Presence of cyclopropane fatty acids in foods and estimation of dietary intake in the Italian population.

Cyclopropane fatty acids (CPFAs) are the most abundant cyclic fatty acids in microorganisms with unknown role(s) regarding their dietary relevance and biological effects in humans. This work was aimed to draw up a list of CPFAs-containing foods for estimating their dietary intake in the Italian population to provide a basis for evaluating their nutritional relevance and potential health-related effects. The CPFAs content of more than 500 food items was investigated and a preliminary dietary intake was assessed (12.0 ± 6.0 mg/day), based on the data reported by the Italian National Food Consumption Survey INRAN-SCAI 2005-06. CPFAs should be considered of dietary relevance in view of their potential physiological activity in humans and their presence in significant amounts in dairy products, as Grana Padano cheese (9.0-30.0 mg/100 g), and in bovine meat (0.7-4.0 mg/100 g). Future studies should elucidate whether this uncommon class of fatty acids has a biological role in human health.

Characterization of a potential nutraceutical ingredient: pomegranate (Punica granatum L.) seed oil unsaponifiable fraction.

The chemical fingerprinting of the unsaponifiable fraction of different Punica granatum seed oils was performed in order to evaluate their potential as a functional food ingredient. Qualitative and quantitative determinations of tocopherol, aliphatic alcohol (including policosanol), squalene, phytosterols and triterpene contents were performed by GC-MS. A high yield (3.1-4.2%) of unsaponifiable matter was obtained and consistent levels of squalene (up to 800 mg/kg) and policosanol (118-185 mg/kg) were noticed. ß-sitosterol (up to 8069 mg/kg) and cycloartenol (5916-7766 mg/kg) were predominant in phytosterol and triterpene fractions, while ß- and δ-tocopherol were the most abundant vitamin E forms. Some minor variations were noticed between samples. From the results obtained, it can be suggested that the seed oil of P. granatum can be considered an interesting alimentary source of substances of nutraceutical value involved in the modulation of cholesterol metabolism.

Volatile fingerprint of unroasted and roasted cocoa beans (Theobroma cacao L.) from different geographical origins.

The aroma characterization of 58 unroasted cocoa beans from 22 different geographical origins was performed by head space solid phase micro-extraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS). Sampling is representative of the average world production (America, Africa, and Southeast Asia). Analysis of cocoa beans before and after roasting were performed to follow the aroma modification with the aim to achieve a cocoa volatile fingerprint and a discrimination model based on beans origin. A total of 57 volatiles was identified in unroasted cocoa beans, while 71 volatiles were identified in roasted cocoa beans. The compounds belong to several chemical groups including esters, alcohols, organic acids, aldehydes, ketones and pyrazines. Datasets were submitted to multivariate statistical analysis (Principal Component Analysis, PCA). Results allowed to discriminate unroasted cocoa beans based on their geographical origin: samples coming from African countries were separated from samples of American regions, whereas samples from Southeast Asia lie between the other two continents suggesting that Asian samples have intermediate characteristics between African and South American cocoa beans. PCA, applied on the corresponding roasted samples, showed that although the same roasting treatment has been applied to all the samples, the differences among the unroasted samples were also maintained in the aromatic profile after roasting. The discrimination model based on volatile fingerprint combined with chemometric tools, showed interesting potential for origin authentication of both unroasted and roasted cocoa beans.

Determination of Cyclopropane Fatty Acids in Food of Animal Origin by 1H NMR.

Cyclopropane fatty acids (CPFAs) are unusual fatty acids of microbial origin, recently detected in milk and dairy products. CPFAs have been demonstrated to be interesting molecular markers for authentication of dairy products obtained without ensiled feeds. Moreover, they can also be recognized as a new secondary component of human diet. Information is lacking on the presence of cyclic fatty acids in other food sources. Cyclopropane fatty acids have been detected by GC-MS analysis in cheese and other animal fats in concentration ranging from 200 to 1000 mg/kg fat, but in some cases, the complex fatty acid profile and the possible presence of interfering peaks make the separation not straightforward and the quantification uneasy. Therefore, a new reliable 1H NMR method was developed to detect and measure CPFA content in different foods of animal origin, based on the detection of the characteristic signals of cyclopropane ring. The 1H NMR (600 MHz) method showed detection limits comparable with those of full scan GC-MS, and it allowed the identification and quantitation of the cyclopropane fatty acids in different foods.

Quantitative 2H NMR spectroscopy with 1H lock extender.

An inexpensive external unit that allows the use of the commercial high-resolution NMR spectrometer for (2)H observation with an (1)H lock system is described. The external unit does not require any tuning, is extremely easy to use, and could be a cheaper and more straightforward alternative to the more expensive (19)F lock configuration. An application for the quantitative determination of the natural isotopic ratio (2)H/(1)H of ethanol and acetic acid is reported.

Determination of 2,3-butanediol and 2-hydroxybutanone stereoisomers in batteries of traditional balsamic vinegar.

The absolute quantities and the stereoisomeric ratios of R, S-2-hydroxybutanone and R, R-, S, S-, R, S ( meso)-2,3-butanediol were determined in batteries of traditional balsamic vinegar (TBV) by gas chromatography-mass spectrometry (GC-MS), using a chiral capillary column, to evaluate if such parameters could be used to differentiate TBV differently aged and from different producers. Results showed that the initial amounts of 2-hydroxybutanone and 2,3-butanediol were quite variable, as a function of the producer of the vinegar; moreover, the 2-hydroxybutanone amount decreased during aging while 2,3-butanediol increased. Initially, the R-2-hydroxybutanone form prevails, and then the R/ S ratio decreased regularly during aging with some exceptions attributed to the addition of new barrels during the battery management. With regard to the 2,3-butanediol isomers, the most abundant was the R, R form, slowly transformed into the R, S and S, S isomers during aging. The GC-MS method used is easy and fast and could allow for a quick control of the maturation level of the vinegar.

GC-MS analysis of hashish samples: a case of adulteration with colophony.

This report describes the analytical characterization of 16 hashish samples confiscated in Italy. The samples were solvent extracted and subjected to GC-MS analysis for the separation and quantitation of the main cannabinoids. One of the analyzed samples was shown to contain extraneous compounds, identified as resin acids characteristic of colophony (rosin). Colophony is a natural resinous product obtained from various species of pine, spruce, and larch; it is a skin sensitizer and its fumes produce nonspecific irritation that can cause bronchial asthma. Similar adulterations of hashish have not been reported previously; therefore, at present there is no information about the effects of smoking colophony or a combination of hashish and colophony. It is presumed, however, that such a combination would be highly detrimental to the health of the user.

Influence of fermentation level and geographical origin on cocoa bean oligopeptide pattern.

Peptides and amino acids generated during cocoa bean fermentation are the most important precursors for the development of cocoa aroma, however cocoa oligopeptide fraction is under-investigated. In this study, we perform a deep investigation of the presence of oligopeptides in unfermented, under fermented, and well-fermented cocoa beans from all of the main producing countries, with the aim to obtain a better definition of cocoa quality and a deeper comprehension of biochemical changes occurring during fermentation. Oligopeptides were determined by UPLC/ESI-MS and 35 low-molecular weight peptides were identified and subjected to semi-quantitative analysis. Results showed that fermented cocoas can be differentiated from unfermented cocoas by their possession of a higher total amount of oligopeptides and a lower ratio of vicilin to 21kDa cocoa seed albumin peptides. A variability in the peptide pattern was observed also among well-fermented cocoa samples of different geographical origin, suggesting diversified proteolytic activities.

Development of a Quantitative GC-MS Method for the Detection of Cyclopropane Fatty Acids in Cheese as New Molecular Markers for Parmigiano Reggiano Authentication.

Cyclopropane fatty acids (CPFA), as lactobacillic acid and dihydrosterculic acid, are components of bacterial membranes and have been recently detected in milk and in dairy products from cows fed with corn silage. In this paper, a specific quantitative gas chromatography-mass spectrometry (GC-MS) method for the detection of CPFA in cheeses was developed, and the quality parameters of the method (limit of detection, limit of quantitation, and intralaboratory precision) were assessed. Limit of detection and quantitation of CPFA were, respectively, 60 and 200 mg/kg of cheese fat, and the intralaboratory precision, determined on three concentration levels, satisfied the Horwitz equation. This method was applied to 304 samples of PDO cheeses of certified origin, including Parmigiano Reggiano (Italy), Grana Padano (Italy), Fontina (Italy), Comté (France), and Gruyère (Switzerland). Results showed that CPFA were absent in all of the cheeses whose Production Specification Rules expressly forbid the use of silages (Parmigiano Reggiano, Fontina, Comté, and Gruyère). CPFA were instead present in variable concentrations (300-830 mg/kg of fat) in all of the samples of Grana Padano cheese (silages admitted). A mix of grated Parmigiano Reggiano and Grana Padano was also prepared, showing that the method is able to detect the counterfeiting of Parmigiano Reggiano with other cheeses up to 10-20% Grana Padano content. These results support the hypothesis that CPFA can be used as a marker of silage feedings for cheeses, and the data reported can be considered a first attempt to create a database for CPFA presence in PDO cheeses.

Metabolic profiling by (1)H NMR of ground beef irradiated at different irradiation doses.

This work describes a metabolic profiling study of non-irradiated and irradiated beef (at 2.5, 4.5 and 8 kGy) using (1)H NMR and chemometrics. The assignment of all major NMR signals of the aqueous/methanolic extracts was performed. A comprehensive multivariate data analysis proved the ability to distinguish between the irradiated and non-irradiated beef. Classification trees revealed that three metabolites (glycerol, lactic acid esters and tyramine or a p-substituted phenolic compound) are important biomarkers for classification of the irradiated and non-irradiated beef samples. Overall, the achieved metabolomic results show that the changes in the metabolic profile of meat provide a valuable insight to be used in detecting irradiated beef. The use of the NMR-based approach simplifies sample preparation and decrease the time required for analysis, compared to available official analytical procedures.

The species-specific ratios of 2,3-butanediol and acetoin isomers as a tool to evaluate wine yeast performance.

The isomers of 2,3-butanediol [R,R; S,S; R,S (meso-form)] and of acetoin (R,S) were determined in laboratory wine fermentations carried out by 50 yeast strains, 10 for each of the following species, Saccharomyces cerevisiae, Kloeckera apiculata, Candida stellata, Metschnikowia pulcherrima and Brettanomyces bruxellensis, in order to evaluate if such parameters might be used to differentiate wines obtained with different yeast species. According to analysis of variance (ANOVA), the strains of the same species behaved similarly, whereas the five yeast species behaved differently so that species-specific profiles were recognized. Moreover, the discriminant analysis grouped the wines into five groups, each including the 10 wines obtained by the 10 yeast strains of the same species. Trials were also included where musts partially fermented by non-Saccharomyces species were inoculated with a selected strain of S. cerevisiae to complete fermentation, and the content in 2,3-butanediol and acetoin isomers was again determined and statistical analysis was performed. Although the final values of these parameters resembled those obtained in pure fermentation with S. cerevisiae, statistical analysis discriminated wines according to the yeast species performing the first fermentation phase.

Presence and racemisation of amino acids in calcium oxalate patinas. A case study from the Baptistery in Parma, Italy.

The organic material present in oxalate patinas, collected from the stone external surfaces of the Baptistery in Parma (Italy), was analysed by GC-MS. The high racemisation levels determined for several specific amino acids indicate that microbial activity could contribute to the formation and modification of the organic material leading to the calcium oxalate patinas.

An overview on the presence of cyclopropane fatty acids in milk and dairy products.

A survey was carried out to determine the presence of cyclopropane fatty acids (CPFA) in various dairy products. CPFA such as lactobacillic acid and dihydrosterculic acid are components of bacterial membranes and have been recently detected in milk from cows fed with maize silage. In this paper about 200 dairy samples comprising cow, sheep, and goat milk, cheese, yogurt/fermented milk, and butter were analyzed. Results showed that cow milks were generally positive to CPFA (0.014-0.105% of total fatty acids), while goat, yak, and sheep milks were negative. Experimental yogurt and fermented milks showed the same CPFA content of the starting milk. Positive to CPFA were also the majority of samples of commercial butter and cheeses, except some PDO cheeses as Parmigiano-Reggiano and Fontina, cheeses from mountain regions, and goat and sheep cheeses. These data suggest that the presence of CPFA in dairy products could be used as a marker of silage feeding.

Application of 1H NMR for the characterisation of cocoa beans of different geographical origins and fermentation levels.

This study reports for the first time the use of (1)H NMR technique combined with chemometrics to study the metabolic profile of cocoa (Theobroma cacao L.) beans of different varieties, origin and fermentation levels. Results of PCA applied to cocoa bean (1)H NMR dataset showed that the main factor influencing the cocoa bean metabolic profile is the fermentation level. In fact well fermented brown beans form a group clearly separated from unfermented, slaty, and underfermented, violet, beans, independently of the variety or geographical origin. Considering only well fermented beans, the metabolic profile obtained by (1)H NMR permitted to discriminate between some classes of samples. The National cocoa of Ecuador, known as Arriba, showed the most peculiar characteristics, while the samples coming from the African region showed some similar traits. The dataset obtained, representative of all the classes of soluble compounds of cocoa, was therefore useful to characterise fermented cocoa beans as a function of their origin and fermentation level.

Application of ¹H NMR for the characterisation and authentication of ''Tonda Gentile Trilobata" hazelnuts from Piedmont (Italy).

The Italian hazelnut (Corylus avellana L.) cultivar "Tonda Gentile Trilobata" (TGT) is covered by protected geographical indication "Nocciola Piemonte" and is well-known as the best-suited hazelnut for the industrial transformation into roasted kernel. The hazelnut cultivar identification is primarily based on morphological characteristics, so there is the need for more objective analytical methods for high quality hazelnut authentication. This study reports the (1)H NMR fingerprinting of raw and roasted hazelnut, with the aim of obtaining hazelnut classification based on their spectroscopic pattern. (1)H NMR analyses were carried out on polar extracts of TGT and other cultivars: the data were analysed with multivariate statistical methods. Results showed that (1)H NMR combined with chemometrics is useful to characterise the hazelnuts as a function of the cultivars, both on raw and roasted form. The classification models allowed identifying molecular markers useful to distinguish TGT from other types, among these trigonelline, amino acids and an unidentified orto-disubstituted aromatic compound.

A simple GC-MS method for the screening of betulinic, corosolic, maslinic, oleanolic and ursolic acid contents in commercial botanicals used as food supplement ingredients.

The occurrence of triterpene pentacyclic acids in plants is extensive, but little is known about their availability in commercial extracts. A simple GC-MS method for the simultaneous determination of betulinic, corosolic, maslinic, oleanolic and ursolic acids was developed and applied to 38 different commercial plant extracts sold as ingredients for dietary supplements. A suitable protocol was set up to perform routine control of a diverse array of samples with different botanical, chemical and physical characteristics. Remarkable quantities of corosolic acid were found in dried extracts from aerial parts of Lagerstroemia speciosa and Ortosiphon stamineus (14233 and 1132 mg/kg, respectively), while oleanolic acid was abundant in O. stamineus and Crataegus monogyna flowers (2774 and 2339 mg/kg); ursolic was identified in O. stamineus, C. monogyna, L. speciosa and Arctostaphylos uva-ursi leaves (7773, 4165, 2108 and 1034 mg/kg). Only L. speciosa was rich in maslinic acid (4958 mg/kg), while minor amounts of betulinic acid (257 and 80 mg/kg) were detected in L. speciosa and C. monogyna extracts. Lower quantities of triterpenic acids were identified in dried extracts of Harpagophyton procumbens root, propolis, Punica granatum root, Styrax benzoin, Vaccinium myrtillus fruits and Vitis vinifera seeds. Decoctions and fluid extracts lacked or contained very low amounts of triterpenic acids. Results are discussed in terms of quality and safety of these ingredients.

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

The combination of (1)H 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). (1)H 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.

Cyclopropyl and ω-cyclohexyl fatty acids as quality markers of cow milk and cheese.

Content of cyclopropyl and ω-cyclohexyl fatty acids of microbial origin, respectively, from silage and rumen, was determined by GC-MS and confirmed by (1)H NMR in more than 200 milk samples from dairy cows fed with different forages. Cyclopropyl fatty acids (about 0.1% of milk fat) were detected for the first time in milk and they were present only in milk samples from cows fed with forages containing maize silage, which is not allowed to produce milk for some PDO cheeses as Parmigiano-Reggiano. Their determination can be proposed as a quality parameter of milk or feed and it can be useful especially to distinguish cheeses sold as Parmigiano Reggiano from others cheeses. The content of ω-cyclohexyl tridecanoic acid varied from 0.0% to 0.15% of milk fat, and it was higher in milk samples from cows fed with diets richer in cereal meals.

Looking beyond sugars: phytochemical profiling and standardization of manna exudates from Sicilian Fraxinus excelsior L.

Different grades of genuine and counterfeit Fraxinus excelsior exudates, marketed as natural sweeteners or mild laxatives, were evaluated for their proximate composition and for saccharidic, organic acids, lipidic and phenolic profile by means of GC-MS and (1)H NMR. Genuine samples contained mannitol (39-48 g/100 g, according to the grade), fructose (9-16 g/100 g), glucose (2-3.7 g/100 g), sorbitol (0,5-0,6 g/100 g), galactose (0.02-0.74 g/100 g), oligosaccharides as mannotriose (13-22 g/100 g) and stachyose (1-11 g/100 g), and traces of myo-inositol, mannose, sucrose. On the contrary, counterfeit samples contained mostly mannitol and sorbitol, with traces of fructose, glucose and mannose. Differences in ash, total polyphenolic content and fatty acid composition allowed a quick identification of counterfeit products, confirmed by a distinct mono-, oligosaccharidic and phenolic pattern. Elenolic acid (63-1628 mg/kg), tyrosol (15-774 mg/kg), homovanillic acid (2,39-52.8 mg/Kg), dopaol (0.8-63 mg/kg), pinoresinol (4.2-18.5 mg/kg) and fraxetin (0.25-11.64 mg/kg), albeit showing a wide concentration range, were the most abundant substances detected in the phenolic fraction of Fraxinus manna, while esculetin, p-hydroxybenzoic acid, 4-hydroxyphenacetic acid, 3,4 hydroxybenzoic acid, hydroxy-pinoresinol, medioresinol and siringaresinol were present in low amounts. The polyphenolic profile may be used as a marker for authentication and should be considered in the evaluation of nutritional and health properties ascribed to Fraxinus manna.

1H NMR study of fermented cocoa (Theobroma cacao L.) beans.

This study reports for the first time the metabolic profile of cocoa (Theobroma cacao L.) beans using the (1)H NMR technique applied to polar extracts of fermented cocoa beans. The simultaneous detection and quantification of amino acids, polyalcohols, organic acids, sugars, methylxanthines, catechins, and phenols were obtained by assigning the major signals of the spectra for different varieties of cocoa beans (Forastero, Criollo, and Trinitario) from different countries (Ecuador, Ghana, Grenada, and Trinidad). The data set obtained, representative of all classes of soluble compounds of cocoa, was useful to characterize the fermented cocoa beans as a function of the variety and geographic origin.