Application of (31)P NMR spectroscopy in food analysis. 1. Quantitative determination of the mono- and diglyceride composition of olive oils

This paper introduces a facile method to determine the amount of mono- and diglycerides in virgin olive oils. This method is based on the phosphorylation of the free hydroxyls of the mono- and diglycerides with 2-chloro-4,4,5,5-tetramethyldioxaphospholane and the integration of the appropriate peaks in the (31)P NMR spectrum. Quantitative (31)P NMR spectroscopy can be extended to the quantification of other constituents of olive oils bearing functional groups with labile protons.     

Protein analysis by 31p NMR spectroscopy in ionic liquid: quantitative determination of enzymatically created cross-links

Cross-linking of β-casein by Trichoderma reesei tyrosinase (TrTyr) and Streptoverticillium mobaraense transglutaminase (Tgase) was analyzed by (31)P nuclear magnetic resonance (NMR) spectroscopy in ionic liquid (IL). According to (31)P NMR, 91% of the tyrosine side chains were cross-linked by TrTyr at high dosages. When Tgase was used, no changes were observed because a different cross-linking mechanism was operational. However, this verified the success of the phosphitylation of phenolics within the protein matrix in the IL. Atomic force microscopy (AFM) in solid state showed that disk-shaped nanoparticles were formed in the reactions with average diameters of 80 and 20 nm for TrTyr and Tgase, respectively. These data further advance the current understanding of the action of tyrosinases on proteins on molecular and chemical bond levels. Quantitative (31)P NMR in IL was shown to be a simple and efficient method for the study of protein modification.     

Determination of the diglyceride content in greek virgin olive oils and some commercial olive oils by employing (31)P NMR spectroscopy

In this study, the diglyceride contents of 96 samples of virgin olive oils from the regions of Crete, Lesvos, Messinia, Pilion, Zakynthos, Halkidiki, and Ilia, 15 samples of commercial extra virgin and pure olive oils, and 3 samples each of refined olive oils and pomace oils were determined by a facile method introduced in a previous publication. This method is based on the phosphitylation of the free hydroxyls of the diglycerides with 2-chloro-4,4,5,5-tetramethyldioxaphospholane and the integration of the appropriate peaks in the (31)P NMR spectra. This preliminary study showed interesting trends in the diglyceride content of the virgin olive oils from the various regions of Greece that can be used as simple criteria to assess the olive oil characteristics. Analysis of variance has been carried out for the diglyceride content of each region in an attempt to detect possible differences in the diglyceride levels among the various regions. Finally, the relationship between the ratio of 1,2-diglycerides to the total amount of diglycerides and the total amount of diglycerides has been used to monitor the quality of virgin olive oils, commercial olive oils, refined olive oils, and pomace oils.     

Excitation-emission fluorescence spectroscopy combined with three-way methods of analysis as a complementary technique for olive oil characterization

This paper shows the potential of excitation-emission fluorescence spectroscopy (EEFS) and three-way methods of analysis [parallel factor analysis (PARAFAC) and multiway partial least-squares (N-PLS) regression] as a complementary technique for olive oil characterization. The fluorescence excitation-emission matrices of a set of Spanish extra virgin, virgin, pure, and olive pomace oils were measured, and the relationship between them and some of the quality parameters of olive oils (peroxide value, K232, and K270) was studied. N-PLS was found to be more suitable than PARAFAC combined with multiple linear regression for correlating fluorescence and quality parameters, yielding better fits and lower prediction errors. The best results were obtained for predicting K270. EEFS allowed detection of extra virgin olive oils highly degraded at early stages (with high peroxide value) and little oxidized pure olive oils (with low K270). The proposed methodology may be used as an aid to analyze doubtful samples.     

Classification of edible oils by employing 31P and 1H NMR spectroscopy in combination with multivariate statistical analysis. A proposal for the detection of seed oil adulteration in virgin olive oils

A combination of (1)H NMR and (31)P NMR spectroscopy and multivariate statistical analysis was used to classify 192 samples from 13 types of vegetable oils, namely, hazelnut, sunflower, corn, soybean, sesame, walnut, rapeseed, almond, palm, groundnut, safflower, coconut, and virgin olive oils from various regions of Greece. 1,2-Diglycerides, 1,3-diglycerides, the ratio of 1,2-diglycerides to total diglycerides, acidity, iodine value, and fatty acid composition determined upon analysis of the respective (1)H NMR and (31)P NMR spectra were selected as variables to establish a classification/prediction model by employing discriminant analysis. This model, obtained from the training set of 128 samples, resulted in a significant discrimination among the different classes of oils, whereas 100% of correct validated assignments for 64 samples were obtained. Different artificial mixtures of olive-hazelnut, olive-corn, olive-sunflower, and olive-soybean oils were prepared and analyzed by (1)H NMR and (31)P NMR spectroscopy. Subsequent discriminant analysis of the data allowed detection of adulteration as low as 5% w/w, provided that fresh virgin olive oil samples were used, as reflected by their high 1,2-diglycerides to total diglycerides ratio (D > or = 0.90).     

Detection of extra virgin olive oil adulteration with lampante olive oil and refined olive oil using nuclear magnetic resonance spectroscopy and multivariate statistical analysis

High-field 31P NMR (202.2 MHz) spectroscopy was applied to the analysis of 59 samples from three grades of olive oils, 34 extra virgin olive oils from various regions of Greece, and from different olive varieties, namely, 13 samples of refined olive oils and 12 samples of lampante olive oils. Classification of the three grades of olive oils was achieved by two multivariate statistical methods applied to five variables, the latter being determined upon analysis of the respective 31P NMR spectra and selected on the basis of one-way ANOVA. The hierarchical clustering statistical procedure was able to classify in a satisfactory manner the three olive oil groups. Subsequent application of discriminant analysis to the five selected variables of oils allowed the grouping of 59 samples according to their quality with no error. Different artificial mixtures of extra virgin olive oil-refined olive oil and extra virgin olive oil-lampante olive oil were prepared and analyzed by 31P NMR spectroscopy. Subsequent discriminant analysis of the data allowed detection of extra virgin olive oil adulteration as low as 5% w/w for refined and lampante olive oils. Further application of the classification/prediction model allowed the estimation of the percent concentration of refined olive oil in six commercial blended olive oils composed of refined and virgin olive oils purchased from supermarkets.     

Novel approach to the detection and quantification of phenolic compounds in olive oil based on 31P nuclear magnetic resonance spectroscopy

31P NMR spectroscopy has been employed to detect and quantify phenolic compounds in the polar fraction of virgin olive oil. This novel analytical method is based on the derivatization of the hydroxyl and carboxyl groups of phenolic compounds with 2-chloro-4,4,5,5-tetramethyldioxaphospholane and the identification of the phosphitylated compounds on the basis of the 31P chemical shifts. Quantification of a large number of phenolic compounds in virgin olive oil can be accomplished by integration of the appropriate signals in the 31P NMR spectrum and the use of the phosphitylated cyclohexanol as internal standard. Finally, the validity of this technique for quantitative measurements was thoroughly examined.     

Kinetics of diglyceride formation and isomerization in virgin olive oils by employing 31P NMR spectroscopy. Formulation of a quantitative measure to assess olive oil storage history

Diacylglycerol isomers and free acidity were determined for five extra virgin olive oils of different initial acidities by employing a facile (31)P NMR methodology as a function of storage time and storage conditions. The kinetic treatment of the hydrolysis of triacylglycerols (TGs) and the isomerization of 1,2-diacylglycerols (1,2-DGs) to 1,3-diacylglycerols (1,3-DGs) during storage of 18 months at ambient temperature in the dark and light and at 5 degrees C in the dark showed that the isomerization is strongly dependent on the rate of the TGs hydrolysis, the initial free acidity (H(0)) of the virgin olive oil samples, and storage conditions. Although the time-evolution of the diacylglycerols (DGs) depends on the TGs hydrolysis, the ratio D of the concentration of 1,2-DGs to the total amount of DGs was found to be independent of this factor. From the kinetic expression of the ratio D, a quantitative measure was formulated that allows the estimation of the storage time or age of virgin olive oils. Application of this quantitative measure to several olive oil samples of known and unknown storage history resulted in a very good agreement with respect to the actual storage time for up to 10-12 months of storage. For a longer storage period, where the isomerization of DGs is close to its equilibrium state, the calculated age index is only indicative.     

Authenticity of the traditional cypriot spirit "zivania" on the basis of 1h NMR spectroscopy diagnostic parameters and statistical analysis

A previous publication (Kokkinofta et al. J. Agric. Food Chem. 2003, 51, 6233-6239) discussed the use of inductively coupled plasma spectroscopy to differentiate between the traditional Cypriot alcoholic beverage zivania and other spirits similar in alcoholic content collected from different countries. In the present paper (1)H NMR spectroscopy is applied to confirm the previous conclusions and to obtain additional physical-chemical characteristics that may be used to differentiate zivania from other similar beverages. NMR spectroscopy gave a satisfactory degree of prediction and classification between zivanias and other distillings. The validity of quantification of the method was tested using comparative GC data. It appears that chemical analysis can be very helpful for identifying the unique geological and climatic conditions existing in the island of Cyprus that lead to an authentic product.     

In vivo 31P and 1H HR-MAS NMR spectroscopy analysis of the unstarved Aporrectodea caliginosa (Lumbricidae)

This study demonstrates that ³¹P and ¹H high resolution-magic angle spinning (HR-MAS) nuclear magnetic resonance (NMR), a recently developed NMR technique, can be applied to the in vivo analysis of metabolites from an unstarved earthworm. The endogeic earthworm Aporrectodea caliginosa was cut into lengths and then the different body parts (anterior, middle, posterior) with the gut content were analyzed. With the HR-MAS NMR, metabolites show well-resolved signals, whereas, with conventional NMR, spectra are highly dependent on the gut content. ³¹P HR-MAS NMR has been used to evaluate the effect of an acute exposure of the earthworm to glyphosate. Our observations support a low toxicity of the herbicide and suggest that glyphosate could be trapped in the cutaneous mucus. Earthworms could therefore play a role in horizontal dispersion and stabilization of glyphosate in the drilosphere. Phosphorylated metabolites, such as phospholombricine and lombricine, were clearly identified and their amount measured during experiments. The ¹H HR-MAS NMR method offers the opportunity to measure, on the same sample and simultaneously, both the hydrosoluble metabolites and lipids. The data on lipid location and relative succinate concentration shed light on the physiological and metabolic functions of the different body parts of the earthworm.     

Influence of olive maturity stage and geographical origin on some minor components in virgin olive oil of the chemlali variety

Minor compounds such as sterols, aliphatic alcohols, tocopherols, and chlorophylls in virgin olive oil from Chemlali cultivar were analyzed during the maturity process. The study concerns oils from the following three different olive growing areas of Tunisia: Sfax, Sidi Bouzid, and Enfidha. Analytical results showed that both the maturity process and the olive provenances influence the evolution of the content of these compounds.     

Geographical characterization of greek virgin olive oils (cv. Koroneiki) using 1H and 31P NMR fingerprinting with canonical discriminant analysis and classification binary trees

This work deals with the prediction of the geographical origin of monovarietal virgin olive oil (cv. Koroneiki) samples from three regions of southern Greece, namely, Peloponnesus, Crete, and Zakynthos, and collected in five harvesting years (2001-2006). All samples were chemically analyzed by means of 1H and 31P NMR spectroscopy and characterized according to their content in fatty acids, phenolics, diacylglycerols, total free sterols, free acidity, and iodine number. Biostatistical analysis showed that the fruiting pattern of the olive tree complicates the geographical separation of oil samples and the selection of significant chemical compounds. In this way the inclusion of the harvesting year improved the classification of samples, but increased the dimensionality of the data. Discriminant analysis showed that the geographical prediction at the level of three regions is very high (87%) and becomes (74%) when we pass to the thinner level of six sites (Chania, Sitia, and Heraklion in Crete; Lakonia and Messinia in Peloponnesus; Zakynthos). The use of classification and binary trees made possible the construction of a geographical prediction algorithm for unknown samples in a self-improvement fashion, which can be readily extended to other varieties and areas.     

Influence of cultivar and concentration of selected phenolic constituents on the in vitro chemiopreventive potential of olive oil extracts

One of the main olive oil phenolic compounds, hydroxytyrosol (3,4-DHPEA), exerts in vitro chemopreventive activities (antiproliferative and pro-apoptotic) on tumor cells through the accumulation of H(2)O(2) in the culture medium. However, the phenol composition of virgin olive oil is complex, and 3,4-DHPEA is present at low concentrations when compared to other secoiridoids. In this study, the in vitro chemopreventive activities of complex virgin olive oil phenolic extracts (VOO-PE, derived from the four Italian cultivars Nocellara del Belice, Coratina, Ogliarola, and Taggiasca) were compared to each other and related to the amount of the single phenolic constituents. A great chemopreventive potential among the different VOO-PE was found following this order: Ogliarola > Coratina > Nocellara > Taggiasca. The antiproliferative and pro-apoptotic activities of VOO-PE were positively correlated to the secoiridoid content and negatively correlated to the concentration of both phenyl alcohols and lignans. All extracts induced H(2)O(2) accumulation in the culture medium, but this phenomenon was not responsible for their pro-apoptotic activity. When tested in a complex mixture, the olive oil phenols exerted a more potent chemopreventive effect compared to the isolated compounds, and this effect could be due either to a synergistic action of components or to any other unidentified extract constituent.     

Enzymatic assay for the determination of olive oil polyphenol content: assay conditions and validation of the method

A new spectrophotometric assay for the determination of the polyphenolic content of olive oil is presented. It is a substrate-recycling assay for phenolic compounds that employs tyrosinase in the presence of excess NADH. The reaction of various phenols with the enzyme produces an o-quinone, which is detected by recycling between reactions with the enzyme and NADH. The method offers some advantages over the classical methods employed to determine the polyphenolic content of olive oil, that is, ease and reproducibility of the analysis, highly increased sensitivity, and selectivity toward phenolic compounds. The amount of total polyphenols was determined in virgin olive oils both with the Folin-Ciocalteu reagent and with the proposed enzymatic method. The results suggest a better estimation of the polyphenol content, as compared with the colorimetric method. This has to be attributed to the different reactivities of the two methods toward phenols and catechols. Finally, the enzymatic method demonstrates that there is a linear relationship between the olive oil phenolic content and the antioxidative capacity of oil extracts.     

Evaluation of soil analytical methods for the characterization of alkaline Technosols: II. Amorphous constituents and carbonates

Purpose

The aims of this study were to identify potential sources of error in common methods for determination of amorphous oxide concentrations and carbonate concentrations, as applied to a Technosolic material (bauxite residue), and where possible, suggest improvements to the methods.

Materials and methods

An acid ammonium oxalate (AAO) extraction was applied to fresh and weathered bauxite residues, at soil to solution ratios varying from 1:100 to 1:800. Two methods for carbonate concentration were compared: the ‘weight loss’ method, and the ‘difference in total C’ method. These were applied to six weathered bauxite residue samples, with CaCO₃ concentrations ranging from 0.1–2 % weight.

Results and discussion

Chemically extractable amorphous content was underreported in bauxite residue at the standard 1:100 extraction ratio, likely due to Al and Si saturation of the oxalate complex. A 1:400 soil: AAO ratio extracted the highest amount of amorphous material. Some crystalline minerals such as sodalite, inherited from the Technosolic parent material, were soluble in the acid ammonium oxalate extractant. The difference in total C method was more precise than the weight loss method for the determination of carbonate concentration in bauxite residues.

Conclusions

The high amorphous content of bauxite residues requires a wider soil to solution ratio (1:400) for acid ammonium oxalate extraction than is used for typical soil materials (1:100). The difference in total C method is recommended for the routine analysis of field samples where small variations in carbonate concentration need to be detected.     

Revealing the metabonomic variation of rosemary extracts using 1H NMR spectroscopy and multivariate data analysis

The molecular compositions of rosemary ( Rosmarinus officinalis L.) extracts and their dependence on extraction solvents, seasons, and drying processes were systematically characterized using NMR spectroscopy and multivariate data analysis. The results showed that the rosemary metabonome was dominated by 33 metabolites including sugars, amino acids, organic acids, polyphenolic acids, and diterpenes, among which quinate, cis-4-glucosyloxycinnamic acid, and 3,4,5-trimethoxyphenylmethanol were found in rosemary for the first time. Compared with water extracts, the 50% aqueous methanol extracts contained higher levels of sucrose, succinate, fumarate, malonate, shikimate, and phenolic acids, but lower levels of fructose, glucose, citrate, and quinate. Chloroform/methanol was an excellent solvent for selective extraction of diterpenes. From February to August, the levels of rosmarinate and quinate increased, whereas the sucrose level decreased. The sun-dried samples contained higher concentrations of rosmarinate, sucrose, and some amino acids but lower concentrations of glucose, fructose, malate, succinate, lactate, and quinate than freeze-dried ones. These findings will fill the gap in the understanding of rosemary composition and its variations.     

New (1)h NMR procedure for the characterization of native and modified food-grade starches

A novel, fast, and straightforward procedure is presented for the characterization of starch (the largest energy component in food) and modified starches (such as octenyl succinic anhydride (OSA)-modified starches used as a dispersing agent in the food industry). The method uses (1)H NMR to measure the degree of branching and also, for modified starches, the degree of chemical substitution. The substrate is dissolved in dimethyl-d(6) sulfoxide; addition of a very low amount of deuterated trifluoroacetic acid (d(1)-TFA) to the medium gives rise to a shift to high frequency of the exchangeable protons of the starch hydroxyl groups, leading to a clear and well-defined (1)H NMR spectrum, which provides an improved way to determine the degrees of both branching and chemical substitution. Measurements of the size and molecular weight distributions by multiple-detector size exclusion chromatography show that degradation by TFA does not affect the accuracy of the method.     

Direct analysis of total antioxidant activity of olive oil and studies on the influence of heating

Aim of this study was to evaluate the total antioxidant activity (TAA) of extra virgin olive oil (EVOO) and the effect of heating on the alpha-tocopherol content and TAA in relation to the presence of polyphenols, heating time, and temperature. Experiments included the measurement by ABTS decolorization assay of antioxidant capacity of alpha-tocopherol and 14 simple phenolic compounds present in EVOO, either dissolved in ethanol or added to refined olive oil, and the evaluation of TAA, total phenols, and alpha-tocopherol of six commercial EVOO and three olive oils. Finally, four experimental oils were prepared from refined olive oil containing a fixed amount (300 ppm) of alpha-tocopherol and increasing amounts of polyphenols (25, 125, 225, and 326 ppm) extracted from EVOO. The thermal stability of experimental oils under domestic heating conditions (heating time from 30 to 120 min, heating temperature from 160 to 190 degrees C) was studied by evaluating the loss of alpha-tocopherol and TAA according to a Latin square design. Results indicate that TAA of commercial oils is mainly due to their phenol and alpha-tocopherol content. Heating experiments suggest that polyphenols from EVOO are effective stabilizers of alpha-tocopherol during olive oil heating, thus contributing to the nutritional value of cooked foods.     

Fluorometric and fluorescent image analysis methods for determination of lipid hydroperoxides in oil models with 3-perylene diphenylphosphine (3-PeDPP)

Fluorometric and fluorescent image analysis methods with 3-perylene diphenylphosphine (3-PeDPP) were developed to measure lipid hydroperoxides in refined tuna oil. The fluorescence intensity of 3-perylene diphenylphosphineoxide (3-PeDPPO, the corresponding peroxidation product of 3-PeDPP) has been used for assessing lipid hydroperoxides in refined tuna oil models, and the results are compared to those of a classical method using ferric-xylenol orange complex. There were good correlations between the lipid hydroperoxide contents determined by the 3-PeDPPO fluorescence and the xylenol orange methods (R(2) = 0.984). Moreover, the novel probe 3-PeDPP and the fluorescent image analysis enabled us to evaluate two-dimensionally the lipid hydroperoxide contents in tuna oil droplets. These results suggest that the fluorometric and fluorescent image analyses using 3-PeDPP would be suitable for batch and two-dimensional determinations of lipid hydroperoxides in oil and oil-containing materials.