fiore Sardo cheese produced with autochthonous adjunct cultures

Fiore Sardo (FS) is a traditional Italian raw ewe’s milk cheese carrying a Protected Designation of Origin (PDO). This study investigated the kinetics of FS cheese ripening by physicochemical parameters, microbial counting, and NMR metabolomics using aqueous extracts. Four Fiore Sardo cheeses, manufactured from milk with deliberately added autochthonous lactic acid bacteria (LAB) or commercial starters were studied during a period of 90 days of ripening. Major differences in the metabolic profiles were observed amongst the samples as a function of the adjunct culture utilised. ¹H NMR metabolomics in combination with multivariate data analysis was able to classify cheese samples on the basis of their maturation age and the type of added cultures. These findings lay the metabolic basis for the authentication of Fiore Sardo cheese produced in compliance with PDO specifications which allow the use of only native LAB cultures.     

The combination of stable isotope abundance ratios of H,C, N and S with Sr/Sr for geographical origin assignment of orange juices

Within the EU-project “Pure Juice” established stable isotope methods (δ²H, δ¹³C, δ¹⁵N) have been applied and improved in order to determine and verify the geographical origin of orange juices. In addition, new approaches employing analyses of δ³⁴S and ⁸⁷Sr/⁸⁶Sr have been developed and tested. Approximately 150 authentic orange juice samples from several regions in North- and South-America, Africa and Europe have been analysed. A discrimination of orange producing regions, based on the results which ultimately depend on geographical, climatic and lithological differences was successfully performed. Furthermore, we demonstrate that blending of single strength juice by adding concentrate can be revealed by comparing ⁸⁷Sr/⁸⁶Sr of soluble and insoluble components of the juices. We conclude that regional assignment of orange juice samples is most successful when single parameters are combined in a “multi-element approach”.     

Oxidation of corn oil at room temperature: Primary and secondary oxidation products and determination of their concentration in the oil liquid matrix from H nuclear magnetic resonance data

The oil liquid matrix of several corn oil samples that have been stored at room temperature in closed receptacles for different periods of time is studied by means of ¹H nuclear magnetic resonance (¹H NMR), in order to further knowledge about this type of edible oil oxidation. As expected, the degradation of linoleic acyl groups predominates. In samples at early oxidation stages the presence of hydroperoxides and of (Z,E) conjugated-dienic systems is demonstrated, the concentration of the first group being higher than that of the second. In addition to these compounds, the presence of hydroxy derivatives supporting (Z,E) conjugated-dienic systems, as well as of hydroperoxy derivatives supporting (E,E) conjugated-dienic systems, in samples at intermediate and advanced oxidation stages, is also shown. Corn oil samples at advanced stages of oxidation also contain aldehydes, among which there are alkanals, (E)-2-alkenals, (E,E)-2,4-alkadienals, 4-hydroxy-(E)-2-alkenals, 4-hydroperoxy-(E)-2-alkenals and 4,5-epoxy-(E)-2-alkenals. The concentrations of the different kinds of intermediate compounds above mentioned as well as of the different kinds of aldehydes present in the oil liquid matrix were determined. These latter compounds can be either free or joined to truncated structures of triglycerides. In addition, a principal component analysis between storage conditions and oxidation level of the samples was carried out.     

Characterisation of fish oil emulsions stabilised by sodium caseinate

Fish oil emulsions varying in sodium caseinate concentration (25% w/w oil and 0.1–1.0% w/w protein, giving oil-to-protein ratios of 250–25) were investigated in terms of their creaming stability, rheological properties, the mobility of oil droplets and the oil/protein interaction at the interface. The presence of excessive protein in an emulsion (i.e., at 1% w/w) caused the aggregation of oil droplets through depletion flocculation, resulting in low creaming stability and high low-shear viscosity. At a lower protein concentration (0.1% w/w), when protein was limited, the emulsion droplets were stabilised by bridging flocculation and showed good stability to creaming. Shear-thinning behaviour was observed for both flocculated emulsions. A reduction in the low-shear viscosity and a Newtonian flow was obtained for the emulsion containing an intermediate concentration of protein (0.25% w/w). At this concentration, there was relatively little excess unadsorbed protein in the continuous phase; thus the emulsion was most stable to creaming. NMR was used to characterise these emulsion systems without dilution. Shorter T₂ values (by low-field ¹H NMR), for the emulsions containing both high (1% w/w) and low (0.1% w/w) amounts of protein, indicated increased restricted mobility of oils, caused by depletion or bridging flocculation. The line broadening in oil signals in the high-field NMR spectra (¹H, ¹³C) indicated increased interaction between oil molecules and proteins at the interface with increasing protein concentration in emulsions. In addition, ³¹P NMR spectra, which reflect the mobility of the casein component only, showed increased line broadening, with reduction in protein content due to the relatively higher proportion of the protein being adsorbed to the interface of the oil droplets, compared to that in the continuous phase (i.e., as the oil-to-protein ratio was increased). The T₂ values of resonances of the individual groups on oil molecules, obtained using high-field ¹H NMR, reflected their different environments within the oil droplet.